U.S. patent application number 15/358219 was filed with the patent office on 2017-09-21 for agonistic antibody to cd27.
The applicant listed for this patent is ADURO BIOTECH HOLDINGS, EUROPE B.V.. Invention is credited to Jannetje Geertruida Borst, Winfried Robert MULDER, Hans VAN EENENNAAM, Aartje Maria Elizabeth Veraar, Paul Maria Frederikus Vink.
Application Number | 20170267771 15/358219 |
Document ID | / |
Family ID | 43016546 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170267771 |
Kind Code |
A1 |
VAN EENENNAAM; Hans ; et
al. |
September 21, 2017 |
AGONISTIC ANTIBODY TO CD27
Abstract
The invention relates to a binding compound, which binds the
same epitope of human CD27 as monoclonal antibody hCD27.15,
produced by hybridoma hCD27.15 which was deposited with the ATCC in
on Jun. 2, 2010 under number PTA-11008. In particular the invention
relates to such a binding compound of claim 1 which may comprise:
an antibody heavy chain variable region which may comprise at least
one CDR selected from the group consisting of SEQ ID NOs : 5, 6 and
7, or a variant of any of said sequences; and/or an antibody light
chain variable region which may comprise at least one CDR selected
from the group consisting of SEQ ID NOs: 8, 9 and 10, or a variant
of any of said sequences.
Inventors: |
VAN EENENNAAM; Hans; (Oss,
NL) ; MULDER; Winfried Robert; (Oss, NL) ;
Borst; Jannetje Geertruida; (Oss, NL) ; Veraar;
Aartje Maria Elizabeth; (Oss, NL) ; Vink; Paul Maria
Frederikus; (Oss, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ADURO BIOTECH HOLDINGS, EUROPE B.V. |
Oss |
|
NL |
|
|
Family ID: |
43016546 |
Appl. No.: |
15/358219 |
Filed: |
November 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13719723 |
Dec 19, 2012 |
9527916 |
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15358219 |
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PCT/EP2011/061557 |
Jul 7, 2011 |
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13719723 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/3955 20130101;
C07K 2317/92 20130101; C07K 16/2878 20130101; A61P 35/00 20180101;
C07K 2319/30 20130101; C07K 2317/75 20130101; A61K 45/06 20130101;
A61P 37/06 20180101; C07K 2317/76 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 9, 2010 |
EP |
10169021.2 |
Claims
1-20. (canceled)
21. A human or humanized antibody or antigen-binding fragment
thereof comprising the following characteristics: (i) competes for
binding to human CD27 with a second antibody or antigen-binding
fragment comprising an antibody heavy chain variable region
comprising the CDRs of SEQ ID NOs: 5, 6 and 7; and an antibody
light chain variable region comprising the CDRs of SEQ ID NOs: 8, 9
and 10; (ii) blocks binding of human CD27 to human CD70; and (iii)
binds human CD27 with a KD of about 100 nM or lower.
22. A human or humanized antibody or antigen-binding fragment
thereof comprising the following characteristics: (i) competes for
binding to human CD27 with a second antibody or antigen-binding
fragment comprising an antibody heavy chain variable region
comprising the CDRs of SEQ ID NOs: 5, 6 and 7; and an antibody
light chain variable region comprising the CDRs of SEQ ID NOs: 8, 9
and 10; (ii) blocks binding of human CD27 to human CD70; (iii)
co-stimulates human CD8+T cells more effectively than the CD27
ligand CD70.
23. The human or humanized antibody or antigen-binding fragment
thereof of claim 21, which is selected from the group consisting of
Fab, Fab', Fab'-SH, Fv, scFv, F(ab').sub.2, bispecific mAb and a
diabody.
24. The human or humanized antibody or antigen-binding fragment
thereof of claim 22, which is selected from the group consisting of
Fab, Fab', Fab'-SH, Fv, scFv, F(ab').sub.2, bispecific mAb and a
diabody.
25. A polynucleotide encoding the human or humanized antibody or
antigen-binding fragment thereof of claim 21.
26. A polynucleotide encoding the human or humanized antibody or
antigen-binding fragment thereof of claim 22.
27. An expression vector comprising the polynucleotide of claim
25.
28. An expression vector comprising the polynucleotide of claim
26.
29. A host cell comprising the expression vector of claim 27.
30. A host cell comprising the expression vector of claim 28.
31. A method of producing an antibody or antigen-binding fragment
thereof, which method comprises: a) culturing a host cell
comprising an expression vector that comprises a polynucleotide
encoding the human or humanized antibody or antigen-binding
fragment thereof of claim 21 under the control of suitable
regulatory sequences in culture medium under conditions wherein the
polynucleotide is expressed, thereby producing polypeptides
comprising the light and heavy chain variable regions; and b)
recovering the polypeptides from the host cell or culture
medium.
32. A method of producing an antibody or antigen-binding fragment
thereof, which method comprises: a) culturing a host cell
comprising an expression vector that comprises a polynucleotide
encoding the human or humanized antibody or antigen-binding
fragment thereof of claim 22 under the control of suitable
regulatory sequences in culture medium under conditions wherein the
polynucleotide is expressed, thereby producing polypeptides
comprising the light and heavy chain variable regions; and b)
recovering the polypeptides from the host cell or culture
medium.
33. A method of stimulating the proliferation and/or survival of
CD27+ cells comprising administering to a subject in need thereof a
human or humanized antibody or antigen-binding fragment thereof
comprising the following characteristics: (i) competes for binding
to human CD27 with a second antibody or antigen-binding fragment
comprising an antibody heavy chain variable region comprising the
CDRs of SEQ ID NOs: 5, 6 and 7; and an antibody light chain
variable region comprising the CDRs of SEQ ID NOs: 8, 9 and 10;
(ii) blocks binding of human CD27 to human CD70; and (iii) binds
human CD27 with a KD of about 100 nM or lower.
34. A method of stimulating the proliferation and/or survival of
CD27+ cells comprising administering to a subject in need thereof a
human or humanized antibody or antigen-binding fragment thereof
comprising the following characteristics: (i) competes for binding
to human CD27 with a second antibody or antigen-binding fragment
comprising an antibody heavy chain variable region comprising the
CDRs of SEQ ID NOs: 5, 6 and 7; and an antibody light chain
variable region comprising the CDRs of SEQ ID NOs: 8, 9 and 10;
(ii) blocks binding of human CD27 to human CD70; and (iii)
co-stimulates human CD8+T cells more effectively than the CD27
ligand CD70.
35. The method of claim 33, wherein the human or humanized antibody
or antigen-binding fragment thereof is selected from the group
consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab').sub.2,
bispecific mAb and a diabody.
36. The method of claim 34, wherein the human or humanized antibody
or antigen-binding fragment thereof is selected from the group
consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab').sub.2,
bispecific mAb and a diabody.
37. The method of claim 33, wherein the CD27+ cell is a CD27+ T
cell.
38. The method of claim 34, wherein the CD27+ cell is a CD27+ T
cell.
39. The human or humanized antibody or antigen-binding fragment
thereof of claim 22 that co-stimulates human CD8+ T cells in
solution more effectively than the CD27 ligand CD70.
40. The human or humanized antibody or antigen-binding fragment
thereof of claim 34 that co-stimulates human CD8+ T cells in
solution more effectively than the CD27 ligand CD70.
Description
RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 13/719,723 filed Dec. 19, 2012, which is a continuation-in-part
application of international patent application Serial No.
PCT/EP2011/061557 filed 7 Jul. 2011, which published as PCT
Publication No. WO 2012/004367 on 12 Jan. 2012, which claims
benefit of European patent application Serial No. 10169021.2 filed
9 Jul. 2010.
[0002] The foregoing applications, and all documents cited therein
or during their prosecution ("appin cited documents") and all
documents cited or referenced in the appin cited documents, and all
documents cited or referenced herein ("herein cited documents"),
and all documents cited or referenced in herein cited documents,
together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products
mentioned herein or in any document incorporated by reference
herein, are hereby incorporated herein by reference, and may be
employed in the practice of the invention. More specifically, all
referenced documents are incorporated by reference to the same
extent as if each individual document was specifically and
individually indicated to be incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to an isolated antibody or
fragments thereof which bind to human CD27, polynucleotides
encoding such antibody and host cells producing said antibody. The
antibody may be used to stimulate lymphocyte proliferation and/or
survival, to treat cancer and to combat autoimmunity or transplant
rejection. In addition, the antibody may be used as a diagnostic
tool and in vitro agent to promote proliferation and/or survival of
CD27.sup.+ cells.
BACKGROUND OF THE INVENTION
[0004] CD27, a TNF receptor family member was identified as a
membrane molecule on human T cells (van Lier et al., 1987, J
Immunol 139:1589-96). According to current evidence, CD27 has a
single ligand, CD70, which is also a TNF family member (Goodwin et
al., 1993, Cell 73:447-56). CD27 and CD70 have also been identified
and cloned in the mouse system (Gravestein et al., 1993, Eur J
Immunol 23:943-50; Tesselaar et al., J Immunol 159:4959-65).
[0005] CD27 is exclusively expressed by hematopoietic cells, in
particular those of the lymphocyte lineage, i.e. T-, B- and NK
cells. In the human system, CD27 expression in the .alpha..beta. T
cell lineage is induced during positive selection of thymocytes and
maintained in naive conventional CD4.sup.+ and CD8.sup.+ T cells
(Vanhecke et al., 1997, J Immunol 159:5973-83). Upon T cell
activation via the T cell antigen receptor (TCR), CD27 expression
increases, in a transient manner (van Lier et al., 1987, J Immunol
139:1589-96). Next, CD27 is shed from the surface of activated T
cells and the soluble form of CD27 can be detected in the serum
marker for (chronic) T cell activation (Hintzen et al., 1991, J
Immunol 147:29-35). Among peripheral T cells, permanent loss of
CD27 expression results from persistent antigenic stimulation and
hallmarks terminally differentiated effector/memory T cells, while
central memory T cells maintain CD27 (Baars et al., 1995, J Immunol
154:17-25; Hamann et al., 1997, J Exp Med 186:1407-18). CD27 is
also expressed on human .gamma..delta. T cells and induced during
thymic development (Offner F et al, 1997, J Immunol 158:4634-41).
Moreover, loss of CD27 expression is a hallmark of chronically
stimulated .gamma..delta. T cells (Gioia C et al., 2002, J Immunol
168:1484-9). Generally, CD27 is an exquisite marker for cellular
activation and differentiation stages and used as such in human
clinical diagnostics and research.
[0006] In the mouse, CD27 was found on hematopoietic stem cells,
multipotent progenitors and common lymphoid precursors (Medina et
al., 2001, Nat Immunol 2:718-24; Wiesmann et al., 2000, Immunity
12:193-9).
[0007] CD27 was originally defined as a human T-cell co-stimulatory
molecule that increments the proliferative response to TCR
stimulation (van Lier et al., 1987, J Immunol 139:1589-96).
Presence of CD70 dictates the timing and persistence of
CD27-mediated co-stimulation. Upon immune activation, dendritic
cells, T-, B- and NK cells transiently express CD70, contingent
upon the presence of antigen, Toll-like receptor agonists or
inflammatory cytokines.
[0008] CD27 stimulation using anti-CD27 mAb CLB-CD27/1 (9F4)
incremented the proliferative response of human T cells to TCR
stimulation (Van Lier et al., 1987, J Immunol 139:1589-96). This
was confirmed using crosslinked anti-CD27 mAb 1A4, or transfectants
expressing CD70. Conversely, antibodies directed to CD27 or CD70
could block this proliferation. Both CD4.sup.+ and CD8.sup.+ T
cells responded to CD27 co-stimulation (Goodwin et al., 1993, Cell
73:447-56; Kobata et al., 1994, J Immunol 153:5422-32; Hintzen et
al., 1995, J Immunol 154:2612-23). Studies in mice unambiguously
support the role of CD27 as a co-stimulatory receptor for naive
CD8.sup.+ and CD4.sup.+ .alpha..beta. T cells. For mouse T cells,
CD27 primarily promotes their survival upon TCR-mediated
activation, but in human T cells, it additionally promotes cell
cycle entry and/or activity (reviewed in Borst et al., 2005, Curr
Op Immunol 17:275-281; Nolte et al., 2009, Immunol Rev
229:216-231).
[0009] Upon its transient engagement as occurs in acute infections
that temporarily upregulate CD70, CD27 supports the generation of a
CD8.sup.+ effector T cell pool in priming organs, its maintenance
at the tissue effector site, its conversion into memory cells and
its potential to exercise memory function (Hendriks et al., 2003, J
Exp Med 198:1369-1380; Hendriks et al. 2005, J Immunol 175:1666-75,
Xiao et al, 2008, J Immunol 181:1071-82). Studies with CD70
blocking antibody in mouse models support the concept that
CD27-CD70 interactions can make an important contribution to
generation of CD8.sup.+ effector T cells, e.g. after protein
immunization, virus infection and allotransplantation (Taraban et
al., 2004, J Immunol 173:6542-46; Bullock and Yagita, 2005, J
Immunol 174:710-17; Yamada et al., 2005, J Immunol 174:1357-1364;
Schildknecht et al., 2007, Eur J Immunol 37:716-28).
[0010] Transgenic expression of CD70 in immature dendritic cells
sufficed to convert immunological tolerance to virus or tumors into
CD8.sup.+ T cell responsiveness upon immunization with MHC class
I-restricted peptide in PBS. Likewise, agonistic soluble CD70
promoted the CD8.sup.+ T cell response upon such peptide
immunization (Rowley et al., 2004, J Immunol 172:6039-6046) and in
CD70 transgenic mice, CD4.sup.+ and CD8.sup.+ effector cell
formation in response to TCR stimulation was greatly facilitated
(Arens et al. 2001, Immunity 15:801-12; Tesselaar et al., 2003, Nat
Immunol 4:49-54; Keller et al. 2008, Immunity 29:334-346). In mouse
lymphoma models, tumor rejection was improved upon CD70
transgenesis or injection of an activating anti-mouse CD27 antibody
(Arens et al., 2003, J Exp Med 199:1595-1605; French et al., 2007,
Blood 109:4810-15; Sakanishi and Yagita, 2010, Biochem. Biophys.
Res. Comm. 393:829-835; WO 2008/051424).
[0011] Generally, CD27 expression on lymphoid cells is associated
with survival potential. Salient examples come from human adoptive
T cell therapies, in cancer and AIDS patients, where long-term
persisting T cells were selected for CD27 expression. In addition,
CD70 expression on tumor-infiltrating lymphocytes was positively
correlated with an anti-tumor immune response, potentially
reflecting effector T cell survival within the tumor (Ochsenbein et
al., 2004, J Exp Med 200:1407-17; Huang et al., 2006, J Immunol
176:7726-35).
[0012] For conventional CD4.sup.+ T cells, CD27 similarly promotes
primary and secondary responses (Hendriks et al., 2000, Nat Immunol
1:433-40; Xiao et al, 2008, J Immunol 181:1071-82). Moreover, CD27
co-stimulation favours an IL-12 independent pathway for T helper-1
development and enables CD4.sup.+ T cells to provide help for
memory programming of CD8.sup.+ T cells (Soares et al., 2007, J Exp
Med 204:1095-106; Xiao et al, 2008, J Immunol 181:1071-8). In
C57BL/6 mice, CD27 stimulation is consistently associated with
Th1-type CD4.sup.+ T cell differentiation (Arens et al. 2001,
Immunity 15:801-12; Soares et al., 2007, J Exp Med 204:1095-106;
Xiao et al, 2008, J Immunol 181:1071-82) and in human CD4.sup.+ T
cells in vitro, CD27 promoted Th1 development in presence of IL-12,
but had no differentiation-inducing effect in presence of IL-4 (van
Oosterwijk et al., 2007, Int Immunol 19:713-18).
[0013] In addition, CD27 stimulation was demonstrated to promote
human regulatory T cell generation and/or function (Jacquot et al.,
1997, Cell Immunol 179:48-54). Amongst natural regulatory T cells
in human, high CD27 expression hallmarks the cells that have the
highest suppressive activity and the CD27 high subpopulation is
preferentially amplified during rapamycin treatment (Koenen et al.,
2005, J Immunol 174:7573-83; Coenen et al., 2006, Blood
107:1018-23). Recent observations suggest that the CD27.sup.+ Treg
subpopulation can differentiate into Th17 cells (Koenen et al.,
2008, Blood 112:2340-52). Interestingly, CD70.sup.+ B lymphoma
cells were found to stimulate Treg formation and impede Th17
differentiation by CD27 triggering on intratumoral T cells (Yang et
al., 2007, Blood 110:2537-44; Yang et al., 2009, Cancer Res
69:5522-30).
[0014] In resting B cells, CD27 expression 1s absent, but it is
induced during B cell activation in germinal centers and in human,
it is subsequently maintained on memory B cells and plasma cells
(Agematsu et al., 2000 Immunol Today 21:204-206; Jung et al., 2000,
Eur J Immunol 30:2437-2443). CD27 also acts as a co-stimulatory
receptor on B cells. In in vitro systems with human B cells,
CD27-CD70 interactions consistently stimulate Ig secretion
(Agematsu et al., 1997, Eur J Immunol 27:2073-79; Jacquot et al.,
1997, J Immunol 159:2652-7).
[0015] Human NK cells can be subdivided into two functional subsets
based on CD27 expression with lack of CD27 expression identifying
the mature effector cells (Sugita et al., 1992, J Immunol
149:1199-203; Vossen et al., 2008, J Immunol 180:3739-45). Data
suggest a similar co-stimulatory role for CD27 in NK cells as for
T-cells (Takeda et al., 2000, J Immunol 164; 1741-1745). The
functional effect of CD27 activation on NK cells was established by
increased NK mediated killing of CD70-expressing tumor cells.
CD27-mediated NK cell activation also promoted the generation of
CD8.sup.+ anti-tumor immunity (Aulwurm et al., 2006, Int J Cancer
11S:1728-1735; Kelly et al., 2002, Nat Immunol 3:83-90). Recently,
NKT cells were shown to promote CD8.sup.+ T cell immunity by
induction of CD70 on dendritic cells (Taraban et al., 2008, J
Immunol 139:1589-96).
[0016] In addition, CD27 is highly expressed on tumor cells derived
from non-Hodgkin's lymphomas and chronic lymphocytic leukemias
(Ranheim et al., 1995, Blood 85:3556-3565; van Oers et al., 1993,
Blood 82:3430-3436). Soluble CD27 is used as a serum marker for
lymphoid malignancy (Van Oers et al., 1993, Blood 82:3430-6).
[0017] In the research that led to the present invention it was
found that the hCD27.15 mAb stimulates the proliferation and/or
survival of CD27.sup.+ cells. Enhanced proliferation and/or
survival of CD27.sup.+ cells forms the basis of different
therapeutic uses. Monoclonal antibodies that activate CD27 are
known. Two activating anti-human CD27 antibodies have been
described (Van Lier et al., 1987, J. Immunol. 1987, 139:1589-96;
Kobata et al., 1994, J. Immunol. 153:5422-5432). In addition,
activating anti-mouse CD27 antibodies have been described (French
et al., 2007, Blood 109:4810-15; WO 2008/051424; Sakanishi and
Yagita, 2010, Biochem. Biophys. Res. Comm. 393:829-835). hCD27.15
is a unique anti-human antibody, which is, in contrast to 1A4 and
9F4 able to activate human CD27 more effectively than its ligand
CD70. These characteristics of hCD27.15 result in a significantly
increased effect on proliferation of CD8.sup.+ and CD4.sup.+
T-cells as compared to 1A4, 9F4 and Fc-CD70. Administration of
hCD27.15 alone or in combination with other agents to a human being
can for example be used in the treatment of cancer.
[0018] Citation or identification of any document in this
application is not an admission that such document is available as
prior art to the present invention.
SUMMARY OF THE INVENTION
[0019] The invention thus relates to binding compounds that bind to
the same epitope as hCD27.15. The hybridoma producing hCD27.15 was
deposited with the ATCC on Jun. 2, 2010 and given the deposit
accession number PTA-11008.
[0020] The invention relates to all molecules that bind the same
epitope as it was found that binding to this particular epitope
stimulates the proliferation and/or survival of a CD27.sup.+
cell.
[0021] In one embodiment, the invention relates to binding
compounds, which bind to CD27 and may comprise: [0022] an antibody
heavy chain variable region which may comprise at least one CDR
selected from the group consisting of SEQ ID NOs: 5, 6 and 7, or a
variant of any of said sequences; and/or [0023] an antibody light
chain variable region which may comprise at least one CDR selected
from the group consisting of SEQ ID NOs: 8, 9 and 10, or a variant
of any of said sequences.
[0024] In one embodiment, the invention relates to binding
compounds, which bind to CD27 and may comprise: [0025] an antibody
heavy chain variable region which may comprise a combination of
[0026] CDRs selected from the group consisting of SEQ ID NOs: 5, 6
and 7, SEQ ID NOs: 5 and 7, SEQ ID NOs: 6 and 7, and SEQ ID NOs: 5
and 6, or a variant of any of said sequences; and/or [0027] an
antibody light chain variable region which may comprise a
combination of CDRs selected from the group consisting of SEQ ID
NOs: 8, 9 and 10, SEQ ID NOs: 8 and 10, SEQ ID NOs: 9 and 10, and
SEQ ID NOs: 8 and 9, or a variant of any of said sequences.
[0028] In one embodiment, the invention relates to any combination
of heavy and light chain variable regions having the combinations
of CDRs disclosed above, in particular the following combinations:
[0029] SEQ ID NOs: 5, 6 and 7 with SEQ ID NOs: 8, 9 and 10, [0030]
SEQ ID NOs: 5, 6 and 7 with SEQ ID NOs: 8 and 10, [0031] SEQ ID
NOs: 5, 6 and 7 with SEQ ID NOs: 9 and 10, [0032] SEQ ID NOs: 5, 6
and 7 with SEQ ID NOs: 8 and 9 [0033] SEQ ID NOs: 5 and 7 with SEQ
ID NOs: 8, 9 and 10, [0034] SEQ ID NOs: 5 and 7 with SEQ ID NOs: 8
and 10, [0035] SEQ ID NOs: 5 and 7 with SEQ ID NOs: 9 and 10,
[0036] SEQ ID NOs: 5 and 7 with SEQ ID NOs: 8 and 9, [0037] SEQ ID
NOs: 6 and 7 with SEQ ID NOs: 8, 9 and 10, [0038] SEQ ID NOs: 6 and
7 with SEQ ID NOs: 8 and 10, [0039] SEQ ID NOs: 6 and 7 with SEQ ID
NOs: 9 and 10, [0040] SEQ ID NOs: 6 and 7 with SEQ ID NOs: 8 and 9,
[0041] SEQ ID NOs: 5 and 6 with SEQ ID NOs: 8, 9 and 10, [0042] SEQ
ID NOs: 5 and 6 with SEQ ID NOs: 8 and 10, [0043] SEQ ID NOs: 5 and
6 with SEQ ID NOs: 9 and 10, [0044] SEQ ID NOs: 5 and 6 with SEQ ID
NOs: 8 and 9.
[0045] In one embodiment, the invention relates to binding
compounds, which bind to CD27 and may comprise: [0046] an antibody
heavy chain variable region which may comprise the CDRs of SEQ ID
NOs: 5, 6 and 7, or a variant of any of said sequences; and/or
[0047] an antibody light chain variable region which may comprise
the CDRs of SEQ ID
[0048] NOs: 8, 9 and 10, or a variant of any of said sequences.
[0049] In one embodiment, the binding molecule binds to CD27 and
comprises: [0050] a heavy chain variable region which may comprise
the amino acid sequence of SEQ ID NO: 3 and a light chain variable
region which may comprise the amino acid sequence of SEQ ID NO:
4.
[0051] In one embodiment, the binding compound is monoclonal
antibody hCD27.15 as produced by hybridoma hCD27.15 (deposit
accession number PTA-11008) or a humanized version thereof.
[0052] In one embodiment the binding compound is a fragment,
variant or derivative of an antibody.
[0053] According to a further aspect thereof, the invention relates
to an isolated polynucleotide encoding a binding compound of the
invention. The invention further relates to an expression vector
which may comprise the said polynucleotide and a host cell which
may comprise the expression vector. In one embodiment, the
invention relates to the isolated polynucleotides of SEQ ID NOs 1
and 2, which encode the heavy and light chain of hCD27.15,
respectively.
[0054] In one embodiment, the binding compound: [0055] binds human
CD27 with a K.sub.D of about 100 nM or lower; and [0056] blocks
binding of human CD27 to human CD70 with an IC.sub.50 of about 10
nM or lower.
[0057] In one embodiment, the invention relates to a binding
compound which competes for a binding epitope on human CD27 with
any of the above binding compounds and has one or more of the
following characteristics: [0058] binds human CD27 with a K.sub.D
of about 100 nM or lower; [0059] binds to human CD27 with about the
same K.sub.D as an antibody having a heavy chain which may comprise
the amino acid sequence of SEQ ID NO: 3 and a light chain which may
comprise the amino acid sequence of SEQ ID NO: 4; [0060] blocks
binding of human CD27 to human CD70 with an IC.sub.50 of about 10
nM or lower.
[0061] The binding compound may be any one of the following: [0062]
a chimeric antibody or a fragment thereof; [0063] a human antibody
or a fragment thereof; [0064] a humanized antibody or a fragment
thereof; or [0065] an antibody fragment selected from the group
consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab').sub.2,
bispecific mAb and a diabody.
[0066] According to another aspect thereof the invention relates to
an isolated polynucleotide encoding the binding compound of the
invention. In one embodiment, the isolated polynucleotide comprises
SEQ ID NOs 1 and 2, which encode the heavy and light chain of
hCD27.15.
[0067] The invention also relates to an expression vector which may
comprise the polynucleotide and to a host cell which may comprise
the expression vector or the polynucleotide.
[0068] According to a further aspect thereof, the invention relates
to a method of producing a binding compound of the invention, which
method comprises: [0069] (a) culturing host cell which may comprise
an expression vector that comprises a polynucleotide encoding a
binding compound of the invention under the control of suitable
regulatory sequences in culture medium under conditions wherein the
polynucleotide is expressed, thereby producing polypeptides which
may comprise the light and heavy chain variable regions; and (b)
recovering the polypeptides from the host cell or culture
medium.
[0070] The invention further relates to a composition which may
comprise a binding compound in combination with a pharmaceutically
acceptable carrier or diluent. Such composition in one embodiment
may comprise more than one binding compound. In one embodiment, the
composition comprises one or more other active compounds in
addition to the one or more binding compounds of the invention.
Such combination compositions may be used for combination therapy,
for example in the treatment of cancer. In that case the binding
compound is combined with one or more of the usual anticancer
drugs. For other combination therapies other additional active
compounds are used. For combination therapy it is not obligatory to
have the two or more active compounds in the same composition.
Thus, also part of the invention is the combined or subsequent use
of the binding compounds and the other active compound, wherein the
binding compound and the other active compound are administered
simultaneously or subsequently.
[0071] The invention further relates to the use of the binding
compounds in therapy and diagnosis and for other, non-therapeutic
purposes.
[0072] In one embodiment, the therapy comprises stimulation of
proliferation and/or survival of CD27.sup.+ cells. In one
embodiment, the therapy comprises the treatment of cancer. In one
embodiment, the therapy comprises the treatment of an autoimmune
disease.
[0073] The binding compound of the invention when used in
non-therapeutic applications may for example be applied in
techniques such as flow-cytometry, Western blotting, enzyme-linked
immunosorbent assay (ELISA) and immunohistochemistry.
[0074] In the following these binding compounds will be referred to
as "binding compounds based on hCD27.15". This phrase is intended
to encompass every compound that binds to the epitope of CD27
recognized by hCD27.15 as described above. Such compounds may be
antibodies that have one or more of the CDR regions of hCD27.15 or
fragments, variants or derivatives thereof, or the monoclonal
antibody hCD27.15 or a humanized version thereof or other molecules
that are capable of binding to this epitope.
[0075] In one embodiment, the therapy of the invention comprises
targeting CD27.sup.+ CD4.sup.+ or CD8.sup.+ T cell subsets such as
Tregs, Th17 cells or Th1 cells with a binding compound based on
hCD27.15, in particular the hCD27.15 mAb. Targeting these
CD27.sup.+ cells with binding compounds based on hCD27.15, in
particular the hCD27.15 mAb, will direct the nature of CD4.sup.+ T
cell cytokine production and CD4.sup.+ T cell help for CD8.sup.+ T
cell responses, which is beneficial in treating various disease
situations, including cancer and auto-immunity. Examples are formed
by, but not restricted to lymphocyte derived tumors such as
non-Hodgkin's lymphoma, Chronic Lymphocytic leukemias; solid tumors
like pancreatic, colon and prostate carcinomas. For this purpose,
hCD27.15 may be dosed directly to subjects, alone or in combination
with other anti-cancer agents. Examples of use in autoimmunity
include, but are not restricted to Rheumatoid Arthritis, Systemic
Lupus Erythematosus and Psoriasis.
[0076] In addition, the therapy may be directed to infections, such
as viral and microbial infections. Examples include, but are not
restricted to administering hCD27.15 alone or in combination with
other anti-infective drugs to a subject who has been infected with
influenza virus or CMV virus.
[0077] In one embodiment, stimulations of the immune system with
binding compounds based on hCD27.15, in particular the hCD27.15
mAb, may be used to increase vaccine responses. Non-limiting
examples of vaccines that may be used in combination with hCD27.15
stimulation include DNA-, cell-based or peptide-based vaccines that
are designed to elicit a CD8.sup.+ T cell response to cancer or
infectious agents. For this purpose, binding compounds based on
hCD27.15, in particular the hCD27.15 antibody, may be administered
before vaccination, at an appropriate time after vaccination, or be
formulated into the vaccine.
[0078] Current state of the art technologies allow the isolation of
CD27.sup.+ cells and the isolation of different cellular subsets.
After stimulation of such isolated subsets of cells outside of the
body of the patient with hCD27.15, they may subsequently be
adoptively transferred to the patient or another patient. In one
embodiment, the subset of cells is formed by CD27.sup.+ regulatory
T-cells (Tregs), which may be isolated from patients suffering from
autoimmune disease and which have been shown to demonstrate
superior suppressive characteristics.
[0079] The monoclonal antibody hCD27.15 promotes the proliferation
and/or survival of CD27.sup.+ cells. State of the art technologies
use the isolation of (subsets of) cells from a wide range of body
fluids and organs. Based on hCD27.15 stimulatory characteristics,
binding compounds based on hCD27.15 may be used in in vitro
cellular systems to promote proliferation and/or survival of
CD27.sup.+ cells. A non-limiting example forms Tregs, which have
been demonstrated to have a short lifespan. Other examples form
memory B-cells and activated T-cells.
[0080] Stimulation and proliferation of CD27.sup.+ cells using
binding compounds based on hCD27.15, in particular the hCD27.15
mAb, may thus be used to increase the Treg populations ex vivo,
which may be adoptively transferred to the patient to suppress the
hyper-activated immune system of the patient. Such approach could
be used to treat patients who suffer from an activated immune
system. This strategy may thus for example be used to prevent
transplant rejections and to treat autoimmune and inflammatory
diseases.
[0081] Another example is the isolation of tumor-associated
lymphocytes. Such lymphocytes harbor anti-tumor activity, but are
suppressed in activation by the tumor and its environment.
Isolation of these lymphocytes, subsequent activation outside of
the body using binding compounds based on hCD27.15, in particular
the hCD27.15 mAb, and adoptive transfer to the patient is expected
to deliver a proficient anti-tumor response.
[0082] The binding compounds based on hCD27.15, in particular the
hCD27.15 antibody, may also be applied in vivo to target CD27.sup.+
tumor cells. The non-modified binding compounds based on hCD27.15,
in particular the hCD27.15 antibody, may for example be injected
into patients with a CD27.sup.+ malignancy to elicit
antibody-dependent cytotoxicity or other immune effector
mechanisms. The binding compounds based on hCD27.15, in particular
the hCD27.15 antibody, may also be conjugated with a toxin or other
appropriate drug to kill the targeted CD27.sup.+ tumor cells.
[0083] The binding compounds based on hCD27.15 may also be useful
in diagnostic assays, e.g., for detecting expression of CD27 on
specific cells, tissues, or in serum. For diagnostic applications,
the binding compounds based on hCD27.15 typically will be labeled
(either directly or indirectly) with a detectable moiety. Numerous
labels are available which may be generally grouped into the
following categories: biotin, fluorochromes, radionucleotides,
enzymes, iodine, and biosynthetic labels.
[0084] Soluble CD27 present in the serum and other body fluids of a
range of different patients has been shown to correlate with
disease severity of the patients. For example, patients suffering
from chronic lymphocytic leukemia, acute lymphoblastic leukemia and
non-Hodgkin's lymphoma demonstrated increased serum levels of
soluble CD27. Based on the demonstrated binding characteristics of
hCD27.15, binding compounds based on hCD27.15 may be used as a
diagnostic tool to detect soluble CD27 in the body fluids.
[0085] The binding compounds based on hCD27.15 of the present
invention may be employed in any known assay method, such as
competitive binding assays, direct and indirect sandwich assays,
and immunoprecipitation assays. Zola, Monoclonal Antibodies. A
Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
[0086] The binding compounds based on hCD27.15 may also be used for
in vivo diagnostic assays. Generally, the binding compound is
labeled with a radionuclide so that the antigen or cells expressing
it may be localized using immunoscintigraphy or positron emission
tomography.
[0087] According to another aspect of the invention, the binding
compounds have other, non-therapeutic uses. The non-therapeutic
uses for these binding compounds based on hCD27.15 include flow
cytometry, western blotting, enzyme linked immunosorbant assay
(ELISA) and immunohistochemistry.
[0088] The binding compounds based on hCD27.15 of this invention
may for example be used as an affinity purification reagent via
immobilization to a Protein A-Sepharose column.
[0089] Accordingly, it is an object of the invention to not
encompass within the invention any previously known product,
process of making the product, or method of using the product such
that Applicants reserve the right and hereby disclose a disclaimer
of any previously known product, process, or method. It is further
noted that the invention does not intend to encompass within the
scope of the invention any product, process, or making of the
product or method of using the product, which does not meet the
written description and enablement requirements of the USPTO (35
U.S.C. .sctn.112, first paragraph) or the EPO (Article 83 of the
EPC), such that Applicants reserve the right and hereby disclose a
disclaimer of any previously described product, process of making
the product, or method of using the product.
[0090] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. Patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. Patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0091] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
Deposits
[0092] The Deposits with the ATCC, under deposit accession number
PTA-11008 were made pursuant to the terms of the Budapest Treaty.
Upon issuance of a patent, all restrictions upon the deposit will
be removed, and the deposit is intended to meet the requirements of
37 CFR .sctn..sctn.1.801-1.809. The deposit will be maintained in
the depository for a period of 30 years, or 5 years after the last
request, or for the effective life of the patent, whichever is
longer, and will be replaced if necessary during that period.
BRIEF DESCRIPTION OF THE DRAWINGS
[0093] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings.
[0094] FIG. 1. Characterization of anti-hCD27 antibody. A. Binding
of hCD27.15 to CHO-K1 that were stably transfected with
pCI-neo-hCD27 (CHO-K1.CD27). Anti-hCD27 57703 (R&D systems) and
anti-hCD27 1A4 (Beckman Coulter) are positive controls. Antibodies
were not reactive with CHO-K1 control cells (data not shown). B.
Effect of hCD27.15 on binding of soluble recombinant hCD27-Fc
fusion protein to CHO-K1 cells that had been stably transfected
with pCI-neo-hCD70 (CHO-K1.CD70). C. Effect of hCD27.15 on binding
of recombinant hCD70-mCD8 fusion protein to CHO-K1.CD27.
[0095] FIG. 2. hCD27.15 induces CD27 signaling leading to
NF-.kappa.B activation. Human embryonic kidney cells (HEK293T) were
transiently transfected to express an NF-.kappa.B-luciferase
reporter construct together with a hCD27 encoding vector or a
control vector. The cells were stimulated for 20 hours in presence
or absence of hCD27.15 mAb (10 .mu.g/ml). Stimulation with hCD27.15
mAb revealed specific CD27-induced NF-.kappa.B activation, as read
out by luciferase activity. (A) Absolute values of luciferase
activity as read out by luciferin bioluminescence after stimulation
of hCD27-expressing HEK293T cells with mAb hCD27.15 or an isotype
control mAb. Data represent triplicate measurements from 1
experiment (+SD). Significance was measured using 2-tailed
Student's t test. (B) Fold induction of luciferase activity after
stimulation of HEK293T cells transfected to express hCD27 or
control vector with hCD27.15 mAb. Data were obtained from 3
independent experiments (N=3+SD). (C) hCD27.15 is superior to other
hCD27 agonists. HEK293T cells expressing CD27 and the
NF-.kappa.B-luciferase reporter were stimulated with soluble
agonistic recombinant CD70 protein (Fc-mCD70, 2 .mu.g/ml), mAb
hCD27.15 (10 .mu.g/ml), or equal concentrations of other mAbs
directed against hCD27. Luciferase activity was read out at the
indicated time points. Data represent triplicate measurements from
1 experiment (+SD). Significance was measured using 2-tailed
Student's t test.
[0096] FIG. 3. hCD27.15 induces proliferation and/or promotes
survival of CD4.sup.+ CD25.sup.- T-cells. CD4.sup.+ CD25.sup.-
T-cells were isolated by MACS (neg. selection) from human PBMC's
and cultured in 96 well-plates at a concentration of
1.times.10.sup.5 cells/well. Stimuli were added as indicated in the
Figure and proliferation was determined by [.sup.3H] thymidine
incorporation.
[0097] FIG. 4A-B. hCD27.15 induces proliferation and/or promotes
survival of naive CD8.sup.+ T-cells. Naive CD8.sup.+ T cells were
purified from human PBMC, labeled with CFSE and stimulated for 6
days with anti-CD3 and anti-CD28 mAbs in presence or absence of
hCD27.15 mAb (10 .mu.g/ml). hCD27.15 mAb stimulates cell division,
as hallmarked by the percentage of cells in each division cycle (A)
and total live cell yield, as hallmarked by the absolute number of
cells in each division cycle (B). Data were obtained from 4
independent experiments with cells of 4 healthy individuals (N=4
+/-SEM).
[0098] FIG. 5. hCD27.15 stimulates CD8.sup.+ T cells to produce
specific cytokines. Naive CDS+ T cells were isolated as indicated
for FIG. 3 and stimulated with anti-CD3 and anti-CD28 mAb in
presence or absence of hCD27.15 mAb (10 .mu.g/ml). (A) After
culture for 72 hours, supernatants of cells were taken and
cytokines were measured by using Luminex assay. (B) Cell numbers
were not significantly different after 72 hours of culture,
indicating a qualitative difference in the secretion of certain
cytokines. (A,B) Data obtained from 3 independent experiments with
cells of 3 healthy individuals (N=3+/-SEM).
[0099] FIG. 6 shows the variable region sequences of hCD27.15.
Panel A and B show the amino acid sequences of the heavy and light
chain variable sequence of hCD27.15, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0100] The term "antibody" refers to any form of antibody that
exhibits the desired biological activity, such as inhibiting
binding of a ligand to its receptor, or by inhibiting
ligand-induced signaling of a receptor. In the present case the
biological activity comprises agonist activity on CD27. Thus,
"antibody" is used in the broadest sense and specifically covers,
but is not limited to, monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, and multispecific
antibodies (e.g., bispecific antibodies).
[0101] "Antibody fragment" and "antibody binding fragment" mean
antigen-binding fragments and analogues of an antibody, typically
including at least a portion of the antigen binding or variable
regions (e.g. one or more CDRs) of the parental antibody. An
antibody fragment retains at least some of the binding specificity
of the parental antibody. Typically, an antibody fragment retains
at least 10% of the parental binding activity when that activity is
expressed on a molar basis. Preferably, an antibody fragment
retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of
the parental antibody's binding affinity for the target. Examples
of antibody fragments include, but are not limited to, Fab, Fab',
F(ab')2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules, e.g., sc-Fv, unibodies (technology
from Genmab); nanobodies (technology from Domantis); domain
antibodies (technology from Ablynx); and multispecific antibodies
formed from antibody fragments. Engineered antibody variants are
reviewed in Bolliger and Hudson, 2005, Nat. Biotechnol.
23:1126-1136.
[0102] An "Fab fragment" is comprised of one light chain and the
CH1 and variable regions of one heavy chain. The heavy chain of a
Fab molecule cannot form a disulfide bond with another heavy chain
molecule.
[0103] An "Fc" region contains two heavy chain fragments which may
comprise the C.sub.H1 and C.sub.H2 domains of an antibody. The two
heavy chain fragments are held together by two or more disulfide
bonds and by hydrophobic interactions of the C.sub.H3 domains.
[0104] An "Fab' fragment" contains one light chain and a portion of
one heavy chain that contains the V.sub.H domain and the C.sub.H1
domain and also the region between the C.sub.H1 and C.sub.H2
domains, such that an interchain disulfide bond may be formed
between the two heavy chains of two Fab' fragments to form a
F(ab').sub.2 molecule.
[0105] An "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H2 domains, such that an interchain
disulfide bond is formed between the two heavy chains. A
F(ab').sub.2 fragment thus is composed of two Fab' fragments that
are held together by a disulfide bond between the two heavy
chains.
[0106] The "Fv region" comprises the variable regions from both the
heavy and light chains, but lacks the constant regions.
[0107] A "single-chain Fv antibody" (or "scFv antibody") refers to
antibody fragments which may comprise the V.sub.H and V.sub.L
domains of an antibody, wherein these domains are present in a
single polypeptide chain. Generally, the Fv polypeptide further
comprises a polypeptide linker between the V.sub.H and V.sub.L
domains which enables the scFv to form the desired structure for
antigen binding. For a review of scFv, see Pluckthun, 1994, The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds. Springer-Verlag, New York, pp. 269-315. See also,
International Patent Application Publication No. WO 88/01649 and
U.S. Pat. Nos. 4,946,778 and 5,260,203.
[0108] A "diabody" is a small antibody fragment with two
antigen-binding sites. The fragments may comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L or
V.sub.L-V.sub.H). By using a linker that is too short to allow
pairing between the two domains on the same chain, the domains are
forced to pair with the complementary domains of another chain and
create two antigen-binding sites. Diabodies are described more
fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al., 1993,
Proc. Natl. Acad. Sci. USA 90:6444-6448.
[0109] A "domain antibody fragment" is an immunologically
functional immunoglobulin fragment containing only the variable
region of a heavy chain or the variable region of a light chain. In
some instances, two or more V.sub.H regions are covalently joined
with a peptide linker to create a bivalent domain antibody
fragment. The two V.sub.H regions of a bivalent domain antibody
fragment may target the same or different antigens.
[0110] As used herein antibody hCD27.15 is a mouse antibody wherein
the heavy chain has the variable region sequence of SEQ ID NO: 3
and is joined to a IgG1 constant region and the light chain has the
variable region sequence of SEQ ID NO: 4 and is joined to the K
constant region. The hybridoma producing the hCD27.15 antibody was
deposited with ATCC on June 2, 2010 under number PTA-11008.
[0111] An antibody fragment of the invention may comprise a
sufficient portion of the constant region to permit dimerization
(or multimerization) of heavy chains that have reduced disulfide
linkage capability, for example where at least one of the hinge
cysteines normally involved in inter-heavy chain disulfide linkage
is altered as described herein. In another embodiment, an antibody
fragment, for example one that comprises the Fe region, retains at
least one of the biological functions normally associated with the
Fe region when present in an intact antibody, such as FcRn binding,
antibody half life modulation, ADCC (antibody dependent cellular
cytotoxicity) function, and/or complement binding (for example,
where the antibody has a glycosylation profile necessary for ADCC
function or complement binding).
[0112] The term "chimeric" antibody refers to antibodies in which a
portion of the heavy and/or light chain is identical with or
homologous to corresponding sequences in antibodies derived from a
particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired biological activity (See, for example, U.S. Pat. No.
4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA
81:6851-6855).
[0113] As used herein, the term "humanized antibody" refers to
forms of antibodies that contain sequences from non-human (e.g.,
murine) antibodies as well as human antibodies. Such antibodies
contain minimal sequence derived from non-human immunoglobulin. In
general, the humanized antibody may comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence. The humanized
antibody optionally also may comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. The humanized forms of rodent antibodies may
essentially comprise the same CDR sequences of the parental rodent
antibodies, although certain amino acid substitutions may be
included to increase affinity, increase stability of the humanized
antibody, or for other reasons. However, as CDR loop exchanges do
not uniformly result in an antibody with the same binding
properties as the antibody of origin, changes in framework residues
(FR), residues involved in CDR loop support, might also be
introduced in humanized antibodies to preserve antigen binding
affinity (Kabat et al., 1991, J Immunol. 147:1709).
[0114] The term "antibody" also includes "fully human" antibodies,
i.e., antibodies that may comprise human immunoglobulin protein
sequences only. A fully human antibody may contain murine
carbohydrate chains if produced in a mouse, in a mouse cell, or in
a hybridoma derived from a mouse cell. Similarly, "mouse antibody"
or "rat antibody" refer to an antibody that may comprise only mouse
or rat immunoglobulin sequences, respectively. A fully human
antibody may be generated in a human being, in a transgenic animal
having human immunoglobulin germline sequences, by phage display or
other molecular biological methods. Also, recombinant
immunoglobulins may also be made in transgenic mice. See Mendez et
al., 1997, Nature Genetics 15:146-156. See also Abgenix and Medarex
technologies.
[0115] The binding compounds of the present invention also include
antibodies with modified (or blocked) Fc regions to provide altered
effector functions. See, e.g. U.S. Pat. No. 5,624,821;
WO2003/086310; WO2005/120571; WO2006/0057702; Presta, 2006, Adv.
Drug Delivery Rev. 58:640-656. Such modification may be used to
enhance or suppress various reactions of the immune system, with
possible beneficial effects in diagnosis and therapy. Alterations
of the Fc region include amino acid changes (substitutions,
deletions and insertions), glycosylation or deglycosylation, and
adding multiple Fc. Changes to the Fc may also alter the half-life
of antibodies in therapeutic antibodies, and a longer half-life
would result in less frequent dosing, with the concomitant
increased convenience and decreased use of material. See Presta,
2005, J Allergy Clin. Immunol. 116:731 at 734-35.
[0116] The binding compounds of the present invention also include
antibodies with intact Fc regions that provide full effector
functions, e.g. antibodies of isotype IgG1, which induce
complement-dependent cytotoxicity (CDC) or antibody dependent
cellular cytotoxicity (ADCC) in the a targeted cell.
[0117] The antibodies may also be conjugated (e.g., covalently
linked) to molecules that improve stability of the antibody during
storage or increase the half-life of the antibody in vivo. Examples
of molecules that increase the half-life are albumin (e.g., human
serum albumin) and polyethylene glycol (PEG). Albumin-linked and
PEGylated derivatives of antibodies may be prepared using
techniques well known in the art. See, e.g. Chapman, 2002, Adv.
Drug Deliv. Rev. 54:531-545; Anderson and Tomasi, 1988, J. Immunol.
Methods 109:37-42; Suzuki et al., 1984, Biochim. Biophys. Acta
788:248-255; and Brekke and Sandlie, 2003, Nature Rev. 2:52-62.
[0118] Binding compounds, in particular antibodies, used in the
present invention will usually bind with at least a KD of about
10.sup.-3 M, more usually at least 10.sup.-6 M, typically at least
10.sup.-7 M, more typically at least 10.sup.-8 M, preferably at
least about 10.sup.-9 M, and more preferably at least 10.sup.-10 M,
and most preferably at least 10.sup.-11 M. See, e.g. Presta, et
al., 2001, Thromb. Haemost. 85:379-389; Yang, et al., 2001, Crit.
Rev. Oncol. Hematol. 38:17-23; Carnahan, et al., 2003, Clin. Cancer
Res. (Suppl.) 9:3982s-3990s. Antibody affinities may be determined
using standard analysis.
[0119] The term "hypervariable region," as used herein, refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region may comprise amino acid
residues from a "complementarity determining region" or "CDR,"
defined by sequence alignment, for example residues 24-34 (L1),
50-56 (L2) and 89-97 (L3) in the light chain variable domain and
31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable
domain (see Kabat et al., 1991, Sequences of proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md.) and/or those residues from a
"hypervariable loop" (HVL), as defined structurally, for example,
residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain
variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the
heavy chain variable domain (see Chothia and Leskl, 1987, J. Mol.
Biol. 196:901-917). "Framework" or "FR" residues are those variable
domain residues other than the hypervariable region residues as
herein defined.
[0120] An "isolated" antibody is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous solutes. In some embodiments,
the antibody will be purified (1) to greater than 95% by weight of
antibody as determined by the Lowry method, and most preferably
more than 99% by weight, (2) to a degree sufficient to obtain at
least 15 residues of N-terminal or internal amino acid sequence by
use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE
under reducing or nonreducing conditions using Coomassie blue or,
preferably, silver stain. Isolated antibody includes the antibody
in situ within recombinant cells since at least one component of
the antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one
purification step.
[0121] An "isolated" nucleic acid molecule is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the antibody nucleic acid. An
isolated nucleic acid molecule is other than in the form or setting
in which it is found in nature. Isolated nucleic acid molecules
therefore are distinguished from the nucleic acid molecule as it
exists in natural cells. However, an isolated nucleic acid molecule
includes a nucleic acid molecule contained in cells that ordinarily
express the antibody where, for example, the nucleic acid molecule
is in a chromosomal location different from that of natural
cells.
[0122] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies which may comprise the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations that typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler et al., 1975, Nature 256:495, or may be made by recombinant
DNA methods (see, for example, U.S. Pat. No. 4,816,567). The
"monoclonal antibodies" may also be isolated from phage antibody
libraries using the techniques described in Clackson et al., 1991,
Nature 352:624-628 and Marks et al., 1991, J. Mol. Biol.
222:581-597, for example. The monoclonal antibodies herein
specifically include "chimeric" antibodies.
[0123] As used herein, the term "immune cell" includes cells that
are of hematopoietic origin and that play a role in the immune
response. Immune cells include lymphocytes, such as B cells and T
cells, natural killer cells, myeloid cells, such as monocytes,
macrophages, eosinophils, mast cells, basophils, and
granulocytes.
[0124] As used herein, an "immunoconjugate" refers to an anti-CD27
antibody, or a fragment thereof, conjugated to a therapeutic
moiety, such as a bacterial toxin, a cytotoxic drug or a
radiotoxin. Toxic moieties may be conjugated to antibodies of the
invention using methods available in the art.
[0125] As used herein, a sequence "variant" refers to a sequence
that differs from the disclosed sequence at one or more amino acid
residues but which retains the biological activity of the resulting
molecule. The invention relates to variants of binding compounds
based on hCD27.15 and to variants of hCD27.15.
[0126] "Conservatively modified variants" or "conservative amino
acid substitution" refers to substitutions of amino acids that are
known to those of skill in this art and may be made generally
without altering the biological activity of the resulting molecule.
Those of skill in this art recognize that, in general, single amino
acid substitutions in non-essential regions of a polypeptide do not
substantially alter biological activity (see, e.g., Watson, et al.,
Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p.
224 (4th Edition 1987)). Such exemplary substitutions are
preferably made in accordance with those set forth below as
follows:
TABLE-US-00001 Exemplary Conservative Amino Acid Substitutions
Original residue Conservative substitution Ala (A) Gly; Ser Arg (R)
Lys, His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q)
Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln Ile (I) Leu; Val
Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu; Ile; Tyr Phe (F)
Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser Trp (W) Tyr; Phe
Tyr (Y) Trp; Phe Val (V) Ile; Leu
[0127] As used herein, the term "about" refers to a value that is
within an acceptable error range for the particular value as
determined by one of ordinary skill in the art, which will depend
in part on how the value is measured or determined, i.e. the
limitations of the measurement system. For example, "about" may
mean within 1 or more than 1 standard deviation per the practice in
the art. Alternatively, "about" or "comprising essentially of" may
mean a range of up to 20%. Furthermore, particularly with respect
to biological systems or processes, the terms may mean up to an
order of magnitude or up to 5-fold of a value. When particular
values are provided in the application and claims, unless otherwise
stated, the meaning of "about" or "comprising essentially of"
should be assumed to be within an acceptable error range for that
particular value.
[0128] "Specifically" binds, when referring to a ligand/receptor,
antibody/antigen, or other binding pair, indicates a binding
reaction which is determinative of the presence of the protein,
e.g., CD27, in a heterogeneous population of proteins and/or other
biologics. Thus, under designated conditions, a specified
ligand/antigen binds to a particular receptor/antibody and does not
bind in a significant amount to other proteins present in the
sample.
[0129] "Administration", "therapy" and "treatment," as it applies
to an animal, human, experimental subject, cell, tissue, organ, or
biological fluid, refers to contact of an exogenous pharmaceutical,
therapeutic, diagnostic agent, or composition to the animal, human,
subject, cell, tissue, organ, or biological fluid.
"Administration", "therapy" and "treatment" may refer, e.g., to
therapeutic, pharmacokinetic, diagnostic, research, and
experimental methods. Treatment of a cell encompasses contact of a
reagent to the cell, as well as contact of a reagent to a fluid,
where the fluid is in contact with the cell. "Administration",
"therapy" and "treatment" also mean in vitro and ex vivo
treatments, e.g., of a cell, by a reagent, diagnostic, binding
composition, or by another cell.
[0130] Monoclonal antibodies may be made according to knowledge and
skill in the art of injecting test subjects with human CD27 antigen
and then generating hybridomas expressing antibodies having the
desired sequence or functional characteristics. DNA encoding the
monoclonal antibodies is readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of the monoclonal antibodies). The hybridoma cells
serve as a preferred source of such DNA. Once isolated, the DNA may
be placed into expression vectors, which are then transfected into
host cells such as E. coli cells, simian COS cells, Chinese hamster
ovary (CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. Recombinant production of
antibodies will be described in more detail below.
[0131] Antibodies or antibody fragments may be isolated from
antibody phage libraries generated using the techniques described
in McCafferty et al., 1990, Nature, 348:552-554. Clackson et al.,
1991, Nature, 352:624-628, and Marks et al., 1991, J. Mol. Biol.
222:581-597 describe the isolation of murine and human antibodies,
respectively, using phage libraries. Subsequent publications
describe the production of high affinity (nM range) human
antibodies by chain shuffling (Marks et al., 1992, Bio/Technology,
10:779-783), as well as combinatorial infection and in vivo
recombination as a strategy for constructing very large phage
libraries (Waterhouse et al., 1993, Nuc. Acids. Res. 21:2265-2266).
Thus, these techniques are viable alternatives to traditional
monoclonal antibody hybridoma techniques for isolation of
monoclonal antibodies.
[0132] The antibody DNA also may be modified, for example, by
substituting the coding sequence for human heavy- and light-chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567; Morrison, et al., 1984, Proc. Natl Acad. Sci.
USA, 81:6851), or by covalently joining to the immunoglobulin
coding sequence all or part of the coding sequence for
non-immunoglobulin material (e.g., protein domains). Typically such
non-immunoglobulin material is substituted for the constant domains
of an antibody, or is substituted for the variable domains of one
antigen-combining site of an antibody to create a chimeric bivalent
antibody which may comprise one antigen-combining site having
specificity for an antigen and another antigen-combining site
having specificity for a different antigen.
[0133] A humanized antibody has one or more amino acid residues
from a source that is non-human. The non-human amino acid residues
are often referred to as "import" residues, and are typically taken
from an "import" variable domain. Humanization may be performed
generally following the method of Winter and co-workers (Jones et
al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature,
332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are antibodies wherein substantially less than an intact
human variable domain has been substituted by the corresponding
sequence from a non-human species. In practice, humanized
antibodies are typically human antibodies in which some CDR
residues and possibly some FR residues are substituted by residues
from analogous sites in non-human, for example, rodent
antibodies.
[0134] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., 1987, J. Immunol. 151:2296;
Chothia et al., 1987, J. Mol. Biol. 196:901). Another method uses a
particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA
89:4285; Presta et al., 1993, J. Immunol. 151:2623).
[0135] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues may be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding.
[0136] Humanization of antibodies is a straightforward protein
engineering task. Nearly all murine antibodies may be humanized by
CDR grafting, resulting in the retention of antigen binding. See,
Lo, Benny, K. C., editor, in Antibody Engineering: Methods and
Protocols, volume 248, Humana Press, N.J., 2004.
[0137] Alternatively, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (JH) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993,
Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology
7:33; and Duchosal et al., 1992, Nature 355:258. Human antibodies
may also be derived from phage-display libraries (Hoogenboom et
al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991,
222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).
[0138] Amino acid sequence variants of humanized anti-CD27
antibodies are prepared by introducing appropriate nucleotide
changes into the humanized anti-CD27 antibodies' DNAs, or by
peptide synthesis. Such variants include, for example, deletions
from, and/or insertions into, and/or substitutions of, residues
within the amino acid sequences shown for the humanized anti-CD27
antibodies. Any combination of deletion, insertion, and
substitution is made to arrive at the final construct, provided
that the final construct possesses the desired characteristics. The
amino acid changes also may alter post-translational processes of
the humanized anti-CD27 antibodies, such as changing the number or
position of glycosylation sites.
[0139] A useful method for identification of certain residues or
regions of the humanized anti-CD27 antibodies polypeptides that are
preferred locations for mutagenesis is called "alanine scanning
mutagenesis," as described by Cunningham and Wells, 1989, Science
244:1081-1085. Here, a residue or group of target residues are
identified (e.g., charged residues such as Arg, Asp, His, Lys, and
Glu) and replaced by a neutral or negatively charged amino acid
(most preferably alanine or polyalanine) to affect the interaction
of the amino acids with CD27 antigen. The amino acid residues
demonstrating functional sensitivity to the substitutions then are
refined by introducing further or other variants at, or for, the
sites of substitution. Thus, while the site for introducing an
amino acid sequence variation is predetermined, the nature of the
mutation per se need not be predetermined. For example, to analyze
the performance of a mutation at a given site, Ala scanning or
random mutagenesis is conducted at the target codon or region and
the expressed humanized anti-CD27 antibodies' variants are screened
for the desired activity.
[0140] Ordinarily, amino acid sequence variants of the humanized
anti-CD27 antibodies will have an amino acid sequence having at
least 75% amino acid sequence identity with the original mouse
antibody amino acid sequences of either the heavy or the light
chain more preferably at least 80%, more preferably at least 85%,
more preferably at least 90%, and most preferably at least 95%, 98%
or 99%. Identity or homology with respect to this sequence is
defined herein as the percentage of amino acid residues in the
candidate sequence that are identical with the humanized residues,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
None of N-terminal, C-terminal, or internal extensions, deletions,
or insertions into the antibody sequence shall be construed as
affecting sequence identity or homology. The percentage of identity
between two sequences may be determined with computer application
such as SeqMan II (DNAstar Inc, version 5.05). Using this program
two sequences may be aligned using the optimal alignment algorithm
of Smith and Waterman (1981) (Journal of Molecular Biology
147:195-197). After alignment of the two sequences the percentage
identity may be calculated by dividing the number of identical
nucleotides between the two sequences by the length of the aligned
sequences minus the length of all gaps.
[0141] Antibodies having the characteristics identified herein as
being desirable in humanized anti-CD27 antibodies may be screened
for increased biologic activity in vitro or suitable binding
affinity. To screen for antibodies that bind to the epitope on
human CD27, a routine cross-blocking assay such as that described
in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory,
Ed Harlow and David Lane (1988), may be performed. Antibodies that
bind to the same epitope are likely to cross-block in such assays,
but not all cross-blocking antibodies will necessarily bind at
precisely the same epitope since cross-blocking may result from
steric hindrance of antibody binding by antibodies at overlapping
epitopes, or even nearby non-overlapping epitopes.
[0142] Alternatively, epitope mapping, e.g., as described in Champe
et al., 1995, J. Biol. Chem. 270:1388-1394, may be performed to
determine whether the antibody binds an epitope of interest.
"Alanine scanning mutagenesis," as described by Cunningham and
Wells, 1989, Science 244:1081-1085, or some other form of point
mutagenesis of amino acid residues in human CD27 may also be used
to determine the functional epitope for anti-CD27 antibodies of the
present invention. Additional antibodies binding to the same
epitope as an antibody of the present invention may be obtained,
for example, by screening of antibodies raised against CD27 for
binding to the epitope, or by immunization of an animal with a
peptide which may comprise a fragment of human CD27 which may
comprise the epitope sequences. Antibodies that bind to the same
functional epitope might be expected to exhibit similar biological
activities, such as blocking receptor binding, and such activities
may be confirmed by functional assays of the antibodies.
[0143] Antibody affinities may be determined using standard
analysis. Preferred binding compounds such as e.g. humanized
antibodies are those that bind human CD27 with a Kd value of no
more than about 1.times.10.sup.-7; preferably no more than about
1.times.10.sup.-8; more preferably no more than about
1.times.10.sup.-9; and most preferably no more than about
1.times.10.sup.-10 or even 1x10.sup.-11 M.
[0144] The humanized antibody may be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably,
the antibody is an IgG antibody. Any isotype of IgG may be used,
including IgG1, IgG2, IgG3, and IgG4. Variants of the IgG isotypes
are also contemplated. The humanized antibody may comprise
sequences from more than one class or isotype. Optimization of the
necessary constant domain sequences to generate the desired
biologic activity is readily achieved by screening the antibodies
in the biological assays described in the Examples.
[0145] Likewise, either class of light chain may be used in the
compositions and methods herein. Specifically, kappa, lambda, or
variants thereof are useful in the present compositions and
methods.
[0146] The antibodies and antibody fragments of the invention may
also be conjugated with cytotoxic payloads such as cytotoxic agents
or radionucleotides such as .sup.99Tc, .sup.90Y, .sup.111In,
.sup.32P, .sup.14C, .sup.125I, .sup.3H, .sup.131I, .sup.11C,
.sup.15O, .sup.13N, .sup.18F, .sup.35S, .sup.51Cr, .sup.57To,
.sup.226Ra, .sup.60Co, .sup.59Fe, .sup.57Se, .sup.152Eu, .sup.67Cu,
.sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc, .sup.109Pd,
.sup.234Th, and .sup.40K, .sup.157Gd, .sup.55Mn, .sup.52Tr and
.sup.56Fe. Such antibody conjugates may be used in immunotherapy to
selectively target and kill cells expressing a target (the antigen
for that antibody) on their surface. Exemplary cytotoxic agents
include ricin, vinca alkaloid, methotrexate, Psuedomonas exotoxin,
saporin, diphtheria toxin, cisplatin, doxorubicin, abrin toxin,
gelonin and pokeweed antiviral protein.
[0147] The antibodies and antibody fragments of the invention may
also be conjugated with fluorescent or chemilluminescent labels,
including fluorophores such as rare earth chelates, fluorescein and
its derivatives, rhodamine and its derivatives, isothiocyanate,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde,
fluorescamine, .sup.152Eu, dansyl, umbelliferone, luciferin,
luminal label, isoluminal label, an aromatic acridinium ester
label, an imidazole label, an acridimium salt label, an oxalate
ester label, an aequorin label, 2,3-dihydrophthalazinediones,
biotin/avidin, spin labels and stable free radicals.
[0148] Any method known in the art for conjugating the antibody
molecules or protein molecules of the invention to the various
moieties may be employed, including those methods described by
Hunter et al., 1962, Nature 144:945; David et al., 1974,
Biochemistry 13:1014; Pain et al., 1981, J. Immunol. Meth. 40:219;
and Nygren, J., 1982, Histochem. and Cytochem. 30:407. Methods for
conjugating antibodies and proteins are conventional and well known
in the art.
[0149] When using recombinant techniques, the antibody may be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., 1992,
Bio/Technology 10:163-167 describe a procedure for isolating
antibodies which are secreted to the periplasmic space of E. coli.
Briefly, cell paste is thawed in the presence of sodium acetate (pH
3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30
min. Cell debris may be removed by centrifugation. Where the
antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0150] The antibody composition prepared from the cells may be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc region that is present
in the antibody. Protein A may be used to purify antibodies that
are based on human Ig.gammal, Ig.gamma2, or Ig.gamma4 heavy chains
(Lindmark et al., 1983, J. Immunol. Meth. 62:1-13). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., 1986, EMBO J 5:1567-1575). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available.
[0151] Mechanically stable matrices such as controlled pore glass
or poly(styrenedivinyl) benzene allow for faster flow rates and
shorter processing times than may be achieved with agarose. Where
the antibody comprises a C.sub.H3 domain, the Bakerbond ABX.TM.
resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0152] In one embodiment, the glycoprotein may be purified using
adsorption onto a lectin substrate (e.g. a lectin affinity column)
to remove fucose-containing glycoprotein from the preparation and
thereby enrich for fucose-free glycoprotein.
[0153] The invention may comprise pharmaceutical formulations of a
CD27 binding compound. To prepare pharmaceutical or sterile
compositions, the binding compound, in particular an antibody or
fragment thereof, is admixed with a pharmaceutically acceptable
carrier or excipient, see, e.g., Remington's Pharmaceutical
Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing
Company, Easton, Pa. (1984). Formulations of therapeutic and
diagnostic agents may be prepared by mixing with physiologically
acceptable carriers, excipients, or stabilizers in the form of,
e.g., lyophilized powders, slurries, aqueous solutions or
suspensions (see, e.g., Hardman, et al., 2001, Goodman and Gilman's
The Pharmacological Basis of Therapeutics, McGraw-Hill, New York,
N.Y.; Gennaro, 2000, Remington: The Science and Practice of
Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis,
et al. (eds.), 1993, Pharmaceutical Dosage Forms: Parenteral
Medications, Marcel Dekker, N.Y.; Lieberman, et al. (eds.), 1990,
Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, N.Y.;
Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms:
Disperse Systems, Marcel Dekker, N.Y.; Weiner and Kotkoskie, 2000,
Excipient Toxicity and Safety, Marcel Dekker, Inc., New York,
N.Y.).
[0154] Toxicity and therapeutic efficacy of the binding compound,
in particular antibody, compositions, administered alone or in
combination with another agent, such as the usual anti-cancer
drugs, may be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., for determining the
LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it may be expressed as the ratio
between LD.sub.50 and ED.sub.50. The data obtained from these cell
culture assays and animal studies may be used in formulating a
range of dosage for use in humans. The dosage of such compounds
lies preferably within a range of circulating concentrations that
include the ED.sub.50 with little or no toxicity. The dosage may
vary within this range depending upon the dosage form employed and
the route of administration utilized.
[0155] Suitable routes of administration include parenteral
administration, such as intramuscular, intravenous, or subcutaneous
administration and oral administration. Administration of binding
compounds such as antibodies, used in the pharmaceutical
composition or to practice the method of the present invention may
be carried out in a variety of conventional ways, such as oral
ingestion, inhalation, topical application or cutaneous,
subcutaneous, intraperitoneal, parenteral, intraarterial or
intravenous injection. In one embodiment, the binding compound of
the invention is administered intravenously. In another embodiment,
the binding compound of the invention is administered
subcutaneously.
[0156] Alternatively, one may administer the binding compound in a
local rather than systemic manner, for example, via injection of
the binding compound directly into the site of action, often in a
depot or sustained release formulation. Furthermore, one may
administer the antibody in a targeted drug delivery system.
[0157] Guidance in selecting appropriate doses of antibodies,
cytokines, and small molecules are available (see, e.g.,
Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd,
Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.),
1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y.; Baert, et al., 2003, New
Engl. J. Med. 348:601-608; Milgrom, et al., 1999, New Engl. J. Med.
341:1966-1973; Slamon, et al., 2001, New Engl. J. Med. 344:783-792;
Beniaminovitz, et al., 2000, New Engl. J. Med. 342:613-619; Ghosh,
et al., 2003, New Engl. J. Med. 348:24-32; Lipsky, et al., 2000,
New Engl. J. Med. 343:1594-1602).
[0158] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the
optimum dose and it is increased by small increments thereafter
until the desired or optimum effect is achieved relative to any
negative side effects. Important diagnostic measures include those
of symptoms of, e.g., the inflammation or level of inflammatory
cytokines produced.
[0159] A preferred dose protocol is one involving the maximal dose
or dose frequency that avoids significant undesirable side effects.
A total weekly dose is generally at least 0.05 .mu.g/kg body
weight, more generally at least 0.2 .mu.g/kg, most generally at
least 0.5 .mu.g/kg, typically at least 1 .mu.g/kg, more typically
at least 10 .mu.g/kg, most typically at least 100 .mu.g/kg,
preferably at least 0.2 mg/kg, more preferably at least 1.0 mg/kg,
most preferably at least 2.0 mg/kg, optimally at least 10 mg/kg,
more optimally at least 25 mg/kg, and most optimally at least 50
mg/kg (see, e.g., Yang, et al., 2003, New Engl. I Med. 349:427-434;
Herold, et al., 2002, New Engl. I Med. 346:1692-1698; Liu, et al.,
1999, J. Neural. Neurosurg. Psych. 67:451-456; Portielji, et al.,
2003, Cancer Immunol. Immunother. 52:133-144). The desired dose of
a small molecule therapeutic, e.g., a peptide mimetic, natural
product, or organic chemical, is about the same as for an antibody
or polypeptide, on a moles/kg basis.
[0160] As used herein, "inhibit" or "treat" or "treatment" includes
a postponement of development of the symptoms associated with
disease and/or a reduction in the severity of such symptoms that
will or are expected to develop with said disease. The terms
further include ameliorating existing symptoms, preventing
additional symptoms, and ameliorating or preventing the underlying
causes of such symptoms. Thus, the terms denote that a beneficial
result has been conferred on a vertebrate subject with a
disease.
[0161] As used herein, the term "therapeutically effective amount"
or "effective amount" refers to an amount of an anti-CD27 antibody
or fragment thereof, that when administered alone or in combination
with an additional therapeutic agent to a cell, tissue, or subject
is effective to prevent or ameliorate the disease or condition to
be treated. A therapeutically effective dose further refers to that
amount of the compound sufficient to result in amelioration of
symptoms, e.g., treatment, healing, prevention or amelioration of
the relevant medical condition, or an increase in rate of
treatment, healing, prevention or amelioration of such conditions.
When applied to an individual active ingredient administered alone,
a therapeutically effective dose refers to that ingredient alone.
When applied to a combination, a therapeutically effective dose
refers to combined amounts of the active ingredients that result in
the therapeutic effect, whether administered in combination,
serially or simultaneously. An effective amount of therapeutic will
decrease the symptoms typically by at least 10%; usually by at
least 20%; preferably at least about 30%; more preferably at least
40%, and most preferably by at least 50%.
[0162] Methods for co-administration or treatment with a second
therapeutic agent are well known in the art, see, e.g., Hardman, et
al. (eds.), 2001, Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 10th ed., McGraw-Hill, New York, N.Y.; Poole and
Peterson (eds.), 2001, Pharmacotherapeutics for Advanced Practice:
A Practical Approach, Lippincott, Williams & Wilkins, Phila.,
Pa.; Chabner and Longo (eds.), 2001, Cancer Chemotherapy and
Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.
[0163] The pharmaceutical composition of the invention may also
contain other agents, including but not limited to a cytotoxic,
chemotherapeutic, cytostatic, anti-angiogenic or antimetabolite
agents, a tumor targeted agent, an immune stimulating or immune
modulating agent or an antibody conjugated to a cytotoxic,
cytostatic, or otherwise toxic agent. The pharmaceutical
composition may also be employed with other therapeutic modalities
such as surgery, chemotherapy and radiation.
[0164] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined in the
appended claims.
[0165] The present invention will be further illustrated in the
following Examples which are given for illustration purposes only
and are not intended to limit the invention in any way.
EXAMPLES
Example 1
Immunization and Selection of Anti-CD27 Antibodies
Immunization of Mice with CD27 cDNA
[0166] To isolate antibodies against the human CD27 protein that
harbour agonistic activity we hypothesized to find such antibodies
among a set of anti-CD27 antibodies, which bind to the ligand
binding site. To generate anti-hCD27 antibodies, the cDNA encoding
the full length open reading frame of hCD27 was subcloned into the
pCI-neo vector (Promega, Madison, Wis.). Expression of the obtained
vector was checked by transient transfection of pCI-neo-hCD27 in
CHO-K1 cells (American Type Culture Collection, Manassas, Va.) and
flow cytometry using 10 .mu.g/ml mouse anti-hCD27 IgG1 (BD
Pharmingen #555439), followed by goat anti-mouse IgG1-FITC (1:100)
(Southern Biotechnology, Birmingham, Ala.).
[0167] Mice were immunized by gene gun immunization using a Helios
Gene gun (BioRad, Hercules, Calif.) and DNA coated gold bullets
(BioRad) following manufacturer's instructions. Briefly, 1 .mu.m
gold particles were coated with pCI-neo-hCD27 cDNA and commercial
expression vectors for mouse Flt3L and mouse GM-CSF in a 2:1:1
ratio (both from Aldevron, Fargo, N.D.). A total of 1 .mu.g of
plasmid DNA was used to coat 500 .mu.g of gold particles.
[0168] Specifically, 7-8 weeks old female BALB/C mice were
immunized in the ears with a gene gun, receiving 3 cycles of a shot
in both ears. Approximately, a 1:4,000 anti-hCD27 titer was
detected by cell-ELISA in mouse serum after two DNA immunizations.
In the cell-ELISA, all incubation steps were followed by a wash
step with PBST (PBS with 0.01% Tween 20). Parental CHO-K1 or
CHO-K1.hCD27 cells were seeded (40,000 cells/well) in tissue
culture plates and incubated overnight at 37.degree. C. The next
day, culture medium was removed and cells were incubated for 1 hour
with (dilutions of) mouse serum at 37.degree. C. Next, cells were
washed with PBST and incubated for 1 hour at 37.degree. C. with
1:1,000 goat-anti-mouse IgG-HRP (Southern Biotechnology, #
1030-05).
[0169] Subsequently, cells were washed 6 times with PBST and
anti-hCD27 immunoreactivity was visualized with 100 .mu.l OptiEIA
TMB substrate (BD Biosciences, Franklin Lake, N.J.). Reactions were
stopped with 100 .mu.l 0.5 M H.sub.2SO.sub.4 and absorbances were
read at 460 and 620 nm. Mice that demonstrated reactivity against
hCD27 were immunized for a final, fourth time and sacrificed four
days later.
[0170] Erythrocyte-depleted spleen cell populations were prepared
as described previously (Steenbakkers et al., 1992, J. Immunol.
Meth. 152:69-77; Steenbakkers et al., 1994, Mol. Biol. Rep.
19:125-134) and frozen at -140.degree. C.
Selection of Anti-hCD27 Antibody Producing B Cells
[0171] To select B cell clones producing anti-hCD27 antibodies,
1.5.times.10.sup.7 erythrocyte-depleted splenocytes were depleted
for monocytes. hCD27-specific B-cells were selected by binding on
irradiated (3,000 RAD) CHO-K1.hCD27 transfectants, which had grown
to confluency in a T25-flask. After extensive washing to delete
non-specific B-cells, bound B-cells were collected by Trypsin
treatment according to the manufacturer's instructions (Invitrogen,
cat. no. 25200-056). Next, B-cells were cultured as described by
Steenbakkers et al., 1994, Mol. Biol. Rep. 19:125-134. Briefly,
selected B-cells were mixed with 7.5% (v/v) T-cell supernatant and
50,000 irradiated (2,500 RAD) EL-4 B5 nursing cells in a final
volume of 200 .mu.l DMEM F12/P/S/10% BCS in 96-well flat-bottom
tissue culture plates.
[0172] On day eight, supernatants were screened for hCD27
reactivity by cell-ELISA as described above. Thirteen
hCD27-reactive supernatants were identified and tested for their
ability to inhibit the interaction between hCD27 and hCD70. In the
cell-ELISA, all incubation steps were followed by a wash step with
PBST (PBS with 0.01% Tween 20). Parental CHO-K1 or CHO-K1.hCD27
cells were seeded (40,000 cells/well) in tissue culture plates and
incubated overnight at 37.degree. C. The next day, culture medium
was removed and cells were incubated for one hour with (dilutions
of) mouse serum at 37.degree. C. Next, cells were washed with PBST
and incubated for one hour at 37.degree. C. with 1:1,000
goat-anti-mouse IgG-HRP (Southern Biotechnology, # 1030-05).
Subsquently, cells were washed 6 times with PBST and anti-hCD27
immunoreactivity was visualized with 100 .mu.l TMB Stabilized
Chromagen (Invitrogen, cat. no. SB02). Reactions were stopped with
100 .mu.l 0.5 M H.sub.2SO.sub.4 and absorbances were read at 460
and 620 nm.
[0173] In addition, blocking properties of the supernatants were
studied using two competition assays. The CHO-K1.CD27 assay works
along the following principles: CHO-K1.CD27 cells were seeded
(40,000 cells/well) in a 96-well plate and incubated overnight at
37.degree. C. After medium removal, 50 .mu.l recombinant hCD70
(CD70 (h)-muCD8 fusion Protein (Ancell, cat. no. ANC-537)) (0.5
.mu.g/ml) and 50 .mu.l anti-hCD27 antibody containing supernatant
were added. After 1 hour incubation at room temperature, the wells
were washed three times with PBST. Next, 100 .mu.l/well
Streptavidin-HRP conjugate (BD Pharmingen, cat. no. 554066)
(1:5,000) was added and cells were incubated for 1 hour at
37.degree. C. After 6 final washes with PBST the ELISA was
developed as outlined above. Positive controls: anti-hCD27, clone
57703 (R&D systems, cat. no. MAB382) and anti-hCD27, clone 1A4
(Beckman Coulter, cat. no. IM2034). The CHO-K1.CD70 assay works
along the following principles: CHO-K1.CD70 cells were seeded in a
96 well plate at a density of 40,000 cells/well. The same amount of
plates was blocked by adding 300 .mu.l medium/well, and all plates
were incubated overnight at 37.degree. C. The following day, the
medium-only containing plates were emptied by flicking the plate,
and 50 .mu.l/well recombinant soluble hCD27-Fc fusion protein
(rhCD27/Fc chimera (0.5 .mu.g/ml) (R&D systems cat. no.
382-CD)) was added. To these plates, 50 .mu.l antibody containing
medium/sera or medium was added. After 1 hour incubation at room
temperature, the 100 .mu.l rhCD27-Fc/antibody mix was transferred
to the CHO-K1/CD70 plate(s) from which the medium had been removed.
These plate(s) were incubated for 1 hour at room temperature and
then washed three times with PBST. 100 .mu.l anti-human Ig
(H+L)-HRP conjugate (1:2,500) was added to every well (Promega,
cat. no. W4031) and the cells were incubated for 1 hour at
37.degree. C. After 6 final washes with PBST, the ELISA was
developed as outlined above.
[0174] Positive controls: anti-hCD27, clone 57703 (R&D systems,
cat. no. MAB382) and anti-hCD27, clone 1A4 (Beckman Coulter, cat.
no. IM2034).
[0175] All supernatants demonstrated to contain antibodies that
blocked the interaction between hCD27 and hCD70. Subsequently, the
B-cell clones from the hCD27 reactive supernatants were
immortalized by mini-electrofusion following published procedures
(Steenbakkers et al., 1992, J. Immunol. Meth. 152:69-77;
Steenbakkers et al., 1994, Mol. Biol. Rep. 19:125-34).
Specifically, B-cells were mixed with 10.sup.6 Sp2/0-Ag14 myeloma
cells, and serum was removed by washing with DMEM F12 media. Cells
were treated with Pronase solution (Calbiochem, cat. no.
4308070.536) for 3 minutes and washed with Electrofusion Isomolar
Buffer (Eppendorf, cat. no. 53702). Electrofusions were performed
in a 50 .mu.l fusion chamber by an alternating electric field of 30
s, 2 MHz, 400 V/cm followed by a square, high field pulse of 10
.mu.s, 3 kV/cm and again by an alternating electric field of 30 s,
2 MHz, 400 V/cm.
[0176] Contents of the chamber were transferred to hybridoma
selective medium and plated in a 96-well plate under limiting
dilution conditions. On day 12 following the fusions, hybridoma
supernatants were screened for hCD27 reactivity and hCD70-blocking
activity, as described above. Seven hybridomas secreting antibodies
in the supernatant which recognized hCD27 and demonstrated blocking
activity were isolated and subcloned by limited dilution to
safeguard their integrity. Antibody hCD27.15 was selected for
further analysis.
Example 2
Purification and Characterization of Anti-hCD27 Antibodies
Stabilization of Anti-hCD27 Producing Hybridomas and Purification
of Anti-hCD27 Antibodies
[0177] Clonal cell populations were obtained for the hCD27.15
hybridoma by two rounds of limiting dilutions. Stable hybridomas
were cultured in serum-free media for 7-10 days; supernatants were
harvested and filtered through a 0.22 .mu.M nitrocellulose
membrane. Antibodies were purified using Prosep A spin columns
according to the manufacturer's instructions (Millipore, cat. no.
LSK2ABA60). Buffer was exchanged for PBS using PD-10 gel-filtration
columns (GE Healthcare). Antibodies were concentrated with Amicon
Ultra-15 centrifugal filter units (Millipore, Billerica, MA) and
quantified using spectrophotometry. Using a mouse monoclonal
antibody isotyping test kit (Roche, #11493027001), the
(sub)-isotype of all hCD27 antibodies was determined to be IgG1,
Kappa.
Binding Analysis
[0178] Cell-based ELISA experiments using purified hCD27 antibodies
were performed to determine binding activities of hCD27 to
cellularly expressed hCD27. In this cell-ELISA, all incubation
steps were followed by a wash step with PEST (PBS with 0.01% Tween
20). CHO-K1.hCD27 cells were seeded (40,000 cells/well) in tissue
culture plates and incubated overnight at 37.degree. C. The next
day, culture medium was removed and cells were incubated for one
hour with (dilutions of) purified antibodies at 37.degree. C. Next,
cells were washed with PBST and incubated for one hour at
37.degree. C. with 1:1,000 goat-anti-mouse IgG-HRP (Southern
Biotechnology, # 1030-05). Subsequently, cells were washed 6 times
with PBST and anti-hCD27 immunoreactivity was visualized with 100
.mu.l TMB Stabilized Chromagen (Invitrogen, cat. no. SB02).
Reactions were stopped with 100 .mu.l 0.5 M H.sub.250.sub.4 and
absorbances were read at 460 and 620 nm. As shown in FIG. 1A, the
different hCD27 antibodies (hCD27.15 and controls) bound to hCD27
with different binding strengths. Calculated EC.sub.50,
representing the concentration at which 50% of the total binding
signal is observed are represented in Table 1.
TABLE-US-00002 TABLE 1 Overview of KD, EC.sub.50 and IC.sub.50
values of hCD27.15 and 1A4CD27 (control). KD .times. EC.sub.50
IC.sub.50 (ng/ml) IC.sub.50 (ng/ml) 1E-9 (M) (ng/ml) CHO-K1.CD27
CHO-K1.CD70 hCD27.15 122 686.5 864 1546 1A4CD27 33 93.7 370
[0179] Blocking properties of the purified antibodies were studied
using two competition assays. The CHO-K1.CD70 assay works along the
following principles: CHO-K1.CD70 cells were seeded in a 96 well
plate at a density of 40,000 cells/well. The same amount of plates
was blocked by adding 300 .mu.l medium/well, and all plates were
incubated overnight at 37.degree. C. The following day, the
medium-only containing plates were emptied by flicking the plate,
and 50 .mu.l/well rhCD27-Fc chimera (0.5 .mu.g/ml) (R&D systems
cat. no. 382-CD) was added. To these plates, 50 .mu.l of different
dilutions of purified hCD27.15 antibodies were added. After 1 hour
incubation at room temperature, the 100 .mu.l rhCD27Fc/antibody mix
was transferred to the CHO-K1/CD70 plate(s) from which the medium
had been removed. These plate(s) were incubated for 1 hour at room
temperature and then washed 3 times with PB ST. 100 .mu.l
anti-human Ig (H+L)-HRP conjugate (1:2,500) was added to every well
(Promega, cat. no. W4031) and the plates were incubated for 1 hour
at 37.degree. C. After 6 final washes with PBST TMB Stabilized
Chromagen (Invitrogen, cat. no. SB02) (100 .mu.l/well) was added
and the ELISA was read out as outlined above. Positive controls:
anti-hCD27, clone 57703 (R&D systems, cat. no. MAB382) and
anti-hCD27, clone 1A4 (Beckman Coulter, cat. no. IM2034). As shown
in FIG. 1B, the purified hCD27.15 antibody blocked the binding of
rhCD27Fc chimera to CHO-K1.CD70 cells. Calculated IC.sub.50 values
of hCD27.15 and the positive control 1A4, which represent the
concentration at which half of the inhibition is observed, are
presented in Table 1.
[0180] The CHO-K1.CD27 assay works along the following principles:
CHO-K1.CD27 cells were seeded (40,000 cells/well) in a 96-well
plate and incubated overnight at 37.degree. C. After medium
removal, 50 .mu.l recombinant mouse CD70 fusion Protein (Fc-mCD70)
(0.5 .mu.g/ml) and 50 .mu.l of different dilutions of purified
anti-hCD27 antibodies were added. Fc-mCD70 is a fusion protein of
murine CD70 (aa 41-195) fused at the C-terminus of the dimerization
domain of human IgG1. A cDNA construct encoding this fusion protein
was constructed as described by Rowley and Al-Shamkhani, 2004, J
Immunol 15:172:6039-46 and used to produce Fc-mCD70 protein in 293T
human embryonic kidney cells. The protein was purified by affinity
chromatography on Protein A Sepharose (GE Health Care).
[0181] After 1 hour incubation at room temperature, the wells were
washed 3 times with PBST. Next, 100 .mu.l/well Streptavidin-HRP
conjugate (BD Pharmingen, cat. no. 554066) (1:5,000) was added and
cells were incubated for one hour at 37.degree. C. After 6 final
washes with PBST TMB Stabilized Chrornagen (Invitrogen, cat. no.
SB02) (100 .mu.l/well) was added. The reaction was stopped by the
addition of 100 .mu.l 0.5 M H.sub.2SO.sub.4. Absorbencies were read
at 460 and 620 nn. Positive controls: anti-hCD27, clone 57703
(R&D systems, cat. no. MAB382) and anti-hCD27, clone 1A4
(Beckman Coulter, cat. no. IM2034). As shown in FIG. 1C, hCD27.15
antibodies blocked the interaction between recombinant human CD70
and CHO-K1.CD27 cells. Calculated IC.sub.50 values are presented in
Table 1.
Kinetic Analysis by Label-Free Surface Plasma Resonance
(Biacore)
[0182] The binding properties of hCD27.15 antibodies were
characterized in more detail using label-free surface plasma
resonance using Biacore 2000 equipment. Low amounts of antibodies
were coupled to a CMS sensor chip using amine coupling at pH=4.5,
with R.sub.max not exceeding 100 RU. This will, in combination with
a high flow level (30 .mu.l/min) yield good fits to the 1:1
Langmuir binding model. A concentration series of rhCD27Fc chimera,
ranging from 0.016 nM to 1 nM, was injected for 1 minute at 30
.mu.l/min. The dissociation was monitored for 5 minutes. The
running buffer is HEPES-buffered saline with 3 mM EDTA and 0.005%
P20 (HBS-EP), pH 7.4. Combination plots were made by subtraction of
the signal obtained at the blank flow cell, using BIAeval 3.2. The
sensor grams were fitted to a 1:1 Langmuir binding model. Antibody
hCD27.15 shows a fast association and dissociation, resulting in a
moderate affinity. The calculated K.sub.D values are presented in
Table 1.
Species Cross-Reactivity
[0183] Binding of hCD27 antibodies to mouse CD27 was determined
using MCF-7 breast carcinoma cells that had been retrovirally
transduced to stably express the full length cDNA encoding human
CD27 or mouse CD27. Empty vector-transduced cells served as a
control. Binding of the antibodies was tested by flow cytometric
analysis, with validated agonistic anti-hCD27 antibodies 1A4 and
CLB CD27/1; and anti-mouse CD27 LG.3A10 (Gravestein et al., 1995,
Int Immunol. 7:551-7) as positive controls. These commercially
available anti-hCD27 antibodies, which have been reported to harbor
agonistic activities, were obtained as described in Table 2.
TABLE-US-00003 TABLE 2 commercially available agonistic anti-CD27
antibodies. Antibody Company Cat no. 9F4 (CLB-CD27/1) Pelicluster
M1455 1A4 Beckman Coulter IM2034
[0184] The hCD27.15 antibodies bound to human CD27, but not to
mouse CD27 as expressed on the MCF-7 cells.
[0185] The binding site of the hCD27.15 antibody was characterized
and compared with the commercially available agonistic antibodies
9F4 and 1A4 using a cross-competition Biacore assay. Using common
amine coupling at pH 4.5, flow cells were immobilized at 25
.mu.g/ml of each antibody to a high immobilization level. Next,
multi flow cell injections of 1 nM of rhCD27Fc chimera (R&D
systems, cat. no. 382-CD) with a speed of 5 .mu.l/min were followed
by 10 nM of the second antibody. The anti-hCD27 antibody hCD27.15
of the invention and the two known agonistic antibodies (1A4 and
9F4) were used as a primary (immobilized) or secondary (free)
antibody. Agonistic anti-CD27 antibody 1A4 was only used as a
second antibody and was not immobilized because of the presence of
BSA in the buffer. Fc-mCD70 is a fusion protein of murine CD70 (aa
41-195) fused at the C-terminus of the dimerization domain of human
IgG1. A cDNA construct encoding this fusion protein was constructed
as described by Rowley and Al-Shamkhani, 2004, J Immunol
15:172:6039-46 and used to produce Fc-mCD70 protein in 293T human
embryonic kidney cells. The protein was purified by affinity
chromatography on Protein A Sepharose (GE Health Care).
[0186] All flow cells were regenerated by a 6-second injection of
10 mM HCl at 50 .mu.l/min. An increase in signal upon injection of
the second antibody means that the second antibody can still bind
and that the primary and secondary bind to different binding sites.
If not, it suggests that both antibodies recognize the same epitope
of hCD27, have overlapping epitopes, or can not bind at the same
time due to steric hindrance. As shown in Table 3 the hCD27.15 and
control antibodies can be divided into different binding
groups.
Example 3
Functional Profiling of Mouse Anti-Human CD27 Antibodies
hCD27.15 Induces CD27 Signaling Leading to NF-.kappa.B
Activation
[0187] The full length human CD27 cDNA was cloned into the pcDNA3
expression vector and transiently expressed by transfection into
HEK293T human embryonic kidney cells (HEK293T), using FuGENE6
transfection reagent (Roche). CD27 expression from this construct
was validated by flow cytometry. To read out hCD27 signaling in
response to binding of hCD27.15 mAb, HEK293 cells were transiently
co-transfected with the hCD27 pcDNA vector or empty control vector
and an NF-.kappa.B-luciferase reporter construct, encoding the
luciferase gene driven by a minimal NF-.kappa.B-responsive promoter
(Bonehill et al., 2008, Mol Ther 16(6):1170-80). At 20 h after
transfection, the cells were stimulated for 4, 8, 20 or 24 hours in
presence or absence of hCD27.15 mAb (10 .mu.g/ml), other CD27
antibodies (described in Table 2, 10 .mu.g/ml) or FcCD70 (2
.mu.g/ml). After stimulation, cells were washed with ice cold PBS
and lysed with Cell Culture Lysis buffer (Promega, Luciferase assay
system, catalog number E1500). Luciferase activity was measured
after substrate was added to cell lysates following protocol of
manufacturer (Luminometer Centro XS3 LB 960, Berthold
Technologies). Data was analysed using Mikrowin 2000 software. FIG.
2 illustrates that hCD27.15 activating CD27 more potently than
other hCD27 antibodies and FcCD70.
hCD27.15 Costimulates Human CD4.sup.+CD25.sup.- T Cells
[0188] The effect of activating CD27 with the hCD27.15 antibody on
naive human CD4.sup.+CD25.sup.- T cells was determined as follows.
PBMCs were isolated from Buffy coat using Ficoll gradient
centrifugation according to the manufacturer's instruction
(Ficoll-Paque.TM. Plus cat. Number 17-1440-03). Untouched
CD4.sup.+CD25.sup.- T-cells were isolated from these PBMC's by MACS
based negative selection using the CD4.sup.+ T-cell isolation kit
II (Miltenyi cat. No 130-091-155) and CD25 microbeads II (Miltenyi
cat. No 130-092-983) according to the manufacturer's instructions.
Purified CD4.sup.+CD25.sup.- cells were seeded in 96 well-plates at
a concentration of 1.times.10.sup.5 cells/well. Prior to culturing
CD4.sup.+CD25.sup.- cells were checked for purity by flow
cytometry. Cells were incubated with different combinations of
anti-CD3 (OKT-3), anti-CD28 (1 .mu.g/ml: clone 15E8, Sanquin),
Fc-CD70 (2 .mu.g/ml), isotype control (MOPC-21, 10 .mu.g/ml) and
hCD27.15 (10 .mu.g/ml), as indicated for FIG. 3. The next day,
proliferation was detected by [.sup.3H] thymidine incorporation.
hCD27.15 stimulated the proliferation of human CD4.sup.+CD25.sup.-
cells under suboptimal stimulation conditions.
hCD27.15 Costimulates Human CD8.sup.+ T Cells
[0189] The effect of activating CD27 by the hCD27.15 antibody on
naive human CD8.sup.+ T cells was examined as follows. PBMCs were
isolated from buffy coat using Ficoll gradient centrifugation.
Untouched naive CD8.sup.+ T-cells were isolated from these PBMC by
MACS-based negative selection using the BD IMag.TM. human naive
CD8.sup.+ T cell enrichment kit (BD cat number 558569), according
to the manufacturer's instructions. The CD8.sup.+ T cells selected
were checked for purity and naivety by flow cytometry using
anti-CD8 and anti-CD45RA antibodies and were labeled with
carboxyfluorescein succinimidyl ester (CF SE, 5 .mu.m) according to
manufacturer's protocol (Invitrogen). Next, they were seeded in 96
well-plates at a concentration of 1.0.times.10.sup.5 cells/well.
Cells were stimulated with soluble anti-CD3 mAb CLB-T3/4E
(Pelicluster) at 10 .mu.g/ml (used s/n of hybridoma culture),
anti-CD28 mAb CLB-CD28/1 (Pelicluster) at 0.02 .mu.g/ml, in
presence of hCD27.15 at 10 .mu.g/ml or isotype control.
[0190] After culture for the indicated number of days, cells were
counted using a CASY cell counter (Scharfe System GmbH), viability
was determined by using propidium iodide (PI) and the number of
cell divisions the T cells had undergone was assessed by flow
cytometric analysis of CFSE fluorescence intensity (FACS Calibur).
FIG. 4 illustrates that hCD27.15 is also promoting survival and
proliferation of CD8.sup.+ cells.
hCD27.15 Stimulates CD8.sup.+ T Cells to Produce Specific
Cytokines
[0191] Human naive CD8.sup.+ T cells were purified and stimulated
as indicated above. Culture supernatants were taken after 72 h of
culture and analyzed for cytokine secretion by 27-Plex Luminex
according to manufacturer's instructions (Biorad, cat. no.
171A11127). As shown in FIG. 5A, hCD27.15 induced the secretion of
TNF-.alpha., IL-2, IFN-.gamma., CXCL10, IL-13 and GM-CSF. In
addition, cells were used to perform intracellular staining for
IL-2 and IFN.gamma.. After 72 hours of culture, cells were cultured
with PMA (conc) and ionomycin (conc) for 4 hours in the presence of
Golgi-Plug (1 .mu.g/ml: BD Biosciences). The total number of
CD8.sup.+ cells is not dramatically different between hCD27.15 and
none stimulated cells indicating that the increased secretion of
cytokines is merely caused by a qualitative increase of cytokines
per cell (FIG. 5B).
Example 4
hCD27.15 Antibody Sequences
Cloning of Immunoglobulin cDNAs
[0192] Degenerate primer PCR-based methods were used to determine
the DNA sequences encoding the variable regions for the mouse
antibody that is expressed by hybridoma hCD27.15. Total RNA was
isolated from 5.times.10.sup.6 hybridoma cells using TRIZOL
(Invitrogen), and gene specific cDNAs for the heavy and light
chains were synthesized using the M-MLV Reverse Transcriptase,
RNase H Minus, point mutant kit (Promega, cat. no. M368C) according
to the manufacturer's instructions. The V.sub.H and V.sub.L genes
were PCR-amplified using a Novagen-based Ig-primer set (Novagen,
San Diego, Calif.) and Taq polymerase (Invitrogen). All PCR
products that matched the expected amplicon size of 500 bp were
cloned into pCR4 TOPO vector (Invitrogen), and the constructs were
transformed in One Shot Competent Top10 E. coli (Invitrogen)
according to the manufacturer's instructions.
[0193] Clones were screened by colony PCR using universal M13
forward and reverse primers, and at least two clones from each
reaction were selected for DNA sequencing analysis. CDRs were
identified following the Kabat rules (Kabat et al., 1991. Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication No. 91-3242). The amino acid sequences were confirmed
by mass spectrometry.
[0194] The sequences are disclosed in the attached Sequence
Listing, FIG. 6 and listed in Table 4.
TABLE-US-00004 TABLE 4 Sequence ID numbers for murine anti-human
hCD27.15 antibody of this invention. SEQ ID NO: Description 1
hCD27.15 heavy chain variable region (DNA) 2 hCD27.15 light chain
variable region (DNA) 3 hCD27.15 heavy chain variable region (AA) 4
hCD27.15 light chain variable region (AA) 5 hCD27.15 heavy chain
CDR1 (AA) 6 hCD27.15 heavy chain CDR2 (AA) 7 hCD27.15 heavy chain
CDR3 (AA) 8 hCD27.15 light chain CDR1 (AA) 9 hCD27.15 light chain
CDR2 (AA) 10 hCD27.15 light chain CDR3 (AA)
[0195] The invention is further described by the following numbered
paragraphs: [0196] 1. A binding compound, which binds the same
epitope of human CD27 as monoclonal antibody hCD27.15, produced by
hybridoma hCD27.15 which was deposited with the ATCC in on Jun. 2,
2010 under number PTA-11008. [0197] 2. The binding compound of
paragraph 1, comprising: [0198] an antibody heavy chain variable
region comprising at least one CDR selected from the group
consisting of SEQ ID NOs: 5, 6 and 7, or a variant of any of said
sequences; and/or [0199] an antibody light chain variable region
comprising at least one CDR selected from the group consisting of
SEQ ID NOs: 8, 9 and 10, or a variant of any of said sequences.
[0200] 3. The binding compound of paragraph 1 or 2, comprising:
[0201] a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 3 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 4. [0202] 4. The
binding compound of paragraph 1 or 2, which binds to CD27 and
comprises: [0203] an antibody heavy chain variable region
comprising the CDRs of SEQ ID NOs: 5, 6 and 7, or a variant of any
of said sequences; and/or [0204] an antibody light chain variable
region comprising the CDRs of SEQ ID NOs: 8, 9 and 10, or a variant
of any of said sequences. [0205] 5. The binding compound of any one
of the paragraphs 1-4, wherein any of said variant(s) comprise up
to three amino acid modifications. [0206] 6. The binding compound
of any one of the paragraphs 1-5, which compound is monoclonal
antibody hCD27.15 as produced by hybridoma hCD27.15 (deposit
accession number PTA-11008) or a humanized version thereof. [0207]
7. The binding compound of any of the preceding paragraphs, wherein
the binding compound: [0208] binds human CD27 with a K.sub.D of
about 100 nM or lower; and blocks binding of human CD27 to human
CD70 with an IC.sub.50 of about 10 nM or lower. [0209] 8. A binding
compound which competes for a binding epitope on human CD27 with
any of the binding compounds of paragraphs 1-7, and has one or more
of the following characteristics: [0210] binds human CD27 with a
K.sub.D of about 100 nM or lower; [0211] binds to human CD27 with
about the same K.sub.D as an antibody having a heavy chain
comprising the amino acid sequence of SEQ ID NO: 3 and a light
chain comprising the amino acid sequence of SEQ ID NO: 4; [0212]
blocks binding of human human CD27 to human CD70 with an IC.sub.50
of about 10 nM or lower. [0213] 9. The binding compound of any one
of the paragraphs 1-8, which is [0214] a chimeric antibody or a
fragment thereof; [0215] a human antibody or a fragment thereof;
[0216] a humanized antibody or a fragment thereof; or [0217] an
antibody fragment selected from the group consisting of Fab, Fab',
Fab'-SH, Fv, scFv, F(ab').sub.2, bispecific mAb and a diabody.
[0218] 10. An isolated polynucleotide encoding the binding compound
of any one of the paragraphs 1-9. [0219] 11. Isolated
polynucleotide of paragraph 10, comprising SEQ ID NOs 1 and 2,
which encode the heavy and light chain of hCD27.15. [0220] 12.
Expression vector comprising the polynucleotide of paragraph 10 or
11. [0221] 13. Host cell comprising the expression vector of
paragraph 11 or the polynucleotide of paragraph 10 or 11. [0222]
14. A method of producing a binding compound of any one of the
paragraphs 1-9, which method comprises: [0223] a) culturing host
cell comprising an expression vector that comprises a
polynucleotide encoding a binding compound of the invention under
the control of suitable regulatory sequences in culture medium
under conditions wherein the polynucleotide is expressed, thereby
producing polypeptides comprising the light and heavy chain
variable regions; and [0224] b) recovering the polypeptides from
the host cell or culture medium. [0225] 15. Composition comprising
a binding compound of any one of the paragraphs 1-9 in combination
with a pharmaceutically acceptable carrier or diluent. [0226] 16.
Composition of paragraph 15, further comprising another active
compound, in particular a therapeutically active compound, more in
particular an anti-cancer drug. [0227] 17. A binding compound of
any one of the paragraphs 1-9 for use in therapy and diagnosis.
[0228] 18. The binding compound of paragraph 17, wherein the
therapy comprises [0229] stimulation of proliferation and/or
survival of CD27 .sup.+ cells; [0230] treatment of cancer; or
[0231] treatment of an autoimmune disease. [0232] 19. A binding
compound of any one of the paragraphs 1-9 for use in
flow-cytometry, Western blotting, enzyme-linked immunosorbent assay
(ELISA) and immunohistochemistry.
[0233] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
TABLE-US-00005 SEQUENCE LISTING SEQ 1: hCD27.15 heavy chain
variable region (DNA) 1 TCAGAGGTTC GGCTGCAGCA GTCTGGGGCA GACCTTGTGA
AGCCAGGGGC 51 CTCAGTCAAG TTGTCCTGCA CAGCTTCTGG CTTCATCATT
AAAGCCACCT 101 ATATGCACTG GGTGAGGCAG AGGCCTGAAC AGGGCCTGGA
GTGGATTGGA 151 AGGATTGATC CTGCGAATGG TGAGACTAAA TATGACCCGA
AGTTCCAGGT 201 CAAGGCCACT ATAACAGCAG ACACATCCTC CAGCACAGCC
TACCTGCAGC 251 TCAACAGCCT GACATCTGAC GACACTGCCG TCTATTACTG
TGCTAGATAC 301 GCCTGGTACT TCGATGTCTG GGGCGCAGGG ACCACGGTCA
CCGTCTCCTC 351 AGCCAAAACG ACACCCCCAY CCGTTTATCC MYTGGYCCCT GGAAGC
SEQ 2: hCD27.15 light chain variable region (DNA) 1 GACATCCAGA
TGACTCAGTC TCCAGCCTCC CTGTCTGCAT CTGTGGGAGA 51 CACTGTCACT
ATCACATGTC GGGCAAGTGA GAATATTTAC AGTTTTTTAG 101 CATGGTATCA
TCAGAAACAG GGAAGGTCTC CGCAACTCCT GGTCTATCAT 151 GCAAAAACCC
TAGCAGAAGG TGTGCCATCA AGGTTCAGTG GCAGTGGATC 201 AGGCACACAG
TTTTCTCTGA AGATCAACAG CCTGCAGGCT GAAGATTTTG 251 GGAGTTATTA
CTGTCAACAT TATTATGGTA GTCCGCTCAC GTTCGGTGCT 301 GGGACCAAGC
TGGAGGTGAA ACGGGCTGAT GCTGCACCAA CTGTATCCAT 351 CTTYCCRCCC
TCCTCWGAGG AGCTAAGCTT G SEQ 3: hCD27.15 heavy chain variable region
(AA) EVRLQQSGADLVKPGASVKLSCTASGFIIKATYMHWVRQRPEQGLEWIGRIDPANGETKY
DPKFQVKATITADTSSSTAYLQLNSLTSDDTAVYYCARYAWYFDVWGAGTTVTVSSAKTT
PPXVYPXXPGS SEQ 4: hCD27.15 light chain variable region (AA)
DIQMTQSPASLSASVGDTVTITCRASENIYSFLAWYHQKQGRSPQLLVYHAKTLAEGVPS
RFSGSGSGTQFSLKINSLQAEDFGSYYCQHYYGSPLTFGAGTKLEVKRADAAPTVSIFPP
SSEELSL SEQ 5: hCD27.15 heavy chain CDR1 (AA) GFIIKATYMH SEQ 6:
hCD27.15 heavy chain CDR2 (AA) RIDPANGETKYDPKFQV SEQ 7: hCD27.15
heavy chain CDR3 (AA) YAWYFDV SEQ 8: hCD27.15 light chain CDR1 (AA)
RASENIYSFLA SEQ 9: hCD27.15 light chain CDR2 (AA) HAKTLAE SEQ 10:
hCD27.15 light chain CDR3 (AA) QHYYGSPLT
Sequence CWU 1
1
101396DNAMus musculus 1tcagaggttc ggctgcagca gtctggggca gaccttgtga
agccaggggc ctcagtcaag 60ttgtcctgca cagcttctgg cttcatcatt aaagccacct
atatgcactg ggtgaggcag 120aggcctgaac agggcctgga gtggattgga
aggattgatc ctgcgaatgg tgagactaaa 180tatgacccga agttccaggt
caaggccact ataacagcag acacatcctc cagcacagcc 240tacctgcagc
tcaacagcct gacatctgac gacactgccg tctattactg tgctagatac
300gcctggtact tcgatgtctg gggcgcaggg accacggtca ccgtctcctc
agccaaaacg 360acacccccay ccgtttatcc mytggyccct ggaagc 3962381DNAMus
musculus 2gacatccaga tgactcagtc tccagcctcc ctgtctgcat ctgtgggaga
cactgtcact 60atcacatgtc gggcaagtga gaatatttac agttttttag catggtatca
tcagaaacag 120ggaaggtctc cgcaactcct ggtctatcat gcaaaaaccc
tagcagaagg tgtgccatca 180aggttcagtg gcagtggatc aggcacacag
ttttctctga agatcaacag cctgcaggct 240gaagattttg ggagttatta
ctgtcaacat tattatggta gtccgctcac gttcggtgct 300gggaccaagc
tggaggtgaa acgggctgat gctgcaccaa ctgtatccat cttyccrccc
360tcctcwgagg agctaagctt g 3813131PRTMus
musculusmisc_feature(123)..(123)Xaa can be any naturally occurring
amino acidmisc_feature(127)..(128)Xaa can be any naturally
occurring amino acid 3Glu Val Arg Leu Gln Gln Ser Gly Ala Asp Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser
Gly Phe Ile Ile Lys Ala Thr 20 25 30 Tyr Met His Trp Val Arg Gln
Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro
Ala Asn Gly Glu Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Val Lys
Ala Thr Ile Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu
Gln Leu Asn Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Tyr Ala Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val
100 105 110 Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Xaa Val Tyr Pro
Xaa Xaa 115 120 125 Pro Gly Ser 130 4127PRTMus musculus 4Asp Ile
Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Phe 20
25 30 Leu Ala Trp Tyr His Gln Lys Gln Gly Arg Ser Pro Gln Leu Leu
Val 35 40 45 Tyr His Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile
Asn Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln
His Tyr Tyr Gly Ser Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys
Leu Glu Val Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile
Phe Pro Pro Ser Ser Glu Glu Leu Ser Leu 115 120 125 510PRTMus
musculus 5Gly Phe Ile Ile Lys Ala Thr Tyr Met His 1 5 10 617PRTMus
musculus 6Arg Ile Asp Pro Ala Asn Gly Glu Thr Lys Tyr Asp Pro Lys
Phe Gln 1 5 10 15 Val 77PRTMus musculus 7Tyr Ala Trp Tyr Phe Asp
Val 1 5 811PRTMus musculus 8Arg Ala Ser Glu Asn Ile Tyr Ser Phe Leu
Ala 1 5 10 97PRTMus musculus 9His Ala Lys Thr Leu Ala Glu 1 5
109PRTMus musculus 10Gln His Tyr Tyr Gly Ser Pro Leu Thr 1 5
* * * * *