U.S. patent application number 13/050615 was filed with the patent office on 2011-07-07 for conjugate of an antibody against ccr5 and an antifusogenic peptide.
Invention is credited to Michael Brandt, Stephan Fischer, Erhard Kopetzki, Suryanarayana Sankuratri, Ralf Schumacher.
Application Number | 20110165182 13/050615 |
Document ID | / |
Family ID | 40085689 |
Filed Date | 2011-07-07 |
United States Patent
Application |
20110165182 |
Kind Code |
A1 |
Brandt; Michael ; et
al. |
July 7, 2011 |
CONJUGATE OF AN ANTIBODY AGAINST CCR5 AND AN ANTIFUSOGENIC
PEPTIDE
Abstract
The current invention is related to a conjugate comprising one
or more antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5)
characterized in that one to eight antifusogenic peptides are each
conjugated to one terminus of the heavy and/or light chains of said
anti-CCR5 antibody and to the pharmaceutical use of said
conjugate.
Inventors: |
Brandt; Michael; (Iffeldorf,
DE) ; Fischer; Stephan; (Polling, DE) ;
Kopetzki; Erhard; (Penzberg, DE) ; Sankuratri;
Suryanarayana; (San Jose, CA) ; Schumacher; Ralf;
(Penzberg, DE) |
Family ID: |
40085689 |
Appl. No.: |
13/050615 |
Filed: |
March 17, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11893899 |
Aug 17, 2007 |
|
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13050615 |
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Current U.S.
Class: |
424/178.1 ;
435/69.6; 530/391.7; 530/391.9 |
Current CPC
Class: |
C07K 16/2866 20130101;
C12N 2740/16122 20130101; A61P 31/12 20180101; C07K 2319/00
20130101; C07K 14/005 20130101; A61P 31/18 20180101 |
Class at
Publication: |
424/178.1 ;
530/391.7; 530/391.9; 435/69.6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 19/00 20060101 C07K019/00; A61P 31/18 20060101
A61P031/18; A61P 31/12 20060101 A61P031/12 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 17, 2006 |
EP |
06017156.8 |
Sep 29, 2006 |
EP |
06020647.1 |
Claims
1. A conjugate comprising an anti-CCR5 antibody (mAb CCR5) having
termini comprising an N-terminus and a C-terminus; and an
antifusogenic peptide, conjugated to said N-terminus or said
C-terminus.
2. The conjugate of claim 1, comprising two antifusogenic peptides,
conjugated to said anti-CCR5 antibody termini.
3. The conjugate according to claim 1, wherein said antifusogenic
peptides are linear peptides that comprise an amino acid sequence
of from 5 to 100 amino acids.
4. The conjugate of claim 1, wherein said antifusogenic peptide is
conjugated to said N-terminus or C-terminus through a peptide
linker.
5. The conjugate of claim 1, wherein said mAb CCR5 comprises two
heavy chains and two light chains.
6. The conjugate of claim 5, characterized by the general formula
mAb CCR5-[linker].sub.m-[antifusogenic peptide].sub.n, wherein m is
independently for each antifusogenic peptide either 0 or 1, and n
is an integer of from 1 to 8.
7. The conjugate of claim 6, comprising a conjugate of heavy and/or
light chain of mAb CCR5 and an antifusogenic peptide(s) ("chain
conjugate"), selected from the group consisting of: [antifusogenic
peptide]-[linker].sub.m-[heavy chain]; (1) [heavy
chain]-[linker].sub.m-[antifusogenic peptide]; (2) [antifusogenic
peptide]-[linker].sub.m-[heavy chain]-[antifusogenic peptide]; (3)
[antifusogenic peptide]-[linker].sub.m-[light chain]; (4) [light
chain]-[linker].sub.m-[antifusogenic peptide]; (5) [antifusogenic
peptide]-[linker].sub.m-[light chain]-[antifusogenic peptide]; (6)
[antifusogenic peptide]-[linker].sub.m-[heavy
chain]-[linker].sub.m-[antifusogenic peptide]; (7) and
[antifusogenic peptide]-[linker].sub.m-[light
chain]-[linker].sub.m-[antifusogenic peptide], (8) wherein each
linker can be the same or different, wherein each m is
independently an integer of 1 or 0.
8. The conjugate of claim 7, which comprises a chain conjugate (2),
(3), (4), or (7).
9. The conjugate of claim 8, which comprises: 2.times.[mAb CCR5
light chain] and 2.times.(2), 2.times.[mAb CCR5 light chain] and
2.times.(3), 2.times.[mAb CCR5 heavy chain] and 2.times.(4), or
2.times.[mAb CCR5 light chain] and 2.times.(7).
10. The conjugate of claim 1, wherein said antifusogenic peptide
has a sequence selected from the group SEQ ID NO:29 to 35.
11. The conjugate according of claim 1, wherein said anti-CCR5
antibody comprises a variable heavy chain domain consisting of an
immunoglobulin framework and a CDR3 region selected from the group
consisting of the heavy chain CDR3 sequences SEQ ID NO:16 and SEQ
ID NO:17.
12. The conjugate of claim 1, wherein said anti-CCR5 antibody
comprises a variable heavy chain domain consisting of an
immunoglobulin framework and a CDR3 region selected from the group
consisting of heavy chain CDR3 sequences SEQ ID NOS:16 and 17; a
CDR2 region selected from the group consisting of heavy chain CDR2
sequences SEQ ID NOS:13, 14, and 15; and a CDR1 region selected
from the group consisting of heavy chain CDR1 sequences SEQ ID
NOS:9, 10, 11, and 12.
13. The conjugate of claim 1, wherein said anti-CCR5 antibody
comprises a heavy chain variable domain selected from the group
consisting of SEQ ID NOS:1, 3, 5, and 7.
14. The conjugate of claim 1, wherein said anti-CCR5 antibody
comprises a variable light chain domain consisting of an
immunoglobulin framework and a CDR1 region selected from SEQ ID
NOS:18, 19, and 20; a CDR2 region selected from SEQ ID NOS:21, 22,
and 23; and a CDR3 region selected from SEQ ID NOS:24 and 25.
15. The conjugate of claim 12, wherein said anti-CCR5 antibody
comprises as heavy chain CDRs the CDRs of SEQ ID NO: 1 and as light
chain CDRs the CDRs of SEQ ID NO: 2, as heavy chain CDRs the CDRs
of SEQ ID NO: 3 and as light chain CDRs the CDRs of SEQ ID NO: 4,
as heavy chain CDRs the CDRs of SEQ ID NO: 5 and as light chain
CDRs the CDRs of SEQ ID NO: 6, or as heavy chain CDRs the CDRs of
SEQ ID NO: 7 and as light chain CDRs the CDRs of SEQ ID NO: 8.
16. The conjugate of claim 15, wherein said anti-CCR5 antibody
comprises a variable heavy chain domain and a variable light chain
domain independently selected from the group consisting of a) the
heavy chain (V.sub.H) variable domain defined by amino acid
sequence SEQ ID NO:1 and the light chain (V.sub.L) variable domain
defined by SEQ ID NO:2; b) the heavy chain variable domain defined
by amino acid sequence SEQ ID NO:3 and the light chain variable
domain defined by SEQ ID NO:4; c) the heavy chain variable domain
defined by amino acid sequence SEQ ID NO:5 and the light chain
variable domain defined by SEQ ID NO:6; d) the heavy chain variable
domain defined by amino acid sequence SEQ ID NO:7 and the light
chain variable domain defined by SEQ ID NO:8.
17. The conjugate of claim 1, wherein said conjugate comprises:
said anti-CCR5 antibody, selected from the group consisting of the
heavy chain (V.sub.H) variable domain defined by amino acid
sequence SEQ ID NO:1 and the light chain (V.sub.L) variable domain
defined by SEQ ID NO:2; the heavy chain variable domain defined by
amino acid sequence SEQ ID NO:3 and the light chain variable domain
defined by amino acid SEQ ID NO:4; the heavy chain variable domain
defined by amino acid sequence SEQ ID NO:5 and the light chain
variable domain defined by amino acid SEQ ID NO:6; and the heavy
chain variable domain defined by amino acid sequence SEQ ID NO:7
and the light chain variable domain defined by amino acid SEQ ID
NO:8; a linker selected from the group consisting of amino acids
glycine (G) and asparagine (N), the tripeptide GST, and SEQ ID NO:
36-62; and an antifusogenic peptide having a sequence selected from
the group consisting of SEQ ID NO:29 to 35.
18. The conjugate of claim 1, wherein said antifusogenic peptide
has a sequence selected from the group consisting of SEQ ID NOS:29
to 35.
19. The conjugate of claim 17, wherein said conjugate comprises two
light chain variable domains of SEQ ID NO:2, two conjugates of type
(2) each comprising a heavy chain variable domain of SEQ ID NO:1, a
linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID
NO:33, in comprising two light chain variable domains of SEQ ID
NO:4, two conjugates of type (2) each comprising a heavy chain
variable domain of SEQ ID NO:3, a linker of SEQ ID NO:40 and an
antifusogenic peptide of SEQ ID NO:33, in comprising two light
chain variable domains of SEQ ID NO:6, two conjugates of type (2)
each comprising a heavy chain variable domain of SEQ ID NO:5, a
linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID
NO:33, or in comprising two light chain variable domains of SEQ ID
NO:8, two conjugates of type (2) each comprising a heavy chain
variable domain of SEQ ID NO:7, a linker of SEQ ID NO:40 and an
antifusogenic peptide of SEQ ID NO:33.
20. The conjugate of claim 1, wherein said anti-CCR5 antibody is of
IgG4 subclass, or is of IgG1 or IgG2 subclass with a mutation in
amino acid 5228, L234, L235, and/or D265, and/or contains the
PVA236 mutation
21. A conjugate according to claim 20, wherein said anti-CCR5
antibody of IgG4 subclass has the mutation S228P and said anti-CCR5
antibody of IgG1 subclass has the mutations L234A and L235A.
22. A method producing a conjugate of claim 1, wherein said method
comprises: a) cultivating a cell containing nucleic acid molecules
encoding a conjugate according to claim 1 under conditions suitable
for the expression of the conjugate; and b) recovering the
conjugate from the cell or the cell culture supernatant.
23. A pharmaceutical composition, comprising an effective amount of
a conjugate of claim 1, together with a pharmaceutically acceptable
excipient or carrier.
24. A method for treating a viral infection, comprising:
administering an effective amount of a conjugate of claim 1 to a
subject in need thereof.
25. The method of claim 24, wherein the viral infection is HIV
infection.
Description
PRIORITY
[0001] This application claims priority under 35 USC .sctn.119 from
European Application EP06017156.8 filed Aug. 17, 2006, and
EP06020647.1 filed Sep. 29, 2006, both of which are incorporated
herein by reference in full.
FIELD OF THE INVENTION
[0002] The present invention relates to a conjugate of an antibody
against CCR5 and an anti-fusogenic peptide wherein one to eight
antifusogenic peptides are each conjugated to one terminus of the
heavy and/or light chains of an anti-CCR5 antibody. The
antifusogenic peptides can be different, similar or identical on
the amino acid level.
BACKGROUND OF THE INVENTION
[0003] The infection of cells by the human immunodeficiency virus
(HIV) is effected by a process in which the membrane of the cells
to be infected and the viral membrane are fused. A general scheme
for this process is proposed: The viral envelope glycoprotein
complex (gp120/gp41) interacts with a cell surface receptor located
on the membrane of the cell to be infected. The binding of gp120
to, e.g., the CD4 receptor in combination with a co-receptor such
as CCR-5 or CXCR-4 causes a change in the conformation of the
gp120/gp41 complex. In consequence of this conformational change
the gp41 protein is able to insert into the membrane of the target
cell. This insertion is the beginning of the membrane fusion
process. It is known that the amino acid sequence of the gp41
protein varies in different HIV strains because of naturally
occurring polymorphisms. But the same domain architecture can be
recognized, more precisely, a fusion signal, two heptad repeat
domains (HR1, HR2) and a transmembrane domain (in N- to C-terminal
direction). It is suggested that the fusion (or fusogenic) domain
is participating in the insertion into and the disintegration of
the cell membrane. The HR regions are built up of multiple
stretches comprising seven amino acids ("heptad") (see e.g. W. Shu,
et al., Biochem (1999) 38:5378-85). Beside the heptads one or more
leucine zipper-like motifs are present. This composition accounts
for the formation of a coiled coil structure of gp41 proteins and
just as well of peptides derived from these domains. Coiled coils
are in general oligomers consisting of two or more interacting
helices. Peptides with amino acid sequences deduced from the HR1 or
the HR2 domain of gp41 are effective in vitro and in vivo
inhibitors of HIV uptake into cells (for example peptides see e.g.
U.S. Pat. No. 5,464,933, U.S. Pat. No. 5,656,480, U.S. Pat. No.
6,258,782, U.S. Pat. No. 6,348,568, or U.S. Pat. No. 6,656,906).
For example, T20 (also known as DP178, Fuzeon.RTM., a HR2 peptide),
T651 (U.S. Pat. No. 6,479,055), and T2635 (WO 2004/029074) are very
potent inhibitors of HIV infection. It has been attempted to
enhance the efficacy of HR2 derived peptides with, for example,
amino acid substitutions or chemical crosslinking (S. K. Sia, et
al., Proc. Natl. Acad. Sci. USA (2002) 99:14664-69; A. Otaka, et
al., Angew. Chem. Int. Ed. (2002) 41:2937-40).
[0004] The conjugation of peptides to certain molecules can change
their pharmacokinetic properties, e.g. the serum half-life of such
peptide conjugates can be increased. Conjugations are reported, for
example, for polyethylene glycol (PEG) and Interleukin-6 (EP 0 442
724), for PEG and Erythropoietin (WO 01/02017), for chimeric
molecules comprising Endostatin and immunoglobulins (US
2005-008649), for secreted antibody based fusion proteins (US
2002-147311), for fusion polypeptides comprising albumin (US
2005-0100991; human serum albumin U.S. Pat. No. 5,876,969), for
PEGylated polypeptides (US 2005-0114037), and for interferon
fusions. Also described in the state of the art are immunotoxins
comprising Gelonin and an antibody (WO 94/26910), modified
transferrin-antibody fusion proteins (US 2003-0226155),
antibody-cytokine fusion proteins (US 2003-0049227), and fusion
proteins consisting of a peptide with immuno-stimulatory, membrane
transport, or homophilic activity and an antibody (US
2003-0103984). In WO 2004/085505 long acting biologically active
conjugates consisting of biologically active compounds chemically
linked to macromolecules, are reported.
[0005] The co-receptor CCR5 is used by most HIV-1 primary isolates
and is critical for the establishment and maintenance of infection.
In addition, CCR5 function is dispensable for human health. A
mutant CCR5 allele, "CCR5.DELTA.32", encodes a truncated,
non-functional protein (M. Samson, et al., Nature (1996)
382:722-25; M. Dean, et al., Science (1996) 273:1856-62).
Individuals homozygous for the mutation lack CCR5 expression and
are strongly protected from HIV-1 infection. They demonstrate no
overt phenotype consequence and are highly resistant to M-tropic
HIV infection, whereas heterozygote individuals present delayed
disease progression (M. K. Schwarz and T. N. Wells, Nat. Rev. Drug
Discov. (2002) 1:347-58). The lack of CCR5 is without apparent
adverse consequences, probably because CCR5 is part of a highly
redundant chemokine network as receptor for the .alpha.-chemokines
MIP-1.alpha., MIP-1.beta., and RANTES, which share many overlapping
functions, and most of which have alternative receptors (D. Rossi
and A. Zlotnik, Ann. Rev. Immunol. (2000) 18:217-42). The
identification of CCR5 as an HIV-1 co-receptor was based on the
ability of its ligands, MIP-1.alpha., MIP-1.beta., and RANTES, to
block infection by R5 but not R5X4 or X4 isolates (F. Cocchi, et
al., Science (1995) 270:1811-15). CCR5 is also a receptor of the
"cluster" chemokines, which are produced primarily during
inflammatory responses and control the recruitment of neutrophils
(CXC chemokines), macrophages and a subset of T cells (T helper Th1
and Th2 cells). Th1 responses are typically those involving
cell-mediated immunity effective against viruses and tumors,
proinflammatory responses responsible for killing intracellular
parasites, and perpetuating autoimmune responses, for example,
whereas Th2 responses are believed to be pivotal in allergies.
Therefore, inhibitors of these chemokine receptors may be useful as
immunomodulators. For Th1 responses, overactive responses are
dampened, for example, in autoimmunity including rheumatoid
arthritis, or, for Th2 responses, asthma attacks or allergic
responses including atopic dermatitis are lessened (see e.g. D.
Schols, Curr. Top. Med. Chem. (2004) 4:883-93; A. Mueller and P. G.
Strange, Int. J. Biochem. Cell Biol. 36:35-38; W. M. Kazmierski et
al., Curr. Drug Targets Infect. Disord. (2002) 2:265-78; T. Lehner,
Trends Immunol. (2002) 23:347-51).
[0006] Antibodies against human CCR5 are e.g. PRO 140 (W. C. Olson
et al., J. Virol. (1999) 73:4145-55), and/or 2D7 (M. Samson et al.,
J. Biol. Chem. (1997) 272:24934-41). Additional antibodies are
mentioned in US 2004-0043033, U.S. Pat. No. 6,610,834, US
2003-0228306, US 2003-0195348, US 2003-0166870, US 2003-0166024, US
2003-0165988, US 2003-0152913, US 2003-0100058, US 2003-0099645, US
2003-0049251, US 2003-0044411, US 2003-0003440, U.S. Pat. No.
6,528,625, US 2002-0147147, US 2002-0146415, US 2002-0106374, US
2002-0061834, US 2002-0048786, US 2001/0000241, EP 1 322 332, EP 1
263 791, EP 1 207 202, EP 1 161 456, EP 1 144 006, WO 2003/072766,
WO 2003/066830, WO 2003/033666, WO 2002/083172, WO 02/22077, WO
01/58916, WO 01/58915, WO 01/43779, WO 01/42308, and EP
05007138.0.
[0007] Polyethylene glycol conjugates of antibodies against CCR5
are known from US 2003-0228306. US 2003-0215421 refers to
chemokine-toxin conjugates. WO 01/43779 refers to conjugates of
anti-CD4 antibodies and anti-CCR5 antibodies and to conjugates of
anti-CD4 antibodies and an HIV-1 fusion inhibiting peptide.
Conjugates of CCR5 antibodies and toxins are mentioned in EP 1 346
731.
SUMMARY OF THE INVENTION
[0008] The invention comprises a conjugate comprising one or more
antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5)
characterized in that one to eight antifusogenic peptides are each
conjugated to one terminus of the heavy and/or light chains of said
anti-CCR5 antibody (a number of eight antifusogenic peptides per
mAb CCR5 is only possible if the mAb CCR5 comprises eight termini,
i.e. is composed e.g. of two heavy chains and two light chains; if
the mAb CCR5 comprises a smaller number of C- and N-termini, e.g.
as a scFv, the corresponding number of antifusogenic peptides
possible at maximum in the conjugate is also reduced, i.e. it is
reduced to less than eight).
[0009] Preferably the carboxy-terminal amino acid of an anti-CCR5
antibody chain is conjugated to the amino-terminal amino acid of
the antifusogenic peptide or the carboxy-terminal amino acid of the
antifusogenic peptide is conjugated to the amino-terminal amino
acid of the antibody chain, preferably by a peptide bond with or
without an intermediate linker.
[0010] Preferably the conjugate is characterized by the general
formula
mAb CCR5-[linker].sub.m-[antifusogenic peptide].sub.n
wherein m is independently for each antifusogenic peptide either 0
(i.e. a peptide bond between mAb CCR5 and antifusogenic peptide) or
1 (i.e. a linker between mAb CCR5 and antifusogenic peptide) and n
is an integer of from 1 to 8.
[0011] A preferred conjugate of a heavy and/or light chain of mAb
CCR5 and an antifusogenic peptide ("chain conjugate") is selected
from the group consisting of:
[antifusogenic peptide]-[linker].sub.m-[heavy chain] (1)
[heavy chain]-[linker].sub.m-[antifusogenic peptide] (2)
[antifusogenic peptide]-[linker].sub.m-[heavy chain]-[antifusogenic
peptide] (3)
[antifusogenic peptide]-[linker].sub.m-[light chain] (4)
[light chain]-[linker].sub.m-[antifusogenic peptide] (5)
[antifusogenic peptide]-[linker].sub.m-[light chain]-[antifusogenic
peptide] (6)
[antifusogenic peptide]-[linker].sub.m-[heavy
chain]-[linker].sub.m-[antifusogenic peptide] (7)
[antifusogenic peptide]-[linker].sub.m-[light
chain]-[linker].sub.m-[antifusogenic peptide] (8)
wherein the linker can be the same or different in (within and
between) said chain conjugates, wherein m is an integer of 1 or 0,
and m can be independently the same or different in (within and
between) said chain conjugates.
[0012] ("Left side" of the peptide or mAb CCR5 chain means
N-terminus, "right side" means C-terminus. In (1) therefore the
C-terminus of the antifusogenic peptide is linked by a peptide bond
or a linker to the N-terminus of the heavy chain of mAb CCR5).
[0013] Preferably the chain conjugates are assembled to conjugates
according to the invention comprising a mAb CCR5 (e.g. consisting
of two light chains and two heavy chains including the constant Fc
domains, a scFv fragment, or a Fab fragment).
[0014] Especially preferred chain conjugates are (2), (3), (4), and
(7). Especially preferred conjugates according to the invention
comprise 2.times.[mAb CCR5 light chain] and 2.times.(2),
2.times.[mAb CCR5 light chain] and 2.times.(3), or 2.times.[mAb
CCR5 heavy chain] and 2.times.(4), or 2.times.[mAb CCR5 light
chain] and 2.times.(7). The heavy and/or light chain comprises
preferably a constant region (Fc).
[0015] Preferably the conjugate is characterized in comprising a
variable heavy chain domain consisting of an immunoglobulin
framework and a CDR3 region selected from the group consisting of
the heavy chain CDR3 sequences SEQ ID NO: 16, 17.
[0016] Preferably the conjugate is characterized in comprising a
variable heavy chain domain consisting of an immunoglobulin
framework and a CDR3 region selected from the group consisting of
CDR3 sequences SEQ ID NO: 16, 17, a CDR2 region selected from the
group consisting of CDR2 sequences SEQ ID NO: 13, 14, 15, and a
CDR1 region selected from the group consisting of CDR1 sequences
SEQ ID NO: 9, 10, 11, 12.
[0017] Preferably the conjugate is characterized in comprising a
heavy chain variable domain selected from the group of heavy chain
variable domains comprising SEQ ID NO: 1, 3, 5, and 7.
[0018] Preferably the conjugate is characterized in comprising a
variable light chain domain consisting of an immunoglobulin
framework and a CDR1 region selected from SEQ ID NO:18, 19, 20, a
CDR2 region selected from SEQ ID NO:21, 22, 23, and a CDR3 region
selected from SEQ ID NO:24, 25.
[0019] Preferably the conjugate is characterized in comprising as
heavy chain CDRs the CDRs of SEQ ID NO:1 and as light chain CDRs
the CDRs of SEQ ID NO:2, as heavy chain CDRs the CDRs of SEQ ID
NO:3 and as light chain CDRs the CDRs of SEQ ID NO:4, as heavy
chain CDRs the CDRs of SEQ ID NO:5 and as light chain CDRs the CDRs
of SEQ ID NO:6, or as heavy chain CDRs the CDRs of SEQ ID NO:7 and
as light chain CDRs the CDRs of SEQ ID NO:8.
[0020] Preferably the conjugate is characterized in comprising a
variable heavy and light chain domain independently selected from
the group consisting of [0021] a) the heavy chain (V.sub.H)
variable domain defined by amino acid sequence SEQ ID NO:1 and the
light chain (V.sub.L) variable domain defined by amino acid
sequence SEQ ID NO:2; [0022] b) the heavy chain variable domain
defined by amino acid sequence SEQ ID NO:3 and the light chain
variable domain defined by amino acid sequence SEQ ID NO:4; [0023]
c) the heavy chain variable domain defined by amino acid sequence
SEQ ID NO:5 and the light chain variable domain defined by amino
acid sequence SEQ ID NO:6; [0024] d) the heavy chain variable
domain defined by amino acid sequence SEQ ID NO:7 and the light
chain variable domain defined by amino acid sequence SEQ ID
NO:8.
[0025] Preferably the conjugate is characterized in comprising the
heavy chain (V.sub.H) variable domain defined by amino acid
sequence SEQ ID NO:1 and the light chain (V.sub.L) variable domain
defined by amino acid sequence SEQ ID NO:2; or the heavy chain
variable domain defined by amino acid sequence SEQ ID NO:3 and the
light chain variable domain defined by amino acid sequence SEQ ID
NO:4; or the heavy chain variable domain defined by amino acid
sequence SEQ ID NO:5 and the light chain variable domain defined by
amino acid sequence SEQ ID NO:6; or the heavy chain variable domain
defined by amino acid sequence SEQ ID NO:7 and the light chain
variable domain defined by amino acid sequence SEQ ID NO:8; a
linker selected from the group consisting of the amino acids
glycine (G) and asparagine (N), the tripeptide GST, and SEQ ID
NO:36-62; and an antifusogenic peptide selected from the group of
peptides defined by SEQ ID NO:29 to 35.
[0026] Preferably the conjugate is characterized in comprising an
antifusogenic peptide selected from the group of peptides
comprising C34, T20, T1249, T651, T2635, N36, and DP107.
[0027] Preferably the conjugate is characterized in comprising an
antifusogenic peptide at each C-terminus of the heavy chains or at
each N-terminus of the light chains (two antifusogenic peptides).
Preferably the conjugate is characterized in that it comprises an
antifusogenic peptide at each C-terminus of the heavy chains and at
each N-terminus of the light chains (four antifusogenic
peptides).
[0028] Preferably the conjugate is characterized in comprising two
light chain variable domains of SEQ ID NO:2, two conjugates of type
(2) each comprising a heavy chain variable domain of SEQ ID NO:1, a
linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID
NO:33, in comprising two light chain variable domains of SEQ ID
NO:4, two conjugates of type (2) each comprising a heavy chain
variable domain of SEQ ID NO:3, a linker of SEQ ID NO:40 and an
antifusogenic peptide of SEQ ID NO:33, in comprising two light
chain variable domains of SEQ ID NO:6, two conjugates of type (2)
each comprising a heavy chain variable domain of SEQ ID NO:5, a
linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID
NO:33, or in comprising two light chain variable domains of SEQ ID
NO:8, two conjugates of type (2) each comprising a heavy chain
variable domain of SEQ ID NO:7, a linker of SEQ ID NO:40 and an
antifusogenic peptide of SEQ ID NO:33.
[0029] Preferably the conjugate is characterized in that said
anti-CCR5 antibody is of IgG1 subclass. It is also preferred, that
said anti-CCR5 antibody is of IgG4 subclass, or of IgG1 or IgG2
subclass, with a mutation in amino acid position 5228, L234, L235,
and/or D265, and/or contains the PVA236 mutation. Preferably the
conjugate is characterized in that said anti-CCR5 antibody of IgG4
subclass has a S228P mutation and said anti-CCR5 antibody of IgG1
subclass has L234A and L235A mutations.
[0030] The invention comprises a method for the production of a
conjugate according to the invention, characterized in that the
method comprises [0031] a) cultivating a cell containing one or
more plasmids containing one or more nucleic acid molecules
encoding a conjugate according to the invention under conditions
suitable for the expression of the conjugate, [0032] b) recovering
the conjugate from the cell or the supernatant.
[0033] In one embodiment are the genes encoding the light and heavy
chains of mAb CCR5 with or without linked antifusogenic peptide
located on the same expression vector or on different expression
vectors.
[0034] The invention comprises a pharmaceutical composition,
containing a conjugate according to the invention, together with a
pharmaceutically acceptable excipient or carrier.
[0035] The invention comprises the use of a conjugate according to
the invention for the manufacture of a medicament for the treatment
of viral infections. Preferably the use is characterized in that
the viral infection is a HIV infection.
[0036] The invention comprises the use of a conjugate according to
the invention for the treatment of a patient in need of an
antiviral treatment, preferably an anti HIV treatment.
DESCRIPTION OF THE INVENTION
[0037] The current invention reports a conjugate comprising one or
more antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5)
characterized in that one to eight antifusogenic peptides are each
conjugated to one terminus of the heavy and/or light chains of said
anti-CCR5 antibody. A number of eight antifusogenic peptides per
mAb CCR5 is only possible if the mAb CCR5 comprises eight termini,
i.e. is composed e.g. of two heavy chains and two light chains. If
the mAb CCR5 comprises a smaller number of C- and N-termini, e.g.
as a scFv, the corresponding number of antifusogenic peptides
possible at maximum in the conjugate is also reduced, i.e. it is
reduced to less than eight.
[0038] An "antifusogenic peptide" is a peptide which inhibits
events associated with membrane fusion or the membrane fusion event
itself, including, among other things, the inhibition of infection
of uninfected cells by a virus due to membrane fusion. These
antifusogenic peptides are preferably linear peptides. For example,
they can be derived from the gp41 ectodomain, e.g. such as DP107,
DP178. Examples of such peptides can be found in U.S. Pat. No.
5,464,933, U.S. Pat. No. 5,656,480, U.S. Pat. No. 6,013,263, U.S.
Pat. No. 6,017,536, U.S. Pat. No. 6,020,459, U.S. Pat. No.
6,093,794, U.S. Pat. No. 6,060,065, U.S. Pat. No. 6,258,782, U.S.
Pat. No. 6,348,568, U.S. Pat. No. 6,479,055, U.S. Pat. No.
6,656,906, WO 1996/19495, WO 1996/40191, WO 1999/59615, WO
2000/69902, and WO 2005/067960. For example, the amino acid
sequences of such peptides comprise or can be selected from the
group of SEQ ID NO:1 to 10 of U.S. Pat. No. 5,464,933; SEQ ID NO:1
to 15 of U.S. Pat. No. 5,656,480; SEQ ID NO:1 to 10 and 16 to 83 of
U.S. Pat. No. 6,013,263; SEQ ID NO:1 to 10, 20 to 83 and 139 to 149
of U.S. Pat. No. 6,017,536; SEQ ID NO:1 to 10, 17 to 83 and 210 to
214 of U.S. Pat. No. 6,093,794; SEQ ID NO:1 to 10, 16 to 83 and 210
to 211 of U.S. Pat. No. 6,060,065; SEQ ID NO:1286 and 1310 of U.S.
Pat. No. 6,258,782; SEQ ID NO:1129, 1278-1309, 1311 and 1433 of
U.S. Pat. No. 6,348,568; SEQ ID NO:1 to 10 and 210 to 238 of U.S.
Pat. No. 6,479,055; SEQ ID NO:1 to 171, 173 to 216, 218 to 219, 222
to 228, 231, 233 to 366, 372 to 398, 400 to 456, 458 to 498, 500 to
570, 572 to 620, 622 to 651, 653 to 736, 739 to 785, 787 to 811,
813 to 823, 825, 827 to 863, 865 to 875, 877 to 883, 885, 887 to
890, 892 to 981, 986 to 999, 1001 to 1003, 1006 to 1018, 1022 to
1024, 1026 to 1028, 1030 to 1032, 1037 to 1076, 1078 to 1079, 1082
to 1117, 1120 to 1176, 1179 to 1213, 1218 to 1223, 1227 to 1237,
1244 to 1245, 1256 to 1268, 1271 to 1275, 1277, 1345 to 1348, 1350
to 1362, 1364, 1366, 1368, 1370, 1372, 1374 to 1376, 1378 to 1379,
1381 to 1385, 1412 to 1417, 1421 to 1426, 1428 to 1430, 1432, 1439
to 1542, 1670 to 1682, 1684 to 1709, 1712 to 1719, 1721 to 1753,
1755 to 1757 of U.S. Pat. No. 6,656,906; or SEQ ID NO:5 to 95 of WO
2005/067960. The antifusogenic peptide has an amino acid sequence
comprising of from 5 to 100 amino acids, preferably of from 10 to
75 amino acids and more preferred of from 15 to 50 amino acids.
Especially preferred antifusogenic peptides are C-34, T-20, T-1249,
T-651, T-2635, N-36, (M. J. Root et al., Curr. Pharm. Des. (2004)
10:1805-25) and DP-107 (C. Wild et al., Proc. Natl. Acad. Sci. USA
(1994) 91:12676-80). One embodiment comprises the conjugate
according to the invention one or more antifusogenic peptides and
an anti-CCR5 antibody (mAb CCR5) wherein i) said antifusogenic
peptides are linear peptides with an amino acid sequence of from 5
to 100 amino acids, and ii) one to eight antifusogenic peptides are
each conjugated to one terminus of the heavy and/or light chains of
said anti-CCR5 antibody. Another embodiment comprises the conjugate
according to the invention having one or more antifusogenic
peptides and an anti-CCR5 antibody (mAb CCR5) wherein i) said
antifusogenic peptides are derived from the gp41 ectodomain, and
ii) one to eight antifusogenic peptides are each conjugated to one
terminus of the heavy and/or light chains of said anti-CCR5
antibody. The term "gp41 ectodomain" denotes the amino acid
sequence starting with amino acid position 561 and ending with
amino acid position 620 of HIV-1 gp160 or starting with amino acid
position 50 and ending with amino acid position 109 of HIV-1 gp41
(SEQ ID NO:66) (see also e.g. S. Bar and M. J. Alizon, Virol.
(2004) 78:811-20).
[0039] The term "antibody" encompasses the various forms of
antibody structures including whole antibodies and antibody
fragments. The antibody according to the invention is preferably a
human antibody, a humanized antibody, a chimeric antibody, a T cell
antigen depleted antibody (WO 98/33523, WO 98/52976, and WO
00/34317). Genetic engineering of antibodies is e.g. described in
S. L. Morrison et al., Proc. Natl. Acad Sci. USA (1984) 81:6851-55;
U.S. Pat. Nos. 5,202,238 and 5,204,244; L. Riechmann et al., Nature
(1988) 332:323-27; M. S. Neuberger et al., Nature (1985)
314:268-70; N. Lonberg, Nat. Biotechnol. (2005) 23:1117-25.
[0040] "Antibody fragments" comprise a portion of a full length
anti-CCR5 antibody, preferably the variable domains thereof or at
least the antigen binding portion thereof. Examples of anti-body
fragments are e.g. single-chain antibody molecules (scFv), Fab,
F(ab).sub.2 fragments, and the like as long as they retain the
binding characteristics of an anti-CCR5 antibody. ScFv antibodies
are, e.g., described in J. S. Huston, Meth. Enzymol. (1991)
203:46-88. Huston also describes linkers and methods for linking of
polypeptides useful for the present invention.
[0041] "CCR5" means human CCR5 as described, e.g., in M. Oppermann,
Cell Signal. (2004) 16:1201-10 and SwissProt P51681. The terms
"antibody binding to CCR5", "anti-CCR5 antibody", or "mAb CCR5",
which are used interchangeably within this application, mean an
antibody specifically binding to CCR5 and preferably inhibiting HIV
fusion with a target cell. Binding can be tested in a cell based in
vitro ELISA assay (CCR5 expressing CHO cells). Binding is found if
the antibody causes an S/N (signal/noise) ratio of 5 or more,
preferably 10 or more at an antibody concentration of 100 ng/ml.
The term "inhibiting HIV fusion with a target cell" refers to
inhibiting HIV fusion with a target cell measured in an assay
comprising contacting said target cell (e.g. PBMC) with the virus
in the presence of the antibody in a concentration effective to
inhibit membrane fusion between the virus and said cell and
measuring e.g. luciferase reporter gene activity or the HIV p24
antigen concentration. The term "membrane fusion" refers to fusion
between a first cell coexpressing CCR5 and CD4 polypeptides and a
second cell or virus expressing an HIV env protein. Membrane fusion
is determined by genetically engineered cells and/or viruses by a
reporter gene assay (e.g. by luciferase reporter gene assay).
[0042] Preferred anti-CCR5 antibodies are mentioned in US
2004-0043033, U.S. Pat. No. 6,610,834, US 2003-0228306, US
2003-0195348, US 2003-0166870, US 2003-0166024, US 2003-0165988, US
2003-0152913, US 2003-0100058, US 2003-0099645, US 2003-0049251, US
2003-0044411, US 2003-0003440, U.S. Pat. No. 6,528,625, US
2002-0147147, US 2002-0146415, US 2002-0106374, US 2002-0061834, US
2002-0048786, US 2001/0000241, EP 1 322 332, EP 1 263 791, EP 1 207
202, EP 1 161 456, EP 1 144 006, WO 2003/072766, WO 2003/066830, WO
2003/033666, WO 2002/083172, WO 02/22077, WO 01/58916, WO 01/58915,
WO 01/43779, WO 01/42308, and WO 2006/103100. Especially preferred
anti-CCR5 antibodies are described in WO 2006/103100. An especially
preferred anti-CCR5 antibody is characterized in that the antibody
comprises a variable heavy chain domain consisting of an
immunoglobulin framework and a CDR3 region selected from the group
consisting of the heavy chain CDR3 sequences SEQ ID NO:16, 17. A
further preferred antibody comprises a variable heavy chain region
consisting of an immunoglobulin framework and a CDR3 region
selected from the group consisting of CDR3 sequences SEQ ID NO:16,
17, a CDR2 region selected from the group consisting of CDR2
sequences SEQ ID NO:13, 14, 15, and a CDR1 region selected from the
group consisting of CDR1 sequences SEQ ID NO:9, 10, 11, 12.
Preferred heavy chain variable domains are shown in SEQ ID NO:1, 3,
5, 7. A preferred anti-CCR5 antibody comprises in addition a
variable light chain domain consisting of an immunoglobulin
framework and a CDR1 region selected from the group consisting of
CDR1 sequences SEQ ID NO:18, 19, 20, a CDR2 region selected from
the group consisting of CDR2 sequences SEQ ID NO:21, 22, 23, and a
CDR3 region selected from the group of CDR3 sequences SEQ ID NO:24,
25. The anti-CCR5 antibody is preferably characterized in
containing as heavy chain CDRs the CDRs of SEQ ID NO:1 and as light
chain CDRs the CDRs of SEQ ID NO:2, as heavy chain CDRs the CDRs of
SEQ ID NO:3 and as light chain CDRs the CDRs of SEQ ID NO:4, as
heavy chain CDRs the CDRs of SEQ ID NO:5 and as light chain CDRs
the CDRs of SEQ ID NO:6, or as heavy chain CDRs the CDRs of SEQ ID
NO:7 and as light chain CDRs the CDRs of SEQ ID NO:8.
[0043] CDR sequences can be determined according to the standard
definition of E. A. Kabat et al., Sequences of Proteins of
Immunological Interest, 5th ed., Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). CDRs of SEQ ID NO:1-8
are shown in SEQ ID NO:9-25.
[0044] The anti-CCR5 antibody comprises preferably a variable heavy
and light chain domain independently selected from the group
consisting of [0045] a) the heavy chain (V.sub.H) variable domain
defined by amino acid sequence SEQ ID NO:1 and the light chain
(V.sub.L) variable domain defined by SEQ ID NO:2; [0046] b) the
heavy chain variable domain defined by amino acid sequence SEQ ID
NO:3 and the light chain variable domain defined by SEQ ID NO:4;
[0047] c) the heavy chain variable domain defined by amino acid
sequence SEQ ID NO:5 and the light chain variable domain defined by
SEQ ID NO:6; [0048] d) the heavy chain variable domain defined by
amino acid sequence SEQ ID NO:7 and the light chain variable domain
defined by SEQ ID NO:8.
[0049] The antibody used in the conjugate according to the
invention is preferably characterized in that the constant domains
are of human origin. Such constant domains are well known in the
state of the art and, e.g., described by Kabat (see e.g. G. Johnson
and T. T. Wu, Nucleic Acids Res. (2000) 28:214-18). For example, a
useful human IgG1 heavy chain constant region
(C.sub.H1-Hinge-C.sub.H2-C.sub.H3) comprises an amino acid sequence
independently selected from the group consisting of SEQ ID NO:26,
27. For example, a useful human kappa (.kappa.) light chain
constant domain comprises an amino acid sequence of a kappa light
chain constant domain (.kappa. light chain constant domain,
C.sub.L) of SEQ ID NO:28. It is further preferred that the
antibody's variable domains are of mouse origin and comprises the
antibody variable domain sequence frame of a mouse antibody
according to Kabat (see e.g. G. Johnson and T. T. Wu, supra).
[0050] A preferred anti-CCR5 antibody shows a binding to the same
epitope(s) of CCR5 as does an antibody selected from the group
consisting of the antibodies A to E or is inhibited in binding to
CCR5 by antibodies A to E due to steric hindrance of binding or
competitive binding. Epitope binding is investigated by using
alanine scanning according to the method described by W. C. Olson
et al. (J. Virol. (1999) 73:4145-55) for epitope mapping. A signal
reduction of 75% or more shows that the mutated amino acid(s)
contribute to the epitope recognized by said antibody. Binding of
the antibody to the same epitope is found, if the amino acids
contributing to the epitope are recognized by the investigated
antibody and antibody A, B, C, D, or E. Antibody C, which shows
lower IC.sub.50 values than antibody 2D7 in HIV assays, binds to an
epitope including amino acids on the ECL2 domain of CCR5 (B. Lee et
al., J. Biol. Chem. (1999) 274:9617-26) which is different from the
epitope recognized by antibody 2D7 (2D7 binds to amino acids K171
and E172 of ECL2A but not to ECL2B amino acids 184-189). Epitope
binding for antibody C is found to be 20% for CCR5 mutant K171A or
E172A (glu 172 is mutated to ala). 100% epitope binding is defined
for wild-type CCR5. A further preferred anti-CCR5 antibody binds to
the same epitope as antibody C binds.
[0051] The term "epitope" means a protein determinant capable of
specific binding to an antibody. Epitopes usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Conformational and non-conformational epitopes are
distinguished in that the binding to the former but not the latter
is lost in the presence of denaturing solvents. Preferably an
antibody according to the invention binds specifically to native
but not to denatured CCR5. Such an antibody comprises preferably
heavy chain CDR3 of SEQ ID NO:17, and preferably in addition heavy
chain CDRs selected from the group of CDRs of SEQ ID NO:10, 11, 12,
14 and/or 15. Preferably such an antibody is antibody B, C, D, or
E, or comprises the variable domains of antibody B, C, D, or E.
Preferably an antibody binding to denatured CCR5 is antibody A or
comprises the variable domains of antibody A.
[0052] The term "variable domain" (variable domain of a light chain
(V.sub.L), variable domain of a heavy chain (V.sub.H)) as used
herein denotes each domain of the pair of light and heavy chain
domains which is involved directly in the binding of the antibody
to the antigen. The variable domains of the light and heavy chain
have the same general structure, i.e. they possess an
"immunoglobulin framework", and each domain comprises four
"framework regions" (FR), whose sequences are widely conserved,
connected by three "hypervariable regions" (or "complementarity
determining regions", CDRs). The framework regions adopt a
.beta.-sheet conformation and the CDRs may form loops connecting
the .beta.-sheet structure. The CDRs in each chain are held in
their three-dimensional structure by the framework regions and form
together with the CDRs from the other chain the antigen binding
site. The antibody heavy and light chain CDR3 regions play a
particularly important role in the binding specificity/affinity of
the antibodies according to the invention and therefore provide a
further object of the invention.
[0053] The terms "antigen-binding portion of an antibody" or
"antigen-binding site of an antibody" when used herein refer to the
amino acid residues of an antibody which are responsible for
antigen-binding. The antigen-binding site of an antibody comprises
amino acid residues from the "complementarity determining regions"
or "CDRs". "Framework" or "FR" regions are those variable domain
regions other than the hypervariable region residues as herein
defined. Therefore, the light and heavy chain variable domains of
an antibody comprise from N- to C-terminus the regions FR1, CDR1,
FR2, CDR2, FR3, CDR3, and FR4 (immunoglobulin framework).
Especially, the CDR3 region of the heavy chain is the region which
contributes most to antigen binding and defines the antibody.
Preferably the antibody according to the invention is characterized
by comprising in its heavy chain variable domain the CDR3 sequence
of SEQ ID NO:16 or SEQ ID NO:17. Complementarity determining (CDR)
and framework (FR) regions are determined according to the standard
definition of E. A. Kabat et al., Sequences of Proteins of
Immunological Interest, 5th ed., Public Health Service, National
Institutes of Health, Bethesda, Md. (1991).
[0054] The "Fc part" of an anti-CCR5 antibody is not involved
directly in binding to CCR5, but exhibit various effector
functions. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies or immunoglobulins are
divided in the classes: IgA, IgD, IgE, IgG, and IgM, and several of
these may be further divided into subclasses (isotypes), e.g. IgG1,
IgG2, IgG3, and IgG4, IgA1 and IgA2. According to the heavy chain
constant regions the different classes of immunoglobulins are
called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The antibodies according to the invention are
preferably of IgG type. An "Fc part of an antibody" is a term well
known to the skilled artisan and defined on basis of papain
cleavage of antibodies. The antibodies according to the invention
contain as Fc part a human Fc part or an Fc part derived from human
origin. In a further embodiment of the invention the Fc part is
either an Fc part of a human antibody of the subclass IgG4 or an Fc
part of a human antibody of the subclass IgG1, IgG2, or IgG3, which
is modified in such a way that no Fc.gamma. receptor (e.g.
Fc.gamma.RIIIa) binding and/or no C1q binding as defined below can
be detected. Preferably the Fc part is a human Fc part and
especially preferred either from human IgG4 subclass or a mutated
Fc part from human IgG1 subclass. Further preferred are Fc parts
from human IgG1 subclass with mutations L234A and L235A. Further
preferred are Fc parts shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ
ID NO: 26 with mutations L234A and L235A, SEQ ID NO: 27 with
mutation S228P. While IgG4 shows reduced Fc.gamma. receptor
(Fc.gamma.RIIIa) binding, antibodies of other IgG subclasses show
strong binding. However Pro238, Asp265, Asp270, Asn297 (loss of Fc
carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253,
Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues
which if altered provide also reduced Fc.gamma. receptor binding
(R. L. Shields et al., J. Biol. Chem. (2001) 276:6591-604; J. Lund
et al., FASEB J. (1995) 9:115-19; A. Morgan et al., Immunol. (1995)
86:319-24; EP 0 307 434). Preferably an antibody according to the
invention is in regard to Fc.gamma. receptor binding of IgG4
subclass or of IgG1 or IgG2 subclass, with a mutation in L234,
L235, and/or D265, and/or contains the PVA236 mutation. Preferred
are the mutations S228P, L234A, L235A, L235E, and/or PVA236 (PVA236
means that the amino acid sequence ELLG (given in one letter amino
acid code) from amino acid position 233 to 236 of IgG1 or EFLG of
IgG4 is replaced by PVA). Especially preferred are the mutations
S228P of IgG4, and L234A and L235A of IgG1. The Fc part of an
antibody is directly involved in ADCC (antibody-dependent
cell-mediated cytotoxicity) and CDC (complement-dependent
cytotoxicity). Complement activation (CDC) is initiated by binding
of complement factor C1q to the Fc part of most IgG antibody
subclasses. Binding of C1q to an antibody is caused by defined
protein-protein interactions at the so called binding site. Such Fc
part binding sites are known in the state of the art and described
e.g. by T. J. Lukas et al., J. Immunol. (1981) 127:2555-60; R.
Brunhouse and J. J. Cebra, Mol. Immunol. (1979) 16:907-17; D. R.
Burton et al., Nature (1980) 288:338-44; J. E. Thommesen et al.,
Mol. Immunol. (2000) 37:995-1004; E. E. Idusogie et al., J.
Immunol. (2000) 164:4178-84; M. Hezareh et al., J. Virol. (2001)
75:12161-68; A. Morgan et al., Immunol. (1995) 86:319-24; and EP 0
307 434. Such Fc part binding sites are, e.g., characterized by the
amino acids L234, L235, D270, N297, E318, K320, K322, P331, and
P329 (numbering according to EU index of Kabat). Antibodies of
subclass IgG1, IgG2, and IgG3 usually show complement activation
including C1q and C3 binding, whereas IgG4 does not activate the
complement system and does not bind C1q and C3. An anti-CCR5
antibody which does not bind Fc.gamma. receptor and/or complement
factor C1q does not elicit antibody-dependent cellular cytotoxicity
(ADCC) and/or complement dependent cytotoxicity (CDC). Preferably,
this antibody is characterized in that it binds CCR5, contains an
Fc part derived from human origin, and does not bind Fc.gamma.
receptors and/or complement factor C1q. More preferably, this
antibody is a human, or humanized, or a T-cell antigen depleted
antibody. C1q binding can be measured according to Idusogie, E. E.,
et al., J. Immunol. 164 (2000) 4178-4184. No "C1q binding" is found
if in such an assay the optical density (OD) at 492-405 nm is for
the test antibody lower than 15% of the value for human C1q binding
of the unmodified wild-type antibody Fc part at an antibody
concentration of 8 .mu.g/ml. ADCC can be measured as binding of the
antibody to human Fc.gamma.RIIIa on human NK cells. Binding is
determined at an antibody concentration of 20 .mu.g/ml. "No
Fc.gamma. receptor binding" or "no ADCC" means a binding of up to
30% to human Fc.gamma.RIIIa on human NK cells at an antibody
concentration of 20 .mu.g/ml compared to the binding of the same
antibody as human IgG1 (SEQ ID NO:26).
[0055] An antibody used in a conjugate according to the invention
include, in addition, such antibodies having "conservative sequence
modifications" (variant antibodies), which are amino acid sequence
modifications which do not affect or alter the above-mentioned
characteristics of the antibody according to the invention.
Modifications can be introduced by standard techniques known in the
art, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Conservative amino acid substitutions include ones in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g. lysine, arginine,
histidine), acidic side chains (e.g. aspartic acid, glutamic acid),
uncharged polar side chains (e.g. glycine, asparagine, glutamine,
serine, threonine, tyrosine, cysteine, tryptophan), non-polar side
chains (e.g. alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine), beta-branched side chains (e.g.
threonine, valine, isoleucine), and aromatic side chains (e.g.
tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted
nonessential amino acid residue in a human anti-CCR5 antibody can
be preferably replaced with another amino acid residue from the
same side chain family. A "variant" anti-CCR5 antibody, refers
therefore herein to a molecule which differs in amino acid sequence
from a "parent" anti-CCR5 antibody's amino acid sequence by up to
ten, preferably from about two to about five, additions, deletions,
and/or substitutions in one or more of the variable domain regions
of the parent antibody outside the heavy chain CDR3 region. Each
other heavy chain CDR region comprises at maximum one single amino
acid addition, deletion, and/or substitution. The invention
comprises a method of modifying the CDR amino acid sequence of a
parent antibody binding to CCR5, characterized in selecting a heavy
chain variable domain from the group of heavy chain variable
domains consisting of SEQ ID NO:1, 3, 5, 7, and/or a light chain
variable domain from the group of light chain variable domains
consisting of SEQ ID NO:2, 4, 6, 8, providing a nucleic acid
encoding said initial variable domain amino acid sequence,
modifying said nucleic acid in that one amino acid is modified in
heavy chain CDR1, one amino acid is modified in heavy chain CDR2,
1-3 amino acid are modified in light chain CDR1, 1-3 amino acids
are modified in light chain CDR2, and/or 1-3 amino acids are
modified in light chain CDR3, expressing and incorporating said
modified variable domain(s) amino acid sequence in an antibody
structure, measuring whether said antibody binds to CCR5 and
selecting said modified variable domain(s)/CDR(s) if the antibody
binds to CCR5. Preferably such modifications are conservative
sequence modifications. Amino acid sequence modifications can be
performed by mutagenesis based on molecular modeling as described
by L. Riechmann et al., Nature (1988) 332:323-27, and C. Queen et
al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-33.
[0056] The term "linker" or "peptidic linker" as used within this
application denotes peptide linkers of natural and/or synthetic
origin. They are building up of a linear amino acid chain wherein
the 20 naturally occurring amino acids are the monomeric building
blocks. The chain has a length of from 1 to 50 amino acids,
preferred between 1 and 28 amino acids, especially preferred
between 3 and 25 amino acids. The linker may contain repetitive
amino acid sequences or sequences of naturally occurring
polypeptides, such as polypeptides with a hinge-function. The
linker has the function to ensure that a peptide conjugated to an
anti-CCR5 antibody can perform its biological activity by allowing
the peptide to fold correctly and to be presented properly.
Preferably the linker is a "synthetic peptidic linker" that is
designated to be rich in glycine, glutamine, and/or serine
residues. These residues are arranged e.g. in small repetitive
units of up to five amino acids, such as GGGGS, QQQQG, or SSSSG.
This small repetitive unit may be repeated for two to five times to
form a multimeric unit. At the amino- and/or carboxy-terminal ends
of the multimeric unit up to six additional arbitrary, naturally
occurring amino acids may be added. Other synthetic peptidic
linkers are composed of a single amino acid, that is repeated
between 10 to 20 times, such as e.g. serine in the linker
SSSSSSSSSSSSSSS. At each of the amino- and/or carboxy-terminal end
up to six additional arbitrary, naturally occurring amino acids may
be present. Preferred linkers are shown in Table 2. Especially
preferred are linkers [GQ.sub.4].sub.3GNN (SEQ ID NO:40), LSLSPGK
(SEQ ID NO:36), LSPNRGEC (SEQ ID NO:37), LSLSGG (SEQ ID NO:61),
LSLSPGG (SEQ ID NO:62). All peptidic linkers can be encoded by a
nucleic acid molecule and therefore can be recombinantly expressed.
As the linkers are themselves peptides, the antifusogenic peptide
is connected to the linker via a peptide bond that is formed
between two amino acids. The peptidic linker is introduced between
the antifusogenic peptide and the anti-CCR5 antibody chain to which
the antifusogenic peptide is to be conjugated. Therefore two or
three, respectively, possible sequences (in amino- to
carboxy-terminal direction) exist: a) antifusogenic
peptide-peptidic linker-anti-CCR5 antibody polypeptide chain, or b)
anti-CCR5 antibody polypeptide chain-peptidic linker-antifusogenic
peptide, or c) antifusogenic peptide-peptidic linker-anti-CCR5
antibody polypeptide chain-peptidic linker-antifusogenic
peptide.
[0057] In one embodiment of the invention the conjugate is
characterized in comprising i) the heavy chain (V.sub.H) variable
domain defined by amino acid sequence SEQ ID NO:1 and the light
chain (V.sub.L) variable domain defined by SEQ ID NO:2; or the
heavy chain variable domain defined by amino acid sequence SEQ ID
NO:3 and the light chain variable domain defined by SEQ ID NO:4; or
the heavy chain variable domain defined by amino acid sequence SEQ
ID NO:5 and the light chain variable domain defined by SEQ ID NO:6;
or the heavy chain variable domain defined by amino acid sequence
SEQ ID NO:7 and the light chain variable domain defined by SEQ ID
NO:8; ii) a linker selected from the group consisting of the amino
acids glycine (G) and asparagine (N), the tripeptide GST, and SEQ
ID NO:36-62; and iii) an antifusogenic peptide selected from the
group of peptides defined by SEQ ID NO:29 to 35.
[0058] A preferred conjugate of a heavy and/or light chain of mAb
CCR5 and an antifusogenic peptide(s) ("chain conjugate") is
selected from the group consisting of the conjugates (1)
[antifusogenic peptide]-[linker].sub.m-[heavy chain], (2) [heavy
chain]-[linker].sub.m-[antifusogenic peptide], (3) [antifusogenic
peptide]-[linker].sub.m-[heavy chain]-[antifusogenic peptide], (4)
[antifusogenic peptide]-[linker].sub.m-[light chain], (5) [light
chain]-[linker].sub.m-[antifusogenic peptide], (6) [antifusogenic
peptide]-[linker].sub.m-[light chain]-[antifusogenic peptide], (7)
[antifusogenic peptide]-[linker].sub.m-[heavy
chain]-[linker].sub.m-[antifusogenic peptide], (8) [antifusogenic
peptide]-[linker].sub.m-[light chain]-[linker].sub.m-[antifusogenic
peptide], wherein the linker can be the same or different both
within and between said chain conjugates, wherein m is an integer
of 1 or 0, and m can be independently the same or different both
within and between said conjugates. For example in a conjugate
comprising a chain conjugate (7) and a mAb CCR5 light chain the two
linkers in chain conjugate (7) can be the same, i.e. have the same
amino acid sequence and length, or can be different, i.e. have
different amino acid sequences and/or lengths, or one or both can
be absent. For example in a conjugate comprising chain conjugates
(2) and (4) the linker contained in chain conjugate (2) and the
linker contained in chain conjugate (4) can be the same, i.e. have
the same amino acid sequence and length, or can be different, i.e.
have different amino acid sequences and/or lengths, or one or both
can be absent. In the chain conjugates the linker(s) can be present
(m=1) or absent (m=0). Preferred chain conjugates are the chain
conjugates (2), (3), (4), and (7). One embodiment of the current
invention is a conjugate comprising 2.times.[mAb CCR5 light chain]
and 2.times. chain conjugate (2). This conjugate comprises two not
conjugate anti-CCR5 antibody light chains and two anti-CCR5
antibody heavy chains conjugated via the C-terminus to the
N-terminus of an antifusogenic peptide, optionally with an
intermediate linker. Another embodiment of the current invention is
a conjugate comprising two mAb CCR5 light chains and two chain
conjugates (3). Still another embodiment is a conjugate comprising
two mAb CCR5 heavy chains and two chain conjugates (4). A further
embodiment of the current invention is a conjugate comprising two
mAb CCR5 light chains and two chain conjugates (7). The heavy
and/or light chain comprises preferably a constant region (Fc).
[0059] The invention further provides a method for the manufacture
of a pharmaceutical composition comprising an effective amount of a
conjugate according to the invention together with a
pharmaceutically acceptable carrier and the use of the conjugate
according to the invention for such a method.
[0060] The invention further provides the use of a conjugate
according to the invention in an effective amount for the
manufacture of a pharmaceutical agent, preferably together with a
pharmaceutically acceptable carrier, for the treatment of a patient
suffering from AIDS.
[0061] The term "amino acid" as used within this application
denotes the group of naturally occurring carboxy .alpha.-amino
acids comprising 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),
tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
[0062] Methods and techniques known to a person skilled in the art,
which are useful for carrying out the current invention, are
described e.g. in F. M. Ausubel, ed., Current Protocols in
Molecular Biology, Volumes Ito III (1997), Wiley and Sons; Sambrook
et al., Molecular Cloning: A Laboratory Manual, Second Edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989).
[0063] In the conjugates according to the invention the
carboxy-terminal amino acid of an anti-CCR5 antibody chain is
conjugated via a peptide bond to the amino-terminal amino acid of
the antifusogenic peptide or the carboxy-terminal amino acid of the
antifusogenic peptide is conjugated via a peptide bond to the
amino-terminal amino acid of an anti-CCR5 antibody chain. In one
embodiment an intermediate linker is present between the
antifusogenic peptide and the anti-CCR5 antibody chain. Thus, the
conjugate according to the invention is characterized by the
general formula
mAb CCR5-[linker].sub.m-[antifusogenic peptide].
wherein m is independently for each antifusogenic peptide either 0
(i.e. a direct peptide bond between mAb CCR5 and the antifusogenic
peptide) or 1 (i.e. a linker is present between mAb CCR5 and
antifusogenic peptide) and n is an integer of from 1 to 8. In one
embodiment n is an integer of from 2 to 8. In another embodiment n
is an integer of from 2 to 4. In another embodiment n is an integer
of 2 or 4. One embodiment of the invention comprises a conjugate
characterized in comprising an antifusogenic peptide at each
C-terminus of the heavy chains or at each N-terminus of the light
chains of the anti-CCR5 antibody. In this embodiment two
antifusogenic peptides are conjugated to one anti-CCR5 antibody. In
an other embodiment is the conjugate characterized by comprising an
antifusogenic peptide at each C-terminus of the heavy chains and at
each N-terminus of the light chains. In this embodiment four
antifusogenic peptides are conjugated to one anti-CCR5
antibody.
[0064] The antifusogenic peptide which is introduced at a terminus
of a mAb CCR5 heavy and/or light chain(s) is small of size compared
to the mAb CCR5. For example, the smallest immunoglobulins,
immunoglobulins of class G, have a molecular weight of
approximately 150 kDa; an antifusogenic peptide has preferably a
size (molecular weight) of less than 12.5 kDa, which is equivalent
to about 100 amino acids, in general less than 7.5 kDa, which is
equivalent to about 60 amino acids. The antifusogenic peptide has
an amino acid sequence of from 5 to 100 amino acid residues,
preferably of from 10 to 75 amino acid residues, more preferably of
from 15 to 50 amino acid residues. The conjugates of the current
invention are useful for pharmaceutical, therapeutical, or
diagnostical applications. The number of antifusogenic peptides,
which can be conjugated to mAb CCR5 heavy and/or light chain(s), is
from one to the combined number of amino- and carboxy-termini of
the anti-CCR5 antibody polypeptide chains. As the current invention
encompasses different anti-CCR5 antibodies the number of
antifusogenic peptides can vary. In case of an anti-CCR5 antibody
comprising two heavy and two light chains the combined number of
amino-termini (N-termini) and carboxy-termini (C-termini) is eight,
which is at the same time the totaling maximum number of conjugated
antifusogenic peptides; in case e.g. of an anti-CCR5 antibody
fragment such as a single chain antibody (scFv) the combined number
of termini and therefore the maximum number of conjugatable
antifusogenic peptides is two. If a single antifusogenic peptide is
conjugated to mAb CCR5, the peptide can occupy any one of the
termini of the anti-CCR5 antibody chains. Likewise, if the maximum
possible number of peptides is conjugated to mAb CCR5, all termini
are occupied by a single peptide. If the number of peptides, which
are conjugated to mAb CCR5, is smaller than the maximum possible
number, different distributions of the peptides at the termini of
the anti-CCR5 antibody chains are possible. For example, if four
peptides are conjugated to an immunoglobulin of the G or E class,
five different combinations are possible (see Table 1). In two
combinations all termini of one kind, i.e. all four amino-termini
or all four carboxy-termini of the anti-CCR5 antibody chains, are
each conjugated to one single antifusogenic peptide. The other
termini are not conjugated. This results in one embodiment in an
allocation of the modifications/conjugations in one area of the
anti-CCR5 antibody. In the other cases the polypeptides are
conjugated to a number of both termini. Within these combinations
the conjugated peptides are allocated to different areas of the
anti-CCR5 antibody. In either case the sum of conjugated termini is
four.
TABLE-US-00001 TABLE 1 Possible combination for the conjugation of
four peptides to the termini of an anti-CCR5 antibody composed of
four polypeptide chains. number of occupied number of occupied
total number of amino-termini carboxy-termini occupied termini 4 0
4 3 1 4 2 2 4 1 3 4 0 4 4
[0065] The current invention preferably comprises conjugates in
which at least two of the termini are conjugated to an
antifusogenic peptide. The amino acid sequences of the
antifusogenic peptides can be different, similar or identical. In
one embodiment the amino acid sequence identity is in the range of
from 90% to less than 100%; these amino acid sequences and the
corresponding peptides are defined as similar. In a preferred
embodiment the antifusogenic peptides are identical, i.e. have an
amino acid identity of 100%.
[0066] The present invention comprises a conjugate comprising one
or more antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5)
wherein one to eight antifusogenic peptides are each conjugated to
one terminus of the heavy and/or light chains of said anti-CCR5
antibody via a peptide bond. In one embodiment the conjugate
according to the invention comprises at least two antifusogenic
peptides and an anti-CCR5 antibody wherein two to eight
antifusogenic peptides are each conjugated to one terminus of the
heavy and/or light chains of said anti-CCR5 antibody.
[0067] In one embodiment the conjugate according to the invention
is characterized i) in comprising two light chain variable domains
of SEQ ID NO:2, two chain conjugates of type (2) each comprising a
heavy chain variable domain of SEQ ID NO:1, a linker of SEQ ID
NO:40 and an antifusogenic peptide of SEQ ID NO:33, ii) in
comprising two light chain variable domains of SEQ ID NO:4, two
chain conjugates of type (2) each comprising a heavy chain variable
domain of SEQ ID NO:3, a linker of SEQ ID NO:40 and an
antifusogenic peptide of SEQ ID NO:33, iii) in comprising two light
chain variable domains of SEQ ID NO:6, two chain conjugates of type
(2) each comprising a heavy chain variable domain of SEQ ID NO:5, a
linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID
NO:33, or iv) in comprising two light chain variable domains of SEQ
ID NO:8, two chain conjugates of type (2) each comprising a heavy
chain variable domain of SEQ ID NO:7, a linker of SEQ ID NO:40 and
an antifusogenic peptide of SEQ ID NO:33.
[0068] The conjugation between the antifusogenic peptide and the
anti-CCR5 antibody is performed on the nucleic acid level.
Therefore a peptide bond is formed between the antifusogenic
peptide and the anti-CCR5 antibody chain with or without an
intermediate linker. Thus either the carboxy-terminal amino acid of
the antifusogenic peptide is conjugated to the amino-terminal amino
acid of an anti-CCR5 antibody chain with or without an intermediate
linker, or a carboxy-terminal amino acid of the anti-CCR5 antibody
chain is conjugated to the amino-terminal amino acid of the
antifusogenic peptide with or without an intermediate linker or
both termini of the anti-CCR5 antibody chain are conjugated to an
antifusogenic peptide each with or without an intermediate linker.
For the recombinant production of the antifusogenic
peptide-anti-CCR5 antibody-conjugate according to the invention one
or more nucleic acid molecules encoding different polypeptides are
required, preferably two to eight nucleic acid molecules are
employed. These nucleic acid molecules encode the different
anti-CCR5 antibody polypeptide chains of the conjugate and are in
the following referred to as structural genes. They can be located
on the same expression plasmid (vector) or can alternatively be
located on different expression plasmids (vectors). The assembly of
the conjugate takes preferably place before secretion of the
conjugate and thus within the expressing cells. Therefore the
nucleic acid molecules encoding the polypeptide chains of the
conjugate are preferably expressed in the same host cell. If after
recombinant expression a mixture of conjugates is obtained, the
conjugates can be separated and purified by methods known to a
person skilled in the art. These methods are well established and
widespread used for immunoglobulin purification and are employed
either alone or in combination. Such methods are, for example,
affinity chromatography using microbial-derived 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
chromatography), 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
preparative electrophoretic methods (such as gel electrophoresis,
capillary electrophoresis) (M. A. Vijayalakshmi, Appl. Biochem.
Biotech. (1998) 75:93-102). With recombinant engineering methods
known to a person skilled in the art the conjugates can be
tailor-made on the nucleic acid/gene level. The nucleic acid
sequences encoding immunoglobulins are known and can be obtained
for example from genomic databases. Likewise the nucleic acid
sequences encoding antifusogenic peptides are known or can easily
be deduced from their amino acid sequence. The elements required
for the construction of an expression plasmid for the expression of
the conjugate of the current invention are, for example, an
expression cassette for the anti-CCR5 antibody light chain in its
natural and/or modified and/or conjugated version, an expression
cassette for the anti-CCR5 antibody heavy chain in its natural
and/or modified and/or conjugated version (alternatively the
anti-CCR5 antibody light chain and the anti-CCR5 antibody heavy
chain can be contained in the same expression cassette, e.g. as
bicistronic expression element), a selection marker, and an E. coli
replication as well as selection unit. These expression cassettes
comprise a promoter, a DNA segment encoding a secretion signal
sequence, the structural gene, and a terminator/polyadenylation
signal. The elements are assembled in an operatively linked form
either on one plasmid encoding all chains of the conjugate, or on
two or more plasmids each encoding one or more chains of the
conjugate. For the expression of the encoded polypeptides the
plasmid(s) is (are) introduced into a suitable host cell. Proteins
are preferably produced in mammalian cells such as CHO cells, NS0
cells, Sp2/0 cells, COS cells, HEK cells, K562 cells, BHK cells,
PER.C6.RTM. cells, and the like. The regulatory elements of the
plasmid have to be selected in a way that they are functional in
the selected host cell. For the expression the host cell containing
the plasmid encoding one or more chains of the conjugate is
cultivated under conditions suitable for the expression of the
chains. The expressed conjugate chains are functionally assembled.
The fully processed antifusogenic peptide-anti-CCR5
antibody-conjugate is secreted into the medium.
[0069] An "expression plasmid" is a nucleic acid encoding a
polypeptide to be expressed in a host cell. Typically, an
expression plasmid comprises a prokaryotic plasmid propagation
unit, e.g. for E. coli, comprising an origin of replication, and a
resistance gene, an eukaryotic selection marker, and one or more
expression cassettes for the expression of the structural gene(s)
of interest comprising a promoter, a structural gene, and a
transcription terminator including a polyadenylation signal. Gene
expression is usually placed under the control of a promoter, and
such a structural gene is said to be "operably linked to" the
promoter. Similarly, a regulatory element and a core promoter are
operably linked if the regulatory element modulates the activity of
the core promoter.
[0070] One aspect of the current invention is thus a method for the
production of a conjugate according to the invention, comprising
the following steps [0071] a) cultivating a cell containing one or
more expression plasmids each comprising one or more nucleic acid
molecules encoding a conjugate according to the invention under
conditions suitable for the expression of the conjugate, [0072] b)
recovering the conjugate from the cell or the supernatant.
[0073] The term "under conditions suitable for the expression of
the conjugate" denotes conditions which are used for the
cultivation of a cell expressing a polypeptide and which are known
to or can easily be determined by a person skilled in the art. It
is known to a person skilled in the art that these conditions may
vary depending on the type of cell cultivated and type of
polypeptide expressed. In general the cell is cultivated at a
temperature, e.g. between 20.degree. C. and 40.degree. C., and for
a period of time sufficient to allow effective production of the
polypeptide conjugate, e.g. for 4 to 28 days.
[0074] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and
absorption/resorption delaying agents, and the like that are
physiologically compatible. Preferably, the carrier is suitable for
injection or infusion. Pharmaceutically acceptable carriers include
sterile aqueous solutions or dispersions and sterile powders for
the preparation of sterile injectable solutions or dispersion. The
use of such media and agents for pharmaceutically active substances
is known in the art. In addition to water, the carrier can be, for
example, an isotonic buffered saline solution.
[0075] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skilled in
the art.
[0076] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention 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,
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.
[0077] The invention preferably comprises the use of a conjugate
according to the invention for the treatment of a patient suffering
from immunodeficiency syndromes such as AIDS.
[0078] The following examples, sequence listing, figures and
deposits are provided to aid the understanding of the present
invention, the true scope of which is set forth in the appended
claims. It is understood that modifications can be made in the
procedures set forth without departing from the spirit of the
invention.
DESCRIPTION OF THE FIGURES
[0079] FIG. 1: Plasmid map of mAb CCR5 .kappa.-light chain
expression vector 4900.
[0080] FIG. 2: Plasmid map of mAb CCR5 .gamma.1-heavy chain
expression vector 4901.
[0081] FIG. 3: Plasmid map of mAb CCR5 .gamma.1-heavy chain
conjugate expression vector 4995.
ANTI-CCR5 ANTIBODY DEPOSITION
[0082] Preferred hybridoma cell lines expressing mAb CCR5 useful in
the conjugates according to the invention were deposited with
Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ),
Germany.
TABLE-US-00002 Cell line Deposition No. Date of Deposit
m<CCR5>Pz01.F3 DSM ACC 2681 18 Aug. 2004
m<CCR5>Pz02.1C11 DSM ACC 2682 18 Aug. 2004
m<CCR5>Pz03.1C5 DSM ACC 2683 18 Aug. 2004
m<CCR5>Pz04.1F6 DSM ACC 2684 18 Aug. 2004
[0083] Antibody Nomenclature
TABLE-US-00003 <CCR5>Pz01.F3: Antibody A SEQ ID NO: 1, 2
<CCR5>Pz02.1C11: Antibody B SEQ ID NO: 3, 4
<CCR5>Pz03.1C5: Antibody C SEQ ID NO: 5, 6
<CCR5>F3.1H12.2E5: Antibody D SEQ ID NO: 7, 8
<CCR5>Pz04.1F6: Antibody E
[0084] CCR 5 Antibody Sequences, Sequences of Antifusogenic
Peptides and Sequences of Peptidic Linkers
TABLE-US-00004 SEQ ID NO: 1 <CCR5>Pz01.F3 heavy chain,
variable domain SEQ ID NO: 2 <CCR5>Pz01.F3 light chain,
variable domain SEQ ID NO: 3 <CCR5>Pz02.1C11 heavy chain,
variable domain SEQ ID NO: 4 <CCR5>Pz02.1C11 light chain,
variable domain SEQ ID NO: 5 <CCR5>Pz03.1C5 heavy chain,
variable domain SEQ ID NO: 6 <CCR5>Pz03.1C5 light chain,
variable domain SEQ ID NO: 7 <CCR5>F3.1H12.2E5 heavy chain,
variable domain SEQ ID NO: 8 <CCR5>F3.1H12.2E5 light chain,
variable domain SEQ ID NO: 9 Heavy chain CDR1 SEQ ID NO: 10 Heavy
chain CDR1 SEQ ID NO: 11 Heavy chain CDR1 SEQ ID NO: 12 Heavy chain
CDR1 SEQ ID NO: 13 Heavy chain CDR2 SEQ ID NO: 14 Heavy chain CDR2
SEQ ID NO: 15 Heavy chain CDR2 SEQ ID NO: 16 Heavy chain CDR3 SEQ
ID NO: 17 Heavy chain CDR3 SEQ ID NO: 18 Light chain CDR1 SEQ ID
NO: 19 Light chain CDR1 SEQ ID NO: 20 Light chain CDR1 SEQ ID NO:
21 Light chain CDR2 SEQ ID NO: 22 Light chain CDR2 SEQ ID NO: 23
Light chain CDR2 SEQ ID NO: 24 Light chain CDR3 SEQ ID NO: 25 Light
chain CDR3 SEQ ID NO: 26 .gamma.1 heavy chain constant region SEQ
ID NO: 27 .gamma.4 heavy chain constant region SEQ ID NO: 28
.kappa. light chain constant domain SEQ ID NO: 29 C34 SEQ ID NO: 30
T20 SEQ ID NO: 31 T1249 SEQ ID NO: 32 T651 SEQ ID NO: 33 T2635 SEQ
ID NO: 34 N36 SEQ ID NO: 35 DP107 SEQ ID NO: 36-62 linker peptides
SEQ ID NO: 63 Amino acid sequence of mature mAb CCR5 .kappa.- light
chain SEQ ID NO: 64 Amino acid sequence of mature mAb CCR5
.gamma.1- heavy chain SEQ ID NO: 65 Amino acid sequence of mature
mAb CCR5 conjugate heavy chain SEQ ID NO: 66 HIV-1 gp41
TABLE-US-00005 TABLE 2 Linker No. Linker peptides SEQ ID NO: 1
LSLSPGK 36 2 LSPNRGEC 37 3 [GQ.sub.4].sub.3 38 4 [GQ.sub.4].sub.3G
39 5 [GQ.sub.4].sub.3GNN 40 6 GGG[SG.sub.4].sub.2SGG 41 7
GGG[SG.sub.4].sub.2SGN 42 8 [SG.sub.4].sub.3 43 9 [SG.sub.4].sub.3G
44 10 G[SG.sub.4].sub.3T 45 11 [SG.sub.4].sub.3GG 46 12
[SG.sub.4].sub.3GGT 47 13 [SG.sub.4].sub.3GGN 48 14
[SG.sub.4].sub.3GAS 49 15 [SG.sub.4].sub.5 50 16 [SG.sub.4].sub.5G
51 17 [SG.sub.4].sub.5GG 52 18 [SG.sub.4].sub.5GAS 53 19
G(S).sub.15G 54 20 G(S).sub.15GAS 55 21 G -- 22 N -- 23 GST -- 24
[(G).sub.4S].sub.3GAS 56 25 [(G).sub.4S].sub.3G 57 26
[(G).sub.4S].sub.5G 58 27 [(G).sub.4S].sub.3GG 59 28
[(G).sub.4S].sub.5GG 60 29 LSLSGG 61 30 LSLSPGG 62
EXAMPLES
Materials & Methods
[0085] General information regarding the nucleotide sequences of
human immunoglobulins light and heavy chains is given in: E. A.
Kabat et al., Sequences of Proteins of Immunological Interest, 5th
ed., Public Health Service, National Institutes of Health,
Bethesda, Md. (1991). Amino acids of antibody chains are numbered
according to EU numbering (G. M. Edelman et al., Proc. Natl. Acad.
Sci. USA (1969) 63:78-85; E. A. Kabat et al., supra).
Recombinant DNA Techniques
[0086] Standard methods were used to manipulate DNA as described in
J. Sambrook et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions.
Gene Synthesis
[0087] Desired gene segments were prepared from oligonucleotides
made by chemical synthesis. The 100-600 by long gene segments,
which are flanked by singular restriction endonuclease cleavage
sites, were assembled by annealing and ligation of oligonucleotides
including PCR amplification and subsequently cloned into the
pCR2.1-TOPO-TA cloning vector (Invitrogen Corp., USA) via
A-overhangs. The DNA sequence of the subcloned gene fragments were
confirmed by DNA sequencing.
Protein Determination
[0088] The protein concentration of the conjugate was determined by
determining the optical density (OD) at 280 nm, using the molar
extinction coefficient calculated on the basis of the amino acid
sequence.
Example 1
Synthesizing the Expression Plasmids
[0089] The gene segments encoding an anti-CCR5 antibody light chain
variable domain (V.sub.L) and the human kappa-light chain constant
domain (C.sub.L) were joined as were gene segments for the
anti-CCR5 antibody heavy chain variable domain (V.sub.H) and the
human .gamma.1-heavy chain constant domains
(C.sub.H1-Hinge-C.sub.H2-C.sub.H3).
[0090] In the case of mAb CCR5 of SEQ ID NO:63/64 the heavy and
light chain variable domains are derived from a mouse antibody and
the heavy and light chain constant domains are derived from a human
antibody (C-kappa and IgG1).
[0091] Subsequently, the gene segment encoding a complete anti-CCR5
antibody light chain was joined at the N- and/or C-terminus with a
nucleic acid encoding an antifusogenic peptide including a
connecting linker sequence and/or the gene segment encoding a
complete anti-CCR5 antibody heavy chain was joined at the N- and/or
C-terminus with a nucleic acid encoding an antifusogenic peptide
including a connecting linker sequence.
a) Vector 4900
[0092] Vector 4900 is an expression plasmid for transient
expression of a mAb CCR5 light chain (genomically organized
expression cassette; exon-intron organization) in HEK293 cells.
[0093] Beside the mAb CCR5 .kappa.-light chain expression cassette
this vector contains: [0094] a hygromycine resistance gene as a
selectable marker, [0095] an origin of replication, oriP, of
Epstein-Barr virus (EBV), [0096] an origin of replication from the
vector pUC18 which allows replication of this plasmid in E. coli,
and [0097] a .beta.-lactamase gene which confers ampicillin
resistance in E. coli.
[0098] The transcription unit of the mAb CCR5 .kappa.-light chain
gene is composed of the following elements: [0099] the immediate
early enhancer and promoter from the human cytomegalovirus, [0100]
a synthetic 5'-untranslated region, [0101] a murine immunoglobulin
heavy chain signal sequence including a signal sequence intron
(signal sequence 1, intron, signal sequence 2 [L1-intron-L2]),
[0102] the murine anti-CCR5 antibody mature variable .kappa.-light
chain encoding segment arranged with a unique BsmI restriction site
at the 5'-end (L2 signal sequence) and a splice donor site and a
unique NotI restriction site at the 3'-end, [0103] a human/mouse
.kappa.-light chain hybrid intron 2, [0104] the human .kappa.-light
gene constant domain, [0105] the human immunoglobulin
.kappa.-polyadenylation ("poly A") signal sequence, and [0106] the
unique restriction sites AscI and FseI at the 5'- and 3'-end,
respectively.
[0107] The plasmid map of the mAb CCR5 .kappa.-light chain
expression vector 4900 is shown in FIG. 1. The amino acid sequence
of the mature (without signal sequence) mAb CCR5 .kappa.-light
chain is shown in SEQ ID NO:63.
b) Vector 4991
[0108] Vector 4991 is an expression plasmid for transient
expression of a mAb CCR5 .gamma.1-heavy chain (genomically
organized expression cassette; exon-intron organization) in HEK293
cells.
[0109] Beside the mAb CCR5 .gamma.1-heavy chain expression cassette
this vector contains: [0110] a hygromycin resistance gene as a
selectable marker, [0111] an origin of replication, oriP, of
Epstein-Barr virus (EBV), [0112] an origin of replication from the
vector pUC18 which allows replication of this plasmid in E. coli,
and [0113] a beta-lactamase gene which confers ampicillin
resistance in E. coli.
[0114] The transcription unit of the mAb CCR5 .gamma.1-heavy chain
is composed of the following elements: [0115] the immediate early
enhancer and promoter from the human cytomegalovirus, [0116] a
synthetic 5'-untranslated region, [0117] a murine immunoglobulin
heavy chain signal sequence including a signal sequence intron
(signal sequence 1, intron, signal sequence 2 [L1-intron-L2]),
[0118] the murine anti-CCR5 antibody mature variable heavy chain
encoding segment arranged with a unique BsmI restriction site at
the 5'-end (L2 signal sequence) and a splice donor site and a
unique NotI restriction site at the 3'-end, [0119] a human/mouse
heavy chain hybrid intron 2 including the mouse heavy chain
enhancer element (part JH.sub.3, JH.sub.4) (Neuberger, M. S., EMBO
J. 2 (1983) 1373-1378), [0120] the genomic human .gamma.1-heavy
gene constant domains, [0121] the human .gamma.1-immunoglobulin
polyadenylation ("poly A") signal sequence, and [0122] the unique
restriction sites AscI and SgrAI at the 5'- and 3'-end,
respectively. The plasmid map of the mAb CCR5 .gamma.1-heavy chain
expression vector 4901 is shown in FIG. 2. The amino acid sequence
of the mature (without signal sequence) mAb CCR5 .gamma.1-heavy
chain is shown in SEQ ID NO:64.
c) Vector 4995
[0123] Vector 4995 is an expression plasmid for transient
expression of a chimeric peptide-anti-CCR5 antibody .gamma.1-heavy
chain conjugate (genomically organized expression cassette;
exon-intron organization) in HEK293 cells.
[0124] The vector 4995 is derived from plasmid 4991 in that way
that the mAb CCR5 .gamma.1-heavy chain is joint at the C-terminus
with a nucleic acid encoding the antifusogenic peptide T-2635 (SEQ
ID NO:33) and the peptidic linker sequence [GQ.sub.4].sub.3GNN (SEQ
ID NO:40).
[0125] Beside the chimeric peptide anti-CCR5 antibody
.gamma.1-heavy chain conjugate expression cassette this vector
contains: [0126] a hygromycin resistance gene as a selectable
marker, [0127] an origin of replication, oriP, of Epstein-Barr
virus (EBV), [0128] an origin of replication from the vector pUC18
which allows replication of this plasmid in E. coli, and [0129] a
beta(.beta.)-lactamase gene which confers ampicillin resistance in
E. coli.
[0130] The transcription unit of the chimeric peptide-anti-CCR5
antibody .gamma.1-heavy chain conjugate is composed of the
following elements: [0131] the immediate early enhancer and
promoter from the human cytomegalovirus, [0132] a synthetic
5'-untranslated region, [0133] a murine immunoglobulin heavy chain
signal sequence including a signal sequence intron (signal sequence
1, intron, signal sequence 2 [L1-intron-L2]), [0134] the murine
anti-CCR5 antibody mature variable heavy chain encoding segment
arranged with a unique BsmI restriction site at the 5'-end (L2
signal sequence) and a splice donor site and a unique NotI
restriction site at the 3'-end, [0135] a human/mouse heavy chain
hybrid intron 2 including the mouse heavy chain enhancer element
(part JH.sub.3, JH.sub.4) (Neuberger, M. S., EMBO J. 2 (1983)
1373-1378), [0136] the genomic human .gamma.1-heavy gene constant
domains, [0137] the antifusogenic peptide T-2635, [0138] the
peptidic linker sequence [GQ.sub.4].sub.3GNN, [0139] the human
.gamma.1-immunoglobulin polyadenylation ("poly A") signal sequence,
and [0140] the unique restriction sites AscI and SgrAI at the 5'-
and 3'-end, respectively.
[0141] The plasmid map of the mAb CCR5 .gamma.1-heavy chain
conjugate expression vector 4995 is shown in FIG. 3. The amino acid
sequence of the mature (without signal sequence) conjugate heavy
chain is shown in SEQ ID NO:65.
Example 2
Making of the Final Expression Plasmids
[0142] The fusion genes (heavy and/or light chain antibody fusion
genes) comprising a mAb CCR5 gene segment, an optional linker gene
segment and an antifusogenic peptide gene segment have been
assembled with known recombinant methods and techniques by
connection of the according nucleic acid segments. The nucleic acid
sequences encoding the peptidic linkers and antifusogenic
polypeptides were each synthesized by chemical synthesis and then
ligated into an E. coli plasmid for amplification. The subcloned
nucleic acid sequences were verified by DNA sequencing.
Example 3
Transient Expression of Immunoglobulins and Immunoglobulin Variants
in HEK293 EBNA Cells
[0143] Recombinant anti-CCR5 antibodies and anti-CCR5
antibody-variants were generated by transient transfection of
adherent growing HEK293-EBNA cells (human embryonic kidney cell
line 293 expressing Epstein-Barr-Virus nuclear antigen; American
type culture collection deposit number ATCC # CRL-10852) cultivated
in DMEM (Dulbecco's modified Eagle's medium, Gibco) supplemented
with 10% ultra-low IgG FCS (fetal calf serum, Gibco), 2 mM
Glutamine (Gibco), 1% volume by volume (v/v) nonessential amino
acids (Gibco) and 250 .mu.g/ml G418 (Roche Molecular Biochemicals).
For transfection FuGENE.TM. 6 Transfection Reagent (Roche Molecular
Biochemicals) was used in a ratio of reagent (.mu.l) to DNA (.mu.g)
ranging from 3:1 to 6:1. Light and heavy chains including
antifusogenic peptide-anti-CCR5 antibody conjugate light and heavy
chains were expressed from two different plasmids using a molar
ratio of light chain to heavy chain encoding plasmid ranging from
1:2 to 2:1, respectively. Antifusogenic peptide-anti-CCR5 antibody
conjugates containing cell culture supernatants were harvested at
day 4 to 11 after transfection. General information regarding the
recombinant expression of human immunoglobulins in e.g. HEK293
cells is given in P. Meissner et al., Biotechnol. Bioeng. (2001)
75:197-203.
Example 4
Expression Analysis Using SDS PAGE, Western Blotting Transfer and
Detection with Immunoglobulin Specific Antibody Conjugates
[0144] The expressed and secreted antifusogenic peptide-anti-CCR5
antibody conjugates were processed by sodium dodecyl sulfate (SDS)
polyacrylamide gel electrophoresis (SDS-PAGE), and the separated
anti-CCR5-antibody and antifusogenic
peptide-anti-CCR5-antibody-conjugate chains were transferred to a
membrane from the gel and subsequently detected by an immunological
method.
SDS-PAGE
[0145] LDS sample buffer, fourfold concentrate (4.times.): 4 g
glycerol, 0.682 g TRIS-Base, 0.666 g TRIS-hydrochloride, 0.8 g LDS
(lithium dodecyl sulfate), 0.006 g EDTA (ethylene diamin tetra
acid), 0.75 ml of a 1% by weight (w/w) solution of Serva Blue G250
in water, 0.75 ml of a 1% by weight (w/w) solution of phenol red,
add water to make a total volume of 10 ml.
[0146] The culture broth containing the secreted antifusogenic
peptide-anti-CCR5 antibody conjugate was centrifuged to remove
cells and cell debris. An aliquot of the clarified supernatant was
admixed with 1/4 volumes (v/v) of 4.times.LDS sample buffer and
1/10 volume (v/v) of 0.5 M 1,4-dithiotreitol (DTT). Then the
samples were incubated for 10 min. at 70.degree. C. and protein
separated by SDS-PAGE. The NuPAGE.RTM. Pre-Cast gel system
(Invitrogen) was used according to the manufacturer's instruction.
In particular, 10% NuPAGE.RTM. Novex.RTM. Bis-TRIS Pre-Cast gels
(pH 6.4) and a NuPAGE.RTM. MOPS running buffer was used.
Western Blot
[0147] Transfer buffer: 39 mM glycine, 48 mM TRIS-hydrochloride,
0.04% by weight (w/w) SDS, and 20% by volume methanol (v/v).
[0148] After SDS-PAGE the separated antifusogenic peptide-anti-CCR5
antibody conjugate chains were transferred electrophoretically to a
nitrocellulose filter membrane (pore size: 0.45 .mu.m) according to
the "Semidry-Blotting-Method" of Burnette (W. N. Burnette, Anal.
Biochem. (1981) 112:195-203).
Immunological Detection
[0149] TBS-buffer: 50 mM TRIS-hydrochloride, 150 mM NaCl, adjusted
to pH 7.5 Blocking solution: 1% (w/v) Western Blocking Reagent
(Roche Molecular Biochemicals) in TBS-buffer
[0150] TBST-Buffer: 1.times.TBS-buffer with 0.05% by volume (v/v)
Tween-20 For immunological detection the western blotting membranes
were incubated with shaking at room temperature two times for 5
minutes in TBS-buffer and once for 90 minutes in blocking
solution.
Detection of the Peptide Immunoglobulin Conjugate Chains
[0151] Heavy chain: For detection of the heavy chain of the
antifusogenic peptide-anti-CCR5 antibody conjugate a purified
rabbit anti-human IgG antibody conjugated to a peroxidase was used
(DAKO, Code No. P 0214).
[0152] Light chain: The light chain of the antifusogenic
peptide-anti-CCR5 antibody conjugate was detected with a purified
peroxidase conjugated rabbit anti-human kappa light chain antibody
(DAKO, Code No. P 0129).
[0153] For visualization of the antibody light and heavy chains
washed and blocked Western blot membranes were first incubated in
case of a heavy chain with a purified rabbit anti-human IgG
antibody conjugated to a peroxidase or in case of a light chain
with a purified peroxidase conjugated rabbit anti-human kappa light
chain antibody in a 1:10,000 dilution in 10 ml blocking solution at
4.degree. C. with shaking over night. After washing the membranes
three times with TBTS-buffer and once with TBS buffer for 10 min.
at room temperature, the Western-blot membranes were developed with
a Luminol/peroxid-solution generating chemiluminescence
(Lumi-Light.sup.PLUS Western Blotting Substrate, Roche Molecular
Biochemicals). Therefore the membranes were incubated in 10 ml
Luminol/peroxid-solution for 10 seconds to 5 minutes and the
emitted light was detected afterwards with a LUMI-Imager F1
Analysator (Roche Molecular Biochemicals) and/or was recorded with
an x-ray-film. The intensity of the spots was quantified with the
LumiAnalyst Software (Version 3.1).
Multiple-Staining of Immunoblots
[0154] The secondary peroxidase-labeled antibody conjugate used for
the detection can be removed from the stained blot by incubating
the membrane for one hour at 70.degree. C. in 1 M
TRIS-hydrochloride-buffer (pH 6.7) containing 100 mM
beta-mercaptoethanol and 20% (w/v) SDS. After this treatment the
blot can be stained with a different secondary antibody a second
time. Prior to the second detection the blot is washed three times
at room temperature with shaking in TBS-buffer for 10 minutes
each.
Example 5
Affinity Purification, Dialysis and Concentration of Peptide
Immunoglobulin Conjugates
[0155] The expressed and secreted antifusogenic peptide-anti-CCR5
antibody conjugates were purified by affinity chromatography using
Protein A-Sepharose.TM. CL-4B (GE Healthcare former Amersham
Bioscience, Sweden) according to known methods. Briefly, after
centrifugation (10,000 g for 10 minutes) and filtration through a
0.45 .mu.m filter the peptide immunoglobulin conjugate containing
clarified culture supernatants were applied on a Protein
A-Sepharose.TM. CL-4B column 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). Unbound proteins were washed out with PBS
equilibration buffer and 0.1 M citrate buffer, pH 5.5. The
antifusogenic peptide-anti-CCR5 antibody conjugates were eluted
with 0.1 M citrate buffer, pH 3.0, and the conjugate containing
fractions were neutralized with 1 M TRIS-Base. Then, the
antifusogenic peptide-anti-CCR5 antibody conjugates were
extensively dialyzed against PBS buffer at 4.degree. C.,
concentrated with a Ultrafree.RTM.-CL Centrifugal Filter Unit
equipped with a Biomax-SK membrane (Millipore Corp., USA) and
stored in an ice-water bath at 0.degree. C. The integrity of the
conjugates was analyzed by SDS-PAGE in the presence and absence of
a reducing agent and staining with Coomassie brilliant blue as
described in example 4. Aggregation of antifusogenic
peptide-anti-CCR5 antibody conjugates was analyzed by analytical
size exclusion chromatography.
Example 6
Deglycosylation of Peptide Immunoglobulin Conjugates
[0156] N-linked carbohydrates of anti-CCR5 antibodies and
antifusogenic peptide-anti-CCR5 antibody conjugates were cleaved
off by enzymatic treatment with Peptide-N-Glycosidase F (PNGaseF,
Roche Molecular Biochemicals, Mannheim, Germany or Prozyme, San
Leandro, Calif.). Therefore, the anti-CCR5 antibodies and
antifusogenic peptide-anti-CCR5 antibody conjugates were incubated
at 37.degree. C. for 12-24 h using 50 mU PNGaseF per mg
N-glycosylated protein in PBS buffer at a protein concentration of
about 2 mg/ml. Thereafter the Peptide-N-Glycosidase F was separated
by preparative gel filtration according to known methods. Briefly,
PNGaseF treated anti-CCR5-antibodies and antifusogenic
peptide-anti-CCR5 antibody conjugates were applied on a
Superose.TM. 12 10/300 GL column (GE Healthcare former Amersham
Bioscience, Sweden) 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) and then eluted with equilibration buffer at a flow
rate of 0.5-1.0 ml/min using the Akta explorer chromatography
system from Amersham Bioscience (GE Healthcare former Amersham
Bioscience, Sweden).
Example 7
Single-Cycle Antiviral Activity Assay
[0157] For the production of pseudotyped NL-Bal viruses, plasmid
pNL4-3.DELTA.env (HIV pNL4-3 genomic construct with a deletion
within the env gene) and pcDNA3.1/NL-BAL env [pcDNA3.1 plasmid
containing NL-Bal env gene (obtained from NIBSC Centralized
Facility for AIDS Reagents)] were co-transfected into the HEK 293FT
cell line (Invitrogen), cultured in Dulbecco's modified minimum
medium (DMEM) containing 10% fetal calf serum (FCS), 100 U/mL
Penicillin, 100 .mu.g/mL Streptomycin, 2 mM L-glutamine and 0.5
mg/mL geniticin (all media from Invitrogen/Gibco). The supernatants
containing pseudotyped viruses were harvested two days following
transfection, and cellular debris was removed by filtration through
a 0.45 .mu.m pore size PES (polyethersulfone) filter (Nalgene) and
stored at -80.degree. C. in aliquots. For normalization in assay
performance, virus stock aliquots were used to infect JC53-BL (US
NIH Aids Reagent Program) cells yielding approximately
1.5.times.10.sup.5 RLU (relative light units) per well. Test
antifusogenic peptide-anti-CCR5 antibody conjugates, reference
antibodies and reference antifusogenic peptides (T-20, T-1249,
T-651 and T-2635) were serially diluted in 96-well plates. The
assay was carried out in quadruplicates. Each plate contained cell
control and virus control wells. The equivalent of
1.5.times.10.sup.5 RLU of virus stocks were added to each well,
then 2.5.times.10.sup.4 JC53-BL cells were added to each well, with
a final assay volume of 200 .mu.l per well. After 3 day incubation
at 37.degree. C., 90% Relative Humidity, and 5% CO.sub.2, media
were aspirated and 50 .mu.l of Steady-Glo.RTM. Luciferase Assay
System (Promega) was added to each well. The assay plates were read
on a Luminometer (Luminoskan, Thermo Electron Corporation) after 10
minutes of incubation at room temperature. Percent inhibition of
luciferase activity was calculated for each dose point after
subtracting the background, and IC.sub.50 and IC.sub.90-values were
determined by using XLfit curve fitting software for Excel (version
3.0.5 Build12; Microsoft). Results are shown in Table 3.
TABLE-US-00006 TABLE 3 Antiviral activity of antifusogenic
polypeptides, antibodies and antifusogenic peptide-anti-CCR5
antibody conjugates Antiviral activity Compound IC.sub.50 (ng/mL)
IC.sub.90 (ng/mL) Reference antibody 1 (inert) inactive inactive
Reference antibody 2 (inert) inactive inactive T-20 206 3955 T-1249
11 90 T-651 11 139 T-2635 14 161 mAb CCR5 (4901/4900) 114 2387
Chimeric peptide mAb CCR5 7 45 conjugate (4995/4900)
Example 8
Cell-Cell Fusion Assay
[0158] At day 1, gp160-expressing HeLa cells (2.times.10.sup.4
cells/50 .mu.l/well) are seeded in a white 96 microtiter plate in
DMEM medium supplemented with 10% FCS and 2 .mu.g/ml doxycycline.
At day 2, 100 .mu.l of supernatant sample or antibody control per
well is added in a clear 96 microtiter plate. Then 100 .mu.l
containing 8.times.10.sup.4 CEM-NKr-Luc suspension cells in medium
are added and incubated 30 min. at 37.degree. C. The HeLa cell
culture medium is aspirated from the 96 well plate, 100 .mu.l from
the 200 .mu.l antibody/CEM-NKr-Luc mixture is added and incubated
overnight at 37.degree. C. At day 3, 100 .mu.l/well Bright-Glo.TM.
Luciferase assay substrate (1,4-dithiothreitol and sodium
dithionite; Promega Corp., USA) is added and luminescence is
measured after a minimum of 15 min. incubation at RT.
Materials:
[0159] HeLa-R5-16 cells (cell line to express HIV gp160 upon
doxycycline induction) are cultured in DMEM medium containing
nutrients and 10% FCS with 400 .mu.g/ml G418 and 200 .mu.g/ml
Hygromycin B. CEM.NKR-CCR5-Luc (Catalog Number: 5198, a T-cell line
available from NIH AIDS Research & Reference Reagent Program
McKesson BioServices Corporation Germantown, Md. 20874, USA). Cell
Type: CEM.NKR-CCR5 (Cat. #4376) is transfected (electroporation) to
express the luciferase gene under the transcriptional control of
the HIV-2 LTR and propagated in RPMI 1640 containing 10% fetal
bovine serum, 4 mM glutamine, penicillin/streptomycin (100 U/mL
Penicillin, 100 .mu.g/mL Streptomycin), and 0.8 mg/ml geniticin
sulfate (G418). Growth Characteristics: Round lymphoid cells,
morphology not very variable. Cells grow in suspension as single
cells, which can form small clumps. Split 1:10 twice weekly.
Special Characteristics: Express luciferase activity after
transactivation of the HIV-2 LTR. Suitable for infection with
primary HIV isolates, for neutralization and drug-sensitivity
assays (C. Spenlehauer et al., Virology (2001) 280:292-300; A.
Trkola et al., J. Virol. (1999) 73:8966-74). The cell line was
obtained through the NIH AIDS Research and Reference Reagent
Program, NIAID, NIH from Drs. John Moore and Catherine Spenlehauer.
Bright-Glo.TM. Luciferase assay buffer (Promega Corp. USA, Part No
E2264B), Bright-Glo.TM., Luciferase assay substrate (Promega Corp.
USA, part No EE26B).
Example 9
Antiviral Activity Assay in Peripheral Blood Mononuclear Cells
(PBMC)
[0160] Human PBMC are isolated from buffy-coats (obtained from the
Stanford Blood Center) by a Ficoll-Paque (Amersham, Piscataway,
N.J., USA) density gradient centrifugation according to
manufacturer's protocol. Briefly, blood is transferred from the
buffy coats in 50 ml conical tubes and diluted with sterile
Dulbecco's phosphate buffered saline (Invitrogen/Gibco) to a final
volume of 50 ml. Twenty-five ml of the diluted blood are
transferred to two 50 ml conical tubes, carefully underlayered with
12.5 ml of Ficoll-Paque Plus (Amersham Biosciences) and centrifuged
at room temperature for 20 min. at 450.times.g without braking. The
white cell layer is carefully transferred to a new 50 ml conical
tube and washed twice with PBS. To remove remaining red blood
cells, cells are incubated for 5 min. at room temperature with ACK
lysis buffer (Biosource) and washed one more time with PBS. PBMC
are counted and incubated at a concentration of 2-4.times.10.sup.6
cells/ml in RPMI1640 containing 10% FCS (Invitrogen/Gibco), 1%
penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium-pyruvate,
and 2 .mu.g/ml Phyto-hemagglutinin (Invitrogen) for 24 h at
37.degree. C. Cells are incubated with 5 Units/ml human IL-2 (Roche
Molecular Biochemicals) for a minimum of 48 h prior to the assay.
In a 96 well round bottom plate, 1.times.10.sup.5 PBMC are infected
with the HIV-1 JR-CSF virus (Y. Koyanagi et al., Science (1987)
236:819-22) in the presence of serially diluted test
peptide-immunoglobulin-conjugates, reference immunoglobulins and
reference peptides (T-20, T-1249, T-651 and T-2635). The amount of
virus used is equivalent to 1.2 ng HIV-1 p24 antigen/well.
Infections are set up in quadruplicates. Plates are incubated for 6
days at 37.degree. C. Virus production is measured at the end of
infection by using p24 ELISA (HIV-1 p24 ELISA #NEK050B, Perkin
Elmer/NEN) using the sigmoid dose-response model with one binding
site in Microsoft Excel Fit (version 3.0.5 Build 12; equation 205;
Microsoft).
Sequence CWU 1
1
661119PRTmouse 1Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys
Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Thr Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys
Arg Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Thr Gly Asp Asn Thr Tyr
Tyr Thr Asp Ser Val Arg 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Arg Asn Ile Leu Tyr Leu65 70 75 80Gln Met Ser Ser Leu Arg Ser
Glu Asp Thr Ala Met Tyr Phe Cys Thr 85 90 95Arg Gly Arg Gly Asp Arg
Gly Asp Leu Phe Gly Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr
Val Ser Ser 1152114PRTmouse 2Asp Ile Val Met Ser Gln Ser Pro Ser
Ser Leu Ala Val Ser Val Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Lys
Ser Ser Gln Ser Leu Leu Tyr Arg 20 25 30Gly Asn Gln Met Asn Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr
Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr65 70 75 80Ile Ser Ser
Val Lys Ala Glu Asp Leu Thr Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr
Thr Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105
110Lys Arg3117PRTmouse 3Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu
Val Arg Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly
Phe Pro Leu Gly Val Phe 20 25 30Gly Val His Trp Val Arg Gln Ser Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Lys Gly Gly Asn
Thr Asp Tyr Asn Ala Ala Phe Met 50 55 60Ser Arg Leu Arg Ile Ser Lys
Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80Arg Met Asn Ser Leu
Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Lys Val Asn Leu
Ala Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110Val Ile
Val Ser Ser 1154108PRTmouse 4Asp Ile Gln Met Thr Gln Ser Pro Ala
Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg
Ser Ser Gly Asn Ile His Gly Tyr 20 25 30Leu Ala Trp Phe Gln Gln Lys
Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Asn Thr Lys Ala Leu
Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Gln Phe Ser Leu Lys Ile Asn Asn Leu Gln Pro65 70 75 80Glu Asp Phe
Gly Ile Tyr Tyr Cys Gln His His Tyr Asp Leu Pro Arg 85 90 95Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 1055117PRTmouse 5Gln
Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Gln1 5 10
15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Pro Leu Gly Ile Phe
20 25 30Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp
Leu 35 40 45Gly Val Ile Trp Lys Gly Gly Asn Thr Asp Tyr Asn Ala Ala
Phe Met 50 55 60Ser Arg Leu Arg Ile Thr Lys Asp Asn Ser Lys Ser Gln
Val Phe Phe65 70 75 80Arg Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
Ile Tyr Tyr Cys Ala 85 90 95Lys Val Asn Leu Ala Asp Ala Met Asp Tyr
Trp Gly Gln Gly Thr Ser 100 105 110Val Ile Val Ser Ser
1156108PRTmouse 6Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser
Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly
Asn Ile His Gly Tyr 20 25 30Leu Ala Trp Phe Gln Gln Lys Gln Gly Lys
Ser Pro Gln Leu Leu Val 35 40 45Tyr Asn Thr Lys Thr Leu Ala Glu Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Ser
Leu Lys Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Gly Asn Tyr
Tyr Cys Gln His His Tyr Asp Leu Pro Arg 85 90 95Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg 100 1057117PRTmouse 7Gln Val Gln Leu
Lys Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Gln1 5 10 15Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Pro Leu Gly Thr Phe 20 25 30Gly Val
His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly
Val Ile Trp Arg Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Met 50 55
60Ser Arg Leu Arg Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65
70 75 80Arg Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys
Ala 85 90 95Lys Val Asn Leu Ala Asp Ala Met Asp Tyr Trp Gly Gln Gly
Thr Ser 100 105 110Val Ile Val Ser Ser 1158108PRTmouse 8Asp Ile Gln
Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr
Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Gly Tyr 20 25 30Leu
Ala Trp Phe Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40
45Tyr Asn Thr Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His His Tyr Asp
Leu Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 10595PRTmouse 9Thr Tyr Ala Met Ser1 5105PRTmouse 10Val Phe Gly
Val His1 5115PRTmouse 11Ile Phe Gly Val His1 5125PRTmouse 12Thr Phe
Gly Val His1 51316PRTmouse 13Ser Ile Ser Thr Gly Asp Asn Thr Tyr
Tyr Thr Asp Ser Val Arg Gly1 5 10 151416PRTmouse 14Val Ile Trp Lys
Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Met Ser1 5 10
151516PRTmouse 15Val Ile Trp Arg Gly Gly Asn Thr Asp Tyr Asn Ala
Ala Phe Met Ser1 5 10 151611PRTmouse 16Gly Arg Gly Asp Arg Gly Asp
Leu Phe Gly Tyr1 5 10179PRTmouse 17Val Asn Leu Ala Asp Ala Met Asp
Tyr1 51816PRTmouse 18Lys Ser Ser Gln Ser Leu Leu Tyr Arg Gly Asn
Gln Met Asn Tyr Leu1 5 10 151911PRTmouse 19Arg Ser Ser Gly Asn Ile
His Gly Tyr Leu Ala1 5 102011PRTmouse 20Arg Ala Ser Gly Asn Ile His
Gly Tyr Leu Ala1 5 10217PRTmouse 21Trp Ala Ser Thr Arg Glu Ser1
5227PRTmouse 22Asn Thr Lys Ala Leu Ala Glu1 5237PRTmouse 23Asn Thr
Lys Thr Leu Ala Glu1 5249PRTmouse 24Gln Gln Tyr Tyr Thr Tyr Pro Arg
Thr1 5259PRTmouse 25Gln His His Tyr Asp Leu Pro Arg Thr1
526330PRTHomo sapiens 26Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 33027327PRTHomo sapiens 27Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65
70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
Ala Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200
205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315
320Leu Ser Leu Ser Leu Gly Lys 32528107PRTHomo sapiens 28Arg 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 1052934PRTArtificialsynthesized 29Trp Met Glu Trp Asp Arg Glu
Ile Asn Asn Tyr Thr Ser Leu Ile His1 5 10 15Ser Leu Ile Glu Glu Ser
Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 20 25 30Leu
Leu3036PRTArtificialsynthesized 30Tyr Thr Ser Leu Ile His Ser Leu
Ile Glu Glu Ser Gln Asn Gln Gln1 5 10 15Glu Lys Asn Glu Gln Glu Leu
Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30Trp Asn Trp Phe
353139PRTArtificialsynthesized 31Trp Gln Glu Trp Glu Gln Lys Ile
Thr Ala Leu Leu Glu Gln Ala Gln1 5 10 15Ile Gln Gln Glu Lys Asn Glu
Tyr Glu Leu Gln Lys Leu Asp Lys Trp 20 25 30Ala Ser Leu Trp Glu Trp
Phe 353236PRTArtificialSynthesized 32Met Thr Trp Met Glu Trp Asp
Arg Glu Ile Asn Asn Tyr Thr Ser Leu1 5 10 15Ile His Ser Leu Ile Glu
Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu 20 25 30Gln Glu Leu Leu
353338PRTArtificialsynthesized 33Thr Thr Trp Glu Ala Trp Asp Arg
Ala Ile Ala Glu Tyr Ala Ala Arg1 5 10 15Ile Glu Ala Leu Ile Arg Ala
Ala Gln Glu Gln Gln Glu Lys Asn Glu 20 25 30Ala Ala Leu Arg Glu Leu
353436PRTArtificialsynthesized 34Ser Gly Ile Val Gln Gln Gln Asn
Asn Leu Leu Arg Ala Ile Glu Ala1 5 10 15Gln Gln His Leu Leu Gln Leu
Thr Val Trp Gly Ile Lys Gln Leu Gln 20 25 30Ala Arg Ile Leu
353538PRTArtificialsynthesized 35Asn Asn Leu Leu Arg Ala Ile Glu
Ala Gln Gln His Leu Leu Gln Leu1 5 10 15Thr Val Trp Gly Ile Lys Gln
Leu Gln Ala Arg Ile Leu Ala Val Glu 20 25 30Arg Tyr Leu Lys Asp Gln
35367PRTArtificiallinker 36Leu Ser Leu Ser Pro Gly Lys1
5378PRTArtificiallinker 37Leu Ser Pro Asn Arg Gly Glu Cys1
53815PRTArtificiallinker 38Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln
Gly Gln Gln Gln Gln1 5 10 153916PRTArtificiallinker 39Gly Gln Gln
Gln Gln Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln Gly1 5 10
154018PRTArtificiallinker 40Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln
Gly Gln Gln Gln Gln Gly1 5 10 15Asn Asn4116PRTArtificiallinker
41Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly1
5 10 154216PRTArtificiallinker 42Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Asn1 5 10 154315PRTArtificiallinker
43Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly1 5 10
154416PRTArtificiallinker 44Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Gly1 5 10 154517PRTArtificiallinker 45Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly1 5 10
15Thr4617PRTArtificiallinker 46Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Gly1 5 10 15Gly4718PRTArtificiallinker
47Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly1
5 10 15Gly Thr4818PRTArtificiallinker 48Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Gly1 5 10 15Gly
Asn4918PRTArtificiallinker 49Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Gly1 5 10 15Ala Ser5025PRTArtificiallinker
50Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1
5 10 15Gly Gly Gly Gly Ser Gly Gly Gly Gly 20
255126PRTArtificiallinker 51Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser1 5 10 15Gly Gly Gly Gly Ser Gly Gly Gly
Gly Gly 20 255227PRTArtificiallinker 52Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15Gly Gly Gly Gly Ser Gly
Gly Gly Gly Gly Gly 20 255328PRTArtificiallinker 53Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15Gly Gly Gly
Gly Ser Gly Gly Gly Gly Gly Ala Ser 20 255417PRTArtificiallinker
54Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser1
5 10 15Gly5519PRTArtificiallinker 55Gly Ser Ser Ser Ser Ser Ser Ser
Ser Ser Ser Ser Ser Ser Ser Ser1 5 10 15Gly Ala
Ser5618PRTArtificiallinker 56Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly1 5 10 15Ala Ser5716PRTArtificiallinker
57Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1
5 10 155826PRTArtificiallinker 58Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 20 255917PRTArtificiallinker 59Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10
15Gly6027PRTArtificiallinker 60Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly 20 25616PRTArtificiallinker 61Leu Ser Leu Ser Gly Gly1
5627PRTArtificiallinker 62Leu Ser Leu Ser Pro Gly Gly1
563214PRTHomo sapiens 63Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu
Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser
Gly Asn Ile His Gly Tyr 20 25 30Leu Ala Trp Phe Gln Gln Lys Gln Gly
Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Asn Thr Lys Thr Leu Ala Glu
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe
Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Gly Asn
Tyr Tyr Cys Gln His His Tyr Asp Leu Pro Arg 85 90 95Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21064447PRTHomo sapiens 64Gln Val Gln Leu Lys Gln Ser Gly Pro
Gly Leu Val Arg Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Pro Leu Gly Ile Phe 20 25 30Gly Val His Trp Val Arg Gln
Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Lys Gly
Gly Asn Thr Asp Tyr Asn Ala Ala Phe Met 50 55 60Ser Arg Leu Arg Ile
Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80Arg Met Asn
Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Lys Val
Asn Leu Ala Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105
110Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val225 230
235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345
350Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 44565503PRTHomo
sapiens 65Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Arg Pro
Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Pro Leu
Gly Ile Phe 20 25 30Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly
Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Lys Gly Gly Asn Thr Asp Tyr
Asn Ala Ala Phe Met 50 55 60Ser Arg Leu Arg Ile Thr Lys Asp Asn Ser
Lys Ser Gln Val Phe Phe65 70 75 80Arg Met Asn Ser Leu Gln Thr Asp
Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Lys Val Asn Leu Ala Asp Ala
Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110Val Ile Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150
155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265
270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390
395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys Gly 435 440 445Gln Gln Gln Gln Gly Gln Gln Gln Gln
Gly Gln Gln Gln Gln Gly Asn 450 455 460Asn Thr Thr Trp Glu Ala Trp
Asp Arg Ala Ile Ala Glu Tyr Ala Ala465 470 475 480Arg Ile Glu Ala
Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn 485 490 495Glu Ala
Ala Leu Arg Glu Leu 50066345PRTHuman immunodeficiency virus 66Ala
Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly1 5 10
15Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln
20 25 30Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala
Ile 35 40 45Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile
Lys Gln 50 55 60Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys
Asp Gln Gln65 70 75 80Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu
Ile Cys Thr Thr Ala 85 90 95Val Pro Trp Asn Ala Ser Trp Ser Asn Lys
Ser Leu Glu Gln Ile Trp 100 105 110Asn His Thr Thr Trp Met Glu Trp
Asp Arg Glu Ile Asn Asn Tyr Thr 115 120 125Ser Leu Ile His Ser Leu
Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys 130 135 140Asn Glu Gln Glu
Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn145 150 155 160Trp
Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys Leu Phe Ile Met 165 170
175Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe Ala Val Leu Ser
180 185 190Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe
Gln Thr 195 200 205His Leu Pro Thr Pro Arg Gly Pro Asp Arg Pro Glu
Gly Ile Glu Glu 210 215 220Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser
Ile Arg Leu Val Asn Gly225 230 235 240Ser Leu Ala Leu Ile Trp Asp
Asp Leu Arg Ser Leu Cys Leu Phe Ser 245 250 255Tyr His Arg Leu Arg
Asp Leu Leu Leu Ile Val Thr Arg Ile Val Glu 260 265 270Leu Leu Gly
Arg Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn Leu 275 280 285Leu
Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu 290 295
300Asn Ala Thr Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Val Ile
Glu305 310 315 320Val Val Gln Gly Ala Cys Arg Ala Ile Arg His Ile
Pro Arg Arg Ile 325 330 335Arg Gln Gly Leu Glu Arg Ile Leu Leu 340
345
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