U.S. patent application number 17/507895 was filed with the patent office on 2022-07-21 for antibodies that bind human cd27 and uses thereof.
The applicant listed for this patent is Celldex Therapeutics, Inc.. Invention is credited to Lizhen HE, Tibor KELER, Henry C. MARSH, Lawrence J. THOMAS, Laura A. VITALE.
Application Number | 20220227877 17/507895 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-21 |
United States Patent
Application |
20220227877 |
Kind Code |
A1 |
KELER; Tibor ; et
al. |
July 21, 2022 |
ANTIBODIES THAT BIND HUMAN CD27 AND USES THEREOF
Abstract
Isolated monoclonal antibodies which bind to human CD27 and
related antibody-based compositions and molecules are disclosed.
Also disclosed are therapeutic and diagnostic methods for using the
antibodies.
Inventors: |
KELER; Tibor; (Pipersville,
PA) ; MARSH; Henry C.; (Reading, MA) ; HE;
Lizhen; (Allentown, PA) ; VITALE; Laura A.;
(Doylestown, PA) ; THOMAS; Lawrence J.; (Easton,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Celldex Therapeutics, Inc. |
Hampton |
NJ |
US |
|
|
Appl. No.: |
17/507895 |
Filed: |
October 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14790247 |
Jul 2, 2015 |
11180566 |
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17507895 |
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13086286 |
Apr 13, 2011 |
9169325 |
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14790247 |
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61471459 |
Apr 4, 2011 |
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61323720 |
Apr 13, 2010 |
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International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. (canceled)
2. An isolated monoclonal antibody which binds to human CD27,
wherein the antibody respectively comprises heavy and light chain
variable region sequences having at least 95% identity to: (a) SEQ
ID NOs: 37 and 43; (b) SEQ ID NOs: 49 and 55; (c) SEQ ID NOs: 103
and 109; (d) SEQ ID NOs: 85 and 91; (e) SEQ ID NOs: 85 and 97; (f)
SEQ ID NOs: 25 and 31; (g) SEQ ID NOs: 73 and 79; (h) SEQ ID NOs: 7
and 13; (i) SEQ ID NOs: 7 and 19; or (j) SEQ ID NOs: 61 and 67.
3. The antibody of claim 2, wherein the antibody comprises heavy
and light chain variable region sequences having at least 95%
identity, 96% identity, 97% identity, 98% identity, or 99% identity
to SEQ ID NOs: 37 and 43, respectively.
4. A composition comprising the antibody of claim 2 and a
carrier.
5. The composition of claim 4, further comprising an adjuvant.
6. The composition of claim 4, further comprising an
immunostimulatory agent.
7. The composition of claim 6, wherein the immunostimulatory agent
is selected from the group consisting of CD40 ligand, FLT 3 ligand,
cytokines, colony-stimulating factors, an anti-CTLA-4 antibody, LPS
(endotoxin), ssRNA, dsRNA, Bacille Calmette-Guerin (BCG),
Levamisole hydrochloride, intravenous immune globulins and a
Toll-like Receptor (TLR) agonist.
8. The composition of claim 7, wherein the Toll-like Receptor
agonist is selected from the group consisting of a TLR3 agonist, a
TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a
TLR 9 agonist.
9. The composition of claim 4, further comprising an
immunosuppressive agent.
10. The composition of claim 4, further comprising another
antibody.
11. The composition of claim 4, further comprising an antigen.
12. The composition of claim 11, wherein the antigen comprises a
tumor antigen, allergen, or an autoantigen.
13. The composition of claim 12, wherein the tumor antigen is
selected from the group consisting of .beta.hCG, gp100 or Pmel17,
HER2/neu, WT1, mesothelin, CEA, gp100, MART1, TRP-2, melan-A,
NY-ESO-1, NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MAGE-3,
MAGE-A3, Tyrosinase, Telomerase, SSX2 antigens, MUC-1 antigens, and
germ cell derived tumor antigens.
14. A bispecific antibody comprising the antibody of claim 2 linked
to a second molecule having a binding specificity which is
different from the antibody.
15. The bispecific molecule of claim 14, wherein the second
molecule binds to a T cell receptor.
16. The bispecific molecule of claim 15, wherein the T cell
receptor is selected from the group consisting of CD3, CD40 and
CD25.
17. The bispecific molecule of claim 14, wherein the second
molecule binds to an NK receptor.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 14/790, 247, which is a divisional of U.S.
Pat. No. 9,169,325, filed Apr. 13, 2011, which claims priority to
U.S. Provisional Application Nos. 61/323,720, filed Apr. 13, 2010;
and 61/471,459, filed on Apr. 4, 2011. The contents of the
aforementioned applications are hereby incorporated by reference in
their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web, and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 22, 2021, is named Sequence_Listing_CDJ367DVCN.txt and is
78,603 bytes in size.
BACKGROUND OF THE INVENTION
[0003] Interactions between T cells and antigen-presenting cells
involve a variety of accessory molecules that facilitate in the
generation of an immune response. One such molecule is CD27, which
binds CD70 and belongs to the tumor necrosis factor receptor
(TNF-R) superfamily (Ranheim E A, et al., Blood. 1995 Jun. 15;
85(12):3556-65). CD27 typically exists as a glycosylated, type I
transmembrane protein, frequently in the form of homodimers with a
disulfide bridge linking the two monomers. The disulfide bridge is
in the extracellular domain close to the membrane (Camerini et al.,
J Immunol. 147:3165-69 (1991). CD27 may also be expressed in a
soluble form (see, e.g., van Oers M H, et al., Blood. 1993 Dec. 1;
82(11):3430-6 and Loenen W A, et al., Eur. J. Immunol. 22:447,
1992). Cross-linking the CD27 antigen on T cells provides a
costimulatory signal that, in concert with T-cell receptor
crosslinking, can induce T-cell proliferation and cellular immune
activation.
[0004] CD27 is expressed on mature thymocytes, on most CD4+ and
CD8+ peripheral blood T cells, natural killer cells and B cells
(Kobata T, et al., Proc. Natl. Acad. Sci. USA. 1995 Nov. 21;
92(24):11249-53). CD27 is also highly expressed on B cell
non-Hodgkin's lymphomas and B cell chronic lymphocytic leukemias
(Ranheim E A, et al., Blood. 1995 Jun. 15; 85(12):3556-65).
Additionally, increased levels of soluble CD27 protein have been
identified in sera or sites of disease activity in parasitic
infection, cytomegalovirus (CMV) infection, sarcoidosis, multiple
sclerosis, and B-cell chronic lymphocytic leukemia (Loenen W A, et
al., Eur. J. Immunol. 22:447, 1992).
[0005] Agonistic monoclonal antibodies against CD27 have recently
been shown to promote T cell responses and show promise as
anti-cancer therapeutics (see e.g., Sakanishi T, et al., Biochem
Biophys. Res. Commun. 2010 Feb. 18 and WO 2008/051424). However,
while the results obtained to date establish CD27 as a useful
target for immunotherapy, it is unknown which particular features
of anti-CD27 monoclonal antibodies are especially advantageous for
therapeutic purposes. As such, there is a need in the art for
further insight into the specific functional properties that make
anti-CD27 antibodies therapeutically effective, as well as improved
therapeutic antibodies against CD27 which are more effective for
treating and/or preventing diseases.
SUMMARY OF THE INVENTION
[0006] The present invention provides inter alia isolated anti-CD27
antibodies having particular functional properties which can be
linked with advantageous and desirable therapeutic effects.
Specifically, anti-CD27 monoclonal antibodies capable of
up-regulating T cell mediated immune responses (e.g., as evidenced
by inducement or enhancement of antigen-specific T cell responses),
which are particularly well-suited for combination with vaccine
therapies, have been generated and characterized by way of the
present invention. In one embodiment, agonist anti-CD27 antibodies
can enhance the immune response against cancers or infectious
diseases by combination with active vaccination, or by enhancing
endogenous immune responses. Such antibodies may also directly or
indirectly induce cytokine expression. Additionally, anti-CD27
antibodies that down-regulate T cell mediated immune responses,
which are particularly well-suited for treating immune disorders,
such as graft rejection, allergy and autoimmune diseases, have been
generated and characterized. Still further, anti-CD27 antibodies
that inhibit growth of CD27 expressing cells by direct cell killing
mechanisms (e.g., antibody dependent cell-mediated cytotoxicity
(ADCC) and/or complement dependent cellular cytotoxicity (CDCC)),
which are particularly effective in treating a wide variety of
diseases involving cell proliferation (e.g., cancers), have been
generated and characterized.
[0007] In one embodiment, the anti-CD27 antibodies of the present
invention exhibit one or more of the following properties:
[0008] (a) blocks binding of sCD70 to CD27 by at least about 70% at
an antibody concentration of 10 .mu.g/ml;
[0009] (b) binds to human CD27 with an equilibrium dissociation
constant Kd of 10.sup.-9 M or less, or alternatively, an
equilibrium association constant Ka of 10.sup.+9 M.sup.-1 or
greater;
[0010] (c) induces specific complement mediated cytotoxicity (CDC)
of CD27 expressing cells of at least 10% at an antibody
concentration of 3 .mu.g/ml and approximately 6% rabbit serum
complement;
[0011] (d) induces antibody dependent cell-mediated cytotoxicity
(ADCC) specific lysis of CD27 expressing cells of at least 10% at
an antibody concentration of 3 .mu.g/ml and ratio of
effector:target cells of 75:1;
[0012] (e) improves median survival by at least 20% in severe
combined immunodeficiency (SCID) mice post tumor cell inoculation
in vivo (5.times.10.sup.5 Raji cells or 1.times.10.sup.6 Daudi
cells) when administered at 0.3 mg (i.p.) at least twice a week for
3 weeks compared to mice to which antibody is not administered;
[0013] (f) induces or enhances antigen-specific immune responses in
combination with a vaccine or endogenous antigen;
[0014] (g) induces or enhances antigen-specific TH1 immune
responses in combination with a vaccine or endogenous antigen;
[0015] (h) induces or enhances antigen-specific T-cell
proliferation or activation in combination with a vaccine or
endogenous antigen;
[0016] (i) reduces or inhibits T-cell proliferation or
activation;
[0017] (j) induces or enhances T-cell activity when combined with
simultaneous, separate or sequential TCR activation;
[0018] (k) blocks binding of sCD70 to CD27 by at least about 70% at
an antibody concentration of 10 .mu.g/ml and reduces or inhibits
T-cell activity when not capable of binding to, or having reduced
binding to Fc receptors;
[0019] (l) results in less than 50% depletion of CD3+ T-cells
(other than NK cells) in macaques when administered at 3 mg/kg
(i.v.) over the period of 29 days immediately following
administration; or
[0020] (m) results in less than 50% depletion of memory B-cells in
macaques when administered at 3 mg/kg (i.v.) over the period of 29
days immediately following administration.
[0021] In a particular embodiment, the antibodies of the invention
exhibit combinations of these functional properties.
[0022] Accordingly, in one aspect, the invention provides anti-CD27
antibodies that induce and/or enhance an immune response (e.g., a T
cell mediated immune response). In a further embodiment, the
antibodies inhibit the binding of CD70 to CD27 on cells. Particular
antibodies having these combinations of properties include mAb 1F5
comprising heavy and/or light chain variable regions sequences
comprising SEQ ID NOs: 37 and/or 43, respectively). Alternatively,
the antibodies do not inhibit the binding of CD70 to CD27 on cells.
Particular antibodies having these combinations of properties
include mAb 3H8 comprising heavy and/or light chain variable
regions sequences comprising SEQ ID NOs: 7 and/or 13, respectively,
or land/or 19, respectively). Such anti-CD27 antibodies also can be
linked to a second molecule (e.g., as a bispecific molecule) having
a binding specificity which is different from the antibody, such as
a T cell receptor (e.g., CD3, CD25, CD137, CD154_), or an Fc
receptor (e.g., Fc.gamma.RI (CD64), Fc.gamma.RIIA (CD32),
Fc.gamma.RIIB1 (CD32), Fc.gamma.RIIB2 (CD32), Fc.gamma.RIIIA
(CD16a), Fc.gamma.RIIIB (CD16b), Fc.epsilon.RI, Fc.epsilon.RII
(CD23), Fc.alpha.RI (CD89), Fc.alpha./.mu.R, and FcRn), or an NK
receptor (e.g. CD56), or a B cell receptor (e.g. CD19, CD20).
[0023] Antibodies intended to be used for induction or enhancement
of immune responses according to the present invention may have a
functional Fc domain permitting binding to Fc receptors, and may
include a mutated Fc domain having increased levels of binding to
Fc receptors.
[0024] In another aspect, the invention provides anti-CD27
antibodies that down-regulate T cell mediated immune responses by
inhibiting the binding of CD27 to CD70 on cells which express these
proteins. In a particular embodiment, the antibodies inhibit the
binding of soluble CD70 (sCD70) to CD27 expressing cells by at
least about 70%. Particular antibodies falling within this class
include, e.g., mAb comprising heavy and/or light chain variable
regions sequences comprising SEQ ID NOs:37 and/or 43 (mAb 1F5), SEQ
ID NOs: 49 and/or 55 (mAb 1H8), or SEQ ID NOs: 103 and/or 109 (mAb
3H12).
[0025] In yet another aspect, the invention provides anti-CD27
antibodies that induce or enhance effector cell function (e.g.,
cell killing via either ADCC and/or CDC). In one embodiment, the
antibody induces at least about 30% specific lysis of CD27
expressing cells via ADCC at an antibody concentration of 10
.mu.g/ml and/or induces at least about 30% CDC of CD27 expressing
cells at a concentration of 10 .mu.g/ml. Particular antibodies
falling within this class exhibiting ADCC effector function
include, e.g., (e.g., mAb comprising heavy and/or light chain
variable region sequences comprising SEQ ID NOs: 61 and/or 67 (mAb
1G5), SEQ ID NOs: 85 and/or 91, 85 and/or 97 (mAb 3A10), SEQ ID
NOs:37 and/or 43 (mAb 1F5), SEQ ID NOs: 7 and/or 13, 7 and/or 19
(mAb 3H8), SEQ ID NOs: 49 and/or 55(mAb 1H8), or SEQ ID NOs: 103
and/or 109 (mAb 3H12). In a further embodiment, the antibody also
inhibits binding of CD70 to CD27 on cells. Particular antibodies
having these combinations of functions include, e.g., (e.g., mAb
comprising heavy and/or light chain variable regions sequences
comprising SEQ ID NOs: 37 and/or 43(mAb 1F5), SEQ ID NOs: 49 and/or
55 (mAb 1H8), SEQ ID NOs:103 and/or 109 (mAb 3H12). Alternatively,
the antibody induces ADCC and/or CDC as described above, but does
not inhibit binding of CD70 to CD27 on cells. Particular antibodies
having these features include, e.g., mAb comprising heavy and/or
light chain variable regions sequences comprising SEQ ID NOs: 61
and/or 67 (mAb 1G5), SEQ ID NOs: 85 and/or 91, 85 and/or 97 (mAb
3A10), SEQ ID NOs:7 and/or 13, 7 and/or 19 (mAb 3H8).
[0026] Anti-CD27 antibodies capable of inducing or enhancing
effector cell function (e.g., ADCC and/or CDC) can also be
constructed to include an Fc region which suitably contributes a
binding specificity for a specific Fc receptor (e.g., Fc.gamma.RI
(CD64), Fc.gamma.RIIA (CD32), Fc.gamma.RIIB1 (CD32), Fc.gamma.RIIB2
(CD32), Fc.gamma.RIIIA (CD16a), Fc.gamma.RIIIB (CD16b),
Fc.epsilon.RI, Fc.epsilon.RII (CD23), Fc.alpha.RI (CD89),
Fc.alpha./.mu.R, and FcRn).
[0027] In a further embodiment there is provided a method for
enhancing an immune response against an antigen in a subject in
need thereof by administering to the subject: i) an anti-CD27
antibody and ii) an antigen, wherein the anti-CD27 antibody is
administered separately from and before the antigen is
administered.
[0028] Typically in such a method the anti-CD27 antibody may be
administered between at least 2 and 96 hours before the antigen.
For example, in such a method, the anti-CD27 antibody may be
administered at least 2 hours before the antigen, for example at
least 12 hours before the antigen, suitably at least 24 hours
before the antigen, at least 48 hours before the antigen or at
least 72 hours before the antigen. wherein the TLR agonist is a
TLR3 agonist.
[0029] In a further embodiment there is provided a method for
enhancing an immune response against an antigen in a subject in
need thereof by simultanously, separately or sequentially
administering to the subject: i) an anti-CD27 antibody; ii) a TLR
agonist; and iii) optionally, the antigen.
[0030] In a preferred embodiment of such a method the TLR agonist
is a TLR3 agonist, for example but not limited to Poly IC:LC.
[0031] CD27 expressing cells include any and all cells that express
CD27, including, but not limited to B cells, NK cells and T cells.
In a particular embodiment, the CD27 expressing cells include
cancer cell lines such as Jurkat cells, Raji cells, Ramos cells and
Daudi cells. In another embodiment, the CD27 expressing cells are
tumor cells or cancer cells. In another embodiment, CD27 expressing
cells include B cells, NK cells, and T cells including T cells that
are found infiltrating tumors, also called tumor infiltrating
lymphocytes.
[0032] Particular antibodies of the invention comprise heavy and
light chain variable regions that utilize particular human
germlines, i.e., are encoded by the germline genes, but include
genetic rearrangements and mutations, e.g., somatic mutations,
which occur during antibody maturation. In one embodiment, the
heavy chain variable region of the antibodies of the present
invention is derived from a human germline 3-7 or 3-33 gene. In
another embodiment, the light chain variable region of the antibody
is derived from a human germline 3-20, 3-11, 24, 1D-16, or 1-13
gene. In a particular embodiment, the heavy chain variable region
of the antibody is derived from a human germline V.sub.H 3-7 or
V.sub.H 3-33 gene and the light chain variable region of the
antibody is derived from a human germline V.sub.K 3-20, V.sub.K
3-11, V.sub.K 1D-16, or V.sub.K 1-13 gene.
[0033] A V.sub.H 3-33 germline sequence is provided (Genbank
Accession No AAP44382) as follows:
TABLE-US-00001 (SEQ ID NO: 3) 1 vqlvesgggv vqpgrslrls caasgftfst
ygmhwvrqap gkglewvaii wfdgsntyya 61 dsvrgrftis rdssrktlyl
emkslrvedt avyycak
[0034] A V.sub.H 3-7 germline sequence is provided (Genbank
Accession No AAP44389) as follows:
TABLE-US-00002 (SEQ ID NO: 4) 1 vqlvesgggl vqpggslrls caasgftfsn
symtwvrqap gkglewvani kpdgsdknyi 61 nsvrgrftis rdnaekssyl
qmnslraedt aiyycvt
[0035] In another embodiment, the heavy chain variable region CDR3
sequence is selected from the group consisting of SEQ ID NOs: 10,
28, 40, 52, 64, 76, 88, 106, and conservative sequence
modifications thereof (e.g., conservative amino acid
substitutions). The antibodies may further include a light chain
variable region CDR3 sequence selected from the group consisting of
SEQ ID NOs: 16, 22, 34, 46, 58, 70, 82, 94, 100, 112, and
conservative sequence modifications thereof. In another embodiment,
the heavy chain CDR2 and CDR1 sequences are selected from SEQ ID
NOs: 9, 27, 39, 51, 63, 75, 87, 105, and SEQ ID NOs: 8, 26, 38, 50,
62, 74, 86, 104, respectively, and conservative sequence
modifications thereof. The light chain CDR2 and CDR1 sequences are
selected from SEQ ID NOs: 15, 21, 33, 45, 57, 69, 81, 93, 99, 111,
and SEQ ID NOs: 14, 20, 32, 44, 56, 68, 80, 92, 98, 110,
respectively, and conservative sequence modifications thereof.
[0036] In still another embodiment, the invention provides an
isolated antibody that binds CD27 and includes heavy and light
chain variable region CDR1, CDR2 and CDR3 sequences selected from
the group consisting of: [0037] (i) a heavy chain variable region
CDR1 comprising SEQ ID NO: 38; [0038] a heavy chain variable region
CDR2 comprising SEQ ID NO: 39; [0039] a heavy chain variable region
CDR3 comprising SEQ ID NO: 40; [0040] a light chain variable region
CDR1 comprising SEQ ID NO: 44; [0041] a light chain variable region
CDR2 comprising SEQ ID NO: 45; [0042] a light chain variable region
CDR3 comprising SEQ ID NO: 46; or [0043] conservative sequence
modifications thereof; [0044] (ii) a heavy chain variable region
CDR1 comprising SEQ ID NO: 50; [0045] a heavy chain variable region
CDR2 comprising SEQ ID NO: 51; [0046] a heavy chain variable region
CDR3 comprising SEQ ID NO: 52; [0047] a light chain variable region
CDR1 comprising SEQ ID NO: 56; [0048] a light chain variable region
CDR2 comprising SEQ ID NO: 57; [0049] a light chain variable region
CDR3 comprising SEQ ID NO: 58; or [0050] conservative sequence
modifications thereof; [0051] (iii) a heavy chain variable region
CDR1 comprising SEQ ID NO: 104; [0052] a heavy chain variable
region CDR2 comprising SEQ ID NO: 105; [0053] a heavy chain
variable region CDR3 comprising SEQ ID NO: 106; [0054] a light
chain variable region CDR1 comprising SEQ ID NO: 110; [0055] a
light chain variable region CDR2 comprising SEQ ID NO: 111; [0056]
a light chain variable region CDR3 comprising SEQ ID NO: 112; or
[0057] conservative sequence modifications thereof; [0058] (iv) a
heavy chain variable region CDR1 comprising SEQ ID NO: 86; [0059] a
heavy chain variable region CDR2 comprising SEQ ID NO: 87; [0060] a
heavy chain variable region CDR3 comprising SEQ ID NO: 88; [0061] a
light chain variable region CDR1 comprising SEQ ID NO: 92 or 98;
[0062] a light chain variable region CDR2 comprising SEQ ID NO: 93
or 99; [0063] a light chain variable region CDR3 comprising SEQ ID
NO: 94 or 100; or [0064] conservative sequence modifications
thereof; [0065] (v) a heavy chain variable region CDR1 comprising
SEQ ID NO: 26; [0066] a heavy chain variable region CDR2 comprising
SEQ ID NO: 27; [0067] a heavy chain variable region CDR3 comprising
SEQ ID NO: 28; [0068] a light chain variable region CDR1 comprising
SEQ ID NO: 32; [0069] a light chain variable region CDR2 comprising
SEQ ID NO: 33; [0070] a light chain variable region CDR3 comprising
SEQ ID NO: 34; or [0071] conservative sequence modifications
thereof; [0072] (vi) a heavy chain variable region CDR1 comprising
SEQ ID NO: 74; [0073] a heavy chain variable region CDR2 comprising
SEQ ID NO: 75; [0074] a heavy chain variable region CDR3 comprising
SEQ ID NO: 76; [0075] a light chain variable region CDR1 comprising
SEQ ID NO: 80; [0076] a light chain variable region CDR2 comprising
SEQ ID NO: 81; [0077] a light chain variable region CDR3 comprising
SEQ ID NO: 82; or [0078] conservative sequence modifications
thereof; [0079] (vii) a heavy chain variable region CDR1 comprising
SEQ ID NO: 8; [0080] a heavy chain variable region CDR2 comprising
SEQ ID NO: 9; [0081] a heavy chain variable region CDR3 comprising
SEQ ID NO: 10; [0082] a light chain variable region CDR1 comprising
SEQ ID NO: 14 or 20; [0083] a light chain variable region CDR2
comprising SEQ ID NO: 15 or 21; [0084] a light chain variable
region CDR3 comprising SEQ ID NO: 16 or 22; or [0085] conservative
sequence modifications thereof; and [0086] (viii) a heavy chain
variable region CDR1 comprising SEQ ID NO: 62; [0087] a heavy chain
variable region CDR2 comprising SEQ ID NO: 63; [0088] a heavy chain
variable region CDR3 comprising SEQ ID NO: 64; [0089] a light chain
variable region CDR1 comprising SEQ ID NO: 68; [0090] a light chain
variable region CDR2 comprising SEQ ID NO: 69; [0091] a light chain
variable region CDR3 comprising SEQ ID NO: 70; or [0092]
conservative sequence modifications thereof.
[0093] In another embodiment, the heavy chain variable region CDR3
sequence comprises an amino acid sequence selected from the
consensus sequence: R (G,E,D) (S,L,G,-) (G,L,T,W,-) (N,A,T,H,-)
(V,T,-) (M,P,-) (G,V,-) (R, -) (G,M, -) (D,H,L,T,W) (A,G,N,W)
(D,F,V,Y) (F,L) (D,E) (H,I,L,Y) (SEQ ID NO: 113), wherein "-"
denotes the option of no amino acid residue being present at that
consensus position. The antibodies may further include a light
chain variable region CDR3 sequence comprising an amino acid
sequence selected from the consensus sequence: Q (F,R,Y) (N,S)
(N,T,S) (Y,W) P (F,L,P,R) T (SEQ ID NO: 114), wherein "-" denotes
the option of no amino acid residue being present at that consensus
position. In another embodiment, the heavy chain variable region
CDR2 sequence comprises an amino acid sequence selected from the
consensus sequence: I (K,W) (Y,N,Q) D G S (E,N) (K,Q) (SEQ ID NO:
115), wherein "-" denotes the option of no amino acid residue being
present at that consensus position, and the light chain variable
region CDR2 sequence comprises an amino acid sequence selected from
the consensus sequence: (A,D) A S (SEQ ID NO: 116). In another
embodiment, the heavy chain variable region CDR1 sequence comprises
an amino acid sequence selected from the consensus sequence: G F
(T,S) (F,L) (S,N) (I,S,H) (Y,H) (SEQ ID NO: 117); and the light
chain variable region CDR1 sequence comprises an amino acid
sequence selected from the consensus sequence: Q (D,G,S) (I,V)
(D,S) (R,S) (A,W,Y) (SEQ ID NO: 118).
[0094] In another embodiment, isolated antibodies of the invention
bind to human CD27 and include a heavy chain variable region
including an amino acid sequence selected from the group consisting
of SEQ ID NOs: 6, 7, 24, 25, 36, 37, 48, 49, 60, 61, 72, 73, 84,
85, 102, 103, and conservative sequence modifications thereof. The
antibody may further include a light chain variable region
including an amino acid sequence selected from the group consisting
of SEQ ID NOs: 12, 13, 18, 19, 30, 31, 42, 43, 54, 55, 66, 67, 78,
79, 90, 91, 96, 97, 108, 109, and conservative sequence
modifications thereof.
[0095] In a still further embodiment, isolated antibodies of the
invention bind to human CD27 and include a heavy chain variable
region and a light chain variable region including the amino acid
sequences selected from the group consisting of:
[0096] (a) SEQ ID NOs: 37 and/or 43, respectively, and conservative
sequence modifications thereof;
[0097] (b) SEQ ID NOs: 49 and/or 55, respectively, and conservative
sequence modifications thereof;
[0098] (c) SEQ ID NOs: 103 and/or 109, respectively, and
conservative sequence modifications thereof;
[0099] (d) SEQ ID NOs: 85 and/or 91 and/or 97, respectively, and
conservative sequence modifications thereof;
[0100] (e) SEQ ID NOs: 25 and/or 31, respectively, and conservative
sequence modifications thereof;
[0101] (f) SEQ ID NOs: 73 and/or 79, respectively, and conservative
sequence modifications thereof;
[0102] (g) SEQ ID NOs: 7 and/or 13 and/or 19, respectively, and
conservative sequence modifications thereof; and
[0103] (h) SEQ ID NOs: 61 and/or 67, respectively, and conservative
sequence modifications thereof;
[0104] Isolated antibodies which include heavy and light chain
variable regions having at least 80%, or at least 85%, or at least
90%, or at least 95%, or at least 96%, or at least 97%, or at least
98%, or at least 99%, or more sequence identity to any of the above
sequences are also included in the present invention. Ranges
intermediate to the above-recited values, e.g., heavy and light
chain variable regions having at least 80-85%, 85-90%, 90-95% or
95-100% sequence identity to any of the above sequences are also
intended to be encompassed by the present invention.
[0105] Also encompassed by the present invention are isolated
antibodies which compete for binding to CD27 with the antibodies of
the invention. In a particular embodiment, the antibody competes
for binding to CD27 with an antibody comprising heavy and/or light
chain variable regions comprising the amino acid sequences set
forth in SEQ ID NOs: 37 and 43, SEQ ID NOs: 49 and 55, SEQ ID NOs:
103 and 109, SEQ ID NOs: 85 and 91, SEQ ID NOs: 85 and 97, SEQ ID
NOs: 25 and 31, SEQ ID NOs: 73 and 79, SEQ ID NOs: 7 and 13, SEQ ID
NOs: 7 and 19, SEQ ID NOs: 61 an 67, respectively, or amino acid
sequences at least 80% identical thereto. In another embodiment,
the antibody competes for binding to CD27 with an antibody
comprising heavy and/or light chain variable regions comprising the
amino acid sequences set forth in SEQ ID NOs: 37 and 43 (1F5), SEQ
ID NOs: 49 and 55 (1H8) or SEQ ID NOs: 103 and 109 (3H12). In
another embodiment, the antibody competes for binding to CD27 with
an antibody comprising heavy and/or light chain variable regions
comprising the amino acid sequences set forth in SEQ ID NOs: 25 and
31 (2C2), SEQ ID NOs: 7 and 13 (3H8), SEQ ID NOs: 7 and 19 (3H8),
SEQ ID NOs: 61 an 67 (1G5) or SEQ ID NOs: 73 and 79 (2G9). In yet
another embodiment, the antibody competes for binding to CD27 with
an antibody comprising heavy and/or light chain variable regions
comprising the amino acid sequences set forth in SEQ ID NOs: 85 and
91 (3A10) or SEQ ID NOs: 85 and 97 (3A10).
[0106] Other antibodies of the invention bind to an epitope on CD27
recognized by the antibodies described herein. In another
particular embodiment, the antibody binds to an epitope on CD27
recognized by an antibody comprising heavy and/or light chain
variable regions comprising the amino acid sequences set forth in
SEQ ID NOs: 37 and 43, SEQ ID NOs: 49 and 55, SEQ ID NOs: 103 and
109, SEQ ID NOs: 85 and 91, SEQ ID NOs: 85 and 97, SEQ ID NOs: 25
and 31, SEQ ID NOs: 73 and 79, SEQ ID NOs: 7 and 13, SEQ ID NOs: 7
and 19, SEQ ID NOs: 61 an 67, respectively, or amino acid sequences
at least 80% identical thereto. In another embodiment, the antibody
binds to an epitope on CD27 recognized by an antibody comprising
heavy and/or light chain variable regions comprising the amino acid
sequences set forth in SEQ ID NOs: 37 and 43 (1F5), SEQ ID NOs: 49
and 55 (1H8) or SEQ ID NOs: 103 and 109 (3H12). In another
embodiment, the antibody binds to an epitope on CD27 recognized by
an antibody comprising heavy and/or light chain variable regions
comprising the amino acid sequences set forth in SEQ ID NOs: 25 and
31 (2C2), SEQ ID NOs: 7 and 13 (3H8), SEQ ID NOs: 7 and 19 (3H8),
SEQ ID NOs: 61 an 67 (1G5) or SEQ ID NOs: 73 and 79 (2G9). In yet
another embodiment, the antibody binds to an epitope on CD27
recognized by an antibody comprising heavy and/or light chain
variable regions comprising the amino acid sequences set forth in
SEQ ID NOs: 85 and 91 (3A10) or SEQ ID NOs: 85 and 97 (3A10).
[0107] The antibodies of the invention can either be full-length,
for example, any of the following isotypes: IgG1, IgG2, IgG3, IgG4,
IgM, IgA1, IgA2, IgAsec, IgD, and IgE. Alternatively, the
antibodies can be fragments such as an antigen-binding portion or a
single chain antibody (e.g., a Fab, F(ab').sub.2, Fv, a single
chain Fv fragment, an isolated complementarity determining region
(CDR) or a combination of two or more isolated CDRs). The
antibodies can be any kind of antibody, including, but not limited
to, human, humanized, and chimeric antibodies.
[0108] Tumor antigens employed by the present invention (e.g., in a
vaccine, used in combination with an anti-CD27 antibody of the
invention) include any antigen or antigenic determinant which is
present on (or associated with) a tumor cell and not typically on
normal cells, or an antigen or antigenic determinant which is
present on or associated with tumor cells in greater amounts than
on normal (non-tumor) cells, or an antigen or antigenic determinant
which is present on tumor cells in a different form than that found
on normal (non-tumor) cells. Such antigens include tumor-specific
antigens, including tumor-specific membrane antigens,
tumor-associated antigens, including tumor-associated membrane
antigens, embryonic antigens on tumors, growth factor receptors,
growth factor ligands, and any other type of antigen that is
associated with cancer. A tumor antigen may be, for example, an
epithelial cancer antigen, (e.g., breast, gastrointestinal, lung),
a prostate specific cancer antigen (PSA) or prostate specific
membrane antigen (PSMA), a bladder cancer antigen, a lung (e.g.,
small cell lung) cancer antigen, a colon cancer antigen, an ovarian
cancer antigen, a brain cancer antigen, a gastric cancer antigen, a
renal cell carcinoma antigen, a pancreatic cancer antigen, a liver
cancer antigen, an esophageal cancer antigen, a head and neck
cancer antigen, or a colorectal cancer antigen. For example, the
antigen may include a tumor antigen, such as .beta.hCG, gp100 or
Pmel17, CEA, gp100, TRP-2, NY-BR-1, NY-CO-58, MN (gp250), idiotype,
Tyrosinase, Telomerase, SSX2, MUC-1, MAGE-A3, and high molecular
weight-melanoma associated antigen (HMW-MAA) MART1, melan-A,
EGFRvIII, NY-ESO-1, MAGE-1, MAGE-3, WT1, Her2,or mesothelin. Other
antigens employed by the present invention (e.g., in a vaccine,
used in combination with an anti-CD27 antibody of the invention)
include antigens from infectious disease pathogens, such as
viruses, bacteria, parasites and fungi, examples of which are
disclosed herein.
[0109] The invention also provides a bispecific molecule comprising
an antibody of the invention linked to a second functional moiety
having a different binding specificity than said antibody. For
example, in one embodiment, the second molecule may bind a T cell
receptor (e.g., CD3, CD40).
[0110] Compositions including an antibody or a bispecific molecule
described herein, formulated with a pharmaceutically acceptable
carrier, are also provided. The compositions may further include an
adjuvant, immunostimulatory agent (e.g., CD40 ligand, FLT 3 ligand,
cytokines, colony-stimulating factors, an anti-CTLA-4 antibody,
anti-PD1 antibody, anti-41BB antibody, anti OX-40 antibody, LPS
(endotoxin), ssRNA, dsRNA, Bacille Calmette-Guerin (BCG),
Levamisole hydrochloride, intravenous immune globulins and a
Toll-like Receptor (TLR) agonist (e.g., TLR3 agonist such as Poly
IC, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist,
and a TLR 9 agonist)), immunosuppressive agent, another antibody,
or an antigen. Exemplary antigens include, but are not limited to,
a component of a pathogen, a tumor antigen (e.g., .beta.hCG, gp100
or Pmel17, HER2/neu, WT1, mesothelin, CEA, gp100, MART1, TRP-2,
melan-A, NY-ESO-1, NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1,
MAGE-3, MAGE-A3, Tyrosinase, Telomerase, SSX2 antigens, MUC-1
antigens, and germ cell derived tumor antigens), an infectious
disease antigen (e.g. viral antigens, bacterial and parasitic
antigens) an allergen, or an autoantigen. Any of the antigens
disclosed herein can be included in a composition of the
invention.
[0111] Nucleic acid molecules encoding the antibodies of the
invention are also encompassed by the invention, as well as
expression vectors comprising such nucleic acids and host cells
comprising such expression vectors. For example, in one embodiment,
the invention provides an isolated monoclonal antibody that binds
human CD27, wherein the antibody comprises a heavy chain variable
region and a light chain variable region encoded by nucleic acid
sequences selected from the group consisting of: (a) SEQ ID NOs: 5
and 11, respectively; (b) SEQ ID NOs: 5 and 17, respectively; (c)
SEQ ID NOs: 23 and 29, respectively; (d) SEQ ID NOs: 35 and 41,
respectively; (e) SEQ ID NOs: 47 and 53, respectively; (f) SEQ ID
NOs: 59 and 65, respectively; (g) SEQ ID NOs: 71 and 77,
respectively; (h) SEQ ID NOs: 83 and 89, (i) SEQ ID NOs: 83 and 95;
(j) SEQ ID NOs: 101 and 107, respectively or nucleic acid sequences
having at least 90% identity to the nucleic acid sequences of
(a)-(h).
[0112] In another embodiment, the present invention provides
methods for inducing or enhancing an immune response (e.g., a T
cell-mediated immune response, and/or an NK-mediated response
and/or a B cell-mediated immune response) against an antigen in a
subject by administering to the subject an effective amount of an
antibody (e.g., a full length antibody), composition or bispecific
molecule described herein. Such methods are particularly
well-suited for use in vaccine therapies.
[0113] The antibodies and other compositions of the present
invention can also be used to inhibit growth of CD27 expressing
cells by contacting the cells with an antibody or composition in an
amount effective to inhibit growth of CD27 expressing cells (e.g.,
in the treatment of cancers). Antibodies useful in inhibiting the
growth of CD27 expressing cells include full length antibodies and
fragments thereof, as well as antibodies that contain a second
binding specificity for an Fc receptor. In one embodiment, the CD27
expressing cells are contacted with an antibody in the presence of
effector cells under conditions sufficient to induce
antibody-dependent cellular cytoxicity (ADCC) of target cells
(e.g., the antibody induces at least about 40% specific lysis of
CD27 expressing cells at a concentration of 10 .mu.g/ml and
comprises SEQ ID NOs:61, 67, 85, 91, 97, 37, and/or 43). In another
embodiment, the cells are contacted with an antibody under
conditions sufficient to induce complement mediated cytotoxicity
(CDC) of the cells (e.g., the antibody induces at least about 40%
complement mediated cytotoxicity (CDC) of CD27 expressing cells at
a concentration of 10 .mu.g/ml and comprises SEQ ID NOs: 7, 13, 19,
49, 55, 103, and/or 109).
[0114] In a further embodiment, the antibody employed to inhibit
growth of CD27 expressing cells may also possess (or lack)
additional functional features. For example, the antibody may also
inhibit the binding of CD70 to CD27 on cells which express these
proteins (e.g., a mAb comprising heavy and/or light chain variable
regions sequences comprising SEQ ID NOs: 37 and/or 43 (mAb 1F5),
SEQ ID NOs: 49 and/or 55 (mAb 1H8), or SEQ ID NOs: 103 and/or 109
(mAb 3H12). Alternatively, the antibody may not inhibit the binding
of CD70 to CD27 on such cells (e.g., a mAb comprising heavy and/or
light chain variable regions sequences comprising SEQ ID NOs: 61
and/or 67 (mAb 1G5), SEQ ID NOs: 85 and/or 91, 85 and/or 97 (mAb
3A10), or SEQ ID NOs:7 and/or 13, 7 and/or 19 (mAb 3H8).
[0115] CD27 expressing cells include any and all cells the express
CD27, including, but not limited to NK cells, B cells and T cells.
In a particular embodiment, the CD27 expressing cells include cell
lines such as Jurkat cells, Raji cells, Ramos cells and Daudi
cells. In another embodiment, the CD27 expressing cells are tumor
cells or cancer cells. In another embodiment, CD27 expressing cells
include B cells, NK cells, T cells that are found infiltrating
tumor or cancer cells, also called tumor infiltrating
lymphocytes.
[0116] The methods of inhibiting the growth of CD27 expressing
cells that are described herein can be used to treat and prevent a
wide variety of diseases and disorders. For example, in one
embodiment, the methods can be used to treat or prevent a cancer
(e.g., a cancer selected from the group consisting of leukemia,
acute lymphocytic leukemia, acute myelocytic leukemia, myeloblasts
promyelocyte myelomonocytic monocytic erythroleukemia, chronic
leukemia, chronic myelocytic (granulocytic) leukemia, chronic
lymphocytic leukemia, mantle cell lymphoma, primary central nervous
system lymphoma, Burkitt's lymphoma, marginal zone B cell lymphoma,
Polycythemia vera Lymphoma, Hodgkin's disease, non-Hodgkin's
disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy
chain disease, solid tumors, sarcomas, and carcinomas,
fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma, osteogenic
sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon sarcoma, colorectal carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's
tumor, cervical cancer, uterine cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, non small cell lung
carcinoma, bladder carcinoma, epithelial carcinoma, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal
carcinoma, esophageal carcinoma, basal cell carcinoma, biliary
tract cancer, bladder cancer, bone cancer, brain and central
nervous system (CNS) cancer, cervical cancer, choriocarcinoma,
colorectal cancers, connective tissue cancer, cancer of the
digestive system, endometrial cancer, esophageal cancer, eye
cancer, head and neck cancer, gastric cancer, intraepithelial
neoplasm, kidney cancer, larynx cancer, liver cancer, lung cancer
(small cell, large cell), melanoma, neuroblastoma; oral cavity
cancer(for example lip, tongue, mouth and pharynx), ovarian cancer,
pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer;
cancer of the respiratory system, sarcoma, skin cancer, stomach
cancer, testicular cancer, thyroid cancer, uterine cancer, and
cancer of the urinary system). Preferred cancers include
CD27-expressing tumors selected from the group consisting of
chronic lymphocytic leukemia, mantle cell lymphoma, primary central
nervous system lymphoma, Burkitt's lymphoma and marginal zone B
cell lymphoma. In another embodiment, the methods can be used to
treat or prevent a bacterial, fungal, viral or parasitic
infection.
[0117] The present invention further provides methods for
inhibiting the binding of CD70 to CD27 on cells in a subject having
a disorder by administering to the subject antibodies or
compositions as described herein, as well as methods for
down-regulating a T cell response in an individual having a
disorder by administering to a subject antibodies or compositions
described herein. These methods are ideally suited for use in the
treatment of immune disorders, such as graft rejection, autoimmune
diseases, and allergy. Antibodies useful in these methods include
Fab fragments, as well as a mutated Fc region so that the antibody
does not bind, or has significantly reduced binding to, Fc
receptors. In a particular embodiment, the antibody comprises heavy
and/or light chain variable regions sequences comprising SEQ ID
NOs: 37 and/or 43 (mAb 1F5), SEQ ID NOs: 49 and/or 55 (mAb 1H8), or
SEQ ID NOs: 103 and/or 109 (mAb 3H12).
[0118] The methods described herein for inhibiting the binding of
CD70 to CD27 on cells and for down-regulating a T cell response can
be used to treat a wide variety of diseases and disorders,
including, but not limited to graft rejection, allergy and
autoimmune diseases. In a particular embodiment, the disease is an
autoimmune disease (e.g., multiple sclerosis, rheumatoid arthritis,
type 1 diabetes, psoriasis, Crohn's disease and other inflammatory
bowel diseases such as ulcerative colitis, systemic lupus
eythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis
(MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus,
Graves disease, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, scleroderma with anti-collagen
antibodies, mixed connective tissue disease, polypyositis,
pernicious anemia, idiopathic Addison's disease, autoimmune
associated infertility, glomerulonephritis, crescentic
glomerulonephritis, proliferative glomerulonephritis, bullous
pemphigoid, Sjogren's syndrome, psoriatic arthritis, insulin
resistance, autoimmune diabetes mellitus, autoimmune hepatitis,
autoimmune hemophilia, autoimmune lymphoproliferative syndrome
(ALPS), autoimmune hepatitis, autoimmune hemophilia, autoimmune
lymphoproliferative syndrome, autoimmune uveoretinitis,
Guillain-Bare syndrome, arteriosclerosis and Alzheimer's
disease).
[0119] The present invention further provides for particular uses
for the antibodies, compositions and bispecific molecules described
herein. For example, in one embodiment, the invention provides for
the use of an antibody, composition or bispecific molecule in the
manufacture of a medicament for inducing or enhancing an immune
response against an antigen in a subject. In further embodiments,
the invention provides for the use of an antibody or composition in
the manufacture of a medicament for inhibiting growth of CD27
expressing cells, the use of an antibody or composition in the
manufacture of a medicament for inhibiting the binding of CD70 to
CD27 on cells in a subject having a disorder, and the use of an
antibody or composition in the manufacture of a medicament for
down-regulating a T cell response in an individual having a
disorder. The present invention further includes an antibody,
composition or bispecific molecule for use in inducing or enhancing
an immune response against an antigen in a subject, an antibody or
composition for use in inhibiting growth of CD27 expressing cells,
an antibody or composition for use in inhibiting the binding of
CD70 to CD27 on cells in a subject having a disorder, and an
antibody or composition for use in down-regulating a T cell
response in an individual having a disorder.
[0120] The present invention also provides methods for detecting
the presence or absence of CD27 in a biological sample by (1)
contacting a biological sample with an antibody described herein
(wherein the antibody is labeled with a detectable substance) and
(2) detecting the antibody bound to CD27.
[0121] Also within the scope of the invention are kits comprising
the compositions (e.g., antibodies and/or bispecific molecules) of
the invention and, optionally, instructions for use. The kit can
further contain a least one additional reagent, such as a cytokine
or complement, or one or more additional antibodies of the
invention.
[0122] Other features and advantages of the instant invention will
be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0123] FIG. 1 depicts the affinity and kinetic parameters for mAbs
1G5, 1H8, 3H12, 3H8, 2G9, 1F5, 3A10, 2C2, ms 1A4, ms 9F4 and ms
M-T271 as determined by Biacore.TM. BiaEvaluation software (Biacore
AB) with recombinant human CD27 immobilized on the chip.
[0124] FIG. 2 is a graph showing the binding of human anti-CD27
antibodies (2C2, 3H8, 1F5, 1G5, 1H8, 2G9, 3A10 and 3H12) to
recombinant purified human CD27 using ELISA.
[0125] FIG. 3 is a graph showing binding by ELISA of 1F5 to
purified recombinant human or monkey (macaque) CD27.
[0126] FIG. 4 is a graph depicting the effect of human anti-CD27
antibodies (2C2, 3H8, 1F5, 1G5, 1H8, 2G9, 3A10 and 3H12) and
MsIgG's (1A4, 9F4, and M-T271) on the binding of soluble CD70
(sCD70) to CD27 protein (shown as % blocking) by ELISA.
[0127] FIG. 5 is a flow cytometric analysis of 1F5 binding to human
lymphoblastoid cell lines, and blocking of sCD70 binding.
[0128] FIGS. 6A-D are graphs showing the binding of human anti-CD27
antibodies (2C2, 3H8, and 1F5) to CD27 on Jurkat cells (FIG. 4A),
Raji cells (FIG. 4B), Ramos cells (FIG. 4C), and Daudi cells (FIG.
4D) as assessed by flow cytometry.
[0129] FIG. 7 is a graph showing the binding of human anti-CD27
antibodies (2C2, 3H8, 1F5, 1G5, 1H8, 2G9, 3A10 and 3H12) to CD27 on
Daudi cells as assessed by flow cytometry.
[0130] FIG. 8 is a bar graph showing the results of an anti-CD27
cross-blocking ELISA experiment, demonstrating that antibodies 1F5,
1H8 and 3H12 are capable of cross-blocking each other and thus bind
to the same epitope.
[0131] FIG. 9 is a bar graph showing the results of an anti-CD27
cross-blocking ELISA experiment, demonstrating that antibodies 2C2,
3H8, 1G5 and 2G9 are capable of cross-blocking each other and thus
bind to the same epitope.
[0132] FIG. 10 is a bar graph showing the results of an anti-CD27
cross-blocking ELISA experiment, demonstrating that the binding of
antibody 3A10 to CD27 is not fully cross-blocked by any of the
other anti-CD27 antibodies tested, thus indicating that 3A10 binds
a unique epitope, but 3A10 binding is partially cross-blocked by
antibodies 1F5, 1H8 and 3H12, indicating that the epitope for 3A10
may be close to the epitope bound by antibodies 1F5, 1H8 and
3H12.
[0133] FIG. 11 is a graph depicting the results of a complement
dependent cellular cytotoxicity (CDCC) assay using mAbs 1F5, 2C2,
3H8, 1G5, 1H8, 2G9, 3A10 and 3H12.
[0134] FIG. 12 is a graph depicting the results of a further
complement dependent cellular cytotoxicity (CDCC) assay using mAb
1F5
[0135] FIG. 13 is a graph depicting the results of an antibody
dependent cell-mediated cytotoxicity (ADCC) assay using mAbs 2C2,
1F5, 3H8, 1G5, 1H8, 2G9, 3A10, 3H12, Rituxan and HuIgG.
[0136] FIG. 14 is a graph depicting the results of a further
antibody dependent cell-mediated cytotoxicity (ADCC) using mAb
1F5
[0137] FIG. 15 is an alignment of the VH sequences of human
anti-CD27 antibodies (1F5, 1G5, 1H8, 2C2, 2G9, 3A10, 3H12 and
3H8).
[0138] FIG. 16 is an alignment of the VL sequences of human
anti-CD27 antibodies (1F5, 1G5, 1H8, 2C2, 2G9, 3A10, 3H12 and
3H8).
[0139] FIGS. 17 and 18 show results from an in vivo non-human
primate study using mAb 1F5. In particular, FIG. 17 shows 1F5 on
circulating lymphocytes after a single dose. FIG. 18 shows 1F5 does
not significantly deplete circulating lymphocytes.
[0140] FIG. 19 depicts the results of a pentamer staining assay on
mouse peripheral blood cells and splenocytes.
[0141] FIG. 20 depicts the results of an ELISPOT assay and enhanced
IFN.gamma. production using anti-CD27 antibodies.
[0142] FIGS. 21 shows enhancement by anti-CD27 mAbs of T cell
responses to a vaccine antigen in a transgenic mouse model by
pentamer staining and IFN ELISPOT.
[0143] FIGS. 22A-C are the protocol for and results of an
experiment showing that anti-CD27 enhances T cell responses to an
APC-targeted vaccine (.alpha.-DEC205-OVA). FIG. 22A shows the
protocol for the experiment. FIG. 22B shows the results of a
tetramer staining experiment to measure antigen-specific T cells.
FIG. 22C shows the results of an IFN-gamma ELISPOT assay to measure
antigen-specific T cells.
[0144] FIGS. 23A-D are the results of an experiment showing that
anti-CD27 in combination with the TLR3 agonist PolyIC (at 25 .mu.g,
50 .mu.g or 100 .mu.g) enhances T cell responses to an APC-targeted
vaccine (a-DEC205-OVA). FIG. 23A is a graph showing the % of
IFN-gamma positive cells among CD8+ T cells for either wild-type
mice treated with poly IC and the anti-CD27 mAb 1F5,
huCD27-transgenic mice treated with poly IC and a control human
IgG1 antibody or huCD27-transgenic mice treated with poly IC and
the anti-CD27 mAb 1F5.
[0145] FIGS. 24 and 25 show results from a study of administration
of ant-CD27 mAb prior to vaccine in the presence or absence of T:LR
agnost and show the significance of timing of the aministration of
the antibody relative to the vaccine.
[0146] FIGS. 26 and 27 show results from administration of
anti-CD27 mAb in combination with TCR activation ion T-cells from
human CD27 transgenic mice, as shown by both proliferation and
cytokine production.
[0147] FIGS. 28A-D are the protocol for and results of an
experiment showing that anti-CD27 enhances the efficacy of an
a-DEC205-OVA vaccine in a MO4 (B16-OVA) melanoma challenge model.
FIG. 24A shows the protocol for the experiment. FIG. 24B is a graph
plotting the tumor size (in mm.sup.2) against number of days post
tumor inoculation in untreated mice. FIG. 24C is a graph plotting
the tumor size (in mm.sup.2) against number of days post tumor
inoculation in mice treated with the vaccine alone. FIG. 24D is a
graph plotting the tumor size (in mm.sup.2) against number of days
post tumor inoculation in mice treated with the vaccine in
combination with an anti-CD27 antibody.
[0148] FIGS. 29A and B are graphs showing prolonged survival of
human CD27-transgenic mice (tumor models) following challenge with
a syngeneic lymphoma and administration of various doses
ofanti-CD27 mAb 1F5.
[0149] FIGS. 30A to 32 show the results of an experiment testing
the effect of anti-CD27 treatment in a Raji xenograft model in SCID
mice.
[0150] FIG. 30A plots the tumor size (in mm.sup.3) against number
of days post tumor inoculation in mice either untreated, treated
with a control human IgG1 antibody or treated with the anti-CD27
1F5and 3H8 antibodies. Arrows indicate days when antibody treatment
was given by i.p. injection.
[0151] FIG. 30B shows survival in a Kaplan-Meier plot.
[0152] FIG. 31A plots the tumor size (in mm.sup.3) against number
of days post tumor inoculation in mice either untreated, treated
with a control human IgG1 antibody or treated with the anti-CD27
1F5 antibody. Arrows indicate days when antibody treatment was
given by i.p. injection. FIG. 31B shows survival in a Kaplan-Meier
plot.
[0153] FIG. 32 shows the results of a further experiment testing
the effect of anti-CD27 treatment in a Raji xenograft model in SCID
mice in a Kaplan-Meier plot.
[0154] FIGS. 33A and 33B show the results of an experiment testing
the effect of anti-CD27 treatment in a Daudi xenograft model in
SCID mice. FIG. 33A plots the tumor size (in mm.sup.3) against
number of days post tumor inoculation in mice either treated with a
control human IgG1 antibody or treated with the anti-CD27 1F5
antibody (0.1 mg or 0.3 mg). Arrows indicate days when antibody
treatment was given by i.p. injection. FIG. 33B shows survival in a
Kaplan-Meier plot.
[0155] FIG. 34 shows the results of an ELISPOT assay and that
enhanced antigen-specific IFNg production using anti-CD27 antibody
is abrogated when the Fc portion of the IgG is unable to engage Fc
receptors.
DETAILED DESCRIPTION OF THE INVENTION
[0156] The present invention provides anti-CD27 antibodies that
exhibit particular functional properties correlating with
significant therapeutic benefits, including upregulation of immune
function (e.g. T cell mediated immune responses as in vaccine
therapies, NK activation in cancer therapies), inhibition of cell
growth (e.g., in cancer therapy) and down-regulation of T cell
mediated immune responses (e.g., in autoimmune therapy). These
functional features include, for example: (1) inhibition of (e.g.,
completely or partially blocks) binding of soluble CD70 to CD27
expressing cells by at least about 70%, further for example by at
least 80% or at least 90% (2) binding to human CD27 with a K.sub.D
of 1.times.10.sup.-9 M or less, (3) induction of at least about 40%
complement mediated cytotoxicity (CDC) of CD27 expressing cells at
a concentration of 10 .mu.g/ml, (4) induction of at least about 40%
specific lysis of CD27 expressing cells by ADCC at a concentration
of 10 .mu.g/ml, (further for example at least about 50%, at least
about 60% or at least about 70% specific lysis)(5) induction or
enhancement of immune responses, especially TH1 responses and/or
(6) induction or enhancement of T-cell activity, especially
specific CD8+ T-cell numbers and/or activity. In other embodiments,
the antibodies include particular heavy and light chain variable
regions and/or CDR sequences.
[0157] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0158] The term "CD27" (also referred to as "CD27 molecule", "CD27L
receptor", "S1521", "T cell activation antigen CD27", "TNFRSF7,"
"MGC20393," "tumor necrosis factor receptor superfamily, member 7",
"T cell activation antigen S152" "Tp55", "Tumor necrosis factor
receptor superfamily member 7", "CD27 antigen", and "T-cell
activation antigen CD27") refers to a receptor that is a member of
the TNF-receptor superfamily, which binds to ligand CD70. CD27 is
required for generation and long-term maintenance of T cell
immunity and plays a key role in regulating B-cell activation and
immunoglobulin synthesis. The term "CD27" includes any variants or
isoforms of CD27 which are naturally expressed by cells (e.g.,
human CD27 deposited with GENBANK.RTM. having accession no.
AAH12160.1). Accordingly, antibodies of the invention may
cross-react with CD27 from species other than human. Alternatively,
the antibodies may be specific for human CD27 and may not exhibit
any cross-reactivity with other species. CD27 or any variants and
isoforms thereof, may either be isolated from cells or tissues
which naturally express them or be recombinantly produced using
well-known techniques in the art and/or those described herein.
Preferably the antibodies are targeted to hCD27 which has a normal
glycosylation pattern.
[0159] Genbank.RTM. (Accession No. AAH12160.1) reports the amino
acid sequence of human CD27 as follows (SEQ ID NO:1):
TABLE-US-00003 1 marphpwwlc vlgtlvglsa tpapkscper hywaqgklcc
qmcepgtflv kdcdqhrkaa 61 qcdpcipgvs fspdhhtrph cescrhcnsg
llvrnctita naecacrngw qcrdkectec 121 dplpnpslta rssqalsphp
qpthlpyvse mleartaghm qtladfrqlp artlsthwpp 181 qrslcssdfi
rilvifsgmf lvftlagalf lhqrrkyrsn kgespvepae pcryscpree 241
egstipiqed yrkpepacsp
[0160] The term "CD70" (also referred to as "CD70 molecule",
"CD27L", "CD27LG", "TNFSF7," "tumor necrosis factor (ligand)
superfamily member 7," "CD27 ligand," "CD70 antigen," "surface
antigen CD70," "tumor necrosis factor ligand superfamily, member
7," "Ki-24 antigen," and "CD27-L") refers to the ligand for CD27
(see, for example, Bowman M R et al., J. Immunol. 1994 Feb. 15;
152(4):1756-61). CD70 is a type II transmembrane protein that
belongs to the tumor necrosis factor (TNF) ligand family. It is a
surface antigen on activated T and B lymphocytes that induces
proliferation of co-stimulated T cells, enhances the generation of
cytolytic T cells, and contributes to T cell activation. It has
also been suggested that CD70 plays a role in regulating B-cell
activation, cytotoxic function of natural killer cells, and
immunoglobulin synthesis (Hintzen R Q et al., J. Immunol. 1994 Feb.
15; 152(4):1762-73).
[0161] Genbank.RTM. (Accession No. NP_001243) reports the amino
acid sequence of human CD70 as follows (SEQ ID NO: 2):
TABLE-US-00004 1 mpeegsgcsv rrrpygcvlr aalvplvagl viclvvciqr
faqaqqqlpl eslgwdvael 61 qlnhtgpqqd prlywqggpa lgrsflhgpe
ldkgqlrihr dgiymvhiqv tlaicsstta 121 srhhpttlav gicspasrsi
sllrlsfhqg ctiasqrltp largdtlctn ltgtllpsrn 181 tdetffgvqw vrp
[0162] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chain thereof. An "antibody" refers, in one
preferred embodiment, to a glycoprotein comprising at least two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds, or an antigen binding portion thereof. Each heavy
chain is comprised of a heavy chain variable region (abbreviated
herein as V.sub.H) and a heavy chain constant region. The heavy
chain constant region is comprised of three domains, CH1, CH2 and
CH3. Each light chain is comprised of a light chain variable region
(abbreviated herein as V.sub.L) and a light chain constant region.
The light chain constant region is comprised of one domain, CL. The
V.sub.H and V.sub.L regions can be further subdivided into regions
of hypervariability, termed complementarity determining regions
(CDR), interspersed with regions that are more conserved, termed
framework regions (FR). Each V.sub.H and V.sub.L is composed of
three CDRs and four FRs, arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. The
constant regions of the antibodies may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (e.g., effector cells) and the first component
(Clq) of the classical complement system.
[0163] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., human CD27). Such "fragments" are, for
example between about 8 and about 1500 amino acids in length,
suitably between about 8 and about 745 amino acids in length,
suitably about 8 to about 300, for example about 8 to about 200
amino acids, or about 10 to about 50 or 100 amino acids in length.
It has been shown that the antigen-binding function of an antibody
can be performed by fragments of a full-length antibody. Examples
of binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, CL and CH1 domains;
(ii) a F(ab').sub.2 fragment, a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge region;
(iii) a Fd fragment consisting of the V.sub.H and CH1 domains; (iv)
a Fv fragment consisting of the V.sub.L and V.sub.H domains of a
single arm of an antibody, (v) a dAb fragment (Ward et al., (1989)
Nature 341:544-546), which consists of a V.sub.H domain; and (vi)
an isolated complementarity determining region (CDR) or (vii) a
combination of two or more isolated CDRs which may optionally be
joined by a synthetic linker. Furthermore, although the two domains
of the Fv fragment, V.sub.L and V.sub.H, are coded for by separate
genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the V.sub.L and V.sub.H regions pair to form
monovalent molecules (known as single chain Fv (scFv); see e.g.,
Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain
antibodies are also intended to be encompassed within the term
"antigen-binding portion" of an antibody. These antibody fragments
are obtained using conventional techniques known to those with
skill in the art, and the fragments are screened for utility in the
same manner as are intact antibodies. Antigen-binding portions can
be produced by recombinant DNA techniques, or by enzymatic or
chemical cleavage of intact immunoglobulins.
[0164] A "bispecific" or "bifunctional antibody" is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin.
Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148,
1547-1553 (1992).
[0165] The term "monoclonal antibody," as used herein, refers to an
antibody which displays a single binding specificity and affinity
for a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to an antibody which displays a single binding
specificity and which has variable and optional constant regions
derived from human germline immunoglobulin sequences. In one
embodiment, human monoclonal antibodies are produced by a hybridoma
which includes a B cell obtained from a transgenic non-human
animal, e.g., a transgenic mouse, having a genome comprising a
human heavy chain transgene and a light chain transgene fused to an
immortalized cell.
[0166] The term "recombinant human antibody," as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as (a) antibodies isolated
from an animal (e.g., a mouse) that is transgenic or
transchromosomal for human immunoglobulin genes or a hybridoma
prepared therefrom, (b) antibodies isolated from a host cell
transformed to express the antibody, e.g., from a transfectoma, (c)
antibodies isolated from a recombinant, combinatorial human
antibody library, and (d) antibodies prepared, expressed, created
or isolated by any other means that involve splicing of human
immunoglobulin gene sequences to other DNA sequences. Such
recombinant human antibodies comprise variable and constant regions
that utilize particular human germline immunoglobulin sequences are
encoded by the germline genes, but include subsequent
rearrangements and mutations which occur, for example, during
antibody maturation. As known in the art (see, e.g., Lonberg (2005)
Nature Biotech. 23(9):1117-1125), the variable region contains the
antigen binding domain, which is encoded by various genes that
rearrange to form an antibody specific for a foreign antigen. In
addition to rearrangement, the variable region can be further
modified by multiple single amino acid changes (referred to as
somatic mutation or hypermutation) to increase the affinity of the
antibody to the foreign antigen. The constant region will change in
further response to an antigen (i.e., isotype switch). Therefore,
the rearranged and somatically mutated nucleic acid molecules that
encode the light chain and heavy chain immunoglobulin polypeptides
in response to an antigen may not have sequence identity with the
original nucleic acid molecules, but instead will be substantially
identical or similar (i.e., have at least 80% identity).
[0167] The term "human antibody" includes antibodies having
variable and constant regions (if present) of human germline
immunoglobulin sequences. Human antibodies of the invention can
include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo)
(see, Lonberg, N. et al. (1994) Nature 368(6474): 856-859);
Lonberg, N. (1994) Handbook of Experimental Pharmacology
113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol.
Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y.
Acad. Sci 764:536-546). However, the term "human antibody" does not
include antibodies in which CDR sequences derived from the germline
of another mammalian species, such as a mouse, have been grafted
onto human framework sequences (i.e., humanized antibodies).
[0168] As used herein, a "heterologous antibody" is defined in
relation to the transgenic non-human organism producing such an
antibody. This term refers to an antibody having an amino acid
sequence or an encoding nucleic acid sequence corresponding to that
found in an organism not consisting of the transgenic non-human
animal, and generally from a species other than that of the
transgenic non-human animal.
[0169] An "isolated antibody," as used herein, is intended to refer
to an antibody which is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds to human CD27 is substantially
free of antibodies that specifically bind antigens other than human
CD27). An isolated antibody that specifically binds to an epitope
of may, however, have cross-reactivity to other CD27 proteins from
different species. However, the antibody preferably always binds to
human CD27. In addition, an isolated antibody is typically
substantially free of other cellular material and/or chemicals. In
one embodiment of the invention, a combination of "isolated"
antibodies having different CD27 specificities is combined in a
well defined composition.
[0170] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which an immunoglobulin or antibody
specifically binds. Epitopes can be formed both from contiguous
amino acids or noncontiguous amino acids juxtaposed by tertiary
folding of a protein. Epitopes formed from contiguous amino acids
are typically retained on exposure to denaturing solvents, whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique
spatial conformation. Methods for determining what epitopes are
bound by a given antibody (i.e., epitope mapping) are well known in
the art and include, for example, immunoblotting and
immunoprecipitation assays, wherein overlapping or contiguous
peptides from CD27 are tested for reactivity with the given
anti-CD27 antibody. Methods of determining spatial conformation of
epitopes include techniques in the art and those described herein,
for example, x-ray crystallography and 2-dimensional nuclear
magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods
in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).
[0171] Also, encompassed by the present invention are antibodies
that bind to an epitope on CD27 which comprises all or a portion of
an epitope recognized by the particular antibodies described herein
(e.g., the same or an overlapping region or a region between or
spanning the region).
[0172] Also encompassed by the present invention are antibodies
that bind the same epitope and/or antibodies that compete for
binding to human CD27 with the antibodies described herein.
Antibodies that recognize the same epitope or compete for binding
can be identified using routine techniques. Such techniques
include, for example, an immunoassay, which shows the ability of
one antibody to block the binding of another antibody to a target
antigen, i.e., a competitive binding assay. Competitive binding is
determined in an assay in which the immunoglobulin under test
inhibits specific binding of a reference antibody to a common
antigen, such as CD27. Numerous types of competitive binding assays
are known, for example: solid phase direct or indirect
radioimmunoassay (RIA), solid phase direct or indirect enzyme
immunoassay (EIA), sandwich competition assay (see Stahli et al.,
Methods in Enzymology 9:242 (1983)); solid phase direct
biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614
(1986)); solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase
direct label RIA using I-125 label (see Morel et al., Mol. Immunol.
25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et
al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer
et al., Scand. J. Immunol. 32:77 (1990)). Typically, such an assay
involves the use of purified antigen bound to a solid surface or
cells bearing either of these, an unlabeled test immunoglobulin and
a labeled reference immunoglobulin. Competitive inhibition is
measured by determining the amount of label bound to the solid
surface or cells in the presence of the test immunoglobulin.
Usually the test immunoglobulin is present in excess. Usually, when
a competing antibody is present in excess, it will inhibit specific
binding of a reference antibody to a common antigen by at least
50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.
[0173] Other techniques include, for example, epitope mapping
methods, such as, x-ray analyses of crystals of antigen:antibody
complexes which provides atomic resolution of the epitope. Other
methods monitor the binding of the antibody to antigen fragments or
mutated variations of the antigen where loss of binding due to a
modification of an amino acid residue within the antigen sequence
is often considered an indication of an epitope component. In
addition, computational combinatorial methods for epitope mapping
can also be used. These methods rely on the ability of the antibody
of interest to affinity isolate specific short peptides from
combinatorial phage display peptide libraries. The peptides are
then regarded as leads for the definition of the epitope
corresponding to the antibody used to screen the peptide library.
For epitope mapping, computational algorithms have also been
developed which have been shown to map conformational discontinuous
epitopes.
[0174] As used herein, the terms "specific binding," "selective
binding," "selectively binds," and "specifically binds," refer to
antibody binding to an epitope on a predetermined antigen.
Typically, the antibody binds with an equilibrium dissociation
constant (K.sub.D) of approximately less than 10.sup.-7 M, such as
approximately less than 10 .sup.-8 M, 10.sup.-9 M or 10.sup.-10 M
or even lower when determined by surface plasmon resonance (SPR)
technology in a BIACORE 2000 instrument using recombinant human
CD27 as the analyte and the antibody as the ligand and binds to the
predetermined antigen with an affinity that is at least two-fold
greater than its affinity for binding to a non-specific antigen
(e.g., BSA, casein) other than the predetermined antigen or a
closely-related antigen. The phrases "an antibody recognizing an
antigen" and "an antibody specific for an antigen" are used
interchangeably herein with the term "an antibody which binds
specifically to an antigen."
[0175] The term "K.sub.D," as used herein, is intended to refer to
the dissociation equilibrium constant of a particular
antibody-antigen interaction. Typically, the human antibodies of
the invention bind to CD27 with a dissociation equilibrium constant
(K.sub.D) of approximately 10.sup.-8 M or less, such as less than
10.sup.-9 M or 10.sup.-10 M or even lower when determined by
surface plasmon resonance (SPR) technology in a BIACORE 2000
instrument using recombinant human CD27 as the analyte and the
antibody as the ligand.
[0176] The term "kd" as used herein, is intended to refer to the
off rate constant for the dissociation of an antibody from the
antibody/antigen complex.
[0177] The term "ka" as used herein, is intended to refer to the on
rate constant for the association of an antibody with the
antigen.
[0178] The term "EC50," as used herein, refers to the concentration
of an antibody or an antigen-binding portion thereof, which induces
a response, either in an in vitro or an in vivo assay, which is 50%
of the maximal response, i.e., halfway between the maximal response
and the baseline.
[0179] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgG1) that is encoded by heavy chain constant region
genes. In one embodiment, a human monoclonal antibody of the
invention is of the IgG1 isotype. In another embodiment, a human
monoclonal antibody of the invention is of the IgG2 isotype.
[0180] The term "binds to immobilized CD27," refers to the ability
of a human antibody of the invention to bind to CD27, for example,
expressed on the surface of a cell or which is attached to a solid
support.
[0181] The term "cross-reacts," as used herein, refers to the
ability of an antibody of the invention to bind to CD27 from a
different species. For example, an antibody of the present
invention which binds human CD27 may also bind another species of
CD27. As used herein, cross-reactivity is measured by detecting a
specific reactivity with purified antigen in binding assays (e.g.,
SPR, ELISA) or binding to, or otherwise functionally interacting
with, cells physiologically expressing CD27. Methods for
determining cross-reactivity include standard binding assays as
described herein, for example, by Biacore.TM. surface plasmon
resonance (SPR) analysis using a Biacore.TM. 2000 SPR instrument
(Biacore AB, Uppsala, Sweden), or flow cytometric techniques.
[0182] As used herein, "isotype switching" refers to the phenomenon
by which the class, or isotype, of an antibody changes from one Ig
class to one of the other Ig classes.
[0183] As used herein, "nonswitched isotype" refers to the isotypic
class of heavy chain that is produced when no isotype switching has
taken place; the CH gene encoding the nonswitched isotype is
typically the first CH gene immediately downstream from the
functionally rearranged VDJ gene. Isotype switching has been
classified as classical or non-classical isotype switching.
Classical isotype switching occurs by recombination events which
involve at least one switch sequence region in the transgene.
Non-classical isotype switching may occur by, for example,
homologous recombination between human .sigma..sub..mu. and human
.SIGMA..sub..mu. (.delta.-associated deletion). Alternative
non-classical switching mechanisms, such as intertransgene and/or
interchromosomal recombination, among others, may occur and
effectuate isotype switching.
[0184] As used herein, the term "switch sequence" refers to those
DNA sequences responsible for switch recombination. A "switch
donor" sequence, typically a .mu. switch region, will be 5' (i.e.,
upstream) of the construct region to be deleted during the switch
recombination. The "switch acceptor" region will be between the
construct region to be deleted and the replacement constant region
(e.g., .gamma., .epsilon., etc.). As there is no specific site
where recombination always occurs, the final gene sequence will
typically not be predictable from the construct.
[0185] As used herein, "glycosylation pattern" is defined as the
pattern of carbohydrate units that are covalently attached to a
protein, more specifically to an immunoglobulin protein. A
glycosylation pattern of a heterologous antibody can be
characterized as being substantially similar to glycosylation
patterns which occur naturally on antibodies produced by the
species of the nonhuman transgenic animal, when one of ordinary
skill in the art would recognize the glycosylation pattern of the
heterologous antibody as being more similar to said pattern of
glycosylation in the species of the nonhuman transgenic animal than
to the species from which the CH genes of the transgene were
derived.
[0186] The term "naturally-occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally-occurring.
[0187] The term "rearranged" as used herein refers to a
configuration of a heavy chain or light chain immunoglobulin locus
wherein a V segment is positioned immediately adjacent to a D-J or
J segment in a conformation encoding essentially a complete V.sub.H
or V.sub.L domain, respectively. A rearranged immunoglobulin gene
locus can be identified by comparison to germline DNA; a rearranged
locus will have at least one recombined heptamer/nonamer homology
element.
[0188] The term "unrearranged" or "germline configuration" as used
herein in reference to a V segment refers to the configuration
wherein the V segment is not recombined so as to be immediately
adjacent to a D or J segment.
[0189] The term "nucleic acid molecule," as used herein, is
intended to include DNA molecules and RNA molecules. A nucleic acid
molecule may be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[0190] The term "isolated nucleic acid molecule," as used herein in
reference to nucleic acids encoding antibodies or antibody portions
(e.g., V.sub.H, V.sub.L, CDR3) that bind to CD27, is intended to
refer to a nucleic acid molecule in which the nucleotide sequences
encoding the antibody or antibody portion are free of other
nucleotide sequences encoding antibodies or antibody portions that
bind antigens other than CD27, which other sequences may naturally
flank the nucleic acid in human genomic DNA. For example, SEQ ID
NOs: 35 and 41, 47 and 53, 101 and 107, 83 and 89, 83 and 95, 23
and 29, 71 and 77 correspond, respectively, to the nucleotide
sequences comprising the heavy chain (V.sub.H) and light chain
(V.sub.L) variable regions of anti-CD27 antibody monoclonal
antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5.
[0191] The present invention also encompasses "conservative
sequence modifications" of the sequences set forth in SEQ ID NOs:
5-112, i.e., nucleotide and amino acid sequence modifications which
do not abrogate the binding of the antibody encoded by the
nucleotide sequence or containing the amino acid sequence, to the
antigen. Such conservative sequence modifications include
conservative nucleotide and amino acid substitutions, as well as,
nucleotide and amino acid additions and deletions. For example,
modifications can be introduced into SEQ ID NOs: 5-112 by standard
techniques known in the art, such as site-directed mutagenesis and
PCR-mediated mutagenesis. Conservative amino acid substitutions
include ones in which the amino acid residue is replaced with an
amino acid residue having a similar side chain. Families of amino
acid residues having similar side chains have been defined in the
art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a predicted nonessential amino acid residue in a
human anti-CD27 antibody is preferably replaced with another amino
acid residue from the same side chain family. Methods of
identifying nucleotide and amino acid conservative substitutions
which do not eliminate antigen binding are well-known in the art
(see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993);
Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et
al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997))
[0192] Alternatively, in another embodiment, mutations can be
introduced randomly along all or part of an anti-CD27 antibody
coding sequence, such as by saturation mutagenesis, and the
resulting modified anti-CD27 antibodies can be screened for binding
activity.
[0193] For nucleic acids, the term "substantial homology" indicates
that two nucleic acids, or designated sequences thereof, when
optimally aligned and compared, are identical, with appropriate
nucleotide insertions or deletions, in at least about 80% of the
nucleotides, usually at least about 90% to 95%, and more preferably
at least about 98% to 99.5% of the nucleotides. Alternatively,
substantial homology exists when the segments will hybridize under
selective hybridization conditions, to the complement of the
strand.
[0194] The percent identity between two sequences is a function of
the number of identical positions shared by the sequences (i.e., %
homology=# of identical positions/total # of positions.times.100),
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences. The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm, as described in the non-limiting examples
below.
[0195] The percent identity between two nucleotide sequences can be
determined using the GAP program in the GCG software package
(available at http://www.gcg.com), using a NWSgapdna.CMP matrix and
a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,
3, 4, 5, or 6. The percent identity between two nucleotide or amino
acid sequences can also be determined using the algorithm of E.
Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. In addition, the percent identity between two amino acid
sequences can be determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6.
[0196] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against public databases to, for example, identify related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to the nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the protein molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
[0197] The nucleic acids may be present in whole cells, in a cell
lysate, or in a partially purified or substantially pure form. A
nucleic acid is "isolated" or "rendered substantially pure" when
purified away from other cellular components or other contaminants,
e.g., other cellular nucleic acids or proteins, by standard
techniques, including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known
in the art. See, F. Ausubel, et al., ed. Current Protocols in
Molecular Biology, Greene Publishing and Wiley Interscience, New
York (1987).
[0198] The nucleic acid compositions of the present invention,
while often in a native sequence (except for modified restriction
sites and the like), from either cDNA, genomic or mixtures thereof
may be mutated, in accordance with standard techniques to provide
gene sequences. For coding sequences, these mutations, may affect
amino acid sequence as desired. In particular, DNA sequences
substantially homologous to or derived from native V, D, J,
constant, switches and other such sequences described herein are
contemplated (where "derived" indicates that a sequence is
identical or modified from another sequence).
[0199] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence. With
respect to transcription regulatory sequences, operably linked
means that the DNA sequences being linked are contiguous and, where
necessary to join two protein coding regions, contiguous and in
reading frame. For switch sequences, operably linked indicates that
the sequences are capable of effecting switch recombination.
[0200] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid,"
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors"(or simply, "expression vectors") In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0201] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which a
recombinant expression vector has been introduced. It should be
understood that such terms are intended to refer not only to the
particular subject cell but to the progeny of such a cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not,
in fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
[0202] As used herein, the term "antigen" refers to any natural or
synthetic immunogenic substance, such as a protein, peptide, or
hapten. Suitable antigens for use in the present invention (e.g.,
in a vaccine in combination with an anti-CD27 antibody of the
invention) include, for example, infectious disease antigens and
tumor antigens, against which protective or therapeutic immune
responses are desired, e.g., antigens expressed by a tumor cell or
a pathogenic organism or infectious disease antigens. For example,
suitable antigens include tumor-associated antigens for the
prevention or treatment of cancers. Examples of tumor-associated
antigens include, but are not limited to, sequences comprising all
or part of the sequences of .beta.hCG, gp100 or Pmel17, HER2/neu,
WT1, mesothelin, CEA, gp100, MART1, TRP-2, melan-A, NY-ESO-1,
NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MAGE-3, MAGE-A3,
Tyrosinase, Telomerase, SSX2 and MUC-1 antigens, and germ cell
derived tumor antigens. Tumor associated antigens also include the
blood group antigens, for example, Le.sup.a, Le.sup.b, LeX, LeY,
H-2, B-1, B-2 antigens. Alternatively, more than one antigen can be
included within the antigen-antibody constructs of the invention.
For example, a MAGE antigen can be combined with other antigens
such as melanin A, tyrosinase, and gp100 along with adjuvants such
as GM-CSF or IL-12, and linked to an anti-APC antibody.
[0203] Other suitable antigens include viral antigens for the
prevention or treatment of viral diseases. Examples of viral
antigens include, but are not limited to, HIV-1 gag, HIV-1 env,
HIV-1 nef, HBV (surface or core antigens), HPV, FAS, HSV-1, HSV-2,
p17, ORF2 and ORF3 antigens. Examples of bacterial antigens
include, but are not limited to, Toxoplasma gondii or Treponema
pallidum. The antibody-bacterial antigen conjugates of the
invention can be in the treatment or prevention of various
bacterial diseases such as Anthrax, Botulism, Tetanus, Chlamydia,
Cholera, Diphtheria, Lyme Disease, Syphilis and Tuberculosis. Other
suitable antigens from infectious disease pathogens, such as
viruses, bacteria, parasites and fungi are disclosed below.
[0204] Sequences of the foregoing antigens are well known in the
art. For example, an example of a MAGE-3 cDNA sequence is provided
in U.S. Pat. No. 6,235,525 (Ludwig Institute for Cancer Research);
examples of NY-ESO-1 nucleic acid and protein sequences are
provided in U.S. Pat. Nos. 5,804,381 and 6,069,233 (Ludwig
Institute for Cancer Research); examples of Melan-A nucleic acid
and protein sequences are provided in U.S. Pat. Nos. 5,620,886 and
5,854,203 (Ludwig Institute for Cancer Research); examples of
NY-BR-1 nucleic acid and protein sequences are provided in U.S.
Pat. Nos. 6,774,226 and 6,911,529 (Ludwig Institute for Cancer
Research) and examples of NY-CO-58 nucleic acid and protein
sequences are provided in WO 02090986 (Ludwig Institute for Cancer
Research); an example of an amino acid sequence for the HER-2/neu
protein is available at GENBANK.RTM. Accession No. AAA58637; and a
nucleotide sequence (mRNA) for human carcinoembryonic antigen-like
1 (CEA-1) is available at GENBANK.RTM. Accession No. NM_020219.
[0205] An HPV antigen that may be used in the compositions and the
methods of the invention may include, for example an HPV-16
antigen, an HPV-18 antigen, an HPV-31 antigen, an HPV-33 antigen
and/or an HPV-35 antigen; and is suitably an HPV-16 antigen and/or
HPV-18 antigen. A genome of HPV-16 is described in Virology,
145:181-185 (1985) and DNA sequences encoding HPV-18 are described
in U.S. Pat. No. 5,840,306, the disclosures of which are
incorporated by reference herein in their entirety. HPV-16 antigens
(e.g., seroreactive regions of the E1 and/or E2 proteins of HPV-16)
are described in U.S. Pat. No. 6,531,127, and HPV-18 antigens
(e.g., seroreactive regions of the L1 and/or L2 proteins of HPV-18)
are described in U.S. Pat. No. 5,840,306, the disclosures of which
are incorporated by reference herein. Similarly, a complete genome
for HBV is available at GENBANK.RTM. Accession No. NC_003977, the
disclosure of which is incorporated herein. The genome of HCV is
described in European Patent Application No. 318 216, the
disclosure of which is incorporated herein. PCT/US90/01348,
incorporated by reference herein, discloses sequence information of
clones of the HCV genome, amino acid sequences of HCV viral
proteins and methods of making and using such compositions for HCV
vaccines comprising HCV proteins and peptides derived there
from.
[0206] Antigenic peptides of proteins (i.e., those containing T
cell epitopes) can be identified in a variety of manners well known
in the art. For example, T cell epitopes can be predicted by
analyzing the sequence of the protein using web-based predictive
algorithms (BIMAS & SYFPEITHI) to generate potential MHC class
I and II-binding peptides that match an internal database of 10,000
well characterized MHC binding peptides previously defined by CTLs.
High scoring peptides can be ranked and selected as "interesting"
on the basis of high affinity to a given MHC molecule.
[0207] Another method for identifying antigenic peptides containing
T cell epitopes is by dividing the protein into non-overlapping
peptides of desired length or overlapping peptides of desired
lengths which can be produced recombinantly, synthetically, or in
certain limited situations, by chemical cleavage of the protein and
tested for immunogenic properties, e.g., eliciting a T cell
response (i.e., proliferation or lymphokine secretion).
[0208] In order to determine precise T cell epitopes of the protein
by, for example, fine mapping techniques, a peptide having T cell
stimulating activity and thus comprising at least one T cell
epitope, as determined by T cell biology techniques, can be
modified by addition or deletion of amino acid residues at either
the amino or carboxy terminus of the peptide and tested to
determine a change in T cell reactivity to the modified peptide. If
two or more peptides which share an area of overlap in the native
protein sequence are found to have human T cell stimulating
activity, as determined by T cell biology techniques, additional
peptides can be produced comprising all or a portion of such
peptides and these additional peptides can be tested by a similar
procedure. Following this technique, peptides are selected and
produced recombinantly or synthetically. Peptides are selected
based on various factors, including the strength of the T cell
response to the peptide (e.g., stimulation index). The physical and
chemical properties of these selected peptides (e.g., solubility,
stability) can then be examined to determine whether the peptides
are suitable for use in therapeutic compositions or whether the
peptides require modification.
[0209] The term "antigen presenting cell" or "APC" is a cell that
displays foreign antigen complexed with MHC on its surface. T-cells
recognize this complex using T-cell receptor (TCR). Examples of
APCs include, but are not limited to, dendritic cells (DCs),
peripheral blood mononuclear cells (PBMC), monocytes (such as
THP-1), B lymphoblastoid cells (such as C1R.A2, 1518 B-LCL) and
monocyte-derived dendritic cells (DCs). Some APCs internalize
antigens either by phagocytosis or by receptor-mediated
endocytosis. Examples of APC receptors include, but are not limited
to C-type lectins, such as, the human Dendritic and Epithelial Cell
205 receptor (CD27), and the human macrophage mannose receptor.
[0210] The term "antigen presentation" refers to the process by
which APCs capture antigens and enables their recognition by
T-cells, e.g., as a component of an MHC-I and/or MHC-II
conjugate.
[0211] "MHC molecules" include two types of molecules, MHC class I
and MHC class II. MHC class I molecules present antigen to specific
CD8+ T cells and MHC class II molecules present antigen to specific
CD4+ T cells. Antigens delivered exogenously to APCs are processed
primarily for association with MHC class II. In contrast, antigens
delivered endogenously to APCs are processed primarily for
association with MHC class I.
[0212] As used herein, the term "immunostimulatory agent" includes
but is not limited to compounds capable of stimulating APCs, such
as DCs and macrophages. For example, suitable immunostimulatory
agents for use in the present invention are capable of stimulating
APCs, so that the maturation process of the APCs is accelerated,
the proliferation of APCs is increased, and/or the recruitment or
release of co-stimulatory molecules (e.g., CD80, CD86, ICAM-1, MHC
molecules and CCR7) and pro-inflammatory cytokines (e.g.,
IL-1.beta., IL-6, IL-12, IL-15, and IFN-.gamma.) is upregulated.
Suitable immunostimulatory agents are also capable of increasing T
cell proliferation. Such immunostimulatory agents include, but are
not be limited to, CD40 ligand; FLT 3 ligand; cytokines, such as
IFN-.alpha., IFN-.beta., IFN-.gamma. and IL-2; colony-stimulating
factors, such as G-CSF (granulocyte colony-stimulating factor) and
GM-CSF (granulocyte-macrophage colony-stimulating factor); an
anti-CTLA-4 antibody, anti-PD1 antibody, anti-41BB antibody, or
anti-OX-40 antibody; LPS (endotoxin); ssRNA; dsRNA; Bacille
Calmette-Guerin (BCG); Levamisole hydrochloride; and intravenous
immune globulins. In one embodiment an immunostimulatory agant may
be a Toll-like Receptor (TLR) agonist. For example the
immunostimulatory agent may be a TLR3 agonist such as
double-stranded inosine:cytosine polynucleotide (Poly I:C, for
example available as Ampligen.TM. from Hemispherx Bipharma, PA, US
or Poly IC:LC from Oncovir) or Poly A:U; a TLR4 agonist such as
monophosphoryl lipid A (MPL) or RC-529 (for example as available
from GSK, UK); a TLRS agonist such as flagellin; a TLR7 or TLR8
agonist such as an imidazoquinoline TLR7 or TLR 8 agonist, for
example imiquimod (eg Aldara.TM.) or resiquimod and related
imidazoquinoline agents (for example as available from 3M
Corporation); or a TLR 9 agonist such as a deoxynucleotide with
unmethylated CpG motifs (so-called "CpGs", for example as available
from Coley Pharmaceutical). A preferred immunostimulatory agent is
a TLR3 agonist, preferably Poly I:C. Such immunostimulatory agents
may be administered simultaneously, separately or sequentially with
the antibodies and constructs of the present invention and may also
be physically linked to the antibodies and constructs.
[0213] As used herein, the term "linked" refers to the association
of two or more molecules. The linkage can be covalent or
non-covalent. The linkage also can be genetic (i.e., recombinantly
fused). Such linkages can be achieved using a wide variety of art
recognized techniques, such as chemical conjugation and recombinant
protein production.
[0214] As used herein, the term antigen "cross-presentation" refers
to presentation of exogenous protein antigens to T cells via MHC
class I and class II molecules on APCs.
[0215] As used herein, the term "T cell-mediated response" refers
to any response mediated by T cells, including effector T cells
(e.g., CD8.sup.+ cells) and helper T cells (e.g., CD4.sup.+ cells).
T cell mediated responses include, for example, T cell cytotoxicity
and proliferation.
[0216] As used herein, the term "cytotoxic T lymphocyte (CTL)
response" refers to an immune response induced by cytotoxic T
cells. CTL responses are mediated primarily by CD8.sup.+ T
cells.
[0217] As used herein, the terms "inhibits" or "blocks" (e.g.,
referring to inhibition/blocking of binding of CD70 to CD27 on
cells) are used interchangeably and encompass both partial and
complete inhibition/blocking. The inhibition/blocking of CD70
preferably reduces or alters the normal level or type of activity
that occurs when CD70 binding occurs without inhibition or
blocking. Inhibition and blocking are also intended to include any
measurable decrease in the binding affinity of CD70 when in contact
with an anti-CD27 antibody as compared to CD70 not in contact with
an anti-CD27 antibody, e.g., inhibits binding of CD70 by at least
about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In a
preferred embodiment, the anti-CD27 antibody inhibits binding of
CD70 by at least about 70%. In another embodiment, the anti-CD27
antibody inhibits binding of CD70 by at least 80%
[0218] As used herein, the term "inhibits growth" (e.g., referring
to cells) is intended to include any measurable decrease in the
growth of a cell, e.g., the inhibition of growth of a cell by at
least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or
100%.
[0219] The terms "inducing an immune response" and "enhancing an
immune response" are used interchangeably and refer the stimulation
of an immune response (i.e., either passive or adaptive) to a
particular antigen. The terms "induce" as used with respect to
inducing CDC or ADCC refer to the stimulation of particular direct
cell killing mechanisms. For example, in one embodiment, the
antibody induces at least about 20, 25, 30, 35, 40, 45, 50, 55, or
60% lysis via CDC of CD27 expressing cells at a concentration of 10
.mu.g/ml. In a preferred embodiment, the antibody induces at least
about 40% lysis via CDC of CD27 expressing cells at a concentration
of 10 .mu.g/ml. In another embodiment, the antibody induces at
least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or
85% lysis via ADCC (i.e., specific lysis) of CD27 expressing cells
at a concentration of 10 .mu.g/ml. In a preferred embodiment, the
antibody induces at least about 40% lysis via ADCC of CD27
expressing cells at a concentration of 10 .mu.g/ml.
[0220] The terms "treat," "treating," and "treatment," as used
herein, refer to therapeutic or preventative measures described
herein. The methods of "treatment" employ administration to a
subject, in need of such treatment, a human antibody of the present
invention, for example, a subject in need of an enhanced immune
response against a particular antigen or a subject who ultimately
may acquire such a disorder, in order to prevent, cure, delay,
reduce the severity of, or ameliorate one or more symptoms of the
disorder or recurring disorder, or in order to prolong the survival
of a subject beyond that expected in the absence of such
treatment.
[0221] The term "effective dose" or "effective dosage" is defined
as an amount sufficient to achieve or at least partially achieve
the desired effect. The term "therapeutically effective dose" is
defined as an amount sufficient to cure or at least partially
arrest the disease and its complications in a patient already
suffering from the disease. Amounts effective for this use will
depend upon the severity of the disorder being treated and the
general state of the patient's own immune system.
[0222] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0223] As used herein, the term "subject" includes any human or
non-human animal. For example, the methods and compositions of the
present invention can be used to treat a subject with an immune
disorder. The term "non-human animal" includes all vertebrates,
e.g., mammals and non-mammals, such as non-human primates, sheep,
dog, cow, chickens, amphibians, reptiles, etc.
[0224] Various aspects of the invention are described in further
detail in the following subsections.
I. Production of Antibodies to CD27
[0225] The present invention encompasses antibodies, e.g., fully
human antibodies, that bind CD27, e.g., human CD27. Exemplary
monoclonal antibodies that bind CD27 include 1F5, 1H8, 3H12, 3A10,
2C2, 2G9, 3H8, and 1G5. Monoclonal antibodies of the invention can
be produced using a variety of known techniques, such as the
standard somatic cell hybridization technique described by Kohler
and Milstein, Nature 256: 495 (1975). Although somatic cell
hybridization procedures are preferred, in principle, other
techniques for producing monoclonal antibodies also can be
employed, e.g., viral or oncogenic transformation of B lymphocytes,
phage display technique using libraries of human antibody
genes.
[0226] Accordingly, in one embodiment, a hybridoma method is used
for producing an antibody that binds human CD27. In this method, a
mouse or other appropriate host animal can be immunized with a
suitable antigen in order to elicit lymphocytes that produce or are
capable of producing antibodies that will specifically bind to the
antigen used for immunization. Alternatively, lymphocytes may be
immunized in vitro. Lymphocytes can then be fused with myeloma
cells using a suitable fusing agent, such as polyethylene glycol,
to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles
and Practice, pp.59-103 (Academic Press, 1986)). Culture medium in
which hybridoma cells are growing is assayed for production of
monoclonal antibodies directed against the antigen. After hybridoma
cells are identified that produce antibodies of the desired
specificity, affinity, and/or activity, the clones may be subcloned
by limiting dilution procedures and grown by standard methods
(Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103
(Academic Press, 1986)). Suitable culture media for this purpose
include, for example, D-MEM or RPMI-1640 medium. In addition, the
hybridoma cells may be grown in vivo as ascites tumors in an
animal. The monoclonal antibodies secreted by the subclones can be
separated from the culture medium, ascites fluid, or serum by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0227] In another embodiment, antibodies and antibody portions that
bind human CD27 can be isolated from antibody phage libraries
generated using the techniques described in, for example,
McCafferty et al., Nature, 348:552-554 (1990). Clackson et al.,
Nature, 352:624-628 (1991), Marks et al., J. Mol. Biol.,
222:581-597 (1991) and Hoet et al (2005) Nature Biotechnology 23,
344-348; U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to
Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et
al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.;
and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;
6,582,915 and 6,593,081 to Griffiths et al. Additionally,
production of high affinity (nM range) human antibodies by chain
shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as
well as combinatorial infection and in vivo recombination as a
strategy for constructing very large phage libraries (Waterhouse et
al., Nuc. Acids. Res., 21:2265-2266 (1993)) may also be used.
[0228] In a particular embodiment, the antibody that binds human
CD27 is produced using the phage display technique described by
Hoet et al., supra. This technique involves the generation of a
human Fab library having a unique combination of immunoglobulin
sequences isolated from human donors and having synthetic diversity
in the heavy-chain CDRs is generated. The library is then screened
for Fabs that bind to human CD27.
[0229] The preferred animal system for generating hybridomas which
produce antibodies of the invention is the murine system. Hybridoma
production in the mouse is well known in the art, including
immunization protocols and techniques for isolating and fusing
immunized splenocytes.
[0230] In one embodiment, antibodies directed against CD27 are
generated using transgenic or transchromosomal mice carrying parts
of the human immune system rather than the mouse system. In one
embodiment, the invention employs transgenic mice, referred to
herein as "HuMAb mice" which contain a human immunoglobulin gene
miniloci that encodes unrearranged human heavy (.mu. and .gamma.)
and .kappa. light chain immunoglobulin sequences, together with
targeted mutations that inactivate the endogenous .mu. and .kappa.
chain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856-859).
Accordingly, the mice exhibit reduced expression of mouse IgM or
.kappa., and in response to immunization, the introduced human
heavy and light chain transgenes undergo class switching and
somatic mutation to generate high affinity human IgG.kappa.
monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed
in Lonberg, N. (1994) Handbook of Experimental Pharmacology
113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol.
Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y.
Acad. Sci 764:536-546). The preparation of HuMAb mice is described
in detail in Section II below and in Taylor, L. et al. (1992)
Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993)
International Immunology 5: 647-656; Tuaillon et al. (1993) Proc.
Natl. Acad. Sci USA 90:3720-3724; Choi et al. (1993) Nature
Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830;
Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Lonberg et al.,
(1994) Nature 368(6474): 856-859; Lonberg, N. (1994) Handbook of
Experimental Pharmacology 113:49-101; Taylor, L. et al. (1994)
International Immunology 6: 579-591; Lonberg, N. and Huszar, D.
(1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F. and
Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546; Fishwild, D. et
al. (1996) Nature Biotechnology 14: 845-851. See further, U.S. Pat.
Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650;
5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to
Lonberg and Kay, and GenPharm International; U.S. Pat. No.
5,545,807 to Surani et al.; International Publication Nos. WO
98/24884, published on Jun. 11, 1998; WO 94/25585, published Nov.
10, 1994; WO 93/1227, published Jun. 24, 1993; WO 92/22645,
published Dec. 23, 1992; WO 92/03918, published Mar. 19, 1992.
Immunizations
[0231] To generate fully human antibodies to CD27, transgenic or
transchromosomal mice containing human immunoglobulin genes (e.g.,
HCo12, HCo7 or KM mice) can be immunized with a purified or
enriched preparation of the CD27 antigen and/or cells expressing
CD27, as described, for example, by Lonberg et al. (1994) Nature
368(6474): 856-859; Fishwild et al. (1996) Nature Biotechnology 14:
845-851 and WO 98/24884. As described herein, HuMAb mice are
immunized either with recombinant CD27 proteins or cell lines
expressing CD27 as immunogens. Alternatively, mice can be immunized
with DNA encoding human CD27. Preferably, the mice will be 6-16
weeks of age upon the first infusion. For example, a purified or
enriched preparation (5-50 .mu.g) of the recombinant CD27 antigen
can be used to immunize the HuMAb mice intraperitoneally. In the
event that immunizations using a purified or enriched preparation
of the CD27 antigen do not result in antibodies, mice can also be
immunized with cells expressing CD27, e.g., a cell line, to promote
immune responses. Exemplary cell lines include CD27-overexpressing
stable CHO and Raji cell lines.
[0232] Cumulative experience with various antigens has shown that
the HuMAb transgenic mice respond best when initially immunized
intraperitoneally (IP) or subcutaneously (SC) with antigen in
complete Freund's adjuvant, followed by every other week IP/SC
immunizations (up to a total of 10) with antigen in incomplete
Freund's adjuvant. The immune response can be monitored over the
course of the immunization protocol with plasma samples being
obtained by retroorbital bleeds. The plasma can be screened by
ELISA (as described below), and mice with sufficient titers of
anti-CD27 human immunoglobulin can be used for fusions. Mice can be
boosted intravenously with antigen 3 days before sacrifice and
removal of the spleen.
Generation of Hybridomas Producing Monoclonal Antibodies to
CD27
[0233] To generate hybridomas producing monoclonal antibodies to
CD27, splenocytes and lymph node cells from immunized mice can be
isolated and fused to an appropriate immortalized cell line, such
as a mouse myeloma cell line. The resulting hybridomas can then be
screened for the production of antigen-specific antibodies. For
example, single cell suspensions of splenic lymphocytes from
immunized mice can be fused to SP2/0-Ag8.653 nonsecreting mouse
myeloma cells (ATCC, CRL 1580) with 50% PEG (w/v). Cells can be
plated at approximately 1.times.10.sup.5 in flat bottom microtiter
plate, followed by a two week incubation in selective medium
containing besides usual reagents 10% fetal Clone Serum, 5-10%
origen hybridoma cloning factor (IGEN) and 1.times. HAT (Sigma).
After approximately two weeks, cells can be cultured in medium in
which the HAT is replaced with HT. Individual wells can then be
screened by ELISA for human anti-CD27 monoclonal IgM and IgG
antibodies, or for binding to the surface of cells expressing CD27,
e.g., a CHO cell line expressing CD27, by FLISA
(fluorescence-linked immunosorbent assay). Once extensive hybridoma
growth occurs, medium can be observed usually after 10-14 days. The
antibody secreting hybridomas can be replated, screened again, and
if still positive for IgG, anti-CD27 monoclonal antibodies can be
subcloned at least twice by limiting dilution. The stable subclones
can then be cultured in vitro to generate antibody in tissue
culture medium for characterization.
Generation of Transfectomas Producing Monoclonal Antibodies to
CD27
[0234] Antibodies of the invention also can be produced in a host
cell transfectoma using, for example, a combination of recombinant
DNA techniques and gene transfection methods as is well known in
the art (Morrison, S. (1985) Science 229:1202).
[0235] For example, in one embodiment, the gene(s) of interest,
e.g., human antibody genes, can be ligated into an expression
vector such as a eukaryotic expression plasmid such as used by GS
gene expression system disclosed in WO 87/04462, WO 89/01036 and EP
338 841 or other expression systems well known in the art. The
purified plasmid with the cloned antibody genes can be introduced
in eukaryotic host cells such as CHO-cells or NSO-cells or
alternatively other eukaryotic cells like a plant derived cells,
fungi or yeast cells. The method used to introduce these genes
could be methods described in the art such as electroporation,
lipofectine, lipofectamine or other. After introducing these
antibody genes in the host cells, cells expressing the antibody can
be identified and selected. These cells represent the transfectomas
which can then be amplified for their expression level and upscaled
to produce antibodies. Recombinant antibodies can be isolated and
purified from these culture supernatants and/or cells.
[0236] Alternatively these cloned antibody genes can be expressed
in other expression systems such as E. coli or in complete
organisms or can be synthetically expressed.
Use of Partial Antibody Sequences to Express Intact Antibodies
[0237] Antibodies interact with target antigens predominantly
through amino acid residues that are located in the six heavy and
light chain complementarity determining regions (CDRs). For this
reason, the amino acid sequences within CDRs are more diverse
between individual antibodies than sequences outside of CDRs.
Because CDR sequences are responsible for most antibody-antigen
interactions, it is possible to express recombinant antibodies that
mimic the properties of specific naturally occurring antibodies by
constructing expression vectors that include CDR sequences from the
specific naturally occurring antibody grafted onto framework
sequences from a different antibody with different properties (see,
e.g., Riechmann, L. et al., 1998, Nature 332:323-327; Jones, P. et
al., 1986, Nature 321:522-525; and Queen, C. et al., 1989, Proc.
Natl. Acad. See. U.S.A. 86:10029-10033). Such framework sequences
can be obtained from public DNA databases that include germline
antibody gene sequences. These germline sequences will differ from
mature antibody gene sequences because they will not include
completely assembled variable genes, which are formed by V(D)J
joining during B cell maturation. Germline gene sequences will also
differ from the sequences of a high affinity secondary repertoire
antibody at individual evenly across the variable region. For
example, somatic mutations are relatively infrequent in the
amino-terminal portion of framework region. For example, somatic
mutations are relatively infrequent in the amino terminal portion
of framework region 1 and in the carboxy-terminal portion of
framework region 4. Furthermore, many somatic mutations do not
significantly alter the binding properties of the antibody. For
this reason, it is not necessary to obtain the entire DNA sequence
of a particular antibody in order to recreate an intact recombinant
antibody having binding properties similar to those of the original
antibody (see PCT/US99/05535 filed on Mar. 12, 1999). Partial heavy
and light chain sequence spanning the CDR regions is typically
sufficient for this purpose. The partial sequence is used to
determine which germline variable and joining gene segments
contributed to the recombined antibody variable genes. The germline
sequence is then used to fill in missing portions of the variable
regions. Heavy and light chain leader sequences are cleaved during
protein maturation and do not contribute to the properties of the
final antibody. To add missing sequences, cloned cDNA sequences can
be combined with synthetic oligonucleotides by ligation or PCR
amplification. Alternatively, the entire variable region can be
synthesized as a set of short, overlapping, oligonucleotides and
combined by PCR amplification to create an entirely synthetic
variable region clone. This process has certain advantages such as
elimination or inclusion or particular restriction sites, or
optimization of particular codons.
[0238] The nucleotide sequences of heavy and light chain
transcripts from a hybridoma are used to design an overlapping set
of synthetic oligonucleotides to create synthetic V sequences with
identical amino acid coding capacities as the natural sequences.
The synthetic heavy and kappa chain sequences can differ from the
natural sequences in three ways: strings of repeated nucleotide
bases are interrupted to facilitate oligonucleotide synthesis and
PCR amplification; optimal translation initiation sites are
incorporated according to Kozak's rules (Kozak, 1991, J. Biol.
Chem. 266:19867-19870); and, HindIII sites are engineered upstream
of the translation initiation sites.
[0239] For both the heavy and light chain variable regions, the
optimized coding, and corresponding non-coding, strand sequences
are broken down into 30-50 nucleotide approximately the midpoint of
the corresponding non-coding oligonucleotide. Thus, for each chain,
the oligonucleotides can be assembled into overlapping double
stranded sets that span segments of 150-400 nucleotides. The pools
are then used as templates to produce PCR amplification products of
150-400 nucleotides. Typically, a single variable region
oligonucleotide set will be broken down into two pools which are
separately amplified to generate two overlapping PCR products.
These overlapping products are then combined by PCR amplification
to form the complete variable region. It may also be desirable to
include an overlapping fragment of the heavy or light chain
constant region (including the BbsI site of the kappa light chain,
or the AgeI site if the gamma heavy chain) in the PCR amplification
to generate fragments that can easily be cloned into the expression
vector constructs.
[0240] The reconstructed heavy and light chain variable regions are
then combined with cloned promoter, leader sequence, translation
initiation, leader sequence, constant region, 3' untranslated,
polyadenylation, and transcription termination, sequences to form
expression vector constructs. The heavy and light chain expression
constructs can be combined into a single vector, co-transfected,
serially transfected, or separately transfected into host cells
which are then fused to form a host cell expressing both
chains.
[0241] Plasmids for use in construction of expression vectors were
constructed so that PCR amplified V heavy and V kappa light chain
cDNA sequences could be used to reconstruct complete heavy and
light chain minigenes. These plasmids can be used to express
completely human IgG.sub.1.kappa. or IgG.sub.4.kappa. antibodies.
Fully human and chimeric antibodies of the present invention also
include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similar
plasmids can be constructed for expression of other heavy chain
isotypes, or for expression of antibodies comprising lambda light
chains.
[0242] Thus, in another aspect of the invention, structural
features of anti-CD27 antibodies of the invention are used to
create structurally related anti-CD27 antibodies that retain at
least one functional property of the antibodies of the invention,
such as, for example, [0243] (1) inhibits (e.g., completely or
partially blocks) binding of CD70 to CD27 expressing cells by at
least about 70% (e.g., by at least about 70%, or by at least 70%,
at an antibody concentration of 10 .mu.g/ml); [0244] (2) binds to
human CD27 with an equilibrium dissociation constant Kd of
10.sup.-9 M or less, or alternatively, an equilibrium association
constant Ka of 10.sup.+9 M.sup.-1 or greater [0245] (3) induces at
least about 30% complement mediated cytotoxicity (CDC) of CD27
expressing cells at a concentration of 10 .mu.g/ml (or induces at
least 30%, or at least about 40% or at least 40% CDC of CD27
expressing cells at a concentration of 10 .mu.g/ml); [0246] (4)
induces at least about 30% specific ADCC-mediated lysis of CD27
expressing cells at a concentration of 10 .mu.g/ml (or induces at
least 30%, or at least about 40% or at least 40% specific
ADCC-mediated lysis of CD27 expressing cells at a concentration of
10 .mu.g/ml); [0247] (5) prevents or inhibits the growth of
CD27-expressing tumor cells in a xenograft model (e.g., reduces
tumor size in severe combined immunodeficiency (SCID) mice by at
least about 50% 20-days post tumor cell inoculation in vivo at 0.5
mg ip on at least 6 days); [0248] (6) induces or enhances antigen
specific immune responses when combined with a vaccine or other
antigen; [0249] (7) induces or enhances immune responses, in
particular but not limited to TH1 immune responses; [0250] (8)
induces or enhances T-cell activity, in particular but not limited
to specific CD8+ T-cell numbers or functional activity, or T cell
proliferation or activation; and/or [0251] (9) reduces or inhibits
T cell proliferation or activation. In one embodiment, one or more
CDR regions of antibodies of the invention can be combined
recombinantly with known framework regions and CDRs to create
additional, recombinantly-engineered, anti-CD27 antibodies of the
invention. The heavy and light chain variable framework regions can
be derived from the same or different antibody sequences. The
antibody sequences can be the sequences of naturally occurring
antibodies or can be consensus sequences of several antibodies. See
Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger
et al., Protein Engineering 6:971 (1993) and Carter et al., WO
92/22653.
[0252] Accordingly, in another embodiment, the invention provides a
method for preparing an anti-CD27 antibody including: preparing an
antibody including (1) heavy chain framework regions and heavy
chain CDRs, where at least one of the heavy chain CDRs includes an
amino acid sequence selected from the amino acid sequences of CDRs
shown in SEQ ID NOs: 8, 9, 10, 26, 27, 28, 38, 39, 40, 50, 51, 52,
62, 63, 64, 74, 75, 76, 86, 87, 88, 104, 105, 106; and (2) light
chain framework regions and light chain CDRs, where at least one of
the light chain CDRs includes an amino acid sequence selected from
the amino acid sequences of CDRs shown in SEQ ID NOs: SEQ ID NOs:
14, 15, 16, 20, 21, 22, 32, 33, 34, 44, 45, 46, 56, 57, 58, 68, 69,
70, 80, 81, 82, 92, 93, 94, 98, 99, 100, 110, 111, 112; where the
antibody retains the ability to bind to CD27. The ability of the
antibody to bind CD27 can be determined using standard binding
assays, such as those set forth in the Examples (e.g., an ELISA or
a FLISA).
[0253] It is well known in the art that antibody heavy and light
chain CDR3 domains play a particularly important role in the
binding specificity/affinity of an antibody for an antigen (see,
Hall et al., J. Imunol., 149:1605-1612 (1992); Polymenis et al., J.
Immunol., 152:5318-5329 (1994); Jahn et al., Immunobiol.,
193:400-419 (1995); Klimka et al., Brit. J. Cancer, 83:252-260
(2000); Beiboer et al., J. Mol. Biol, 296:833-849 (2000); Rader et
al., Proc. Natl. Acad. Sci. USA, 95:8910-8915 (1998); Barbas et
al., J. Am. Chem. Soc., 116:2161-2162 (1994); Ditzel et al., J.
Immunol., 157:739-749 (1996)). Accordingly, the recombinant
antibodies of the invention prepared as set forth above preferably
comprise the heavy and/or light chain CDR3s of antibodies 1F5, 1H8,
3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. The antibodies further can
comprise the CDR2s of antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9,
3H8, and 1G5. The antibodies further can comprise the CDR1s of
antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. The
antibodies can further comprise any combinations of the CDRs.
[0254] Accordingly, in another embodiment, the invention further
provides anti-CD27 antibodies comprising: (1) heavy chain framework
regions, a heavy chain CDR1 region, a heavy chain CDR2 region, and
a heavy chain CDR3 region, wherein the heavy chain CDR3 region is
selected from the CDR3s of 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and
1G5 and (2) light chain framework regions, a light chain CDR1
region, a light chain CDR2 region, and a light chain CDR3 region,
wherein the light chain CDR3 region is selected from the CDR3s of
1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5, wherein the antibody
binds CD27. The antibody may further include the heavy chain CDR2
and/or the light chain CDR2 of antibodies 1F5, 1H8, 3H12, 3A10,
2C2, 2G9, 3H8, and 1G5. The antibody may further comprise the heavy
chain CDR1 and/or the light chain CDR1 of antibodies 1F5, 1H8,
3H12, 3A10, 2C2, 2G9, 3H8, and 1G5.
Generation of Antibodies Having Modified Sequences
[0255] In another embodiment, the variable region sequences, or
portions thereof, of the anti-CD27 antibodies of the invention are
modified to create structurally related anti-CD27 antibodies that
retain binding (i.e., to the same epitope as the unmodified
antibody) and, thus, are functionally equivalent. Methods for
identifying residues that can be altered without removing antigen
binding are well-known in the art (see, e.g., Marks et al.
(Biotechnology (1992) 10(7):779-83 (monoclonal antibodies
diversification by shuffling light chain variable regions, then
heavy chain variable regions with fixed CDR3 sequence changes),
Jespers et al. (1994) Biotechnology 12(9):899-903 (selection of
human antibodies from phage display repertoires to a single epitope
of an antigen), Sharon et al. (1986) PNAS USA 83(8):2628-31
(site-directed mutagenesis of an invariant amino acid residue at
the variable-diversity segments junction of an antibody); Casson et
al. (1995) J. Immunol. 155(12):5647-54 (evolution of loss and
change of specificity resulting from random mutagenesis of an
antibody heavy chain variable region).
[0256] Accordingly, in one aspect of the invention, the CDR1, 2,
and/or 3 regions of the engineered antibodies described above can
comprise the exact amino acid sequence(s) as those of antibodies
1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5 disclosed herein.
However, in other aspects of the invention, the antibodies comprise
derivatives from the exact CDR sequences of 1F5, 1H8, 3H12, 3A10,
2C2, 2G9, 3H8, and 1G5 yet still retain the ability of to bind CD27
effectively. Such sequence modifications may include one or more
amino acid additions, deletions, or substitutions, e.g.,
conservative sequence modifications as described above. Sequence
modifications may also be based on the consensus sequences
described above for the particular CDR1, CDR2, and CDR3 sequences
of antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5.
[0257] Accordingly, in another embodiment, the engineered antibody
may be composed of one or more CDRs that are, for example, 90%,
95%, 98% or 99.5% identical to one or more CDRs of antibodies 1F5,
1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. Ranges intermediate to the
above-recited values, e.g., CDRs that are 90-95%, 95-98%, or
98-100% identical identity to one or more of the above sequences
are also intended to be encompassed by the present invention.
[0258] In another embodiment, one or more residues of a CDR may be
altered to modify binding to achieve a more favored on-rate of
binding, a more favored off-rate of binding, or both, such that an
idealized binding constant is achieved. Using this strategy, an
antibody having ultra high binding affinity of, for example,
10.sup.10 M.sup.-1 or more, can be achieved. Affinity maturation
techniques, well known in the art and those described herein, can
be used to alter the CDR region(s) followed by screening of the
resultant binding molecules for the desired change in binding.
Accordingly, as CDR(s) are altered, changes in binding affinity as
well as immunogenicity can be monitored and scored such that an
antibody optimized for the best combined binding and low
immunogenicity are achieved.
[0259] In addition to or instead of modifications within the CDRs,
modifications can also be made within one or more of the framework
regions, FR1, FR2, FR3 and FR4, of the heavy and/or the light chain
variable regions of a antibody, so long as these modifications do
not eliminate the binding affinity of the antibody. For example,
one or more non-germline amino acid residues in the framework
regions of the heavy and/or the light chain variable region of a
antibody of the invention, is substituted with a germline amino
acid residue, i.e., the corresponding amino acid residue in the
human germline sequence for the heavy or the light chain variable
region, which the antibody has significant sequence identity with.
For example, an antibody chain can be aligned to a germline
antibody chain which it shares significant sequence identity with,
and the amino acid residues which do not match between antibody
framework sequence and the germline chain framework can be
substituted with corresponding residues from the germline sequence.
When an amino acid differs between a antibody variable framework
region and an equivalent human germline sequence variable framework
region, the antibody framework amino acid should usually be
substituted by the equivalent human germline sequence amino acid if
it is reasonably expected that the amino acid falls within one of
the following categories:
[0260] (1) an amino acid residue which noncovalently binds antigen
directly,
[0261] (2) an amino acid residue which is adjacent to a CDR
region,
[0262] (3) an amino acid residue which otherwise interacts with a
CDR region (e.g., is within about 3-6 .ANG. of a CDR region as
determined by computer modeling), or
[0263] (4) an amino acid reside which participates in the VL-VH
interface.
[0264] Residues which "noncovalently bind antigen directly" include
amino acids in positions in framework regions which have a good
probability of directly interacting with amino acids on the antigen
according to established chemical forces, for example, by hydrogen
bonding, Van der Waals forces, hydrophobic interactions, and the
like. Accordingly, in one embodiment, an amino acid residue in the
framework region of a antibody of the invention is substituted with
the corresponding germline amino acid residue which noncovalently
binds antigen directly.
[0265] Residues which are "adjacent to a CDR region" include amino
acid residues in positions immediately adjacent to one or more of
the CDRs in the primary sequence of the antibody, for example, in
positions immediately adjacent to a CDR as defined by Kabat, or a
CDR as defined by Chothia (see e.g., Chothia and Lesk J. Mol. Biol.
196:901 (1987)). Accordingly, in one embodiment, an amino acid
residue within the framework region of an antibody of the invention
is substituted with a corresponding germline amino acid residue
which is adjacent to a CDR region.
[0266] Residues that "otherwise interact with a CDR region" include
those that are determined by secondary structural analysis to be in
a spatial orientation sufficient to affect a CDR region. Such amino
acids will generally have a side chain atom within about 3 angstrom
units (.ANG.) of some atom in the CDRs and must contain an atom
that could interact with the CDR atoms according to established
chemical forces, such as those listed above. Accordingly, in one
embodiment, an amino acid residue within the framework region of an
antibody of the invention is substituted with the corresponding
germline amino acid residue which otherwise interacts with a CDR
region.
[0267] The amino acids at several positions in the framework are
known to be important for determining CDR confirmation (e.g.,
capable of interacting with the CDRs) in many antibodies (Chothia
and Lesk, supra, Chothia et al., supra and Tramontano et al., J.
Mol. Biol. 215:175 (1990), all of which are incorporated herein by
reference). These authors identified conserved framework residues
important for CDR conformation by analysis of the structures of
several known antibodies. The antibodies analyzed fell into a
limited number of structural or "canonical" classes based on the
conformation of the CDRs. Conserved framework residues within
members of a canonical class are referred to as "canonical"
residues. Canonical residues include residues 2, 25, 29, 30, 33,
48, 64, 71, 90, 94 and 95 of the light chain and residues 24, 26,
29, 34, 54, 55, 71 and 94 of the heavy chain. Additional residues
(e.g., CDR structure-determining residues) can be identified
according to the methodology of Martin and Thorton (1996) J. Mol.
Biol. 263:800. Notably, the amino acids at positions 2, 48, 64 and
71 of the light chain and 26-30, 71 and 94 of the heavy chain
(numbering according to Kabat) are known to be capable of
interacting with the CDRs in many antibodies. The amino acids at
positions 35 in the light chain and 93 and 103 in the heavy chain
are also likely to interact with the CDRs. Additional residues
which may effect conformation of the CDRs can be identified
according to the methodology of Foote and Winter (1992) J. Mol.
Biol. 224:487. Such residues are termed "vernier" residues and are
those residues in the framework region closely underlying (i.e.,
forming a "platform" under) the CDRs.
[0268] Residues which "participate in the VL-VH interface" or
"packing residues" include those residues at the interface between
VL and VH as defined, for example, by Novotny and Haber, Proc.
Natl. Acad. Sci. USA, 82:4592-66 (1985) or Chothia et al,
supra.
[0269] Occasionally, there is some ambiguity about whether a
particular amino acid falls within one or more of the
above-mentioned categories. In such instances, alternative variant
antibodies are produced, one of which has that particular
substitution, the other of which does not. Alternative variant
antibodies so produced can be tested in any of the assays described
herein for the desired activity, and the preferred antibody
selected.
[0270] Additional candidates for substitution within the framework
region are amino acids that are unusual or "rare" for an antibody
at that position. These amino acids can be substituted with amino
acids from the equivalent position of the human germline sequence
or from the equivalent positions of more typical antibodies. For
example, substitution may be desirable when the amino acid in a
framework region of the antibody is rare for that position and the
corresponding amino acid in the germline sequence is common for
that position in immunoglobulin sequences; or when the amino acid
in the antibody is rare for that position and the corresponding
amino acid in the germline sequence is also rare, relative to other
sequences. It is contemplated that by replacing an unusual amino
acid with an amino acid from the germline sequence that happens to
be typical for antibodies, the antibody may be made less
immunogenic.
[0271] The term "rare", as used herein, indicates an amino acid
occurring at that position in less than about 20%, preferably less
than about 10%, more preferably less than about 5%, even more
preferably less than about 3%, even more preferably less than about
2% and even more preferably less than about 1% of sequences in a
representative sample of sequences, and the term "common", as used
herein, indicates an amino acid occurring in more than about 25%
but usually more than about 50% of sequences in a representative
sample. For example, all light and heavy chain variable region
sequences are respectively grouped into "subgroups" of sequences
that are especially homologous to each other and have the same
amino acids at certain critical positions (Kabat et al., supra).
When deciding whether an amino acid in an antibody sequence is
"rare" or "common" among sequences, it will often be preferable to
consider only those sequences in the same subgroup as the antibody
sequence.
[0272] In general, the framework regions of antibodies are usually
substantially identical, and more usually, identical to the
framework regions of the human germline sequences from which they
were derived. Of course, many of the amino acids in the framework
region make little or no direct contribution to the specificity or
affinity of an antibody. Thus, many individual conservative
substitutions of framework residues can be tolerated without
appreciable change of the specificity or affinity of the resulting
immunoglobulin. Thus, in one embodiment the variable framework
region of the antibody shares at least 85% sequence identity to a
human germline variable framework region sequence or consensus of
such sequences. In another embodiment, the variable framework
region of the antibody shares at least 90%, 95%, 96%, 97%, 98% or
99% sequence identity to a human germline variable framework region
sequence or consensus of such sequences.
[0273] In addition to simply binding CD27, an antibody may be
selected for its retention of other functional properties of
antibodies of the invention, such as, for example: [0274] (1)
inhibits (e.g., completely or partially blocks) binding of CD70 to
CD27 expressing cells by at least about 70%; [0275] (2) binds to
human CD27 with an equilibrium dissociation constant Kd of
10.sup.-9 M or less, or alternatively, an equilibrium association
constant Ka of 10.sup.+9 M.sup.-1 or greater ; [0276] (3) induces
at least about 40% complement mediated cytotoxicity (CDC) of CD27
expressing cells at a concentration of 10 .mu.g/ml; and/or [0277]
(4) induces at least about 40% ADCC mediated specific lysis of CD27
expressing cells at a concentration of 10 .mu.g/ml.
Characterization of Monoclonal Antibodies to CD27
[0278] Monoclonal antibodies of the invention can be characterized
for binding to CD27 using a variety of known techniques. Generally,
the antibodies are initially characterized by ELISA. Briefly,
microtiter plates can be coated with purified CD27 in PBS, and then
blocked with irrelevant proteins such as bovine serum albumin (BSA)
diluted in PBS. Dilutions of plasma from CD27-immunized mice are
added to each well and incubated for 1-2 hours at 37.degree. C. The
plates are washed with PBS/Tween 20 and then incubated with a
goat-anti-human IgG Fc-specific polyclonal reagent conjugated to
alkaline phosphatase for 1 hour at 37.degree. C. After washing, the
plates are developed with ABTS substrate, and analyzed at OD of
405. Preferably, mice which develop the highest titers will be used
for fusions.
[0279] An ELISA assay as described above can be used to screen for
antibodies and, thus, hybridomas that produce antibodies that show
positive reactivity with the CD27 immunogen. Hybridomas that bind,
preferably with high affinity, to CD27 can then be subcloned and
further characterized. One clone from each hybridoma, which retains
the reactivity of the parent cells (by ELISA), can then be chosen
for making a cell bank, and for antibody purification.
[0280] To purify anti-CD27 antibodies, selected hybridomas can be
grown in roller bottles, two-liter spinner-flasks or other culture
systems. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-Sepharose (Pharmacia,
Piscataway, N.J.) to purify the protein. After buffer exchange to
PBS, the concentration can be determined by OD.sub.280 using 1.43
extinction coefficient or preferably by nephelometric analysis. IgG
can be checked by gel electrophoresis and by antigen specific
method.
[0281] To determine if the selected anti-CD27 monoclonal antibodies
bind to unique epitopes, each antibody can be biotinylated using
commercially available reagents (Pierce, Rockford, Ill.).
Biotinylated MAb binding can be detected with a streptavidin
labeled probe. To determine the isotype of purified antibodies,
isotype ELISAs can be performed using art recognized techniques.
For example, wells of microtiter plates can be coated with 10
.mu.g/ml of anti-Ig overnight at 4.degree. C. After blocking with
5% BSA, the plates are reacted with 10 .mu.g/ml of monoclonal
antibodies or purified isotype controls, at ambient temperature for
two hours. The wells can then be reacted with either IgG1 or other
isotype specific conjugated probes. Plates are developed and
analyzed as described above.
[0282] To test the binding of monoclonal antibodies to live cells
expressing CD27, flow cytometry can be used. Briefly, cell lines
and/or human PBMCs expressing membrane-bound CD27 (grown under
standard growth conditions) are mixed with various concentrations
of monoclonal antibodies in PBS containing 0.1% BSA at 4.degree. C.
for 1 hour. After washing, the cells are reacted with
Fluorescein-labeled anti-IgG antibody under the same conditions as
the primary antibody staining. The samples can be analyzed by
FACScan instrument using light and side scatter properties to gate
on single cells and binding of the labeled antibodies is
determined. An alternative assay using fluorescence microscopy may
be used (in addition to or instead of) the flow cytometry assay.
Cells can be stained exactly as described above and examined by
fluorescence microscopy. This method allows visualization of
individual cells, but may have diminished sensitivity depending on
the density of the antigen.
[0283] Anti-CD27 IgGs can be further tested for reactivity with the
CD27 antigen by Western blotting. Briefly, cell extracts from cells
expressing CD27 can be prepared and subjected to sodium dodecyl
sulfate polyacrylamide gel electrophoresis. After electrophoresis,
the separated antigens will be transferred to nitrocellulose
membranes, blocked with 20% mouse serum, and probed with the
monoclonal antibodies to be tested. IgG binding can be detected
using anti-IgG alkaline phosphatase and developed with BCIP/NBT
substrate tablets (Sigma Chem. Co., St. Louis, Mo.).
[0284] Methods for analyzing binding affinity, cross-reactivity,
and binding kinetics of various anti-CD27 antibodies include
standard assays known in the art, for example, Biacore.TM. surface
plasmon resonance (SPR) analysis using a Biacore.TM. 2000 SPR
instrument (Biacore AB, Uppsala, Sweden), as described in Example 2
herein.
II. Immunotoxins
[0285] In another embodiment, the antibodies of the present
invention are linked to a therapeutic moiety, such as a cytotoxin,
a drug or a radioisotope. When conjugated to a cytotoxin, these
antibody conjugates are referred to as "immunotoxins." A cytotoxin
or cytotoxic agent includes any agent that is detrimental to (e.g.,
kills) cells. Examples include taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine). An antibody of the present invention
can be conjugated to a radioisotope, e.g., radioactive iodine, to
generate cytotoxic radiopharmaceuticals for treating a
dendritic-related disorder, such as an autoimmune or inflammatory
disease, or graft versus host disease.
[0286] The antibody conjugates of the invention can be used to
modify a given biological response, and the drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, an enzymatically active toxin, or active
fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor or
interferon-.gamma.; or, biological response modifiers such as, for
example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating
factor ("G-CSF"), or other growth factors.
[0287] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
III. Compositions
[0288] In another embodiment, the present invention provides a
composition, e.g., a composition, containing one or a combination
of monoclonal antibodies of the present invention, formulated
together with a carrier (e.g., a pharmaceutically acceptable
carrier). Compositions containing bispecific molecules which
comprise an antibody of the present invention are also provided. In
one embodiment, the compositions include a combination of multiple
(e.g., two or more) isolated antibodies of the invention.
Preferably, each of the antibodies of the composition binds to a
distinct, pre-selected epitope of CD27.
[0289] Pharmaceutical compositions of the invention also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include a
composition of the present invention with at least one or more
additional therapeutic agents, such as anti-inflammatory agents,
DMARDs (disease-modifying anti-rheumatic drugs), immunosuppressive
agents, and chemotherapeutics. The pharmaceutical compositions of
the invention can also be administered in conjunction with
radiation therapy. Co-administration with other antibodies is also
encompassed by the invention.
[0290] As used herein, the terms "carrier" and "pharmaceutically
acceptable carrier" includes any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically
compatible. Preferably, the carrier is suitable for intravenous,
intramuscular, subcutaneous, parenteral, spinal or epidermal
administration (e.g., by injection or infusion). Depending on the
route of administration, the active compound, i.e., antibody,
bispecific and multispecific molecule, may be coated in a material
to protect the compound from the action of acids and other natural
conditions that may inactivate the compound.
[0291] Examples of adjuvants which may be used with the antibodies
and constructs of the present invention include: Freund's
Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories,
Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc.,
Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.);
aluminum salts such as aluminum hydroxide gel (alum) or aluminum
phosphate; salts of calcium, iron or zinc; an insoluble suspension
of acylated tyrosine; acylated sugars; cationically or anionically
derivatised polysaccharides; polyphosphazenes; biodegradable
microspheres; cytokines, such as GM-CSF, interleukin-2, -7, -12,
and other like factors; 3D-MPL; CpG oligonucleotide; and
monophosphoryl lipid A, for example 3-de-O-acylated monophosphoryl
lipid A.
[0292] MPL adjuvants are available from Corixa Corporation
(Seattle, Wash; see, for example, U.S. Pat. Nos. 4,436,727;
4,877,611; 4,866,034 and 4,912,094). CpG-containing
oligonucleotides (in which the CpG dinucleotide is unmethylated)
are well known and are described, for example, in WO 96/02555, WO
99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462.
Immunostimulatory DNA sequences are also described, for example, by
Sato et al., Science 273:352, 1996.
[0293] Further alternative adjuvants include, for example,
saponins, such as Quil A, or derivatives thereof, including QS21
and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin;
Digitonin; or Gypsophila or Chenopodium quinoa saponins; Montanide
ISA 720 (Seppic, France); SAF (Chiron, Calif., United States);
ISCOMS (CSL), MF-59 (Chiron); the SBAS series of adjuvants (e.g.,
SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart,
Belgium); Detox (Enhanzyn.TM.) (Corixa, Hamilton, Mont.); RC-529
(Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide
4-phosphates (AGPs); polyoxyethylene ether adjuvants such as those
described in WO 99/52549A1; synthetic imidazoquinolines such as
imiquimod [S-26308, R-837], (Harrison, et al., Vaccine 19:
1820-1826, 2001; and resiquimod [S-28463, R-848] (Vasilakos, et
al., Cellular immunology 204: 64-74, 2000; Schiff bases of
carbonyls and amines that are constitutively expressed on antigen
presenting cell and T-cell surfaces, such as tucaresol (Rhodes, J.
et al., Nature 377: 71-75, 1995); cytokine, chemokine and
co-stimulatory molecules as either protein or peptide, including
for example pro-inflammatory cytokines such as Interferon, GM-CSF,
IL-1 alpha, IL-1 beta, TGF-alpha and TGF-beta, Th1 inducers such as
interferon gamma, IL-2, IL-12, IL-15, IL-18 and IL-21, Th2 inducers
such as IL-4, IL-5, IL-6, IL-10 and IL-13 and other chemokine and
co-stimulatory genes such as MCP-1, MIP-1 alpha, MIP-1 beta,
RANTES, TCA-3, CD80, CD86 and CD40L; immunostimulatory agents
targeting ligands such as CTLA-4 and L-selectin, apoptosis
stimulating proteins and peptides such as Fas; synthetic lipid
based adjuvants, such as vaxfectin, (Reyes et al., Vaccine 19:
3778-3786, 2001) squalene, alpha-tocopherol, polysorbate 80, DOPC
and cholesterol; endotoxin, [LPS], (Beutler, B., Current Opinion in
Microbiology 3: 23-30, 2000); ligands that trigger Toll receptors
to produce Th1-inducing cytokines, such as synthetic Mycobacterial
lipoproteins, Mycobacterial protein p19, peptidoglycan, teichoic
acid and lipid A; and CT (cholera toxin, subunits A and B) and LT
(heat labile enterotoxin from E. coli, subunits A and B), heat
shock protein family (HSPs), and LLO (listeriolysin O; WO
01/72329). These and various further Toll-like Receptor (TLR)
agonists are described for example in Kanzler et al, Nature
Medicine, May 2007, Vol 13, No 5. A preferred immunostimulatory
agent for use in combination with an anti-CD27 antibody of the
invention is a TLR3 agonist, such as Poly IC.
[0294] A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the parent compound and
does not impart any undesired toxicological effects (see e.g.,
Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of
such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, phosphorous and the like, as well as from
nontoxic organic acids such as aliphatic mono- and dicarboxylic
acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,
aromatic acids, aliphatic and aromatic sulfonic acids and the like.
Base addition salts include those derived from alkaline earth
metals, such as sodium, potassium, magnesium, calcium and the like,
as well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0295] A composition of the present invention can be administered
by a variety of methods known in the art. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results. The active compounds can
be prepared with carriers that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
[0296] To administer a compound of the invention by certain routes
of administration, it may be necessary to coat the compound with,
or co-administer the compound with, a material to prevent its
inactivation. For example, the compound may be administered to a
subject in an appropriate carrier, for example, liposomes, or a
diluent. Acceptable diluents include saline and aqueous buffer
solutions. Liposomes include water-in-oil-in-water CGF emulsions as
well as conventional liposomes (Strejan et al. (1984) J.
Neuroimmunol. 7:27).
[0297] Carriers include sterile aqueous solutions or dispersions
and sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. The use of such media and
agents for pharmaceutically active substances is known in the art.
Except insofar as any conventional media or agent is incompatible
with the active compound, use thereof in the pharmaceutical
compositions of the invention is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
[0298] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0299] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0300] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. For example, the antibodies of the invention may be
administered once or twice weekly by subcutaneous or intramuscular
injection or once or twice monthly by subcutaneous or intramuscular
injection.
[0301] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0302] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0303] For the therapeutic compositions, formulations of the
present invention include those suitable for oral, nasal, topical
(including buccal and sublingual), rectal, vaginal and/or
parenteral administration. The formulations may conveniently be
presented in unit dosage form and may be prepared by any methods
known in the art of pharmacy. The amount of active ingredient which
can be combined with a carrier material to produce a single dosage
form will vary depending upon the subject being treated, and the
particular mode of administration. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will generally be that amount of the composition which
produces a therapeutic effect. Generally, out of one hundred per
cent, this amount will range from about 0.001 per cent to about
ninety percent of active ingredient, preferably from about 0.005
per cent to about 70 per cent, most preferably from about 0.01 per
cent to about 30 per cent.
[0304] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate. Dosage forms for the
topical or transdermal administration of compositions of this
invention include powders, sprays, ointments, pastes, creams,
lotions, gels, solutions, patches and inhalants. The active
compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0305] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0306] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0307] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0308] When the compounds of the present invention are administered
as pharmaceuticals, to humans and animals, they can be given alone
or as a pharmaceutical composition containing, for example, 0.001
to 90% (more preferably, 0.005 to 70%, such as 0.01 to 30%) of
active ingredient in combination with a pharmaceutically acceptable
carrier.
[0309] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0310] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts. A physician or veterinarian having ordinary skill in
the art can readily determine and prescribe the effective amount of
the pharmaceutical composition required. For example, the physician
or veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved. In general, a suitable daily dose of a composition of the
invention will be that amount of the compound which is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above. It is
preferred that administration be intravenous, intramuscular,
intraperitoneal, or subcutaneous, preferably administered proximal
to the site of the target. If desired, the effective daily dose of
a therapeutic composition may be administered as two, three, four,
five, six or more sub-doses administered separately at appropriate
intervals throughout the day, optionally, in unit dosage forms.
While it is possible for a compound of the present invention to be
administered alone, it is preferable to administer the compound as
a pharmaceutical formulation (composition).
[0311] Therapeutic compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
therapeutic composition of the invention can be administered with a
needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335,
5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of
well-known implants and modules useful in the present invention
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4.,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Pat. No. 4,447,233,
which discloses a medication infusion pump for delivering
medication at a precise infusion rate; U.S. Pat. No. 4,447,224,
which discloses a variable flow implantable infusion apparatus for
continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses
an osmotic drug delivery system having multi-chamber compartments;
and U.S. Pat. No. 4,475,196, which discloses an osmotic drug
delivery system. Many other such implants, delivery systems, and
modules are known to those skilled in the art.
[0312] In certain embodiments, the antibodies of the invention can
be formulated to ensure proper distribution in vivo. For example,
the blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds of the
invention cross the BBB (if desired), they can be formulated, for
example, in liposomes. For methods of manufacturing liposomes, see,
e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties which are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides
(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);
antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M.
Owais et al. (1995) Antimicrob. Agents Chemother. 39:180);
surfactant protein A receptor (Briscoe et al. (1995) Am. J.
Physiol. 1233:134), different species of which may comprise the
formulations of the inventions, as well as components of the
invented molecules; p120 (Schreier et al. (1994) J. Biol. Chem.
269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.
346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In
one embodiment of the invention, the therapeutic compounds of the
invention are formulated in liposomes; in a more preferred
embodiment, the liposomes include a targeting moiety. In a most
preferred embodiment, the therapeutic compounds in the liposomes
are delivered by bolus injection to a site proximal to the tumor or
infection. The composition must be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and
fungi.
[0313] The ability of a compound to inhibit cancer can be evaluated
in an animal model system predictive of efficacy in human tumors.
Alternatively, this property of a composition can be evaluated by
examining the ability of the compound to inhibit, such inhibition
in vitro by assays known to the skilled practitioner. A
therapeutically effective amount of a therapeutic compound can
decrease tumor size, or otherwise ameliorate symptoms in a subject.
One of ordinary skill in the art would be able to determine such
amounts based on such factors as the subject's size, the severity
of the subject's symptoms, and the particular composition or route
of administration selected.
[0314] The composition must be sterile and fluid to the extent that
the composition is deliverable by syringe. In addition to water,
the carrier can be an isotonic buffered saline solution, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyetheylene glycol, and the like), and suitable mixtures thereof.
Proper fluidity can be maintained, for example, by use of coating
such as lecithin, by maintenance of required particle size in the
case of dispersion and by use of surfactants. In many cases, it is
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol or sorbitol, and sodium chloride in
the composition. Long-term absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate or gelatin.
[0315] When the active compound is suitably protected, as described
above, the compound may be orally administered, for example, with
an inert diluent or an assimilable edible carrier.
IV. Uses and Methods of the Invention
[0316] In one embodiment, the antibodies, bispecific molecules, and
compositions of the present invention can be used to treat and/or
prevent (e.g., immunize against) a variety of diseases and
conditions.
[0317] One of the primary disease indications that can be treated
is cancer. In particular, an anti-CD27 antibody that induces or
enhances an immune response can be used in the treatment of cancer.
Types of cancers include, but are not limited to, leukemia, acute
lymphocytic leukemia, acute myelocytic leukemia, myeloblasts
promyelocyte myelomonocytic monocytic erythroleukemia, chronic
leukemia, chronic myelocytic (granulocytic) leukemia, chronic
lymphocytic leukemia, mantle cell lymphoma, primary central nervous
system lymphoma, Burkitt's lymphoma and marginal zone B cell
lymphoma, Polycythemia vera Lymphoma, Hodgkin's disease,
non-Hodgkin's disease, multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, solid tumors, sarcomas, and
carcinomas, fibrosarcoma, myxosarcoma, liposarcoma,
chrondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal
carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, uterine cancer,
testicular tumor, lung carcinoma, small cell lung carcinoma, non
small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma,
retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma,
basal cell carcinoma, biliary tract cancer, bladder cancer, bone
cancer, brain and central nervous system (CNS) cancer, cervical
cancer, choriocarcinoma, colorectal cancers, connective tissue
cancer, cancer of the digestive system, endometrial cancer,
esophageal cancer, eye cancer, head and neck cancer, gastric
cancer, intraepithelial neoplasm, kidney cancer, larynx cancer,
liver cancer, lung cancer (small cell, large cell), melanoma,
neuroblastoma; oral cavity cancer(for example lip, tongue, mouth
and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma,
rhabdomyosarcoma, rectal cancer; cancer of the respiratory system,
sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid
cancer, uterine cancer, and cancer of the urinary system. Preferred
cancers include CD27-expressing tumors selected from the group
consisting of chronic lymphocytic leukemia, mantle cell lymphoma,
primary central nervous system lymphoma, Burkitt's lymphoma and
marginal zone B cell lymphoma.
[0318] Other disease indications for use of an anti-CD27 antibody
that induces or enhances an immune response include bacterial,
fungal, viral and parasitic infectious diseases. Other disease
indications for use of an anti-CD27 antibody that inhibits or
reduces an immune response include graft rejection, allergy and
autoimmune diseases.
[0319] Exemplary autoimmune diseases include, but are not limited
to, multiple sclerosis, rheumatoid arthritis, type 1 diabetes,
psoriasis, Crohn's disease and other inflammatory bowel diseases
such as ulcerative colitis, systemic lupus eythematosus (SLE),
autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's
thyroiditis, Goodpasture's syndrome, pemphigus, Graves disease,
autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura,
scleroderma with anti-collagen antibodies, mixed connective tissue
disease, polypyositis, pernicious anemia, idiopathic Addison's
disease, autoimmune associated infertility, glomerulonephritis,
crescentic glomerulonephritis, proliferative glomerulonephritis,
bullous pemphigoid, Sjogren's syndrome, psoriatic arthritis,
insulin resistance, autoimmune diabetes mellitus, autoimmune
hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative
syndrome (ALPS), autoimmune hepatitis, autoimmune hemophilia,
autoimmune lymphoproliferative syndrome, autoimmune uveoretinitis,
Guillain-Bare syndrome, arteriosclerosis and Alzheimer's disease.
Exemplary allergic disorders include, but are not limited to
allergic conjunctivitis, vernal conjunctivitis, vernal
keratoconjunctivitis, and giant papillary conjunctivitis; nasal
allergic disorders, including allergic rhinitis and sinusitis; otic
allergic disorders, including eustachian tube itching; allergic
disorders of the upper and lower airways, including intrinsic and
extrinsic asthma; allergic disorders of the skin, including
dermatitis, eczema and urticaria; and allergic disorders of the
gastrointestinal tract.
[0320] In another aspect, an antibody of the invention is
administered in combination with a vaccine, to enhance the immune
response against the vaccine antigen, for example a tumor antigen
(to thereby enhance the immune response against the tumor) or an
antigen from an infectious disease pathogen (to thereby enhance the
immune response against the infectious disease pathogen).
Accordingly, in this embodiment, a vaccine antigen can comprise,
for example, an antigen or antigenic composition capable of
eliciting an immune response against a tumor or against an
infectious disease pathogen such as a virus, a bacteria, a parasite
or a fungus. The antigen or antigens can be, for example,
peptides/proteins, polysaccharides and/or lipids. The antigen or
antigens be derived from tumors, such as the various tumor antigens
previously disclosed herein. Alternatively, the antigen or antigens
can be derived from pathogens such as viruses, bacteria, parasites
and/or fungi, such as the various pathogen antigens previously
disclosed herein. Additional examples of suitable pathogen antigens
include, but are not limited to, the following:
[0321] Viral antigens or antigenic determinants can be derived
from, for example,: Cytomegalovirus (especially Human, such as gB
or derivatives thereof); Epstein Barr virus (such as gp350);
flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borne
encephalitis virus, Japanese Encephalitis Virus); hepatitis virus
such as hepatitis B virus (for example Hepatitis B Surface antigen
such as the PreS1, PreS2 and S antigens described in EP-A-414 374;
EP-A-0304 578, and EP-A-198474), hepatitis A virus, hepatitis C
virus and hepatitis E virus; HIV-1, (such as tat, nef, gp120 or
gp160); human herpes viruses, such as gD or derivatives thereof or
Immediate Early protein such as ICP27 from HSV1 or HSV2; human
papilloma viruses (for example HPV6, 11, 16, 18); Influenza virus
(whole live or inactivated virus, split influenza virus, grown in
eggs or MDCK cells, or Vero cells or whole flu virosomes (as
described by Gluck, Vaccine, 1992,10, 915-920) or purified or
recombinant proteins thereof, such as NP, NA, HA, or M proteins);
measles virus; mumps virus; parainfluenza virus; rabies virus;
Respiratory Syncytial virus (such as F and G proteins); rotavirus
(including live attenuated viruses); smallpox virus; Varicella
Zoster Virus (such as gpl, II and IE63); and the HPV viruses
responsible for cervical cancer (for example the early proteins E6
or E7 in fusion with a protein D carrier to form Protein D-E6 or E7
fusions from HPV 16, or combinations thereof; or combinations of E6
or E7 with L2 (see for example WO 96/26277).
[0322] Bacterial antigens or antigenic determinants can be derived
from, for example,: Bacillus spp., including B. anthracis (eg
botulinum toxin); Bordetella spp, including B. pertussis (for
example pertactin, pertussis toxin, filamenteous hemagglutinin,
adenylate cyclase, fimbriae); Borrelia spp., including B.
burgdorferi (eg OspA, OspC, DbpA, DbpB), B. garinii (eg OspA, OspC,
DbpA, DbpB), B. afzelii (eg OspA, OspC, DbpA, DbpB), B. andersonii
(eg OspA, OspC, DbpA, DbpB), B. hermsii; Campylobacter spp,
including C. jejuni (for example toxins, adhesins and invasins) and
C. coli; Chlamydia spp., including C. trachomatis (eg MOMP,
heparin-binding proteins), C. pneumonie (eg MOMP, heparin-binding
proteins), C. psittaci; Clostridium spp., including C. tetani (such
as tetanus toxin), C. botulinum (for example botulinum toxin), C.
difficile (eg clostridium toxins A or B); Corynebacterium spp.,
including C. diphtheriae (eg diphtheria toxin); Ehrlichia spp.,
including E. equi and the agent of the Human Granulocytic
Ehrlichiosis; Rickettsia spp, including R. rickettsii; Enterococcus
spp., including E. faecalis, E. faecium; Escherichia spp, including
enterotoxic E. coli (for example colonization factors, heat-labile
toxin or derivatives thereof, or heat-stable toxin),
enterohemorragic E. coli, enteropathogenic E. coli (for example
shiga toxin-like toxin); Haemophilus spp., including H. influenzae
type B (eg PRP), non-typable H. influenzae, for example OMP26, high
molecular weight adhesins, P5, P6, protein D and lipoprotein D, and
fimbrin and fimbrin derived peptides (see for example U.S. Pat. No.
5,843,464); Helicobacter spp, including H. pylori (for example
urease, catalase, vacuolating toxin); Pseudomonas spp, including P.
aeruginosa; Legionella spp, including L. pneumophila; Leptospira
spp., including L. interrogans; Listeria spp., including L.
monocytogenes; Moraxella spp, including M. catarrhalis, also known
as Branhamella catarrhalis (for example high and low molecular
weight adhesins and invasins); Morexella aatarrhalis (including
outer membrane vesicles thereof, and OMP106 (see for example
WO97/41731)); Mycobacterium spp., including M. tuberculosis (for
example ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M.
avium, M. paratuberculosis, M. smegmatis; Neisseria spp, including
N. gonorrhea and N. meningitidis (for example capsular
polysaccharides and conjugates thereof, transferrin-binding
proteins, lactoferrin binding proteins, PilC, adhesins); Neisseria
mengitidis B (including outer membrane vesicles thereof, and NspA
(see for example WO 96/29412); Salmonella spp, including S. typhi,
S. paratyphi, S. choleraesuis, S. enteritidis; Shigella spp,
including S. sonnei, S. dysenteriae, S. flexnerii; Staphylococcus
spp., including S. aureus, S. epidermidis; Streptococcus spp,
including S. pneumonie (eg capsular polysaccharides and conjugates
thereof, PsaA, PspA, streptolysin, choline-binding proteins) and
the protein antigen Pneumolysin (Biochem Biophys Acta,
1989,67,1007; Rubins et al., Microbial Pathogenesis, 25,337-342),
and mutant detoxified derivatives thereof (see for example WO
90/06951; WO 99/03884); Treponema spp., including T. pallidum (eg
the outer membrane proteins), T. denticola, T. hyodysenteriae;
Vibrio spp, including V. cholera (for example cholera toxin); and
Yersinia spp, including Y. enterocolitica (for example a Yop
protein), Y. pestis, Y. pseudotuberculosis.
[0323] Parasitic/fungal antigens or antigenic determinants can be
derived from, for example,: Babesia spp., including B. microti;
Candida spp., including C. albicans; Cryptococcus spp., including
C. neoformans; Entamoeba spp., including E. histolytica; Giardia
spp., including; G. lamblia; Leshmania spp., including L. major;
Plasmodium faciparum (MSP1, AMA1, MSPS, EBA, GLURP, RAP1, RAP2,
Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25,
Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and their analogues in
Plasmodium spp.); Pneumocystis spp., including P. carinii;
Schisostoma spp., including S. mansoni; Trichomonas spp., including
T. vaginalis; Toxoplasma spp., including T. gondii (for example
SAG2, SAGS, Tg34); Trypanosoma spp., including T. cruzi.
[0324] It will be appreciated that in accordance with this aspect
of the present invention antigens and antigenic determinants can be
used in many different forms. For example, antigens or antigenic
determinants can be present as isolated proteins or peptides (for
example in so-called "subunit vaccines") or, for example, as
cell-associated or virus-associated antigens or antigenic
determinants (for example in either live or killed pathogen
strains). Live pathogens will preferably be attenuated in known
manner. Alternatively, antigens or antigenic determinants may be
generated in situ in the subject by use of a polynucleotide coding
for an antigen or antigenic determinant (as in so-called "DNA
vaccination"), although it will be appreciated that the
polynucleotides which can be used with this approach are not
limited to DNA, and may also include RNA and modified
polynucleotides as discussed above.
[0325] In one embodiment, a vaccine antigen can also be targeted,
for example to particular cell types or to particular tissues. For
example, the vaccine antigen can be targeted to Antigen Presenting
Cells (APCs), for example by use of agents such as antibodies
targeted to APC-surface receptors such as DEC-205, for example as
discussed in WO 2009/061996 (Celldex Therapeutics, Inc), or the
Mannose Receptor (CD206) for example as discussed in WO 03040169
(Medarex, Inc.).
[0326] For use in therapy, the antibodies of the invention can be
administered to a subject directly (i.e., in vivo), either alone or
with other therapies such as an immunostimulatory agent, a vaccine,
chemotherapy or radiation therapy. In all cases, the antibodies,
bispecifics, compositions, and immunostimulatory agents and other
therapies are administered in an effective amount to exert their
desired therapeutic effect. The term "effective amount" refers to
that amount necessary or sufficient to realize a desired biologic
effect. For example, an effective amount could be that amount
necessary to eliminate a tumor, cancer, or bacterial, viral or
fungal infection. The effective amount for any particular
application can vary depending on such factors as the disease or
condition being treated, the particular antibody being
administered, the size of the subject, or the severity of the
disease or condition. One of ordinary skill in the art can
empirically determine the effective amount of a particular molecule
without necessitating undue experimentation.
[0327] Preferred routes of administration for vaccines include, for
example, injection (e.g., subcutaneous, intravenous, parenteral,
intraperitoneal, intrathecal). The injection can be in a bolus or a
continuous infusion. Other routes of administration include oral
administration.
[0328] Antibodies and bispecific molecules of the invention also
can be coadministered with adjuvants and other therapeutic agents.
It will be appreciated that the term "coadministered" as used
herein includes any or all of simultaneous, separate, or sequential
administration of the antibodies and conjugates of the present
invention with adjuvants and other agents, including administration
as part of a dosing regimen. The antibodies are typically
formulated in a carrier alone or in combination with such agents.
Examples of such carriers include solutions, solvents, dispersion
media, delay agents, emulsions and the like. The use of such media
for pharmaceutically active substances is well known in the art.
Any other conventional carrier suitable for use with the molecules
falls within the scope of the instant invention.
[0329] Suitable agents for co-administration with the antibodies,
conjugates, bispecifics, and compositions include other antibodies,
cytotoxins and/or drugs, as well as adjuvants, immunostimulatory
agents and/or immunosuppressive agents. In one embodiment, the
agent is a chemotherapeutic agent. The antibodies, bispecifics, and
compositions can be administered in combination with radiation.
[0330] Chemotherapeutic agents suitable for coadministration with
the antibodies and conjugates of the present invention in the
treatment of tumors include, for example: taxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof. Further agents include, for example,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine) and temozolomide.
[0331] Agents that delete or inhibit immunosuppressive activities,
for example, by immune cells (for example regulatory T-cells, NKT
cells, macrophages, myeloid-derived suppressor cells, immature or
suppressive dendritic cells) or suppressive factors produced by the
tumor or host cells in the local microenvironment of the tumor (for
example, TGFbeta, indoleamine 2,3 dioxygenase--IDO), may also be
administered with the antibodies and conjugates of the present
invention. Such agents include antibodies and small molecule drugs
such as IDO inhibitors such as 1 methyl tryptophan or
derivatives.
[0332] Suitable agents for coadministration with the antibodies and
bispecifics of the present invention for treatment of such immune
disorders include for example, immunosuppressive agents such as
rapamycin, cyclosporin and FK506; anti-TNFa agents such as
etanercept, adalimumab and infliximab; and steroids. Examples of
specific natural and synthetic steroids include, for example:
aldosterone, beclomethasone, betamethasone, budesonide, cloprednol,
cortisone, cortivazol, deoxycortone, desonide, desoximetasone,
dexamethasone, difluorocortolone, fluclorolone, flumethasone,
flunisolide, fluocinolone, fluocinonide, fluocortin butyl,
fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone,
fluticasone, halcinonide, hydrocortisone, icomethasone,
meprednisone, methylprednisolone, paramethasone, prednisolone,
prednisone, tixocortol and triamcinolone.
[0333] Suitable agents for coadministration with the antibodies and
bispecifics of the present invention for inducement or enhancement
of an immune response include, for example, adjuvants and/or
immunostimulatory agents, non-limiting examples of which have been
disclosed hereinbefore. A preferred immunostimulatory agent is a
TLR3 agonist, such as Poly IC.
[0334] The present invention is further illustrated by the
following examples which should not be construed as further
limiting. The contents of Sequence Listing, figures and all
references, patents and published patent applications cited
throughout this application are expressly incorporated herein by
reference.
EXAMPLES
Example 1
Generation of CD27-Specific Human Monoclonal Antibodies
[0335] Human anti-CD27 monoclonal antibodies were generated by
immunizing the HC2/KCo7 strain of HuMAb.RTM. transgenic mice
("HuMAb" is a Trade Mark of Medarex, Inc., Princeton, N.J.) with a
soluble human CD27 antigen. HC2/KCo7 HuMAb mice were generated as
described in U.S. Pat. Nos. 5,770,429 and 5,545,806, the entire
disclosures of which are hereby incorporated by reference.
[0336] Antigen and Immunization: The antigen was a soluble fusion
protein comprising a CD27 extracellular domain fused with an
antibody Fc domain (recombinant human CD27-Fc chimeric protein
(R&D Systems). The antigen was mixed with Complete Freund's
(Sigma) adjuvant for the first immunization. Thereafter, the
antigen was mixed with Incomplete Freund's (Sigma). Additional mice
were immunized with the soluble CD27 protein in RIBI MPL plus TDM
adjuvant system (Sigma). 5-25 micrograms soluble recombinant CD27
antigen in PBS or 5.times.10.sup.6 CHO cells transfected for
surface expression of human CD27 in PBS were mixed 1:1 with the
adjuvant. Mice were injected with 100 microliters of the prepared
antigen into the peritoneal cavity every 14 days. Animals that
developed anti-CD27 titers were given an iv injection of 10
micrograms soluble recombinant CD27 antigen three to four days
prior to fusion. Mouse spleens were harvested, and the isolated
splenocytes used for hybridoma preparation.
[0337] Hybridoma Preparation: The P3x63Ag8.653 murine myeloma cell
line (ATCC CRL 1580) was used for the fusions. RPMI 1640
(Invitrogen) containing 10% FBS, and was used to culture the
myeloma cells. Additional media supplements were added to the
Hybridoma growth media, which included: 3% Origen-Hybridoma Cloning
Factor (Igen), 10% FBS (Sigma), L-glutamine (Gibco) 0.1% gentamycin
(Gibco), 2-mercaptoethanol (Gibco), HAT (Sigma; 1.0.times.10.sup.4
M hypoxanthine, 4.0.times.10.sup.-7 M aminopterin,
1.6.times.10.sup.-5 M thymidine), or HT (Sigma; 1.0.times.10.sup.-4
M hypoxanthine, 1.6.times.10.sup.-5 M thymidine) media.
[0338] Spleen cells were mixed with the P3x63Ag8.653myeloma cells
in a 6:1 ratio and pelleted by centrifugation. Polyethylene glycol
was added dropwise with careful mixing to facilitate fusion.
Hybridomas were allowed to grow out for one to two weeks until
visible colonies become established. Supernatant was harvested and
used for initial screening for human IgG via ELISA using a human
kappa chain specific capture and a human Fc specific detection. IgG
positive supernatants were then assayed for CD27 specificity via
flow cytometry or using a ELISA to detect anti-CD27.
[0339] Hybridomas producing specific human monoclonal antibodies
(human mAbs; IgG) were subcloned and expanded. The human mAbs
produced were then purified by protein A column chromatography
according to standard conditions which led to the isolation of a
number of antibodies of particular interest, which were designated
as 4B7-1B3 (also referred to herein as 4B7), 3H12-1C8 (also
referred to herein as 3H12), 1F5-1H5 (also referred to herein as
1F5), 2C2-1A10 (also referred to herein as 2C2), 2G9-1D11 (also
referred to herein as 2G9), 1H8-B4 (also referred to herein as
1H8), 3H12-1E12 (also referred to herein as 3H12), 3G1-1A11 (also
referred to herein as 3G1) (1B10), 4A2-B11 (also referred to herein
as 4A2), 3A10-G10 (also referred to herein as 3A10), 2G11-B5 (also
referred to herein as 2G11), 4H11-G11 (also referred to herein as
4H11), 2H3-E8 (also referred to herein as 2H3), 4A7-B3 (also
referred to herein as 4A7), 3H8-1B11 (also referred to herein as
3H8)and 1G5-1B9 (also referred to herein as 1G5).
[0340] The hybridomas were also screened for cross-reactivity with
rhesus macaque CD27 and all were positive for binding.
Example 2
[0341] Determination of Affinity and Rate Constants of Human mAbs
by Surface Plasmon Resonance (SPR)
[0342] Binding affinity and binding kinetics of various human
anti-CD27 antibodies from Example 1 were examined by Biacore.TM.
surface plasmon resonance (SPR) analysis using a Biacore.TM. 2000
SPR instrument (Biacore AB, Uppsala, Sweden) according to the
manufacturer's guidelines.
[0343] Purified recombinant human CD27/TNFRSF7/Fc chimera (R&D
Systems Catalog No. 382-CD) protein was covalently linked to a
Biacore.TM. CM5 sensor chip (carboxymethylated dextran covalently
attached to a gold surface; Biacore Product No. BR-1000-14) using
standard amine coupling chemistry with an Amine Coupling Kit
provided by Biacore according to the manufacturer's guidelines
(BIAcore Product No. BR-1000-50, comprising coupling reagents
N-hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC)). Low levels of ligand were
immobilised to limit any effects of mass transport of analyte on
kinetic parameters, such that the R.sub.MAX observed was in the
order of 100-400 RU.
[0344] Binding was measured by flowing the antibodies over the
sensor chip in HBS-EP buffer (HBS-EP buffer, Biacore Product No.
BR-1001-88: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) 0.01M, sodium chloride 0.15M ethylenediaminetetraacetic
acid (EDTA) 3 mM, Surfactant P20 0.005%), at concentrations ranging
from 1.56 to 50 nM and at a flow rate of 30 .mu.l/minute for 180
seconds. The antigen-antibody association and dissociation kinetics
were followed for either 480 seconds or 1200 seconds for antibodies
with slower dissociation rates.
[0345] Corresponding controls were conducted in each case using a
blank flowcell with no protein immobilized for "background"
subtraction. Sequential injections of 10 mM HCl for 10 seconds at
50 .mu.l/min followed by 10 mM Glycine pH 2.0 for 30 seconds at 50
.mu.l/min was used as the regeneration conditions throughout the
study.
[0346] Biacore's BIAevaluation software version 3.2 (Biacore AB,
Uppsala, Sweden) was used in each case to derive kinetic parameters
from the concentration series of analyte diluted in HBS-EP running
buffer. The association and dissociation curves were fitted to a
1:1 Langmuir binding model using Biacore.TM. BIAevaluation software
(Biacore AB) according to the manufacturer's guidelines.
[0347] The affinity and kinetic parameters (with background
subtracted) as determined are shown in FIG. 1.
[0348] For each antibody, the figures shown are the mean of two
separate series of experiments, using separately prepared flow
cells in each case, (where ka=rate constant of association, kd=rate
constant of dissociation, K.sub.D=dissociation equilibrium constant
(measure of affinity), K.sub.A=association equilibrium constant,
Rmax=maximum SPR response signal).
Example 3
ELISA Assay to Determine Human mAb Binding Characteristics on
CD27
[0349] Microtiter plates were coated with soluble or recombinant
human or macaque CD27in PBS, and then blocked with 5% bovine serum
albumin in PBS. Protein A purified human mAbs and an isotype
control were added at saturating concentrations and incubated at
37.degree. C. The plates were washed with PBS/Tween and then
incubated with a goat-anti-human IgG Fc-specific polyclonal reagent
conjugated to alkaline phosphatase at 37.degree. C. After washing,
the plates were developed with pNPP substrate (1 mg/ml), and
analyzed at OD 405-650 using a microtiter plate reader.
Representatives binding curves are shown in FIG. 2. The results
were also used to estimate the 50% saturating concentration (C
value in 4-parameter fit curve) as shown in Table 1 below.
[0350] To establish that cynomolgus macques are a relevant model
for testing anti-CD27 mAbs, various concentrations of purified
macaque CD27 or human CD27 were captured to ELISA plates with
anti-Flag antibody, followed by incubation with anti-human CD27
mAb. A goat anti-human IgG Fc-HRP antibody and substrate Super Blue
TMB were used for detection. Results are shown in Table 1, which
indicate similar binding to CD27 from macque and human.
Representative binding curves for antibody 1F5 are also shown in
FIG. 3.
TABLE-US-00005 TABLE 1 Characterization of selected anti-CD27 mAb
Half -max Half -max Half-max binding to binding to binding to human
human monkey mAb CD27(M)* CO27(.mu.g/ml)** CD270.1(.mu.g/ml)** 1G5
4.9E-10 0.074 0.065 1H8 4.3E-10 0.064 0.105 3H12 5.7E-10 0.085
0.123 3H8 4.6E-10 0.069 0.065 2G9 4.3E-10 0.064 0.069 1F5 3.9E-10
0.059 0.12 3A10 6.3E-10 0.094 no binding 2C2 3.0E-10 0.045 0.034 **
estimated by binding to CD27 coated plates in an ELISA format
[0351] In a further experiment, to establish similar distribution
of 1F5 binding to peripheral blood cells, PBMCs were isolated from
whole blood of 3 humans and 3 cynomolgus macaques. The cells were
stained with 1F5 mAb together with markers to delineate the major T
cell and B cell populations that express CD27. The following table
(Table 2) summarizes the mean.+-.standard error of results for
human and macaque cells with respect to the percent of cells
expressing CD27 and the intensity of expression (MFI). These data
establish a similar distribution of 1F5 binding to peripheral blood
cells from human and monkey.
TABLE-US-00006 TABLE 2 CD4+ T cells CD8+ T cells B cells (CD20+) NK
cells Analysis human monkey human monkey human monkey human monkey
% CD27+.sup.b 84 .+-. 5 81 .+-. 1 70 .+-. 12 90 .+-. 1 37 .+-. 4 15
.+-. 1 11 .+-. 4 88 .+-. 6 MFI.sup.c 1517 .+-. 123 416 .+-. 14 1415
.+-. 153 519 .+-. 11 893 .+-. 101 491 .+-. 113 667 .+-. 28 1050
.+-. 42
Example 4
[0352] 4A: Blocking of sCD70 Binding by ELISA
[0353] The effect of the human mAbs from Example 1 on the binding
of soluble CD70 (sCD70) to CD27 protein was measured by ELISA. A
microtiter plate was coated with 1 .mu.g/ml soluble recombinant
human CD27/Fc chimera from R&D Systems at 1 .mu.g/mL., then
blocked with 5% PBA. The anti-CD27 antibodies ([final]=25 .mu.g/mL)
were premixed with soluble human recombinant CD70-biotin from US
Biologicals ([final]=0.5 .mu.g/mL) and added to the plate.
CD27-captured rCD70 was detected with streptavidin-HRP and
substrate Super Blue TMB. The results (shown as % blocking) are
shown in FIG. 4 with controls as indicated. These results show that
several of the antibodies (including 1F5, 1H8, 3H12 and 1A4) had
the property of blocking or at least significantly inhibiting the
binding of sCD70.
4B: CD27 mAb Binds to CD27 on Human Lymphoblastoid Cell Lines and
Blocks Ligand (CD70) Binding
[0354] Binding of anti-human CD27 mAb 1F5 to human lymphoblastoid
cell lines, and blocking of sCD70 binding, was analysed by flow
cytometry using a Becton Dickinson FACSCanto II flow cytometer. The
results are shown in FIG. 5, and show that 1F5 effectively binds to
a variety of cell lines and competitively inhibits binding of
sCD70.
Example 5
[0355] Binding of Human mAbs to Cells Expressing Human CD27
[0356] The ability of anti-CD27 human mAbs to bind to CD27 on cells
expressing human CD27 on their surface was investigated by flow
cytometry as follows.
[0357] Antibodies were tested for binding to human cell lines
expressing human D27 on their surface. Protein A purified human
mAbs 2C2, 3H8 and 1F5 were incubated with Jurkat, Raji, Ramos and
Daudi cells expressing human CD27, as well as control cells at
4.degree. C. All antibodies were used at saturating concentrations.
After 1 hour, the cells were washed with PBS containing 0.1% BSA
and 0.05% NaN.sub.3 (PBA) and the bound antibodies were detected by
incubating the cells with a PE labeled goat anti-human IgG
Fc-specific probe, at 4.degree. C. The excess probe was washed from
the cells with PBA and the cell associated fluorescence was
determined by analysis using a LSR.TM. instrument (BD Biosciences,
NJ, USA) according to the manufacturer's directions.
[0358] As shown in FIGS. 6 and 7, the human mAbs demonstrated high
level binding to cells expressing human CD27. These data
demonstrate that these antibodies bind efficiently and specifically
to human CD27 expressed on live cells compared to the control
cells.
Example 6
[0359] Cross-Blocking/Competition of Human mAbs Determined by
ELISA
[0360] A microtiter plate was coated with a recombinant human
CD27-Fc chimeric fusion protein, then blocked with 5% BSA in PBS.
Unconjugated human mAbs (20 .mu.g/mL) were mixed with horseradish
peroxidase labeled secondary antibodies (0.5 .mu.g/mL), then added
to the plate and incubated at 37.degree. C. The plates were washed
with PBS/Tween and developed with TMB substrate and analyzed at OD
450 using a microtiter plate reader. The results, shown in FIGS. 8
to 10, indicate that a first set of the human mAbs (comprising mAbs
1F5, 1H8 and 3H12) cross-competed with each other (see FIG. 8),
that a further set of the human mAbs (comprising mAbs 2C2, 3H8,
1G5, and 2G9) also cross-competed with each other (see FIG. 9), and
that human mAb 3A10 binds a unique epitope but may bind at a site
distinct from but possibly close to the binding sites of mAbs 1F5,
1H8, and 3H12 since these antibodies were able to partially
cross-block the binding of 3A10 to CD27 (see FIG. 10).
Example 7
Complement Dependent Cellular Cytotoxicity (CDCC or CDC)
[0361] Target cells (Lymphoma Raji cells) were cultured (in AIM-V
medium) for 1-2 hours at 37.degree. C., 5% CO.sub.2 in the presence
of anti-CD27 antibodies and rabbit complement (final dilution of
1:15) in a final volume of 150 ul. Appropriate controls with a leak
signal (targets only) and MAX signal (targets with Lysol.TM.
detergent at 12% for a final concentration of 4%) were included as
well. Cells were adjusted to 1.times.10.sup.6/ml and 50 ul were
added to each well (to give 50,000 cells/well). Wells were then
resuspended and 100 ul of the cell suspension was transferred to an
opaque, white plate. To each of these wells, 100 uL of Promega
CellTiter Glo reagent was added and the plate was mixed for 2
minutes at room temperature. The plate was allowed to incubate for
10 minutes to stabilize the luminescent signal. Luminescence was
recorded on a Perkin Elmer Victor X4 plate reader. Cytotoxicity was
determined with the following formula:
(100-((sample-MAX)/(leak-MAX))).times.100. Results (shown as %
lysis) are shown in FIG. 11 from which it can be seen that a number
of the anti-CD27 antibodies displayed significant CDCC
activity.
[0362] In a further experiment, target cells (Ramos cells) were
washed and loaded with Calcein AM (Molecular Probes). Loaded cells
were then washed again and resuspended to 1.times.10.sup.6/ml in
culture media (RPMI+10% FBS).Target cells were cultured for 2 hours
at 37.degree. C., 5% CO.sub.2 in the presence of anti-CD27
antibodies and rabbit complement (final dilution of 1:15) in a
final volume of 150 ul. Appropriate controls with a leak signal
(targets only) and MAX signal (targets with Triton X-100 at 20% for
a final concentration of 2%) were included as well. Following the
incubation, 75 ul of supernatant from the wells were transferred
into an opaque, black plate. Fluorescence (Ex 485; Em 535) was
recorded on a Perkin Elmer Victor X4 plate reader. Specific
cytotoxicity was determined using the following formula:
(experimental-spontaneous lysis)/(maximum lysis-spontaneous
lysis).times.100. The results, shown in FIG. 12, indicate that
anti-CD27 mAb (1F5) showed at least 10% CDC activity in Ramos cells
at an antibody concentration of 3 .mu.g/ml.
Example 8
Antibody Dependent Cell-Mediated Cytotoxicity (ADCC)
[0363] Targets cells (Lymphoma Raji cells) were washed and loaded
with BATDA reagent (Perkin Elmer). Loaded cells were then washed
again and resuspended to 2.times.10.sup.5/ml in culture media
(RPMI+10% FBS). Effector cells were prepared and adjusted to
appropriate concentrations in culture media to yield desired
effector:target ratios (100:1-50:1). In a round bottom plate,
target cells, effector cells and antibody were added in a final
volume of 150 ul. Appropriate controls were used, including a leak
signal (targets only), a spontaneous lysis signal
(targets+effectors), and a maximum lysis signal (targets+Lysol.TM.
detergent at 12% for a final concentration of 4%). Cells were
pelleted in the plate and incubated for 2 hours at 37.degree. C.,
5% CO.sub.2. Following the incubation, 20 ul of supernatant from
the wells were transferred into an opaque, white plate. To each of
these wells, 200 ul of Europium solution (Perkin Elmer) was added
and the plate was mixed for 15 minutes. Time-resolved fluorescence
was recorded on a Perkin Elmer Victor X4 plate reader. Specific
cytotoxicity was determined using the following formula:
(experimental-spontaneous lysis)/(maximum lysis-spontaneous
lysis).times.100. Results (shown as % lysis) are shown in FIG. 13
from which it can be seen that a number of the anti-CD27 antibodies
displayed significant ADCC activity.
[0364] In a further experiment, target cells (Lymphoma Ramos and
Daudi cells) were washed and loaded with Calcein AM (Molecular
Probes) Loaded cells were then washed again and resuspended to
1.times.10.sup.5/ml in culture media (RPMI+10% FBS). Effector cells
were prepared and adjusted to appropriate concentrations in culture
media to yield desired effector:target ratios (75:1). In a round
bottom plate, target cells, effector cells and antibody were added
in a final volume of 150 ul. Appropriate controls were used,
including a leak signal (targets only), a spontaneous lysis signal
(targets+effectors), and a maximum lysis signal (targets+Triton
X-100 at 20% for a final concentration of 2%). Cells were pelleted
in the plate and incubated for 4 hours at 37.degree. C., 5%
CO.sub.2. Following the incubation, 75 ul of supernatant from the
wells were transferred into an opaque, black plate. Fluorescence
(Ex 485; Em 535) was recorded on a Perkin Elmer Victor X4 plate
reader. Specific cytotoxicity was determined using the following
formula: (experimental-spontaneous lysis)/(maximum
lysis-spontaneous lysis).times.100. The results, shown in FIG. 14
indicate that anti-CD27 mAb (1F5) showed at least 10% ADCC activity
(measured as specific cytotoxicity) in Daudi and Ramos cells at an
antibody concentration of 3 .mu.g/ml and ratio of effector:target
cells of 75:1.
Example 9
Antibody Sequencing
[0365] As described above in Example 1, human mAbs from hybridomas
producing specific human mAbs IgG were purified by protein A column
chromatography which led to the isolation of a panel of antibodies
(human mAbs) of particular interest. The V.sub.H and V.sub.L coding
regions of human mAbs 4B7, 3H12, 1F5, 2C2, 2G9, 1H8, 3H12, 3G1
(1B10) 4A2, 3A10, 2G11, 4H11, 2H3, 4A7, 3H8 and 1G5 were identified
using RNA from the corresponding hybridomas. RNA was reverse
transcribed to cDNA, the V coding regions were amplified by PCR and
the PCR product was sequenced. The following are the nucleic and
amino acid sequences of the V.sub.H and V.sub.L regions of the
human mAbs (in the case of the amino acid sequences, the
Complementarity Determining Regions (CDRs) are underlined).
TABLE-US-00007 3H8 VH (VH 3-7; D7-27; JH2) V.sub.H nucleic acid
sequence (SEQ ID NO: 5)
atggagttggggctgagctgggttttccttgttgctattttagaaggtgtccagtgtgaggtgcagctggtgga-
gtctgggggaggcttg
gtccagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagtagttattggatggcctg-
ggtccgccaggctcca
gggaaagggctggagtggctgggcaatataaagcaagatggaagtgagaaatactatgtggactctgtgaaggg-
ccgattcaccatc
tccagagacaacgccaagaactcactgtatctacaaatgaacagcctgagagccgaggacacggctgtgtatta-
ctgtgtgagggaa
ctggggatggactggtacttcgatctctggggccgtggcaccctggtcactgtctcctca
V.sub.H amino acid sequence (SEQ ID NO: 6) (including signal
peptide in underlined italics):
MELGLSWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMAWVR
QAPGKGLEWLGNIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY
YCVRELGMDWYFDLWGRGTLVTVSS V.sub.H "mature" amino acid sequence (SEQ
ID NO: 7) excluding signal peptide:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMAWVRQAPGKGLEWLGNIKQDG
SEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRELGMDWYFDLWG RGTLVTVSS
V.sub.H CDR1 (SEQ ID NO: 8): GFTFSSYW V.sub.H CDR2 (SEQ ID NO: 9):
IKQDGSEK V.sub.H CDR3 (SEQ ID NO: 10): VRELGMDWYFDL 3H8 VK #2 (VK
3-11; JK1) V.sub.L nucleic acid sequence (SEQ ID NO: 11)
Atggaagccccagctcagcttctcttcctcctgctactctggctcccagataccaccggagaaattgtgttgac-
acagtctccagccac
cctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttgacagctacttagcct-
ggtaccaacagaaac
ctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggc-
agtgggtctggga
cagacttcactctcaccatcagcaacctagagcctgaagattttgcagtttattactgtcagcagcgtagcaac-
tggcctccgacgttcg gccaagggaccaaggtggaaatcaaa V.sub.L amino acid
sequence (SEQ ID NO: 12) (including signal peptide in underlined
italics):
MEAPAQLLFILLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVDSYLAWYQQ
KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISNLEPEDFAVYYCQQRSNWPP
TFGQGTKVEIK V.sub.L amino acid sequence (SEQ ID NO: 13) excluding
signal peptide:
EIVLTQSPATLSLSPGERATLSCRASQSVDSYLAWYQQKPGQAPRLLIYDASNRATGI
PARFSGSGSGTDFTLTISNLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK V.sub.L CDR1 (SEQ
ID NO: 14): QSVDSY V.sub.L CDR2 (SEQ ID NO: 15): DAS V.sub.L CDR3
(SEQ ID NO: 16): QQRSNWPPT 3H8 VK #3 (VK 3-11; JK1) A further light
chain was also found to be active as follows (though only the above
light chain (3H8-1B11 VK #2) was used in the above Examples):
V.sub.L nucleic acid sequence (SEQ ID NO: 17)
Atggaagccccagctcagcttctcttcctcctgctactctggctcccagataccaccggagaaattgtgttgac-
acagtctccagccac
cctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcct-
ggtaccaacagaaac
ctggccaggctcccaggctcctcatctatgatgcatccagcagggccactggcatcccagacaggttcagtggc-
agtgggtctggga
cagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagcgtagcaac-
tggcctccgacgttcg gccaagggaccaaggtggaaatcaaa V.sub.L amino acid
sequence (SEQ ID NO: 18) (including signal peptide in underlined
italics):
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK
PGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPPTF
GQGTKVEIK V.sub.L amino acid sequence (SEQ ID NO: 19) excluding
signal peptide:
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASSRATGIP
DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK V.sub.L CDR1 (SEQ
ID NO: 20): QSVSSY V.sub.L CDR2 (SEQ ID NO: 21): DAS V.sub.L CDR3
(SEQ ID NO: 22): QQRSNWPPT 2C2 VH (VH 3-33; D1-7; JH4) V.sub.H
nucleic acid sequence (SEQ ID NO: 23)
Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcaactggtgga-
gtctgggggaggcgtg
gtccagcctgggaggtccctgcgactctcctgtgcagcgtctggattcaccttcagtagctatgacatacactg-
ggtccgccaggctcc
aggcaaggggctggagtgggtggcagttatatggaatgatggaagtaataaatactatgcagactccgtgaagg-
gccgattcaccatc
tccagagacaattccacgaactcgctgtttctgcaaatgaacagcctgagagccgaggacacggctgtgtatta-
ttgtgtgggaggaac tgctgaccttgaacactgggaccagggaaccctggtcaccgtctcctca
V.sub.H amino acid sequence (SEQ ID NO: 24) (including signal
peptide in underlined italics):
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDIHWVR
QAPGKGLEWVAVIWNDGSNKYYADSVKGRFTISRDNSTNSLFLQMNSLRAEDTAVY
YCVGGTADLEHWDQGTLVTVSS V.sub.H amino acid sequence (SEQ ID NO: 25)
excluding signal peptide:
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDIHWVRQAPGKGLEWVAVIWNDGS
NKYYADSVKGRFTISRDNSTNSLFLQMNSLRAEDTAVYYCVGGTADLEHWDQGTL VTVSS
V.sub.H CDR1 (SEQ ID NO: 26): GFTFSSYD V.sub.H CDR2 (SEQ ID NO:
27): IWNDGSNK V.sub.H CDR3 (SEQ ID NO: 28): VGGTADLEHWDQ 2C2 VK (VK
1D-16; JK4) V.sub.L nucleic acid sequence (SEQ ID NO: 29)
Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac-
ccagtctccatcctc
actgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattagcagctggttagcct-
ggtatcagcagaaac
cagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggc-
agtggatctgggac
agatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatagtt-
accctctcactttcggcg gagggaccaaggtggagatcaaa V.sub.L amino acid
sequence (SEQ ID NO: 30) (including signal peptide in underlined
italics):
MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQ
KPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLT
FGGGTKVEIK V.sub.L amino acid sequence (SEQ ID NO: 31) excluding
signal peptide:
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK V.sub.L CDR1 (SEQ
ID NO: 32): QGISSW V.sub.L CDR2 (SEQ ID NO: 33): AAS V.sub.L CDR3
(SEQ ID NO: 34): QQYNSYPLT 1F5 VH (VH 3-33; D7-27; JH4) V.sub.H
nucleic acid sequence (SEQ ID NO: 35)
Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtgga-
gtctgggggaggcgt
ggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagttatgacatgcact-
gggtccgccaggctc
caggcaaggggctggagtgggtggcagttatatggtatgatggaagtaataaatactatgcagactccgtgaag-
ggccgattcaccat
ctccagagacaattccaagaacacgctgtatctccaaatgaacagcctgagagccgaggacacggctgtgtatt-
actgtgcgagaggt
agtggtaactggggtttctttgactactggggccagggaaccctggtcaccgtctcctca
V.sub.H amino acid sequence (SEQ ID NO: 36) (including signal
peptide in underlined italics):
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWV
RQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA
VYYCARGSGNWGFFDYWGQGTLVTVSS V.sub.H amino acid sequence (SEQ ID NO:
37) excluding signal peptide:
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDG
SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWG QGTLVTVSS
V.sub.H CDR1 (SEQ ID NO: 38): GFTFSSYD V.sub.H CDR2 (SEQ ID NO:
39): IWYDGSNK V.sub.H CDR3 (SEQ ID NO: 40): ARGSGNWGFFDY 1F5 VK #2
(VK 1D-16; JK1) V.sub.L nucleic acid sequence (SEQ ID NO: 41)
Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac-
ccagtctccatcctc
actgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattagcaggtggttagcct-
ggtatcagcagaaac
cagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggc-
agtggatctgggac
agatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatactt-
accctcggacgttcggcc aagggaccaaggtggaaatcaaa V.sub.L amino acid
sequence (SEQ ID NO: 42) (including signal peptide in underlined
italics):
MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQ
KPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRT
FGQGTKVEIK V.sub.L amino acid sequence (SEQ ID NO: 43) excluding
signal peptide:
DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK V.sub.L CDR1 (SEQ
ID NO: 44): QGISRW
V.sub.L CDR2 (SEQ ID NO: 45): AAS V.sub.L CDR3 (SEQ ID NO: 46):
QQYNTYPRT 1H8 VH (VH 3-33; D7-27; JH4) V.sub.H nucleic acid
sequence (SEQ ID NO: 47)
Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtgga-
gtctgggggaggcgt
ggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcaatatctatgacatgcact-
gggtccgccaggctc
caggcaaggggctggagtgggtggcagttatatggtatgatggaagtaatcaatactatgcagactccgtgaag-
ggccgattcaccat
ctccagagacaattccaagaacacgctgtatctgcaaatgaacattttgagagccgaggacacggctgtgtatt-
actgtgcgagaggta
ctcactgggggtactttgactactggggccagggaaccctggtcaccgtctcctca V.sub.H
amino acid sequence (SEQ ID NO: 48) (including signal peptide in
underlined italics):
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFNIYDMHWVR
QAPGKGLEWVAVIWYDGSNQYYADSVKGRFTISRDNSKNTLYLQMNILRAEDTAV
YYCARGTHWGYFDYWGQGTLVTVSS V.sub.H amino acid sequence (SEQ ID NO:
49) excluding signal peptide:
QVQLVESGGGVVQPGRSLRLSCAASGFTFNIYDMHWVRQAPGKGLEWVAVIWYDG
SNQYYADSVKGRFTISRDNSKNTLYLQMNILRAEDTAVYYCARGTHWGYFDYWGQ GTLVTVSS
V.sub.H CDR1 (SEQ ID NO: 50): GFTFNIYD V.sub.H CDR2 (SEQ ID NO:
51): IWYDGSNQ V.sub.H CDR3 (SEQ ID NO: 52): ARGTHWGYFDY 1H8 VK (VK
1D-16; JK1) V.sub.L nucleic acid sequence (SEQ ID NO: 53)
Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac-
ccagtctccatcctc
actgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattagcagctggttagcct-
ggtatcagcagaaac
cagagaaagcccctaagtccctgatctatgctgcatccaatttgcaaagtggggtcccatcaaggttcagcggc-
agtggatctgggac
agatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatagtt-
accctcggacgttcggcc aagggaccaaggtggaaatcaaa V.sub.L amino acid
sequence (SEQ ID NO: 54) (including signal peptide in underlined
italics):
MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQ
KPEKAPKSLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRT
FGQGTKVEIK V.sub.L amino acid sequence (SEQ ID NO: 55) excluding
signal peptide:
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASNLQSGV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK V.sub.L CDR1 (SEQ
ID NO: 56): QGISSW V.sub.L CDR2 (SEQ ID NO: 57): AAS V.sub.L CDR3
(SEQ ID NO: 58): QQYNSYPRT 1G5 VH (VH 3-33; D6-19; JH2) V.sub.H
nucleic acid sequence (SEQ ID NO: 59)
Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcaactggtgga-
gtctgggggaggcgtg
gtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcagcttcagtagctatggcatgcactg-
ggtccgccaggctcc
aggcaagggactggagtgggtggcacttctatggtatgatggtagccataaagactttgcagactccgtgaagg-
gccgattcaccatct
ccagagacaattccaagaacacgctagatctgcaaatgaacagcctgagagccgaggacacggctgtgtattac-
tgtgcgagagag
ggtttagcagtacctggtcactggtacttcgatctctggggccgtggcaccctggtcactgtctcctca
V.sub.H amino acid sequence (SEQ ID NO: 60) (including signal
peptide in underlined italics):
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVR
QAPGKGLEWVALLWYDGSHKDFADSVKGRFTISRDNSKNTLDLQMNSLRAEDTAV
YYCAREGLAVPGHWYFDLWGRGTLVTVSS V.sub.H amino acid sequence (SEQ ID
NO: 61) excluding signal peptide:
QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEWVALLWYDG
SHKDFADSVKGRFTISRDNSKNTLDLQMNSLRAEDTAVYYCAREGLAVPGHWYFDL
WGRGTLVTVSS V.sub.H CDR1 (SEQ ID NO: 62): GFSFSSYG V.sub.H CDR2
(SEQ ID NO: 63): LWYDGSHK V.sub.H CDR3 (SEQ ID NO: 64):
AREGLAVPGHWYFDL 1G5 VK (VK 1-13; JK1) V.sub.L nucleic acid sequence
(SEQ ID NO: 65)
Atgagggtccccgctcagctcctggggcttctgctgctctggctcccaggtgccagatgtgccatccagttgac-
ccagtctccatcctc
cctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagggcattagcagtgctttagcct-
ggtatcagcagaaac
cagggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggggtcccatcaaggttcagcggc-
agtggatctgggac
agatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagtttaatactt-
accctcggacgttcggcc aagggaccaaggtggaaatcaaa V.sub.L amino acid
sequence (SEQ ID NO: 66) (including signal peptide in underlined
italics):
MRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQK
PGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNTYPRTF
GQGTKVEIK V.sub.L amino acid sequence (SEQ ID NO: 67) excluding
signal peptide:
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNTYPRTFGQGTKVEIK V.sub.L CDR1 (SEQ
ID NO: 68): QGISSA V.sub.L CDR2 (SEQ ID NO: 69): DAS V.sub.L CDR3
(SEQ ID NO: 70): QQFNTYPRT 2G9 VH (VH 3-33; D1-7; JH4) V.sub.H
nucleic acid sequence (SEQ ID NO: 71)
Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagttggtgga-
gtctgggggaggcgtg
gtccagcctgggaggtccctgcgactctcctgtgcagcgtctggattcaccctcagtagccatgacatacactg-
ggtccgccaggctc
caggcaaggggctggagtgggtggcagttatatggaatgatggaagtaataaatactatgcagactccgtgaag-
ggccgattcaccat
ctccagagacaattccacgaactcgctgtttctgcaaatgaacagcctgagagccgaggacacggctgtgtatt-
attgtgtgagaggaa ctgctgaccttgaacactgggaccagggaaccctggtcaccgtctcctca
V.sub.H amino acid sequence (SEQ ID NO: 72) (including signal
peptide in underlined italics):
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTLSSHDIHWVR
QAPGKGLEWVAVIWNDGSNKYYADSVKGRFTISRDNSTNSLFLQMNSLRAEDTAVY
YCVRGTADLEHWDQGTLVTVSS V.sub.H amino acid sequence (SEQ ID NO: 73)
excluding signal peptide:
QVQLVESGGGVVQPGRSLRLSCAASGFTLSSHDIHWVRQAPGKGLEWVAVIWNDGS
NKYYADSVKGRFTISRDNSTNSLFLQMNSLRAEDTAVYYCVRGTADLEHWDQGTL VTVSS
V.sub.H CDR1 (SEQ ID NO: 74): GFTLSSHD V.sub.H CDR2 (SEQ ID NO:
75): IWNDGSNK V.sub.H CDR3 (SEQ ID NO: 76): VRGTADLEHWDQ 2G9 VK (VK
1D-16; JK4) V.sub.L nucleic acid sequence (SEQ ID NO: 77)
Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac-
ccagtctccatcctc
actgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattagcagctggttagcct-
ggtatcagcagaaac
cagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggc-
agtggatctgggac
agatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatagtt-
accctctcactttcggcg gagggaccaaggtggagatcaaa V.sub.L amino acid
sequence (SEQ ID NO: 78) (including signal peptide in underlined
italics):
MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQ
KPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLT
FGGGTKVEIK V.sub.L amino acid sequence (SEQ ID NO: 79) excluding
signal peptide:
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK V.sub.L CDR1 (SEQ
ID NO: 80): QGISSW V.sub.L CDR2 (SEQ ID NO: 81): AAS V.sub.L CDR3
(SEQ ID NO: 82): QQYNSYPLT 3A10 VH (VH 3-33; D3-10; JH3) V.sub.H
nucleic acid sequence (SEQ ID NO: 83)
Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtgga-
gtctgggggaggcgt
ggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtcattatggcatgcact-
gggtccgccaggctc
caggcaaggggccggagtgggtggcaattatatggtatgatggaagtaataaatactatgcagactccgtgaag-
ggccgattcaccat
ctccagagacaattccaagaacacgctggatctgcaaatgaacagcctgagagccgaggacacggctgtgtatt-
actgtgcgagaga
tggatggactactatggttcggggacttaatgatttgatatctggggccaagggacaatggtcaccgtctcttc-
a V.sub.H amino acid sequence (SEQ ID NO: 84) (including signal
peptide in underlined italics):
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSHYGMHWV
RQAPGKGPEWVAIIWYDGSNKYYADSVKGRFTISRDNSKNTLDLQMNSLRAEDTAV
YYCARDGWTTMVRGLNVFDIWGQGTMVTVSS V.sub.H amino acid sequence (SEQ ID
NO: 85) excluding signal peptide:
QVQLVESGGGVVQPGRSLRLSCAASGFTFSHYGMHWVRQAPGKGPEWVAIIWYDG
SNKYYADSVKGRFTISRDNSKNTLDLQMNSLRAEDTAVYYCARDGWTTMVRGLNV
FDIWGQGTMVTVSS V.sub.H CDR1 (SEQ ID NO: 86): GFTFSHYG V.sub.H CDR2
(SEQ ID NO: 87): IWYDGSNK V.sub.H CDR3 (SEQ ID NO: 88):
ARDGWTTMVRGLNVFDI 3A10 VK #1 (VK 1D-16; JK5) V.sub.L nucleic acid
sequence (SEQ ID NO: 89)
Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac-
ccagtctccatcctc
actgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcaggatattagcagctggttagcct-
ggtatcagcagaaac
cagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggc-
agtggatctgggac
agatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatagtt-
accctcccaccttcggcc aagggacacgactggagattaaa V.sub.L amino acid
sequence (SEQ ID NO: 90) (including signal peptide in underlined
italics):
MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQDISSWLAWYQQ
KPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPT
FGQGTRLEIK V.sub.L amino acid sequence (SEQ ID NO: 91) excluding
signal peptide:
DIQMTQSPSSLSASVGDRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGQGTRLEIK V.sub.L CDR1 (SEQ
ID NO: 92): QDISSW V.sub.L CDR2 (SEQ ID NO: 93): AAS V.sub.L CDR3
(SEQ ID NO: 94): QQYNSYPPT 3A10 VK #4 (VK 1-13; JK5) A futher light
chain was also found to be active as follows (though only the above
light chain (3H8-1B11 VK #1) was used in the above Examples):
V.sub.L nucleic acid sequence (SEQ ID NO: 95)
Atgagggtcctcgctcagctcctggggcttctgctgctctggctcccaggtgccagatgtgccatccagttgac-
ccagtctccatcctc
cctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagggcattagcagtgctttagcct-
ggtatcagcagaaac
cagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggc-
agtggatctgggac
agatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatagtt-
accctcccaccttcggcc aagggacacgactggagattaaa V.sub.L amino acid
sequence (SEQ ID NO: 96) (including signal peptide in underlined
italics):
MRVLAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQK
PEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTF
GQGTRLEIK V.sub.L amino acid sequence (SEQ ID NO: 97) excluding
signal peptide:
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPEKAPKSLIYAASSLQSGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGQGTRLEIK V.sub.L CDR1 (SEQ
ID NO: 98): QGISSA V.sub.L CDR2 (SEQ ID NO: 99): AAS V.sub.L CDR3
(SEQ ID NO: 100): QQYNSYPPT 3H12 VH (VH 3-33; D7-27; JH4) V.sub.H
nucleic acid sequence (SEQ ID NO: 101)
atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtgga-
gtctgggggaggcgtg
gtccagcctgggaggtccctgagactctcctgtgcaacgtctggattcaccttcagtagctatgacatgcactg-
ggtccgccaggctcc
aggcaaggggctggagtgggtggcagttatttggtatgatggaagtaataaatactatgcagactccgtgaagg-
gccgattcaccatct
ccagagacaattccaagaacacgctgtatctccaaatgaacagcctgggagacgaggacacggctgtgtattac-
tgtgcgagaggta
gtggtaactggggtttctttgactactggggccagggaaccctggtcaccgtctcctca V.sub.H
amino acid sequence (SEQ ID NO: 102) (including signal peptide in
underlined italics):
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCATSGFTFSSYDMHWVR
QAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLGDEDTAV
YYCARGSGNWGFFDYWGQGTLVTVSS V.sub.H amino acid sequence (SEQ ID NO:
103) excluding signal peptide: QVQLVESGGGVVQP
GRSLRLSCATSGFTFSSYDMHWVRQAPGKGLEWVAVIWYDG
SNKYYADSVKGRFTISRDNSKNTLYLQMNSLGDEDTAVYYCARGSGNWGFFDYWG QGTLVTVSS
V.sub.H CDR1 (SEQ ID NO: 104): GFTFSSYD V.sub.H CDR2 (SEQ ID NO:
105): IWYDGSNK V.sub.H CDR3 (SEQ ID NO: 106): ARGSGNWGFFDY 3H12 VK
#2 (VK 1D-16; JK1) V.sub.L nucleic acid sequence (SEQ ID NO: 107)
Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac-
ccagtctccatcctc
actgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattagcaggtggttagcct-
ggtatcagcagaaac
cagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggc-
agtggatctgggac
agatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatactt-
accctcggacgttcggcc aagggaccaaggtggaaatcaaa V.sub.L amino acid
sequence (SEQ ID NO: 108) (including signal peptide in underlined
italics):
MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQ
KPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRT
FGQGTKVEIK V.sub.L amino acid sequence (SEQ ID NO: 109) excluding
signal peptide:
DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK V.sub.L CDR1 (SEQ
ID NO: 110): QGISRW V.sub.L CDR2 (SEQ ID NO: 111): AAS V.sub.L CDR3
(SEQ ID NO: 112): QQYNTYPRT ALIGNMENTS ARE SHOWN IN FIGS. 15 AND
16
Example 10
In Vivo Non-Human Primate Study
[0366] To assess tolerance of anti-human CD27 mAb 1F5 in non-human
primates, 3 cynomolgus monkeys were treated with one i.v. dose of
1, 3 or 10 mg/kg of 1F5. Animals were followed for 29 days. Total
lymphocytes (based on side and forward scatter size), memory B
cells (CD20+ and CD95 bright), and monocytes (based on side and
forward scatter size) were stained with anti-human IgG antibody
(bold line) and compared to unstained controls (shaded histogram).
The results are shown in FIG. 17. These results show that the 1F5
mAb was bound to the surface of circulating lymphocytes know to
express CD27 for the entire duration of the study. Monocytes, cells
that do not express CD27 did not have 1F5 binding.
[0367] In addition, to determine the effect of 1F5 on circulating
lymphocyte populations, lymphocytes were stained with subset
markers and the % positive cells plotted vs time in FIG. 17 for
each animal treated at the different doses (square points=1 mg/kg;
circle points=3 mg/kg; triangle points=10 mg/kg). Results are shown
in FIG. 18.
[0368] Collectively, the results from these studies, shown in FIGS.
17 and 18, demonstrate that 1F5 was well tolerated and did not
significantly deplete circulating lymphocytes (other than some
transient depletion of NK cells) after a single 1-10 mg/kg dose. In
addition, there was no elevation in body temperatures and no
detectable levels of TNF-.alpha., IL-6 or IL-1.beta..
Example 11
[0369] Anti-CD27 mAbs Enhanced Antigen-Specific CD8+ T-Cell
Proliferation and Activation Pentamer Staining on Mouse Peripheral
Blood Cells and Splenocytes
[0370] Human CD27 transgenic mice (huCD27-Tg) were intravenously
injected with 5 mg of chicken ovalbumin and the panel of fully
human antibodies recognizing human CD27 generated as in Example 1
(CD27 human mAbs). Rat anti-mouse CD27 clone AT124 and irrelevant
human IgG1 were included as positive and negative controls,
respectively. Each antibody (250 .mu.g) was co-injected with
ovalbumin on day 0 and an additional 250 .mu.g of antibody alone on
day 1. Peripheral blood and spleen cells were harvested on day 7.
Splenocytes (1.times.10.sup.6) or whole blood (100 .mu.l) were used
for staining. After Fc-receptor blocking, cells were stained with
10 .mu.l of H-2Kb/SIINFEKL, a tetrameric complex of mouse MHC
complexed with the peptide T cell epitope from ovalbumin (Beckman
Coulter), or a similar pentameric complex (ProImmune), anti-CD8
(eBioscience) and anti-huCD27 mAb or anti-msCD27 mAb (BD
Biosciences) at room temperature for 30 minutes. Cells were then
RBC-lysed, washed and fixed, and at least 100,000 events were
acquired in Flow Cytometer LSR (BD Biosciences). The fraction of
tetramer- or pentamer-positive cells in the CD8.sup.+ or CD27.sup.+
gated population was determined. Results are shown in FIGS. 19 and
21 from which it can be seen that the anti-CD27 antibodies
significantly enhanced immune responses.
Example 12
ELISPOT Assay
[0371] Splenocytes (2.5.times.10.sup.5 and 0.5.times.10.sup.5) from
the pentamer staining preparation of Example 8 above were placed on
an anti-IFN.gamma. monoclonal antibody (mAb) coated plate in
triplicate wells after RBC lysis. SIINFEKL peptide was added at a
final concentration of 2 .mu.g/ml. A background control was set up
for each sample in triplicate in the absence of peptide. The
stimulation was maintained at 37.degree. C. in a tissue culture
incubator overnight. ELISPOT detection was performed using an
ELISPOT kit (BD Biosciences) following the manufacturer's protocol.
IFN.gamma.-spot number was counted. Results are shown in FIGS. 20
and 21, from which it can be seen that anti-CD27 mAbs significantly
enhanced T-cell activity.
Example 13
Anti-CD27 mAb Enhances T Cell Responses to a Vaccine Antigen
[0372] HuCD27 transgenic mice were immunized with 5 .mu.g (s.c.) of
the APC-targeted vaccine comprising an anti-mouse DEC-205 IgG
antibody fused to ovalbumin (OVA) (referred to as
.alpha.-mDEC-205-OVA), in combination with the anti-CD27 human mAb
1F5 (i.p.) at various doses (25, 50, 100, 200 or 400 .mu.g). One
week later splenocytes were analyzed for CD8+ T cell reactivity to
the OVA SIINFEKL peptide (OVA peptide 257-264) by tetramer staining
(% tetramer positive of all CD8+ shown) and IFN-.gamma. ELISPOT by
the procedure as generally described in Examples 8 and 9
respectively. The results are shown in FIGS. 22A-C, wherein FIG.
22A shows the protocol used, FIG. 22B shows the results of the
tetramer staining experiment and FIG. 22C shows the results of the
IFN-gamma ELISPOT experiment. These results indicate that the
co-administered human mAb 1F5 significantly enhanced the T-cell
responses to the vaccine component administered.
Example 14
Synergistic Effect of Anti-CD27 mAb (1F5) and TLR Agonist (Poly IC)
on T Cell Responses to Vaccine (Anti-DEC205-OVA)
[0373] huCD27-Tg (transgenic) and wild-type (WT) mouse littermates
were intraperitoneally injected with anti-CD27 mAb 1F5 (50 .mu.g)
on day -3, and then subcutaneously via 4 paws injected with
anti-mDec205-OVA (5 .mu.g) plus Poly IC 0, 25, 50 or 100 .mu.g on
day 0. Spleens were collected on day 7 and assessed by tetramer
staining, IFN.gamma.-ELISPOT and IFN.gamma.-ICS. Mean.+-.SD of
positive IFN.gamma.-ICS among gated CD8 T cells from 3 mice per
group were calculated and a panel of representative Dot Plots was
collected. The results, shown in FIGS. 23A-D, indicate that the the
anti-CD27 human mAb acted synergistically with TLR3 agonist Poly IC
to enhance the T-cell responses to the vaccine component
administered.
Example 15
Administration of Anti-CD27 mAb Prior to Dec205-Targeted Vaccine in
the Absence or Presence of TLR Agonist
[0374] huCD27-Tg mice and wild type (WT) littermates were
intraperitoneally injected with anti-CD27 mAb (50 .mu.g) on various
days related to vaccine as indicated in FIG. 28, and subcutaneously
via 4 paws injected with anti-mDec205-OVA (5 .mu.g) plus or minus
TLR agonist Poly IC-LC (20 .mu.g) on day 0. Spleens were collected
on day 7 and assessed by tetramer staining and IFN.gamma.-ELISPOT.
A representative panel of tetramer staining among gated CD8 T cells
is shown in FIGS. 24 and 25. IFN.gamma.-ELISPOT showed a similar
pattern.
[0375] These results show that, surprisingly, when anti-CD27
antibody is administered in combination with a vaccine in the
presence or absence of TLR agonist, T-cell activation is greater
when the antibody is administered before the vaccine, for example a
day or more before the vaccine (antigen) is administered.
Example 16
[0376] Anti-CD27 mAb Combined with TCR Activation Activates T-Cells
from Human CD27 Transgenic Mice
[0377] To assess the T cell activation capability of 1F5 mAb, T
cells were purified from spleen of hCD27-Tg mice by negative
selection with beads. Cells were labeled with CFSE and incubated
with 0.2 .mu.g/ml of anti-CD27 human mAb 1F5 or isotype control for
3 days. The cross-linking anti-human IgG was passed through an
endotoxin removal column before use. IFN.gamma.-ICS and CSFE
dilution among CD8 and CD4 T cells are shown in FIGS. 26 and 27.
TNFa-ICS showed the same pattern as IFNg. As shown in FIGS. 26 and
27, when combined with TCR activation, 1F5 mAb effectively induces
proliferation and cytokine production from T cells in vitro. The
data show that both cross-linking with anti-human IgG and T cell
receptor activation with anti-CD3 mAb were required for 1F5 induced
proliferation and cytokine production.
Example 17
Anti-CD27 mAb Enhances the Efficacy of Vaccine in a MO4 Melanoma
Challenge Model
[0378] To assess in vivo anti-tumor activity, huCD27-Tg and WT
control, 8 mice per group, were subcutaneously inoculated with
0.3.times.10.sup.5 MO4 cells on day 0. On day 5 and 12, these mice
were intraperitoneally injected with anti-CD27 mAb 1F5 (50 .mu.g);
on day 8 and 15, additional doses of CD27 HuMab (50 .mu.g) and
vaccinated with anti-mDec205-OVA (5 .mu.g). Tumor growth was
measured with calipers 2 times a week. Results are shown in FIG.
24.
[0379] In a further study, huCD27-Tg and WT control, 5 mice per
group, were subcutaneously inoculated with 1.times.10.sup.5 MO4
cells on day 0. On day 5 and 12, these mice were vaccinated with
anti-mDec205-OVA (5 .mu.g) plus the TLR agonist Poly IC-LC (10
.mu.g) intraperitoneally. On days 6 and 13, mice were injected with
anti-CD27 mAb 1F5 (50 .mu.g). Tumor growth was measured with
calipers 2 times a week as indicated in the protocol illustrated in
FIG. 285A. The results, shown in FIGS. 28B, C and D, show the
effect of no treatment (FIG. 28B), vaccine treatment alone (FIG.
28C), or vaccine treatment in combination with anti-CD27 treatment
(FIG. 28D) on tumor size in the mice as a function of the number of
days post tumor inoculation. The results demonstrate that the
combination treatment with anti-CD27 mAb (1F5) significantly
prolonged survival of tumor challenged mice.
Example 18
1F5 Exhibits Potent Anti-Tumor Activity in a Syngeneic Transgenic
Mouse Tumor Challenge Model of the BCL.sub.1 B-Lymphoma
[0380] To assess in vivo anti-tumor activity of 1F5, groups of 9-10
hCD27 transgenic mice (Balb/c background) were challenged with
10.sup.7 BCL.sub.1 B-lymphoma cells administered intravenously on
Day 0. Animals were then treated with 5 doses of anti-human CD27
mAb 1F5 as indicated. As shown in FIGS. 29A and 29B, significantly
prolonged survival of tumor challenged mice, in a dose dependent
manner.
Example 19
Tumor Killing in Raji Xenograph SCID Mouse Model
[0381] CB.17 SCID mice (purchased from Taconic) were maintained in
a pathogen-free mouse facility. Lymphoma Raji cells
(1.times.10.sup.5) were subcutaneously injected into SCID mice, 4
mice per group. On day 6, these mice were treated with CD27 human
mAbs via intraperitoneal administration, 0.5 mg per dose and dosed
twice a week for 3 weeks. Tumor growth was measured with calipers 3
times a week. Results of tumor growth and Kaplan-Meier analysis are
shown in FIGS. 30A and 30B, from which is can be seen that the
anti-CD27 mAbs significantly prolonged survival of the tumor
challenged mice. In a further experiment, CB.17 SCID mice
(purchased from Taconic) were maintained in a pathogen-free mouse
facility. Human lymphoma Raji cells (5.times.10.sup.5) were
subcutaneously injected into SCID mice on day 0, 6 mice per group.
On day 5, these mice were treated with anti-CD27 mAb 1F5 via
intraperitoneal administration, 0.033, 0.1 or 0.3 mg per dose and
dosed twice a week for 3 weeks. Tumor growth was measured with
calipers 2 times a week.
[0382] The results, shown in FIG. 31A, indicate that the anti-CD27
mAb (1F5) significantly inhibited tumor growth and thus
significantly prolonged survival of the tumor challenged mice. A
Kaplan-Meier survival plot of the data is also provided in FIG.
31B, which shows that median survival was increased by at least 10
days in mice from the treated group compared to the control group.
A further xenograft experiment was conducted with 1G5 and results
are shown in FIG. 32.
Example 20
Tumor Killing in Daudi Xenograft SCID Mouse Model
[0383] CB.17 SCID mice (purchased from Taconic) were maintained in
a pathogen-free mouse facility. Human lymphoma Daudi cells
(1.times.10.sup.6) were subcutaneously injected into SCID mice on
day 0, 6 mice per group. On day 5, these mice were treated with
anti-CD27 human mAb 1F5 via intraperitoneal administration, 0.033,
0.1 or 0.3 mg per dose and dosed twice a week for 3 weeks. Tumor
growth was measured with calipers 2 times a week.
[0384] The results, shown in FIG. 33, indicate that the anti-CD27
mAb (1F5) significantly inhibited tumor gowth (FIG. 33A) and thus
significantly prolonged survival of the tumor challenged mice
(Kaplan-Meier plot in FIG. 33B).
Example 21
Anti-CD27 mAb Engineered to Not Bind Fc Receptors Does Not Enhance
T Cell Responses to a Vaccine Antigen
[0385] HuCD27 transgenic mice were immunized with 5 .mu.g (s.c.) of
APC-targeted vaccine comprising an anti-mouse DEC-205 IgG antibody
fused to ovalbumin (OVA) (referred to as .alpha.-mDEC-205-OVA), in
combination with the anti-CD27 human mAb 1F5 (i.p.) or mAb 1F5
mutant (Fc portion mutated to prevent Fc receptor binding) or
contol IgG mAb. One week later, splenocytes were analyzed for CD8+
T cell reactivity to the OVA SIINFEKL peptide (OVA peptide 257-264)
by IFN-.gamma. ELISPOT by the procedure as generally described.
[0386] The results, shown in FIG. 34, demonstrate that the altered
human mAb 1F5 does not enhance the T-cell responses to the vaccine,
and thus would be an effective agent for blocking the CD70/CD27
pathway.
Equivalents
[0387] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
TABLE-US-00008 SUMMARY OF SEQUENCE LISTING SEQ ID NO: DESCRIPTION 1
Human CD27 (GenBank Accession No.: AAH12160.1) 2 Human CD70
(GenBank Accession No.: NP 001243) 3 VH 3-33 germline sequence is
provided (Genbank Accession No AAP44382) 4 VH 3-7 germline sequence
is provided (Genbank Accession No AAP44389) 5 3H8-1B11 VH nucleic
acid 6 3H8-1B11 VH amino acid including signal peptide 7 3H8-1B11
VH "mature" amino acid excluding signal peptide 8 3H8-1B11 VH CDR1
amino acid 9 3H8-1B11 VH CDR2 amino acid 10 3H8-1B11 VH CDR3 amino
acid 11 3H8-1B11 VL #2 nucleic acid 12 3H8-1B11 VL #2 amino acid
including signal peptide 13 3H8-1B11 VL #2 "mature" amino acid
excluding signal peptide 14 3H8-1B11 VL #2 CDR1 amino acid 15
3H8-1B11 VL #2 CDR2 amino acid 16 3H8-1B11 VL #2 CDR3 amino acid 17
3H8-1B11 VL #3 nucleic acid 18 3H8-1B11 VL #3 amino acid including
signal peptide 19 3H8-1B11 VL #3 "mature" amino acid excluding
signal peptide 20 3H8-1B11 VL #3 CDR1 amino acid 21 3H8-1B11 VL #3
CDR2 amino acid 22 3H8-1B11 VL #3 CDR3 amino acid 23 2C2-1A10 VH
nucleic acid 24 2C2-1A10 VH amino acid including signal peptide 25
2C2-1A10 VH "mature" amino acid excluding signal peptide 26
2C2-1A10 VH CDR1 amino acid 27 2C2-1A10 VH CDR2 amino acid 28
2C2-1A10 VH CDR3 amino acid 29 2C2-1A10 VL nucleic acid 30 2C2-1A10
VL amino acid including signal peptide 31 2C2-1A10 VL "mature"
amino acid excluding signal peptide 32 2C2-1A10 VL CDR1 amino acid
33 2C2-1A10 VL CDR2 amino acid 34 2C2-1A10 VL CDR3 amino acid 35
1F5-1H5 VH nucleic acid 36 1F5-1H5 VH amino acid including signal
peptide 37 1F5-1H5 VH "mature" amino acid excluding signal peptide
38 1F5-1H5 VH CDR1 amino acid 39 1F5-1H5 VH CDR2 amino acid 40
1F5-1H5 VH CDR3 amino acid 41 1F5-1H5 VL #2 nucleic acid 42 1F5-1H5
VL #2 amino acid including signal peptide 43 1F5-1H5 VL #2 "mature"
amino acid excluding signal peptide 44 1F5-1H5 VL #1 CDR1 amino
acid 45 1F5-1H5 VL #2 CDR2 amino acid 46 1F5-1H5 VL #2 CDR3 amino
acid 47 1H8-B4 VH nucleic acid 48 1H8-B4 VH amino acid including
signal peptide 49 1H8-B4 VH "mature" amino acid excluding signal
peptide 50 1H8-B4 VH CDR1 amino acid 51 1H8-B4 VH CDR2 amino acid
52 1H8-B4 VH CDR3 amino acid 53 1H8-B4 VL nucleic acid 54 1H8-B4 VL
amino acid including signal peptide 55 1H8-B4 VL "mature" amino
acid excluding signal peptide 56 1H8-B4 VL CDR1 amino acid 57
1H8-B4 VL CDR2 amino acid 58 1H8-B4 VL CDR3 amino acid 59 1G5-1B9
VH nucleic acid 60 1G5-1B9 VH amino acid including signal peptide
61 1G5-1B9 VH "mature" amino acid excluding signal peptide 62
1G5-1B9 VH CDR1 amino acid 63 1G5-1B9 VH CDR2 amino acid 64 1G5-1B9
VH CDR3 amino acid 65 1G5-1B9 VL nucleic acid 66 1G5-1B9 VL amino
acid including signal peptide 67 1G5-1B9 VL "mature" amino acid
excluding signal peptide 68 1G5-1B9 VL CDR1 amino acid 69 1G5-1B9
VL CDR2 amino acid 70 1G5-1B9 VL CDR3 amino acid 71 2G9-1D11 VH
nucleic acid 72 2G9-1D11 VH amino acid including signal peptide 73
2G9-1D11 VH "mature" amino acid excluding signal peptide 74
2G9-1D11 VH CDR1 amino acid 75 2G9-1D11 VH CDR2 amino acid 76
2G9-1D11 VH CDR3 amino acid 77 2G9-1D11 VL nucleic acid 78 2G9-1D11
VL amino acid including signal peptide 79 2G9-1D11 VL "mature"
amino acid excluding signal peptide 80 2G9-1D11 VL CDR1 amino acid
81 2G9-1D11 VL CDR2 amino acid 82 2G9-1D11 VL CDR3 amino acid 83
3A10-1G10 VH nucleic acid 84 3A10-1G10 VH amino acid including
signal peptide 85 3A10-1G10 VH "mature" amino acid excluding signal
peptide 86 3A10-1G10 VH CDR1 amino acid 87 3A10-1G10 VH CDR2 amino
acid 88 3A10-1G10 VH CDR3 amino acid 89 3A10-1G10 VL #1 nucleic
acid 90 3A10-1G10 VL #1 amino acid including signal peptide 91
3A10-1G10 VL #1 "mature" amino acid excluding signal peptide 92
3A10-1G10 VL #1 CDR1 amino acid 93 3A10-1G10 VL #1 CDR2 amino acid
94 3A10-1G10 VL #1 CDR3 amino acid 95 3A10-1G10 VL #4 nucleic acid
96 3A10-1G10 VL #4 amino acid including signal peptide 97 3A10-1G10
VL +1904 "mature" amino acid excluding signal peptide 98 3A10-1G10
VL #4 CDR1 amino acid 99 3A10-1G10 VL #4 CDR2 amino acid 100
3A10-1G10 VL #4 CDR3 amino acid 101 3H12-1E12 VH nucleic acid 102
3H12-1E12 VH amino acid including signal peptide 103 3H12-1E12 VH
"mature" amino acid excluding signal peptide 104 3H12-1E12 VH CDR1
amino acid 105 3H12-1E12 VH CDR2 amino acid 106 3H12-1E12 VH CDR3
amino acid 107 3H12-1E12 VL #2 nucleic acid 108 3H12-1E12 VL #2
amino acid including signal peptide 109 3H12-1E12 VL #2 "mature"
amino acid excluding signal peptide 110 3H12-1E12 VL #2 CDR1 amino
acid 111 3H12-1E12 VL #2 CDR2 amino acid 112 3H12-1E12 VL #2 CDR3
amino acid 113 VH CDR3 consensus 114 VL CDR3 consensus 115 VH CDR2
consensus 116 VL CDR2 consensus 117 VH CDR1 consensus 118 VL CDR1
consensus
Sequence CWU 1
1
1201260PRTHomo sapiens 1Met Ala Arg Pro His Pro Trp Trp Leu Cys Val
Leu Gly Thr Leu Val1 5 10 15Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser
Cys Pro Glu Arg His Tyr 20 25 30Trp Ala Gln Gly Lys Leu Cys Cys Gln
Met Cys Glu Pro Gly Thr Phe 35 40 45Leu Val Lys Asp Cys Asp Gln His
Arg Lys Ala Ala Gln Cys Asp Pro 50 55 60Cys Ile Pro Gly Val Ser Phe
Ser Pro Asp His His Thr Arg Pro His65 70 75 80Cys Glu Ser Cys Arg
His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys 85 90 95Thr Ile Thr Ala
Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys 100 105 110Arg Asp
Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu 115 120
125Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His
130 135 140Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly
His Met145 150 155 160Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala
Arg Thr Leu Ser Thr 165 170 175His Trp Pro Pro Gln Arg Ser Leu Cys
Ser Ser Asp Phe Ile Arg Ile 180 185 190Leu Val Ile Phe Ser Gly Met
Phe Leu Val Phe Thr Leu Ala Gly Ala 195 200 205Leu Phe Leu His Gln
Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser 210 215 220Pro Val Glu
Pro Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu225 230 235
240Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro
245 250 255Ala Cys Ser Pro 2602193PRTHomo sapiens 2Met Pro Glu Glu
Gly Ser Gly Cys Ser Val Arg Arg Arg Pro Tyr Gly1 5 10 15Cys Val Leu
Arg Ala Ala Leu Val Pro Leu Val Ala Gly Leu Val Ile 20 25 30Cys Leu
Val Val Cys Ile Gln Arg Phe Ala Gln Ala Gln Gln Gln Leu 35 40 45Pro
Leu Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His 50 55
60Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala65
70 75 80Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln
Leu 85 90 95Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val
Thr Leu 100 105 110Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His
Pro Thr Thr Leu 115 120 125Ala Val Gly Ile Cys Ser Pro Ala Ser Arg
Ser Ile Ser Leu Leu Arg 130 135 140Leu Ser Phe His Gln Gly Cys Thr
Ile Ala Ser Gln Arg Leu Thr Pro145 150 155 160Leu Ala Arg Gly Asp
Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu 165 170 175Pro Ser Arg
Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg 180 185
190Pro397PRTHomo sapiens 3Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg Ser1 5 10 15Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Thr Tyr Gly 20 25 30Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala 35 40 45Ile Ile Trp Phe Asp Gly Ser
Asn Thr Tyr Tyr Ala Asp Ser Val Arg 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Ser Ser Arg Lys Thr Leu Tyr Leu65 70 75 80Glu Met Lys Ser
Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95Lys497PRTHomo sapiens 4Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser1 5 10 15Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asn Ser Tyr 20 25 30Met Thr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala 35 40 45Asn Ile Lys Pro Asp Gly Ser
Asp Lys Asn Tyr Ile Asn Ser Val Arg 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Glu Lys Ser Ser Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Val 85 90
95Thr5414DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 5atggagttgg ggctgagctg ggttttcctt
gttgctattt tagaaggtgt ccagtgtgag 60gtgcagctgg tggagtctgg gggaggcttg
gtccagcctg gggggtccct gagactctcc 120tgtgcagcct ctggattcac
ctttagtagt tattggatgg cctgggtccg ccaggctcca 180gggaaagggc
tggagtggct gggcaatata aagcaagatg gaagtgagaa atactatgtg
240gactctgtga agggccgatt caccatctcc agagacaacg ccaagaactc
actgtatcta 300caaatgaaca gcctgagagc cgaggacacg gctgtgtatt
actgtgtgag ggaactgggg 360atggactggt acttcgatct ctggggccgt
ggcaccctgg tcactgtctc ctca 4146138PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 6Met Glu Leu Gly Leu
Ser Trp Val Phe Leu Val Ala Ile Leu Glu Gly1 5 10 15Val Gln Cys Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45Ser Ser
Tyr Trp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu
Trp Leu Gly Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val65 70 75
80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
85 90 95Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val 100 105 110Tyr Tyr Cys Val Arg Glu Leu Gly Met Asp Trp Tyr Phe
Asp Leu Trp 115 120 125Gly Arg Gly Thr Leu Val Thr Val Ser Ser 130
1357119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ala Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Asn Ile Lys Gln Asp Gly Ser
Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Glu Leu
Gly Met Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 11588PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Gly Phe Thr Phe Ser Ser Tyr
Trp1 598PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Ile Lys Gln Asp Gly Ser Glu Lys1
51012PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Val Arg Glu Leu Gly Met Asp Trp Tyr Phe Asp
Leu1 5 1011381DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 11atggaagccc cagctcagct
tctcttcctc ctgctactct ggctcccaga taccaccgga 60gaaattgtgt tgacacagtc
tccagccacc ctgtctttgt ctccagggga aagagccacc 120ctctcctgca
gggccagtca gagtgttgac agctacttag cctggtacca acagaaacct
180ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 240aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcaa cctagagcct 300gaagattttg cagtttatta ctgtcagcag
cgtagcaact ggcctccgac gttcggccaa 360gggaccaagg tggaaatcaa a
38112127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu
Leu Leu Trp Leu Pro1 5 10 15Asp Thr Thr Gly Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser 20 25 30Leu Ser Pro Gly Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser 35 40 45Val Asp Ser Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60Arg Leu Leu Ile Tyr Asp Ala
Ser Asn Arg Ala Thr Gly Ile Pro Ala65 70 75 80Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Asn Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser 100 105 110Asn Trp
Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120
12513107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Asp Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Asn Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105146PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Gln
Ser Val Asp Ser Tyr1 5153PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 15Asp Ala
Ser1169PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Gln Gln Arg Ser Asn Trp Pro Pro Thr1
517381DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 17atggaagccc cagctcagct tctcttcctc
ctgctactct ggctcccaga taccaccgga 60gaaattgtgt tgacacagtc tccagccacc
ctgtctttgt ctccagggga aagagccacc 120ctctcctgca gggccagtca
gagtgttagc agctacttag cctggtacca acagaaacct 180ggccaggctc
ccaggctcct catctatgat gcatccagca gggccactgg catcccagac
240aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag
actggagcct 300gaagattttg cagtgtatta ctgtcagcag cgtagcaact
ggcctccgac gttcggccaa 360gggaccaagg tggaaatcaa a
38118127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 18Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu
Leu Leu Trp Leu Pro1 5 10 15Asp Thr Thr Gly Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser 20 25 30Leu Ser Pro Gly Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser 35 40 45Val Ser Ser Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60Arg Leu Leu Ile Tyr Asp Ala
Ser Ser Arg Ala Thr Gly Ile Pro Asp65 70 75 80Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Arg Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser 100 105 110Asn Trp
Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120
12519107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 19Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105206PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Gln
Ser Val Ser Ser Tyr1 5213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 21Asp Ala
Ser1229PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22Gln Gln Arg Ser Asn Trp Pro Pro Thr1
523405DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 23atggagtttg ggctgagctg ggttttcctc
gttgctcttt taagaggtgt ccagtgtcag 60gtgcaactgg tggagtctgg gggaggcgtg
gtccagcctg ggaggtccct gcgactctcc 120tgtgcagcgt ctggattcac
cttcagtagc tatgacatac actgggtccg ccaggctcca 180ggcaaggggc
tggagtgggt ggcagttata tggaatgatg gaagtaataa atactatgca
240gactccgtga agggccgatt caccatctcc agagacaatt ccacgaactc
gctgtttctg 300caaatgaaca gcctgagagc cgaggacacg gctgtgtatt
attgtgtggg aggaactgct 360gaccttgaac actgggacca gggaaccctg
gtcaccgtct cctca 40524135PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 24Met Glu Phe Gly Leu Ser
Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15Val Gln Cys Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30Pro Gly Arg Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45Ser Ser Tyr
Asp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp
Val Ala Val Ile Trp Asn Asp Gly Ser Asn Lys Tyr Tyr Ala65 70 75
80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Thr Asn
85 90 95Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val 100 105 110Tyr Tyr Cys Val Gly Gly Thr Ala Asp Leu Glu His Trp
Asp Gln Gly 115 120 125Thr Leu Val Thr Val Ser Ser 130
13525116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 25Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Asp Ile His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Asn Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Thr Asn Ser Leu Phe65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Gly Gly Thr
Ala Asp Leu Glu His Trp Asp Gln Gly Thr Leu Val 100 105 110Thr Val
Ser Ser 115268PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 26Gly Phe Thr Phe Ser Ser Tyr Asp1
5278PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Ile Trp Asn Asp Gly Ser Asn Lys1
52812PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Val Gly Gly Thr Ala Asp Leu Glu His Trp Asp
Gln1 5 1029381DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 29atgagggtcc tcgctcagct
cctggggctc ctgctgctct gtttcccagg tgccagatgt 60gacatccaga tgacccagtc
tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 120atcacttgtc
gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca
180gagaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 240aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 300gaagattttg caacttatta ctgccaacag
tataatagtt accctctcac tttcggcgga 360gggaccaagg tggagatcaa a
38130127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 30Met Arg Val Leu Ala Gln Leu Leu Gly Leu Leu
Leu Leu Cys Phe Pro1 5 10 15Gly Ala Arg Cys Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly 35 40 45Ile Ser Ser Trp Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro 50 55 60Lys Ser Leu Ile Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn 100 105 110Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 115 120 12531107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 31Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105326PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Gln Gly Ile Ser Ser Trp1 5333PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 33Ala
Ala Ser1349PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Gln Gln Tyr Asn Ser Tyr Pro Leu Thr1
535414DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 35atggagtttg ggctgagctg ggttttcctc
gttgctcttt taagaggtgt ccagtgtcag 60gtgcagctgg tggagtctgg gggaggcgtg
gtccagcctg ggaggtccct gagactctcc 120tgtgcagcgt ctggattcac
cttcagtagt tatgacatgc actgggtccg ccaggctcca 180ggcaaggggc
tggagtgggt ggcagttata tggtatgatg gaagtaataa atactatgca
240gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac
gctgtatctc 300caaatgaaca gcctgagagc cgaggacacg gctgtgtatt
actgtgcgag aggtagtggt 360aactggggtt tctttgacta ctggggccag
ggaaccctgg tcaccgtctc ctca 41436138PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
36Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1
5 10 15Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln 20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe 35 40 45Ser Ser Tyr Asp Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 50 55 60Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn
Lys Tyr Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn 85 90 95Thr Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Gly Ser
Gly Asn Trp Gly Phe Phe Asp Tyr Trp 115 120 125Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 130 13537119PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 37Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val
Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115388PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Gly
Phe Thr Phe Ser Ser Tyr Asp1 5398PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 39Ile Trp Tyr Asp Gly Ser
Asn Lys1 54012PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 40Ala Arg Gly Ser Gly Asn Trp Gly Phe
Phe Asp Tyr1 5 1041381DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 41atgagggtcc
tcgctcagct cctggggctc ctgctgctct gtttcccagg tgccagatgt 60gacatccaga
tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc
120atcacttgtc gggcgagtca gggtattagc aggtggttag cctggtatca
gcagaaacca 180gagaaagccc ctaagtccct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 240aggttcagcg gcagtggatc tgggacagat
ttcactctca ccatcagcag cctgcagcct 300gaagattttg caacttatta
ctgccaacag tataatactt accctcggac gttcggccaa 360gggaccaagg
tggaaatcaa a 38142127PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 42Met Arg Val Leu Ala Gln
Leu Leu Gly Leu Leu Leu Leu Cys Phe Pro1 5 10 15Gly Ala Arg Cys Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly 35 40 45Ile Ser Arg
Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro 50 55 60Lys Ser
Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser65 70 75
80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn 100 105 110Thr Tyr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 115 120 12543107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 43Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Trp 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Arg
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105446PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Gln Gly Ile Ser Arg Trp1 5453PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Ala
Ala Ser1469PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Gln Gln Tyr Asn Thr Tyr Pro Arg Thr1
547411DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 47atggagtttg ggctgagctg ggttttcctc
gttgctcttt taagaggtgt ccagtgtcag 60gtgcagctgg tggagtctgg gggaggcgtg
gtccagcctg ggaggtccct gagactctcc 120tgtgcagcgt ctggattcac
cttcaatatc tatgacatgc actgggtccg ccaggctcca 180ggcaaggggc
tggagtgggt ggcagttata tggtatgatg gaagtaatca atactatgca
240gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac
gctgtatctg 300caaatgaaca ttttgagagc cgaggacacg gctgtgtatt
actgtgcgag aggtactcac 360tgggggtact ttgactactg gggccaggga
accctggtca ccgtctcctc a 41148137PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 48Met Glu Phe Gly Leu
Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15Val Gln Cys Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30Pro Gly Arg
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45Asn Ile
Tyr Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu
Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Gln Tyr Tyr Ala65 70 75
80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
85 90 95Thr Leu Tyr Leu Gln Met Asn Ile Leu Arg Ala Glu Asp Thr Ala
Val 100 105 110Tyr Tyr Cys Ala Arg Gly Thr His Trp Gly Tyr Phe Asp
Tyr Trp Gly 115 120 125Gln Gly Thr Leu Val Thr Val Ser Ser 130
13549118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 49Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Ile Tyr 20 25 30Asp Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Ser
Asn Gln Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ile
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Thr
His Trp Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val
Thr Val Ser Ser 115508PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 50Gly Phe Thr Phe Asn Ile Tyr
Asp1 5518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Ile Trp Tyr Asp Gly Ser Asn Gln1
55211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Ala Arg Gly Thr His Trp Gly Tyr Phe Asp Tyr1 5
1053381DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 53atgagggtcc tcgctcagct cctggggctc
ctgctgctct gtttcccagg tgccagatgt 60gacatccaga tgacccagtc tccatcctca
ctgtctgcat ctgtaggaga cagagtcacc 120atcacttgtc gggcgagtca
gggtattagc agctggttag cctggtatca gcagaaacca 180gagaaagccc
ctaagtccct gatctatgct gcatccaatt tgcaaagtgg ggtcccatca
240aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 300gaagattttg caacttatta ctgccaacag tataatagtt
accctcggac gttcggccaa 360gggaccaagg tggaaatcaa a
38154127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 54Met Arg Val Leu Ala Gln Leu Leu Gly Leu Leu
Leu Leu Cys Phe Pro1 5 10 15Gly Ala Arg Cys Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly 35 40 45Ile Ser Ser Trp Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro 50 55 60Lys Ser Leu Ile Tyr Ala Ala
Ser Asn Leu Gln Ser Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn 100 105 110Ser Tyr
Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120
12555107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 55Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu
Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Asn Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Arg 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105566PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 56Gln
Gly Ile Ser Ser Trp1 5573PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 57Ala Ala
Ser1589PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 58Gln Gln Tyr Asn Ser Tyr Pro Arg Thr1
559423DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 59atggagtttg ggctgagctg ggttttcctc
gttgctcttt taagaggtgt ccagtgtcag 60gtgcaactgg tggagtctgg gggaggcgtg
gtccagcctg ggaggtccct gagactctcc 120tgtgcagcgt ctggattcag
cttcagtagc tatggcatgc actgggtccg ccaggctcca 180ggcaagggac
tggagtgggt ggcacttcta tggtatgatg gtagccataa agactttgca
240gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac
gctagatctg 300caaatgaaca gcctgagagc cgaggacacg gctgtgtatt
actgtgcgag agagggttta 360gcagtacctg gtcactggta cttcgatctc
tggggccgtg gcaccctggt cactgtctcc 420tca 42360141PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
60Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1
5 10 15Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln 20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ser Phe 35 40 45Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 50 55 60Glu Trp Val Ala Leu Leu Trp Tyr Asp Gly Ser His
Lys Asp Phe Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn 85 90 95Thr Leu Asp Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Glu Gly
Leu Ala Val Pro Gly His Trp Tyr Phe 115 120 125Asp Leu Trp Gly Arg
Gly Thr Leu Val Thr Val Ser Ser 130 135 14061122PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
61Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser
Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Leu Leu Trp Tyr Asp Gly Ser His Lys Asp Phe Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Asp65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Leu Ala Val Pro Gly
His Trp Tyr Phe Asp Leu Trp 100 105 110Gly Arg Gly Thr Leu Val Thr
Val Ser Ser 115 120628PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 62Gly Phe Ser Phe Ser Ser Tyr
Gly1 5638PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63Leu Trp Tyr Asp Gly Ser His Lys1
56415PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Ala Arg Glu Gly Leu Ala Val Pro Gly His Trp Tyr
Phe Asp Leu1 5 10 1565381DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 65atgagggtcc
ccgctcagct cctggggctt ctgctgctct ggctcccagg tgccagatgt 60gccatccagt
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
120atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca
gcagaaacca 180gggaaagctc ctaagctcct gatctatgat gcctccagtt
tggaaagtgg ggtcccatca 240aggttcagcg gcagtggatc tgggacagat
ttcactctca ccatcagcag cctgcagcct 300gaagattttg caacttatta
ctgtcaacag tttaatactt accctcggac gttcggccaa 360gggaccaagg
tggaaatcaa a 38166127PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 66Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro1 5 10 15Gly Ala Arg Cys Ala
Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly 35 40 45Ile Ser Ser
Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60Lys Leu
Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser65 70 75
80Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn 100 105 110Thr Tyr
Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120
12567107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 67Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Ala 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Phe Asn Thr Tyr Pro Arg 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105686PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 68Gln
Gly Ile Ser Ser Ala1 5693PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 69Asp Ala
Ser1709PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 70Gln Gln Phe Asn Thr Tyr Pro Arg Thr1
571405DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 71atggagtttg ggctgagctg ggttttcctc
gttgctcttt taagaggtgt ccagtgtcag 60gtgcagttgg tggagtctgg gggaggcgtg
gtccagcctg ggaggtccct gcgactctcc 120tgtgcagcgt ctggattcac
cctcagtagc catgacatac actgggtccg ccaggctcca 180ggcaaggggc
tggagtgggt ggcagttata tggaatgatg gaagtaataa atactatgca
240gactccgtga agggccgatt caccatctcc agagacaatt ccacgaactc
gctgtttctg 300caaatgaaca gcctgagagc cgaggacacg gctgtgtatt
attgtgtgag aggaactgct 360gaccttgaac actgggacca gggaaccctg
gtcaccgtct cctca 40572135PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 72Met Glu Phe Gly Leu Ser
Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15Val Gln Cys Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30Pro Gly Arg Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu 35 40 45Ser Ser His
Asp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp
Val Ala Val Ile Trp Asn Asp Gly Ser Asn Lys Tyr Tyr Ala65 70 75
80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Thr Asn
85 90 95Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val 100 105 110Tyr Tyr Cys Val Arg Gly Thr Ala Asp Leu Glu His Trp
Asp Gln Gly 115 120 125Thr Leu Val Thr Val Ser Ser 130
13573116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 73Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Leu Ser Ser His 20 25 30Asp Ile His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Asn Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Thr Asn Ser Leu Phe65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Gly Thr
Ala Asp Leu Glu His Trp Asp Gln Gly Thr Leu Val 100 105 110Thr Val
Ser Ser 115748PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 74Gly Phe Thr Leu Ser Ser His Asp1
5758PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 75Ile Trp Asn Asp Gly Ser Asn Lys1
57612PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 76Val Arg Gly Thr Ala Asp Leu Glu His Trp Asp
Gln1 5 1077381DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 77atgagggtcc tcgctcagct
cctggggctc ctgctgctct gtttcccagg tgccagatgt 60gacatccaga tgacccagtc
tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 120atcacttgtc
gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca
180gagaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 240aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 300gaagattttg caacttatta ctgccaacag
tataatagtt accctctcac tttcggcgga 360gggaccaagg tggagatcaa a
38178127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 78Met Arg Val Leu Ala Gln Leu Leu Gly Leu Leu
Leu Leu Cys Phe Pro1 5 10 15Gly Ala Arg Cys Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly 35 40 45Ile Ser Ser Trp Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro 50 55 60Lys Ser Leu Ile Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn 100 105 110Ser Tyr
Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120
12579107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 79Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu
Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105806PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 80Gln
Gly Ile Ser Ser Trp1 5813PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 81Ala Ala
Ser1829PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 82Gln Gln Tyr Asn Ser Tyr Pro Leu Thr1
583429DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 83atggagtttg ggctgagctg ggttttcctc
gttgctcttt taagaggtgt ccagtgtcag 60gtgcagctgg tggagtctgg gggaggcgtg
gtccagcctg ggaggtccct gagactctcc 120tgtgcagcgt ctggattcac
cttcagtcat tatggcatgc actgggtccg ccaggctcca 180ggcaaggggc
cggagtgggt ggcaattata tggtatgatg gaagtaataa atactatgca
240gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac
gctggatctg 300caaatgaaca gcctgagagc cgaggacacg gctgtgtatt
actgtgcgag agatggatgg 360actactatgg ttcggggact taatgttttt
gatatctggg gccaagggac aatggtcacc 420gtctcttca 42984143PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
84Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1
5 10 15Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln 20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe 35 40 45Ser His Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Pro 50 55 60Glu Trp Val Ala Ile Ile Trp Tyr Asp Gly Ser Asn
Lys Tyr Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn 85 90 95Thr Leu Asp Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Asp Gly
Trp Thr Thr Met Val Arg Gly Leu Asn 115 120 125Val Phe Asp Ile Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135
14085124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 85Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser His Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Pro Glu Trp Val 35 40 45Ala Ile Ile Trp Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Asp65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly
Trp Thr Thr Met Val Arg Gly Leu Asn Val Phe Asp 100 105 110Ile Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120868PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 86Gly
Phe Thr Phe Ser His Tyr Gly1 5878PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 87Ile Trp Tyr Asp Gly Ser
Asn Lys1 58817PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 88Ala Arg Asp Gly Trp Thr Thr Met Val
Arg Gly Leu Asn Val Phe Asp1 5 10 15Ile89381DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
89atgagggtcc tcgctcagct cctggggctc ctgctgctct gtttcccagg tgccagatgt
60gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc
120atcacttgtc gggcgagtca ggatattagc agctggttag cctggtatca
gcagaaacca 180gagaaagccc ctaagtccct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 240aggttcagcg gcagtggatc tgggacagat
ttcactctca ccatcagcag cctgcagcct 300gaagattttg caacttatta
ctgccaacag tataatagtt accctcccac cttcggccaa 360gggacacgac
tggagattaa a 38190127PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 90Met Arg Val Leu Ala Gln
Leu Leu Gly Leu Leu Leu Leu Cys Phe Pro1 5 10 15Gly Ala Arg Cys Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45Ile Ser Ser
Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro 50 55 60Lys Ser
Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser65 70 75
80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn 100 105 110Ser Tyr Pro Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu
Ile Lys 115 120 12591107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 91Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
105926PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 92Gln Asp Ile Ser Ser Trp1 5933PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 93Ala
Ala Ser1949PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 94Gln Gln Tyr Asn Ser Tyr Pro Pro Thr1
595381DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 95atgagggtcc tcgctcagct cctggggctt
ctgctgctct ggctcccagg tgccagatgt 60gccatccagt tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 120atcacttgcc gggcaagtca
gggcattagc agtgctttag cctggtatca gcagaaacca 180gagaaagccc
ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca
240aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 300gaagattttg caacttatta ctgccaacag tataatagtt
accctcccac cttcggccaa 360gggacacgac tggagattaa a
38196127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 96Met Arg Val Leu Ala Gln Leu Leu Gly Leu Leu
Leu Leu Trp Leu Pro1 5 10 15Gly Ala Arg Cys Ala Ile Gln Leu Thr Gln
Ser Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly 35 40 45Ile Ser Ser Ala Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro 50 55 60Lys Ser Leu Ile Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn 100 105 110Ser Tyr
Pro Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 115 120
12597107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 97Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Ala 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu
Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro 85 90 95Thr Phe Gly Gln
Gly Thr Arg Leu Glu Ile Lys 100 105986PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 98Gln
Gly Ile Ser Ser Ala1 5993PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 99Ala Ala
Ser11009PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 100Gln Gln Tyr Asn Ser Tyr Pro Pro Thr1
5101414DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 101atggagtttg ggctgagctg ggttttcctc
gttgctcttt taagaggtgt ccagtgtcag 60gtgcagctgg tggagtctgg gggaggcgtg
gtccagcctg ggaggtccct gagactctcc 120tgtgcaacgt ctggattcac
cttcagtagc tatgacatgc actgggtccg ccaggctcca 180ggcaaggggc
tggagtgggt ggcagttatt tggtatgatg gaagtaataa atactatgca
240gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac
gctgtatctc 300caaatgaaca gcctgggaga cgaggacacg gctgtgtatt
actgtgcgag aggtagtggt 360aactggggtt tctttgacta ctggggccag
ggaaccctgg tcaccgtctc ctca 414102138PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
102Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1
5 10 15Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln 20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe
Thr Phe 35 40 45Ser Ser Tyr Asp Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 50 55 60Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn
Lys Tyr Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn 85 90 95Thr Leu Tyr Leu Gln Met Asn Ser Leu
Gly Asp Glu Asp Thr Ala Val
100 105 110Tyr Tyr Cys Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp
Tyr Trp 115 120 125Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130
135103119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 103Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Thr Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly
Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Gly Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 1151048PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 104Gly Phe Thr Phe Ser Ser
Tyr Asp1 51058PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 105Ile Trp Tyr Asp Gly Ser Asn Lys1
510612PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 106Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp
Tyr1 5 10107381DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 107atgagggtcc tcgctcagct
cctggggctc ctgctgctct gtttcccagg tgccagatgt 60gacatccaga tgacccagtc
tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 120atcacttgtc
gggcgagtca gggtattagc aggtggttag cctggtatca gcagaaacca
180gagaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 240aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 300gaagattttg caacttatta ctgccaacag
tataatactt accctcggac gttcggccaa 360gggaccaagg tggaaatcaa a
381108127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 108Met Arg Val Leu Ala Gln Leu Leu Gly Leu
Leu Leu Leu Cys Phe Pro1 5 10 15Gly Ala Arg Cys Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly 35 40 45Ile Ser Arg Trp Leu Ala Trp
Tyr Gln Gln Lys Pro Glu Lys Ala Pro 50 55 60Lys Ser Leu Ile Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn 100 105 110Thr
Tyr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120
125109107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 109Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Arg Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 1051106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 110Gln
Gly Ile Ser Arg Trp1 51113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 111Ala Ala
Ser11129PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 112Gln Gln Tyr Asn Thr Tyr Pro Arg Thr1
511316PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus sequenceMOD_RES(2)..(2)Gly, Glu, or
AspMOD_RES(3)..(3)Ser, Leu, Gly, or absentMOD_RES(4)..(4)Gly, Leu,
Thr, Trp, or absentMOD_RES(5)..(5)Asn, Ala, Thr, His, or
absentMOD_RES(6)..(6)Val, Thr, or absentMOD_RES(7)..(7)Met, Pro, or
absentMOD_RES(8)..(8)Gly, Val, or absentMOD_RES(9)..(9)May or may
not be presentMOD_RES(10)..(10)Gly, Met, or
absentMOD_RES(11)..(11)Asp, His, Leu, Thr, or
TrpMOD_RES(12)..(12)Ala, Gly, Asn, or TrpMOD_RES(13)..(13)Asp, Phe,
Val, or TyrMOD_RES(14)..(14)Phe or LeuMOD_RES(15)..(15)Asp or
GluMOD_RES(16)..(16)His, Ile, Leu, or Tyr 113Arg Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10
151148PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus sequenceMOD_RES(2)..(2)Phe, Arg, or
TyrMOD_RES(3)..(3)Asn or SerMOD_RES(4)..(4)Asn, Thr, or
SerMOD_RES(5)..(5)Tyr or TrpMOD_RES(7)..(7)Phe, Leu, Pro, or Arg
114Gln Xaa Xaa Xaa Xaa Pro Xaa Thr1 51158PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
sequenceMOD_RES(2)..(2)Lys or TrpMOD_RES(3)..(3)Tyr, Asn, or
GlnMOD_RES(7)..(7)Glu or AsnMOD_RES(8)..(8)Lys or Gln 115Ile Xaa
Xaa Asp Gly Ser Xaa Xaa1 51163PRTArtificial SequenceDescription of
Artificial Sequence Synthetic consensus sequenceMOD_RES(1)..(1)Ala
or Asp 116Xaa Ala Ser11177PRTArtificial SequenceDescription of
Artificial Sequence Synthetic consensus sequenceMOD_RES(3)..(3)Thr
or SerMOD_RES(4)..(4)Phe or LeuMOD_RES(5)..(5)Ser or
AsnMOD_RES(6)..(6)Ile, Ser, or HisMOD_RES(7)..(7)Tyr or His 117Gly
Phe Xaa Xaa Xaa Xaa Xaa1 51186PRTArtificial SequenceDescription of
Artificial Sequence Synthetic consensus sequenceMOD_RES(2)..(2)Asp,
Gly, or SerMOD_RES(3)..(3)Ile or ValMOD_RES(4)..(4)Asp or
SerMOD_RES(5)..(5)Arg or SerMOD_RES(6)..(6)Ala, Trp, or Tyr 118Gln
Xaa Xaa Xaa Xaa Xaa1 51198PRTGallus sp. 119Ser Ile Ile Asn Phe Glu
Lys Leu1 5120127PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 120Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp Leu Pro1 5 10 15Gly Ala Arg Cys Ala Ile Gln
Leu Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly 35 40 45Ile Ser Ser Ala Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60Lys Leu Leu Ile
Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser65 70 75 80Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn 100 105
110Ser Tyr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 115
120 125
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References