U.S. patent application number 15/348551 was filed with the patent office on 2017-03-02 for human antibodies that bind cd22 and uses thereof.
The applicant listed for this patent is E. R. SQUIBB & SONS, L.L.C.. Invention is credited to David John King, Heidi N. LEBLANC, Asna MASOOD, David Passmore, Chetana RAO-NAIK, Sarah R. REED, Tim SPROUL, Dawn M. TANAMACHI, Richard THEOLIS, Kristopher TOY, Alison WITTE, Mark YAMANAKA, Kyra D. ZENS.
Application Number | 20170058031 15/348551 |
Document ID | / |
Family ID | 39493006 |
Filed Date | 2017-03-02 |
United States Patent
Application |
20170058031 |
Kind Code |
A1 |
King; David John ; et
al. |
March 2, 2017 |
HUMAN ANTIBODIES THAT BIND CD22 AND USES THEREOF
Abstract
The present disclosure provides isolated monoclonal antibodies
that specifically bind to CD22 with high affinity, particularly
human monoclonal antibodies. Nucleic acid molecules encoding the
antibodies of this disclosure, expression vectors, host cells and
methods for expressing the antibodies of this disclosure are also
provided. Antibody-partner molecule conjugates, bispecific
molecules and pharmaceutical compositions comprising the antibodies
of this disclosure are also provided. This disclosure also provides
methods for detecting CD22, as well as methods for treating various
cancers and inflammatory and autoimmune disorders using an
anti-CD22 antibody of this disclosure.
Inventors: |
King; David John; (Solana
Beach, CA) ; WITTE; Alison; (Rogue River, OR)
; LEBLANC; Heidi N.; (Mountain View, CA) ;
THEOLIS; Richard; (Santa Cruz, CA) ; MASOOD;
Asna; (Saratoga, CA) ; YAMANAKA; Mark;
(Pleasanton, CA) ; ZENS; Kyra D.; (San Mateo,
CA) ; REED; Sarah R.; (Santa Cruz, CA) ;
SPROUL; Tim; (Livermore, CA) ; RAO-NAIK; Chetana;
(Walnut Creek, CA) ; Passmore; David; (San Carlos,
CA) ; TANAMACHI; Dawn M.; (San Carlos, CA) ;
TOY; Kristopher; (San Jose, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
E. R. SQUIBB & SONS, L.L.C. |
Princeton |
NJ |
US |
|
|
Family ID: |
39493006 |
Appl. No.: |
15/348551 |
Filed: |
November 10, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13772063 |
Feb 20, 2013 |
9499632 |
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15348551 |
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12517183 |
Feb 17, 2010 |
8481683 |
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PCT/US2007/086152 |
Nov 30, 2007 |
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13772063 |
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60868231 |
Dec 1, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/21 20130101;
C07K 2317/56 20130101; C07K 2317/734 20130101; A61P 19/02 20180101;
C07K 16/3061 20130101; A61P 37/04 20180101; A61P 37/06 20180101;
A61K 2039/505 20130101; C07K 2317/92 20130101; A61P 35/00 20180101;
A61P 37/02 20180101; C07K 16/2803 20130101; C07K 2317/732 20130101;
C07K 2317/70 20130101; A61P 37/00 20180101; A61K 47/6829 20170801;
A61K 45/06 20130101; A61K 47/6849 20170801; A61K 39/39558 20130101;
A61P 35/02 20180101; A61K 51/1027 20130101; A61P 29/00 20180101;
C07K 2317/77 20130101; C07K 2317/565 20130101; C07K 2317/41
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 47/48 20060101 A61K047/48; A61K 39/395 20060101
A61K039/395; C07K 16/30 20060101 C07K016/30; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method of treating a CD22-expressing cancer, comprising
administering to a subject a human monoclonal antibody which
specifically binds to CD22, or antigen-binding portion thereof, in
an amount effective to treat the cancer.
2. The method of claim 1, wherein the cancer is a B cell
lymphoma.
3. The method of claim 2, wherein the B cell lymphoma is a
non-Hodgkin's lymphoma.
4. The method of claim 1, wherein the cancer is selected from
Burkitt's lymphoma and B cell chronic lymphocytic leukemia.
5. The method of claim 1, wherein the subject is human.
6. A method of inhibiting growth of a CD22-expressing tumor cell,
the method comprising contacting the CD22-expressing tumor cell
with a human monoclonal antibody which specifically binds to CD22,
or antigen-binding portion thereof, such that growth of the
CD22-expressing tumor cell is inhibited.
7-8. (canceled)
9. The method of claim 1, wherein the antibody, or antigen-binding
portion thereof, comprises: (a) a heavy chain variable region CDR1
comprising SEQ ID NO:2; (b) a heavy chain variable region CDR2
comprising SEQ ID NO:6 or SEQ ID NO:60; (c) a heavy chain variable
region CDR3 comprising SEQ ID NO: 10; (d) a light chain variable
region CDR1 comprising SEQ ID NO:14; (e) a light chain variable
region CDR2 comprising SEQ ID NO:20; and (f) a light chain variable
region CDR3 comprising SEQ ID NO:26.
10. The method of claim 9, wherein the antibody, or antigen-binding
portion thereof, comprises: (a) a heavy chain variable region CDR1
comprising SEQ ID NO:2; (b) a heavy chain variable region CDR2
comprising SEQ ID NO:6; (c) a heavy chain variable region CDR3
comprising SEQ ID NO: 10; (d) a light chain variable region CDR1
comprising SEQ ID NO:14; (e) a light chain variable region CDR2
comprising SEQ ID NO:20; and (f) a light chain variable region CDR3
comprising SEQ ID NO:26.
11. The method of claim 9, wherein the antibody, or antigen-binding
portion thereof, comprises: (a) a heavy chain variable region CDR1
comprising SEQ ID NO:2; (b) a heavy chain variable region CDR2
comprising SEQ ID NO:60; (c) a heavy chain variable region CDR3
comprising SEQ ID NO: 10; (d) a light chain variable region CDR1
comprising SEQ ID NO:14; (e) a light chain variable region CDR2
comprising SEQ ID NO:20; and (f) a light chain variable region CDR3
comprising SEQ ID NO:26.
12. The method of claim 9, wherein the antibody, or antigen-binding
portion thereof, comprises: (a) a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO:32 or 61; and (b) a
light chain variable region comprising the amino acid sequence of
SEQ ID NO:36.
13. The method of claim 12, wherein the antibody, or
antigen-binding portion thereof, comprises: (a) a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:32;
and (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO:36.
14. The method of claim 12, wherein the antibody, or
antigen-binding portion thereof, comprises: (a) a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:61;
and (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO:36.
15. The method of claim 1, wherein the antibody, or antigen-binding
portion thereof, cross-competes for binding to CD22 with a
reference antibody, wherein the reference antibody comprises: (a) a
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO:32 or 61; and (b) a light chain variable region
comprising the amino acid sequence of SEQ ID NO:36.
16. The method of claim 1, wherein the antibody is a full-length
antibody of an IgG1 isotype or an IgG4 isotype.
17. The method of claim 1, wherein the antibody or antigen-binding
portion thereof, is linked to a therapeutic agent.
18. The method of claim 17, wherein the therapeutic agent is a
cytotoxin or a radioactive agent.
19. The method of claim 17, wherein the therapeutic agent is
conjugated to the antibody by a chemical linker.
20. The method of claim 19, wherein the chemical linker is selected
from the group consisting of peptidyl linkers, hydrazine linkers,
and disulfide linkers.
21. The method of claim 1, wherein the antibody is administered by
a route selected from subcutaneously, intravenously,
intramuscularly, intradermally, and intraperitoneally.
22. The method of claim 1, further comprising administering to the
subject a second anti-neoplastic agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 13/772,063, filed Feb. 20, 2013, which is Divisional of U.S.
application Ser. No. 12/517,183, filed Feb. 17, 2010 (now U.S. Pat.
No. 8,481,683, issued Jul. 9, 2013), which is a national stage of
International Application Serial No. PCT/US2007/086152, filed Nov.
30, 2007, which claims priority of U.S. Provisional Application
Ser. No. 60/868,231, filed on Dec. 1, 2006, each of which is herein
incorporated by reference.
BACKGROUND
[0002] CD22 is a cell-surface type I glycoprotein of the
sialoadhesin family. CD22 is also known in the art as BL-CAM, B3,
Leu-14 and Lyb-8, among other names. CD22 was initially
characterized by the antibodies anti-S-HCL-1 (Schwarting, R. et al.
(1985) Blood 65:974-983), HD39 (Dorken, B. et al. (1986) J.
Immunol. 136:4470-4479) and RFB4 (Campana, D. et al. (1985) J.
Immunol. 134:1524-1530). CD22 has been established as a lectin-like
adhesion molecule that binds alpha2,6-linked sialic acid-bearing
ligands and as a regulator of B cell antigen receptor (BCR)
signaling. Structurally, there are several splice variants of CD22
that exist, but the predominant form in humans has an extracellular
region containing seven immunoglobulin-like domains.
[0003] CD22 has been shown to be specifically expressed by B
lymphocytes and is functionally important as a negative regulator
of B lymphocyte activation (reviewed by Nitschke, L. (2005) Curr.
Opin. Immunol. 17:290-297 and Tedder, T. F. et al (2005) Adv.
Immunol. 88:1-50). In studies that utilized gene-targeted mice that
expressed mutant CD22 molecules that do not interact with
alpha2,6-linked sialic acid ligands, it was determined that certain
functions (such as expression of cell surface CD22, IgM and MHC
Class II on mature B cells, maintenance of marginal zone B cell
populations, optimal B cell antigen receptor-induced proliferation
and B cell turnover rates) were regulated by CD22 ligand binding,
whereas other functions (such as CD22 phosphorylation, CD22
negative regulation of calcium mobilization after BCR ligation,
recruitment of SHP-1 to CD22 and B cell migration) did not require
ligand engagement (Poe, J. C. et al. (2004) Nat. Immunol.
5:1078-1087).
[0004] CD22 is considered to be an inhibitory co-receptor that
downmodulates BCR signalling by setting a signalling threshold that
prevents overstimulation of B cells. Activation of such an
inhibitory co-receptor occurs by phosphorylation on cytoplasmic
ITIMs (immunoreceptor tyrosine-based inhibition motifs), followed
by recruitment of the tyrosine phosphatase SHP-1 or the lipid
phosphatase SHIIP (reviewed in by Nitschke, L. (2005) Curr. Opin.
Immunol. 17:290-297). Additionally, CD22 has been found to play a
central role in a regulatory loop controlling the
CD19/CD21-Src-family protein tyrosine kinase (PTK) amplification
pathway that regulates basal signaling thresholds and intensifies
Src-family kinase activation after BCR ligation (reviewed in
Tedder, T. F. et al (2005) Adv. Immunol. 88:1-50).
[0005] Approximately 60-80% of B cell malignancies express CD22,
thereby making it a potential target for passive immunotherapy (see
e.g., Cesano, A. and Gayko, U. (2003) Semin. Oncol. 30:253-257).
Moreover, selective modulation of B cell activity via targeting of
CD22 has been suggested as a means for treatment of autoimmune
diseases (see e.g., Steinfeld, S. D. and Youinou, P. (2006) Expert.
Opin. Biol. Ther. 6:943-949). A humanized anti-CD22 monoclonal
antibody, epratuzumab, has been described (Coleman, M. et al.
(2003) Clin. Cancer Res. 9:3991S-3994S). However, additional
anti-CD22 reagents are still needed.
SUMMARY
[0006] The present disclosure provides isolated monoclonal
antibodies, in particular human monoclonal antibodies, that bind to
human CD22 and that exhibit numerous desirable properties. These
properties include high affinity binding to CD22, the ability to
internalize into CD22+ cells, the ability to mediate antibody
dependent cellular cytotoxicity (ADCC), the ability to enhance cell
death of Ramos cells induced by B cell receptor (BCR) stimulation,
and/or inhibits growth of CD22-expressing cells in vivo when
conjugated to a cytotoxin. The antibodies of the invention can be
used, for example, to treat CD22+ B cell malignancies and/or to
treat various inflammatory or autoimmune disorders.
[0007] In one aspect, the instant disclosure pertains to an
isolated human monoclonal antibody, or an antigen-binding portion
thereof, wherein the antibody binds to human CD22 and exhibits at
least one of the following properties:
[0008] (a) internalizes into CD22.sup.+ cells;
[0009] (b) exhibits antibody dependent cellular cytotoxicity (ADCC)
against CD22.sup.+ cells;
[0010] (c) enhances cell death of Ramos cells induced by B cell
receptor (BCR) stimulation; and
[0011] (d) inhibits growth of CD22-expressing cells in vivo when
conjugated to a cytotoxin.
In another embodiment, the antibody exhibits at least two of
properties (a), (b), (c) and (d). In yet another embodiment, the
antibody exhibits three of properties (a), (b), (c) and (d). In
another embodiment, the antibody exhibits all four of properties
(a), (b), (c), and (d). In certain embodiments, the antibody does
not have a direct anti-proliferative effect on Ramos cells. In
certain embodiments, the antibody does not induce calcium flux in
Ramos cells. In certain embodiments, the antibody does not mediate
complement dependent cytotoxicity (CDC) on Ramos cells. Preferably,
the antibody binds to human CD22 with high affinity, e.g., with a
K.sub.D of 1.times.10.sup.-7 M or less or a K.sub.D of
1.times.10.sup.-8 M or less or a K.sub.D of 1.times.10.sup.-9 M or
less or a K.sub.D of 1.times.10.sup.-10 or less or a K.sub.D of
7.times.10.sup.-11 or less.
[0012] In another aspect, the invention pertains to an isolated
human monoclonal antibody, or antigen binding portion thereof,
wherein the antibody cross-competes for binding to CD22 with a
reference antibody, wherein the reference antibody comprises:
[0013] (a) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:31 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:35; or
[0014] (b) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:32 or 61 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:36; or
[0015] (c) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:33 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:37 or 38; or
[0016] (d) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:34 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:39 or 40; or
[0017] (e) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:81 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:84 or 85; or
[0018] (f) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:82 or 83 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:86.
wherein the antibody specifically binds human CD22
[0019] In yet another aspect, the invention pertains to an isolated
monoclonal antibody, or an antigen-binding portion thereof,
comprising a heavy chain variable region that is the product of or
derived from a human V.sub.H 7-4.1 gene, a human V.sub.H 4-34 gene,
a human V.sub.H 5-51 gene, or a human VH 1-69 gene, wherein the
antibody specifically binds human CD22. In yet another aspect, the
invention pertains to an isolated monoclonal antibody, or an
antigen-binding portion thereof, comprising a light chain variable
region that is the product of or derived from a human V.sub..lamda.
2b2 gene, a human V.sub.K L6 gene, a human V.sub.K A27 gene, a
human V.sub.K A10 gene, or a human L18 gene, wherein the antibody
specifically binds human CD22. In still another aspect, the
invention pertains to an isolated antibody, or antigen-binding
portion thereof, comprising:
[0020] (a) a heavy chain variable region that is the product of or
derived from a human V.sub.H 7-4.1 gene and a light chain variable
region that is the product of or derived from a human V.sub..lamda.
2b2 gene; or
[0021] (b) a heavy chain variable region that is the product of or
derived from a human V.sub.H 4-34 gene and a light chain variable
region that is the product of or derived from a human V.sub.K L6
gene; or
[0022] (c) a heavy chain variable region that is the product of or
derived from a human V.sub.H 5-51 gene and a light chain variable
region that is the product of or derived from a human V.sub.K A27
or A10 gene;
(d) a heavy chain variable region that is the product of or derived
from a human V.sub.H 1-69 gene and a light chain variable region
that is the product of or derived from a human V.sub.K L6 gene;
or
[0023] (e) a heavy chain variable region that is the product of or
derived from a human V.sub.H 1-69 gene and a light chain variable
region that is the product of or derived from a human V.sub.K L18
or A27 gene;
wherein the antibody specifically binds human CD22.
[0024] In another aspect, this disclosure provides an isolated
monoclonal antibody, or antigen binding portion thereof,
comprising: [0025] a heavy chain variable region that comprises
CDR1, CDR2, and CDR3 sequences; [0026] and a light chain variable
region that comprises CDR1, CDR2, and CDR3 sequences, wherein:
[0027] (a) the heavy chain variable region CDR3 sequence comprises
an amino acid sequence selected from the group consisting of amino
acid sequences of SEQ ID NOs: 9-12 and 69-71, and conservative
modifications thereof;
[0028] (b) the light chain variable region CDR3 sequence comprises
an amino acid sequence selected from the group consisting of amino
acid sequence of SEQ ID NOs: 25-30, 78-80, and conservative
modifications thereof; and
[0029] (c) the antibody binds to human CD22.
[0030] In preferred embodiments, this antibody also has one or more
of the following characteristics: internalizes into CD22+ cells,
mediates ADCC activity and/or enhances cell death of Ramos cells
induced by BCR stimulation, and/or inhibits growth of
CD22-expressing cells in vivo when conjugated to a cytotoxin.
[0031] Preferably, the heavy chain variable region CDR2 sequence
comprises an amino acid sequence selected from the group consisting
of amino acid sequences of SEQ ID NOs: 5-8, 60, 66-68, and
conservative modifications thereof; and the light chain variable
region CDR2 sequence comprises an amino acid sequence selected from
the group consisting of amino acid sequences of SEQ ID NOs: 19-24,
75-77, and conservative modifications thereof.
[0032] Preferably, the heavy chain variable region CDR1 sequence
comprises an amino acid sequence selected from the group consisting
of amino acid sequences of SEQ ID NOs: 1-4, 63-65, and conservative
modifications thereof; and the light chain variable region CDR1
sequence comprises an amino acid sequence selected from the group
consisting of amino acid sequences of SEQ ID NOs: 13-18, 72-74, and
conservative modifications thereof.
[0033] A preferred combination comprises:
[0034] (a) a heavy chain variable region CDR1 comprising SEQ ID NO:
1;
[0035] (b) a heavy chain variable region CDR2 comprising SEQ ID
NO:5;
[0036] (c) a heavy chain variable region CDR3 comprising SEQ ID
NO:9;
[0037] (d) a light chain variable region CDR1 comprising SEQ ID
NO:13;
[0038] (e) a light chain variable region CDR2 comprising SEQ ID
NO:19; and
[0039] (f) a light chain variable region CDR3 comprising SEQ ID
NO:25.
[0040] Another preferred combination comprises:
[0041] (a) a heavy chain variable region CDR1 comprising SEQ ID
NO:2;
[0042] (b) a heavy chain variable region CDR2 comprising SEQ ID
NO:6 or 60;
[0043] (c) a heavy chain variable region CDR3 comprising SEQ ID NO:
10;
[0044] (d) a light chain variable region CDR1 comprising SEQ ID NO:
14;
[0045] (e) a light chain variable region CDR2 comprising SEQ ID
NO:20; and
[0046] (f) a light chain variable region CDR3 comprising SEQ ID
NO:26.
[0047] Another preferred combination comprises:
[0048] (a) a heavy chain variable region CDR1 comprising SEQ ID
NO:3;
[0049] (b) a heavy chain variable region CDR2 comprising SEQ ID
NO:7;
[0050] (c) a heavy chain variable region CDR3 comprising SEQ ID NO:
11;
[0051] (d) a light chain variable region CDR1 comprising SEQ ID
NO:15 or 16;
[0052] (e) a light chain variable region CDR2 comprising SEQ ID
NO:21 or 22; and
[0053] (f) a light chain variable region CDR3 comprising SEQ ID
NO:27 or 28.
[0054] Another preferred combination comprises:
[0055] (a) a heavy chain variable region CDR1 comprising SEQ ID
NO:4;
[0056] (b) a heavy chain variable region CDR2 comprising SEQ ID
NO:8;
[0057] (c) a heavy chain variable region CDR3 comprising SEQ ID NO:
12;
[0058] (d) a light chain variable region CDR1 comprising SEQ ID
NO:17 or 18;
[0059] (e) a light chain variable region CDR2 comprising SEQ ID
NO:23 or 24; and
[0060] (f) a light chain variable region CDR3 comprising SEQ ID
NO:29 or 30.
[0061] Another preferred combination comprises:
[0062] (a) a heavy chain variable region CDR1 comprising SEQ ID
NO:63;
[0063] (b) a heavy chain variable region CDR2 comprising SEQ ID
NO:66;
[0064] (c) a heavy chain variable region CDR3 comprising SEQ ID
NO:69;
[0065] (d) a light chain variable region CDR1 comprising SEQ ID
NO:72 or 73;
[0066] (e) a light chain variable region CDR2 comprising SEQ ID
NO:75 or 76; and
[0067] (f) a light chain variable region CDR3 comprising SEQ ID
NO:78 or 79.
[0068] Another preferred combination comprises:
[0069] (a) a heavy chain variable region CDR1 comprising SEQ ID
NO:64 or 65;
[0070] (b) a heavy chain variable region CDR2 comprising SEQ ID
NO:67 or 68;
[0071] (c) a heavy chain variable region CDR3 comprising SEQ ID
NO:70 or 71;
[0072] (d) a light chain variable region CDR1 comprising SEQ ID
NO:74;
[0073] (e) a light chain variable region CDR2 comprising SEQ ID
NO:77; and
[0074] (f) a light chain variable region CDR3 comprising SEQ ID
NO:80.
[0075] Other preferred antibodies of this disclosure, or antigen
binding portions thereof, comprise: [0076] (a) a heavy chain
variable region comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs: 31-34, 61 and 81-83; and [0077] (b)
a light chain variable region comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 35-40 and 84-86;
[0078] wherein the antibody specifically binds human CD22.
[0079] A preferred combination comprises: (a) a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:31;
and (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO:35.
[0080] Another preferred combination comprises: (a) a heavy chain
variable region comprising the amino acid sequence of SEQ ID NOS:
32 or 61; and (b) a light chain variable region comprising the
amino acid sequence of SEQ ID NO:36.
[0081] Another preferred combination comprises: (a) a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:33;
and (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO:37 or 38.
[0082] Another preferred combination comprises: (a) a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:34;
and (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO:39 or 40.
[0083] Another preferred combination comprises: (a) a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:81;
and (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO:84 or 85.
[0084] Another preferred combination comprises: (a) a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:82
or 83; and (b) a light chain variable region comprising the amino
acid sequence of SEQ ID NO:86.
[0085] In another aspect of this disclosure, antibodies, or
antigen-binding portions thereof, are provided that compete for
binding to CD22 with any of the aforementioned antibodies.
[0086] The antibodies of this disclosure can be, for example,
full-length antibodies, for example of an IgG1 or IgG4 isotype.
Alternatively, the antibodies can be antibody fragments, such as
Fab, Fab' or Fab'2 fragments, or single chain antibodies.
[0087] This disclosure also provides an immunoconjugate comprising
an antibody of this disclosure, or antigen-binding portion thereof,
linked to a therapeutic agent, such as a cytotoxin or a radioactive
isotope. In a particularly preferred embodiment, the invention
provides an immunoconjugate comprising an antibody of this
disclosure, or antigen-binding portion thereof, linked to the
compound "Cytotoxin A" (e.g., via a thiol linkage). This disclosure
also provides a bispecific molecule comprising an antibody, or
antigen-binding portion thereof, of this disclosure, linked to a
second functional moiety having a different binding specificity
than said antibody, or antigen binding portion thereof.
[0088] Compositions comprising an antibody, or antigen-binding
portion thereof, or immunoconjugate or bispecific molecule of this
disclosure and a pharmaceutically acceptable carrier are also
provided.
[0089] Nucleic acid molecules encoding the antibodies, or
antigen-binding portions thereof, of this disclosure are also
encompassed by this disclosure, as well as expression vectors
comprising such nucleic acids and host cells comprising such
expression vectors. Methods for preparing anti-CD22 antibodies
using the host cells comprising such expression vectors are also
provided and may include the steps of (i) expressing the antibody
in the host cell and (ii) isolating the antibody from the host
cell.
[0090] Another aspect of this disclosure pertains to methods of
inhibiting growth of a CD22-expressing tumor cell. The method
comprises contacting the CD22-expressing tumor cell with an
antibody, or antigen-binding portion thereof, of the invention such
that growth of the CD22-expressing tumor cell is inhibited. The
tumor cell can be, for example, a B cell lymphoma, such as a
non-Hodgkin's lymphoma. In certain embodiments, the antibody, or
antigen-binding portion thereof, is conjugated to a therapeutic
agent, such as a cytotoxin.
[0091] Another aspect of this disclosure pertains to methods of
treating an inflammatory or autoimmune disorder in a subject. The
method comprises administering to the subject an antibody, or
antigen-binding portion thereof, of the invention such that the
inflammatory or autoimmune disorder in the subject is treated. The
autoimmune disorder can be, for example, systemic lupus
erythematosus or rheumatoid arthritis.
[0092] The present disclosure also provides isolated anti-CD22
antibody-partner molecule conjugates that specifically bind to CD22
with high affinity, particularly those comprising human monoclonal
antibodies. Certain of such antibody-partner molecule conjugates
are capable of being internalized into CD22-expressing cells and
are capable of mediating antibody dependent cellular cytotoxicity.
This disclosure also provides methods for treating cancers, such as
a B cell lymphoma, such as a non-Hodgkin's lymphoma, using an
anti-CD22 antibody-partner molecule conjugate disclosed herein.
[0093] In another aspect, the invention provides a method of
treating an inflammatory or autoimmune disorder in a subject. The
method comprises administering to the subject an antibody, or
antigen-binding portion thereof, of the invention such that the
inflammatory or autoimmune disorder in the subject is treated.
Non-limiting examples of preferred autoimmune disorders include
systemic lupus erythematosus and rheumatoid arthritis. Other
examples of autoimmune disorders include inflammatory bowel disease
(including ulcerative colitis and Crohn's disease), Type I
diabetes, multiple sclerosis, Sjogren's syndrome, autoimmune
thyroiditis (including Grave's disease and Hashimoto's
thyroiditis), psoriasis and glomerulonephritis.
[0094] Compositions comprising an antibody, or antigen-binding
portion thereof, conjugated to a partner molecule of this
disclosure are also provided. Partner molecules that can be
advantageously conjugated to an antibody in an antibody partner
molecule conjugate as disclosed herein include, but are not limited
to, molecules as drugs, toxins, marker molecules (e.g.,
radioisotopes), proteins and therapeutic agents. Compositions
comprising antibody-partner molecule conjugates and
pharmaceutically acceptable carriers are also disclosed herein.
[0095] In one aspect, such antibody-partner molecule conjugates are
conjugated via chemical linkers. In some embodiments, the linker is
a peptidyl linker, and is depicted herein as (L4)p-F-(L1)m. Other
linkers include hydrazine and disulfide linkers, and is depicted
herein as (L4)p-H-(L1)m or (L4)p-J-(L1)m, respectively. In addition
to the linkers being attached to the partner, the present invention
also provides cleavable linker arms that are appropriate for
attachment to essentially any molecular species.
[0096] In another aspect, the invention pertains to a method of
inhibiting growth of a CD22-expressing tumor cell. The method
comprises contacting the CD22-expressing tumor cell with an
antibody-partner molecule conjugate of the disclosure such that
growth of the CD22-tumor cell is inhibited. In a preferred
embodiment, the partner molecule is a therapeutic agent, such as a
cytotoxin. Particularly preferred CD22-expressing tumor cells are B
cell lymphomas, such as non-Hodgkin's lymphoma. Other types of
CD22-expressing tumor cells include Burkitt's lymphomas and B cell
chronic lymphocytic leukemias. In still other embodiments, the
CD22-expressing tumor cell is from a cancer selected from the group
consisting of Burkitt's lymphomas and B cell chronic lymphocytic
leukemias.
[0097] In another aspect, the invention pertains to a method of
treating cancer in a subject. The method comprises administering to
the subject an antibody-partner molecule conjugate of the
disclosure such that the cancer is treated in the subject. In a
preferred embodiment, the partner molecule is a therapeutic agent,
such as a cytotoxin. Particularly preferred cancers for treatment
are B cell lymphomas, such as a non-Hodgkin's lymphoma. Other types
of cancers include Burkitt's lymphomas and B cell chronic
lymphocytic leukemias.
[0098] Other features and advantages of the instant disclosure will
be apparent from the following detailed description and examples,
which should not be construed as limiting. The contents of all
references, Genbank entries, patents and published patent
applications cited throughout this application are expressly
incorporated herein by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0099] FIG. 1A shows the nucleotide sequence (SEQ ID NO:41) and
amino acid sequence (SEQ ID NO:31) of the heavy chain variable
region of the 12C5 human monoclonal antibody. The CDR1 (SEQ ID
NO:1), CDR2 (SEQ ID NO:5) and CDR3 (SEQ ID NO:9) regions are
delineated and the V, D and J germline derivations are
indicated.
[0100] FIG. 1B shows the nucleotide sequence (SEQ ID NO:45) and
amino acid sequence (SEQ ID NO:35) of the lambda light chain
variable region of the 12C5 human monoclonal antibody. The CDR1
(SEQ ID NO:13), CDR2 (SEQ ID NO:19) and CDR3 (SEQ ID NO:25) regions
are delineated and the V and J germline derivations are
indicated.
[0101] FIG. 2A shows the nucleotide sequence (SEQ ID NO:42) and
amino acid sequence (SEQ ID NO:32) of the heavy chain variable
region of the 19A3 human monoclonal antibody, and the nucleotide
sequence and amino acid sequence of the heavy chain variable region
of the CD22.1 recombinant antibody. The sequences of the heavy
chain variable region of 19A3 are identical to that of CD22.1. The
CDR1 (SEQ ID NO:2), CDR2 (SEQ ID NO:6) and CDR3 (SEQ ID NO:10)
regions are delineated and the V, D and J germline derivations are
indicated.
[0102] FIG. 2B shows the nucleotide sequence (SEQ ID NO:46) and
amino acid sequence (SEQ ID NO:36) of the kappa light chain
variable region of the 19A3 human monoclonal antibody, and the
nucleotide and amino acid sequences of the kappa light chain
variable region of the CD22.1 recombinant human monoclonal
antibody. The sequences of the kappa light chain variable region of
both CD22.1 and CD22.2 are identical to those of 19A3. The CDR1
(SEQ ID NO:14), CDR2 (SEQ ID NO:20) and CDR3 (SEQ ID NO:26) regions
are delineated and the V and J germline derivations are
indicated.
[0103] FIG. 2C shows the nucleotide sequence (SEQ ID NO:62) and
amino acid sequence (SEQ ID NO:61) of the heavy chain variable
region variable region of the CD22.2 recombinant human monoclonal
antibody. The CDR1 (SEQ ID NO:2), CDR2 (SEQ ID NO:60) and CDR3 (SEQ
ID NO:10) regions are delineated and the V and J germline
derivations are indicated.
[0104] FIG. 3A shows the nucleotide sequence (SEQ ID NO:43) and
amino acid sequence (SEQ ID NO:33) of the heavy chain variable
region of the 16F7 human monoclonal antibody. The CDR1 (SEQ ID
NO:3), CDR2 (SEQ ID NO:7) and CDR3 (SEQ ID NO: 11) regions are
delineated and the V, D and J germline derivations are
indicated.
[0105] FIG. 3B shows the nucleotide sequence (SEQ ID NO:47) and
amino acid sequence (SEQ ID NO:37) of the V.sub.K.1 kappa light
chain variable region of the 16F7 human monoclonal antibody. The
CDR1 (SEQ ID NO:15), CDR2 (SEQ ID NO 21:) and CDR3 (SEQ ID NO 27:)
regions are delineated and the V and J germline derivations are
indicated.
[0106] FIG. 3C shows the nucleotide sequence (SEQ ID NO:48) and
amino acid sequence (SEQ ID NO:38) of the V.sub.K.2 kappa light
chain variable region of the 16F7 human monoclonal antibody. The
CDR1 (SEQ ID NO:16), CDR2 (SEQ ID NO:22) and CDR3 (SEQ ID NO:28)
regions are delineated and the V and J germline derivations are
indicated.
[0107] FIG. 4A shows the nucleotide sequence (SEQ ID NO:44) and
amino acid sequence (SEQ ID NO:34) of the heavy chain variable
region of the 23C6 human monoclonal antibody. The CDR1 (SEQ ID
NO:4), CDR2 (SEQ ID NO:8) and CDR3 (SEQ ID NO:12) regions are
delineated and the V, D and J germline derivations are
indicated.
[0108] FIG. 4B shows the nucleotide sequence (SEQ ID NO:49) and
amino acid sequence (SEQ ID NO:39) of the V.sub.K.1 kappa light
chain variable region of the 23C6 human monoclonal antibody. The
CDR1 (SEQ ID NO:17), CDR2 (SEQ ID NO:23) and CDR3 (SEQ ID NO:29)
regions are delineated and the V and J germline derivations are
indicated.
[0109] FIG. 4C shows the nucleotide sequence (SEQ ID NO:50) and
amino acid sequence (SEQ ID NO:40) of the V.sub.K.2 kappa light
chain variable region of the 23C6 human monoclonal antibody. The
CDR1 (SEQ ID NO:18), CDR2 (SEQ ID NO:24) and CDR3 (SEQ ID NO:30)
regions are delineated and the V and J germline derivations are
indicated.
[0110] FIG. 5A shows the alignment of the amino acid sequence of
the heavy chain variable regions of 12C5 (SEQ ID NO:31) with the
human germline V.sub.H 7-4.1 amino acid sequence (SEQ ID
NO:51).
[0111] FIG. 5B shows the alignment of the amino acid sequence of
the light chain variable region of 12C5 (SEQ ID NO:35) with the
human germline V.sub..lamda. 2b2 amino acid sequence (SEQ ID
NO:55).
[0112] FIG. 6A shows the alignment of the amino acid sequence of
the heavy chain variable regions of 19A3/CD22.1 (SEQ ID NO:32) with
the human germline V.sub.H 4-34 amino acid sequence (SEQ ID
NO:52).
[0113] FIG. 6B shows the alignment of the amino acid sequence of
the light chain variable regions of 19A3/CD22.1/CD22.2 (SEQ ID
NO:36) with the human germline V.sub.K L6 amino acid sequence (SEQ
ID NO:56).
[0114] FIG. 6C shows the alignment of the amino acid sequence of
the heavy chain variable region of CD22.2 (SEQ ID NO:61) with the
human germline V.sub.H 4-34 amino acid sequence (SEQ ID NO:52).
[0115] FIG. 7A shows the alignment of the amino acid sequence of
the heavy chain variable regions of 16F7 (SEQ ID NO:33) with the
human germline V.sub.H 5-51 amino acid sequence (SEQ ID NO:53).
[0116] FIG. 7B shows the alignment of the amino acid sequence of
the V.sub.K.1 light chain variable region of 16F7 (SEQ ID NO:37)
with the human germline V.sub.K A27 amino acid sequence (SEQ ID
NO:57).
[0117] FIG. 7C shows the alignment of the amino acid sequence of
the V.sub.K.2 light chain variable region of 16F7 (SEQ ID NO:38)
with the human germline V.sub.K A10 amino acid sequence (SEQ ID
NO:57).
[0118] FIG. 8A shows the alignment of the amino acid sequence of
the heavy chain variable regions of 23C6 (SEQ ID NO:34) with the
human germline V.sub.H 1-69 amino acid sequence (SEQ ID NO:54).
[0119] FIG. 8B shows the alignment of the amino acid sequence of
the V.sub.K.1 light chain variable region of 23C6 (SEQ ID NO:39)
and the V.sub.K.2 light chain variable region of 23C6 (SEQ ID
NO:40) with the human germline V.sub.K L6 amino acid sequence (SEQ
ID NO:56).
[0120] FIG. 9 is a bar graph showing the internalization of
anti-CD22 human antibodies 12C5, 19A3, 16F7 and 23C6 into Raji
cells.
[0121] FIG. 10A is a graph showing ADCC activity (as measured by %
lysis) of anti-CD22 human antibodies 12C5, 19A3, 16F7 and 23C6
against Daudi cells.
[0122] FIG. 10B is a graph showing ADCC activity (as measured by %
lysis) of anti-CD22 human antibodies 12C5, 19A3, 16F7 and 23C6
against Raji cells.
[0123] FIG. 11 is a bar graph showing the effect of immobilized
anti-CD22 human antibodies 12C5, 19A3, 16F7 and 23C6 on
BCR-stimulated Ramos cells, as measured by % cell death.
[0124] FIG. 12 is a graph showing CD22 ECD binding by anti-CD22
recombinant human antibodies CD22.1 and CD22.2 as compared to that
of the 19A3 parent human antibody.
[0125] FIG. 13 is a graph showing binding of CD22 expressed on the
surface of CHO cells by the by anti-CD22 recombinant human
antibodies CD22.1 and CD22.2.
[0126] FIG. 14 is a graph showing binding of CD22 expressed on the
surface of Raji cells by the by anti-CD22 recombinant human
antibodies CD22.1 and CD22.2.
[0127] FIG. 15 is a bar graph showing binding of the CD22 ECD
amino-terminal domains 1 and 2 by anti-CD22 antibodies 12C5, 19A3,
16F7 and 23C6, and by recombinant human antibodies CD22.1 and
CD22.2.
[0128] FIG. 16 shows the in-vivo effect of antibody-drug conjugates
CD22.1-Cytotoxin A and CD22.2-Cytotoxin A on Raji-cell tumor size
in SCID mice.
[0129] FIG. 17A shows the nucleotide sequence (SEQ ID NO:87) and
amino acid sequence (SEQ ID NO:81) of the 4G6 human antibody. The
CDR1 (SEQ ID NO:63), CDR2 (SEQ ID NO:66) and CDR3 (SEQ ID NO:69)
regions are delineated and the V, D and J germline derivations are
indicated.
[0130] FIG. 17B shows the nucleotide sequence (SEQ ID NO:90) and
amino acid sequence (SEQ ID NO:84) of the V.sub.K1 kappa light
chain variable region of the 4G6 human monoclonal antibody. The
CDR1 (SEQ ID NO:72), CDR2 (SEQ ID NO:75) and CDR3 (SEQ ID NO:78)
regions are delineated and the V and J germline derivations are
indicated.
[0131] FIG. 17C shows the nucleotide sequence (SEQ ID NO:91) and
amino acid sequence (SEQ ID NO:85) of the V.sub.K2 kappa light
chain variable region of the 4G6 human monoclonal antibody. The
CDR1 (SEQ ID NO:73), CDR2 (SEQ ID NO:76) and CDR3 (SEQ ID NO:79)
regions are delineated and the V and J germline derivations are
indicated.
[0132] FIG. 18A shows the nucleotide sequence (SEQ ID NO:88) and
amino acid sequence (SEQ ID NO:82) of the V.sub.H1 heavy chain
variable region of the 21F6 human monoclonal antibody. The CDR1
(SEQ ID NO:64), CDR2 (SEQ ID NO:67) and CDR3 (SEQ ID NO:70) regions
are delineated and the V, D and J germline derivations are
indicated.
[0133] FIG. 18B shows the nucleotide sequence (SEQ ID NO:89) and
amino acid sequence (SEQ ID NO:83) of the V.sub.H2 heavy chain
variable region of the 21F6 human monoclonal antibody. The CDR1
(SEQ ID NO:65), CDR2 (SEQ ID NO:68) and CDR3 (SEQ ID NO:71) regions
are delineated and the V, D and J germline derivations are
indicated.
[0134] FIG. 18C shows the nucleotide sequence (SEQ ID NOs:92) and
amino acid sequence (SEQ ID NO:86) of the kappa light chain
variable region of the 21F6 human monoclonal antibody. The CDR1
(SEQ ID NO:74), CDR2 (SEQ ID NO:77) and CDR3 (SEQ ID NO:80) regions
are delineated and the V and J germline derivations are
indicated.
[0135] FIG. 19A shows the alignment of the amino acid sequence of
the heavy chain variable regions of 4G6 (SEQ ID NO:81) with the
human germline V.sub.H 1-69 amino acid sequence (SEQ ID NO:54).
[0136] FIG. 19B shows the alignment of the amino acid sequence of
the V.sub.K1 kappa light chain variable region of 4G6 (SEQ ID
NO:84) with the human germline V.sub.K L18 amino acid sequence (SEQ
ID NO:93).
[0137] FIG. 19C shows the alignment of the amino acid sequence of
the V.sub.K2 kappa light chain variable region of 4G6 (SEQ ID
NO:85) with the human germline V.sub.K A27 amino acid sequence (SEQ
ID NO:57).
[0138] FIG. 20A shows the alignment of the amino acid sequence of
the V.sub.H1 heavy chain variable regions of 21F6 (SEQ ID NO:82)
with the human germline V.sub.H 4-34 amino acid sequence (SEQ ID
NO:52).
[0139] FIG. 20B shows the alignment of the amino acid sequence of
the V.sub.H2 heavy chain variable regions of 21F6 (SEQ ID NO:83)
with the human germline V.sub.H 4-34 amino acid sequence (SEQ ID
NO:52).
[0140] FIG. 20C shows the alignment of the amino acid sequence of
the kappa light chain variable region of 21F6 (SEQ ID NO:86) with
the human germline V.sub.K L6 amino acid sequence (SEQ ID
NO:56).
[0141] FIG. 21 is a graph showing binding of CD22 expressed on the
surface of CHO cells by the anti-CD22 human antibody 4G6.
[0142] FIG. 22 is a graph showing binding of CD22 expressed on the
surface of CHO cells by anti-CD22 human antibody 21F6.
[0143] FIG. 23 is a graph showing binding of CD22 expressed on the
surface of Raji cells by anti-CD22 human antibody 21F6.
DETAILED DESCRIPTION OF THIS DISCLOSURES
[0144] The present disclosure relates to isolated monoclonal
antibodies, particularly human monoclonal antibodies that bind
specifically to human CD22 with high affinity. In certain
embodiments, the antibodies of this disclosure are derived from
particular heavy and light chain germline sequences and/or comprise
particular structural features such as CDR regions comprising
particular amino acid sequences. This disclosure provides isolated
antibodies, immuno-partner molecule conjugates, bispecific
molecules, affibodies, domain antibodies, nanobodies and unibodies,
methods of making said molecules, and pharmaceutical compositions
comprising said molecules and pharmaceutical carriers. The
invention also relates to methods of using the molecules, such as
to detect CD22, as well as to modulate B cell activity in diseases
or disorders associated with expression of CD22 or involving B cell
regulation, such as CD22+ tumors and inflammatory or autoimmune
disorders. This disclosure also provides methods of using the
anti-CD22 antibodies of this invention to inhibit the growth of
CD22+ tumor cells, for example, to treat B cell lymphomas.
Additionally, this disclosure provides methods of using the
anti-CD22 antibodies of this disclosure to treat inflammatory or
autoimmune disorders.
[0145] 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.
[0146] The terms "CD22," "BL-CAM," "B3," "Leu-14" and "Lyb-8" are
used interchangeably, and include variants, isoforms, and species
homologs of CD22. Accordingly, human antibodies of this disclosure
may, in certain cases, cross-react with CD22 from species other
than human. In certain embodiments, the antibodies may be
completely specific for human CD22 and may not exhibit species or
other types of non-human cross-reactivity. The complete amino acid
sequence of an exemplary human CD22 has Genbank accession number
NP_001762 (SEQ ID NO:59).
[0147] The human CD22 sequence may differ from human CD22 of SEQ ID
NO:59 by having, for example, conserved mutations or mutations in
non-conserved regions and the CD22 has substantially the same
biological function as the human CD22 of SEQ ID NO:59. For example,
a biological function of human CD22 is having an epitope in the
extracellular domain of CD22 that is specifically bound by an
antibody of the instant disclosure or a biological function of
human CD22 is modulation of BCR signalling.
[0148] A particular human CD22 sequence will generally be at least
90% identical in amino acids sequence to human CD22 of SEQ ID NO:59
and contains amino acid residues that identify the amino acid
sequence as being human when compared to CD22 amino acid sequences
of other species (e.g., murine). In certain cases, a human CD22 may
be at least 95%, or even at least 96%, 97%, 98%, or 99% identical
in amino acid sequence to CD22 of SEQ ID NO:59. In certain
embodiments, a human CD22 sequence will display no more than 10
amino acid differences from the CD22 of SEQ ID NO:59. In certain
embodiments, the human CD22 may display no more than 5, or even no
more than 4, 3, 2, or 1 amino acid difference from the CD22 of SEQ
ID NO:59. Percent identity can be determined as described
herein.
[0149] The term "immune response" refers to the action of, for
example, lymphocytes, antigen presenting cells, phagocytic cells,
granulocytes, and soluble macromolecules produced by the above
cells or the liver (including antibodies, cytokines, and
complement) that results in selective damage to, destruction of, or
elimination from the human body of invading pathogens, cells or
tissues infected with pathogens, cancerous cells, or, in cases of
autoimmunity or pathological inflammation, normal human cells or
tissues.
[0150] A "signal transduction pathway" refers to the biochemical
relationship between a variety of signal transduction molecules
that play a role in the transmission of a signal from one portion
of a cell to another portion of a cell. As used herein, the phrase
"cell surface receptor" includes, for example, molecules and
complexes of molecules capable of receiving a signal and the
transmission of such a signal across the plasma membrane of a cell.
An example of a "cell surface receptor" of the present disclosure
is the CD22 protein.
[0151] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chains thereof. An "antibody" refers 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, C.sub.H1, C.sub.H2 and C.sub.H3. 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, C.sub.L. 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 (C1q) of the
classical complement system.
[0152] 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., CD22). 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, C.sub.L and C.sub.H1 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 Fab' fragment, which is essentially an Fab with part of the
hinge region (see, Fundamental Immunology (Paul ed., 3.sup.rd ed.
1993); (iv) a Fd fragment consisting of the V.sub.H and C.sub.H1
domains; (v) a Fv fragment consisting of the V.sub.L and V.sub.H
domains of a single arm of an antibody, (vi) a dAb fragment (Ward
et al., (1989) Nature 341:544-546), which consists of a V.sub.H
domain; (vii) an isolated complementarity determining region (CDR);
and (viii) a nanobody, a heavy chain variable region containing a
single variable domain and two constant domains. 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.
[0153] The abbreviation "V.sub.K", as used herein, refers to the
variable domain of a kappa light chain, whereas the abbreviation
"V.sub..lamda.", as used herein, refers to the variable domain of a
lambda light chain. The abbreviation "V.sub.L", as used herein,
refers to the variable domain of an immunoglobulin light chain and
thus encompasses both V.sub.K and V.sub..lamda..quadrature. light
chains.
[0154] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds CD22 is substantially free of
antibodies that specifically bind antigens other than CD22). An
isolated antibody that specifically binds CD22 may, however, have
cross-reactivity to other antigens, such as CD22 molecules from
other species. Moreover, an isolated antibody may be substantially
free of other cellular material and/or chemicals.
[0155] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope.
[0156] The term "human antibody", as used herein, is intended to
include antibodies having variable regions in which both the
framework and CDR regions are derived from human germline
immunoglobulin sequences. Furthermore, if the antibody contains a
constant region, the constant region also is derived from human
germline immunoglobulin sequences. The human antibodies of this
disclosure may 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). However, the term "human antibody", as used herein, is
not intended to 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.
[0157] The term "human monoclonal antibody" refers to antibodies
displaying a single binding specificity, which have variable
regions in which both the framework and CDR regions are derived
from human germline immunoglobulin sequences. In one embodiment,
the human monoclonal antibodies are produced by a hybridoma which
includes a B cell obtained from a transgenic nonhuman 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.
[0158] 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 (described further below), (b) antibodies
isolated from a host cell transformed to express the human
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 have
variable regions in which the framework and CDR regions are derived
from human germline immunoglobulin sequences. In certain
embodiments, however, such recombinant human antibodies can be
subjected to in vitro mutagenesis (or, when an animal transgenic
for human Ig sequences is used, in vivo somatic mutagenesis) and
thus the amino acid sequences of the V.sub.H and V.sub.L regions of
the recombinant antibodies are sequences that, while derived from
and related to human germline V.sub.H and V.sub.L sequences, may
not naturally exist within the human antibody germline repertoire
in vivo.
[0159] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgG1) that is encoded by the heavy chain constant
region genes.
[0160] 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."
[0161] The term "human antibody derivatives" refers to any modified
form of the human antibody, e.g., a conjugate of the antibody and
another agent or antibody.
[0162] The term "humanized antibody" is intended to refer to
antibodies in which CDR sequences derived from the germline of
another mammalian species, such as a mouse, have been grafted onto
human framework sequences. Additional framework region
modifications may be made within the human framework sequences.
[0163] The term "chimeric antibody" is intended to refer to
antibodies in which the variable region sequences are derived from
one species and the constant region sequences are derived from
another species, such as an antibody in which the variable region
sequences are derived from a mouse antibody and the constant region
sequences are derived from a human antibody.
[0164] The term "antibody mimetic" is intended to refer to
molecules capable of mimicking an antibody's ability to bind an
antigen, but which are not limited to native antibody structures.
Examples of such antibody mimetics include, but are not limited to,
Affibodies, DARPins, Anticalins, Avimers, and Versabodies, all of
which employ binding structures that, while they mimic traditional
antibody binding, are generated from and function via distinct
mechanisms.
[0165] As used herein, the term "partner molecule" refers to the
entity which is conjugated to an antibody in an antibody-partner
molecule conjugate. Examples of partner molecules include drugs,
toxins, marker molecules (including, but not limited to peptide and
small molecule markers such as fluorochrome markers, as well as
single atom markers such as radioisotopes), proteins and
therapeutic agents.
[0166] As used herein, an antibody that "specifically binds to
human CD22" is intended to refer to an antibody that binds to human
CD22 (and possibly CD22 from one or more non-human species) but
does not substantially bind to non-CD22 proteins. In certain
embodiments, an antibody of the instant disclosure specifically
binds to human CD22 of SEQ ID NO:59 or a variant thereof.
Preferably, the antibody binds to human CD22 with a K.sub.D of
1.times.10.sup.-7 M or less, more preferably 1.times.10.sup.-8 M or
less, more preferably 5.times.10.sup.-9 M or less, more preferably
1.times.10.sup.-9 M or less, even more preferably
5.times.10.sup.-10 M or less, and even more preferably
7.times.10.sup.-11 or less.
[0167] The term "does not substantially bind" to a protein or
cells, as used herein, means does not bind or does not bind with a
high affinity to the protein or cells, i.e. binds to the protein or
cells with a K.sub.D of 1.times.10.sup.-6 M or more, more
preferably 1.times.10.sup.-5 M or more, more preferably
1.times.10.sup.-4 M or more, more preferably 1.times.10.sup.-3 M or
more, even more preferably 1.times.10.sup.-2 M or more.
[0168] The term "K.sub.assoc" or "K.sub.a", as used herein, is
intended to refer to the association rate of a particular
antibody-antigen interaction, whereas the term "K.sub.dis" or
"K.sub.d," as used herein, is intended to refer to the dissociation
rate of a particular antibody-antigen interaction. The term
"K.sub.D", as used herein, is intended to refer to the dissociation
constant, which is obtained from the ratio of K.sub.d to K.sub.a
(i.e., K.sub.d/K.sub.a) and is expressed as a molar concentration
(M). K.sub.D values for antibodies can be determined using methods
well established in the art. A preferred method for determining the
K.sub.D of an antibody is by using surface plasmon resonance,
preferably using a biosensor system such as a Biacore.RTM.
system.
[0169] As used herein, the term "high affinity" for an IgG antibody
refers to an antibody having a K.sub.D of 1.times.10.sup.-7 M or
less, more preferably 5.times.10.sup.-8 M or less, even more
preferably 1.times.10.sup.-8 M or less, even more preferably
5.times.10.sup.-9 M or less and even more preferably
1.times.10.sup.-9 M or less for a target antigen. However, "high
affinity" binding can vary for other antibody isotypes. For
example, "high affinity" binding for an IgM isotype refers to an
antibody having a K.sub.D of 10.sup.-6 M or less, more preferably
10.sup.-7 M or less, even more preferably 10.sup.-8 M or less.
[0170] As used herein, the term "subject" includes any human or
nonhuman animal. The term "nonhuman animal" includes all
vertebrates, e.g., mammals and non-mammals, such as nonhuman
primates, sheep, dogs, cats, horses, cows, chickens, amphibians,
reptiles, etc.
[0171] The symbol "--", whether utilized as a bond or displayed
perpendicular to a bond, indicates the point at which the displayed
moiety is attached to the remainder of the molecule, solid support,
etc.
[0172] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
may be fully saturated, mono- or polyunsaturated and can include
di- and multivalent radicals, having the number of carbon atoms
designated (i.e. C.sub.1-C.sub.10 means one to ten carbons).
Examples of saturated hydrocarbon radicals include, but are not
limited to, groups such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,
(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for
example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An
unsaturated alkyl group is one having one or more double bonds or
triple bonds. Examples of unsaturated alkyl groups include, but are
not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The
term "alkyl," unless otherwise noted, is also meant to include
those derivatives of alkyl defined in more detail below, such as
"heteroalkyl." Alkyl groups, which are limited to hydrocarbon
groups are termed "homoalkyl".
[0173] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified, but not limited, by
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and further includes those
groups described below as "heteroalkylene." Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred in the
present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having eight or fewer
carbon atoms.
[0174] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon radical, or combinations
thereof, consisting of the stated number of carbon atoms and at
least one heteroatom selected from the group consisting of O, N,
Si, and S, and wherein the nitrogen, carbon and sulfur atoms may
optionally be oxidized and the nitrogen heteroatom may optionally
be quaternized. The heteroatom(s) O, N, S, and Si may be placed at
any interior position of the heteroalkyl group or at the position
at which the alkyl group is attached to the remainder of the
molecule. Examples include, but are not limited to,
--CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Similarly, the term
"heteroalkylene" by itself or as part of another substituent means
a divalent radical derived from heteroalkyl, as exemplified, but
not limited by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). The terms
"heteroalkyl" and "heteroalkylene" encompass poly(ethylene glycol)
and its derivatives (see, for example, Shearwater Polymers Catalog,
2001). Still further, for alkylene and heteroalkylene linking
groups, no orientation of the linking group is implied by the
direction in which the formula of the linking group is written. For
example, the formula --C(O).sub.2R'-- represents both
--C(O).sub.2R'-- and --R'C(O).sub.2--.
[0175] The term "lower" in combination with the terms "alkyl" or
"heteroalkyl" refers to a moiety having from 1 to 6 carbon
atoms.
[0176] The terms "alkoxy," "alkylamino," "alkylsulfonyl," and
"alkylthio" (or thioalkoxy) are used in their conventional sense,
and refer to those alkyl groups attached to the remainder of the
molecule via an oxygen atom, an amino group, an SO.sub.2 group or a
sulfur atom, respectively. The term "arylsulfonyl" refers to an
aryl group attached to the remainder of the molecule via an
SO.sub.2 group, and the term "sulfhydryl" refers to an SH
group.
[0177] In general, an "acyl substituent" is also selected from the
group set forth above. As used herein, the term "acyl substituent"
refers to groups attached to, and fulfilling the valence of a
carbonyl carbon that is either directly or indirectly attached to
the polycyclic nucleus of the compounds of the present
invention.
[0178] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of substituted or unsubstituted "alkyl" and
substituted or unsubstituted "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include, but are not limited
to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),
1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,
3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,
2-piperazinyl, and the like. The heteroatoms and carbon atoms of
the cyclic structures are optionally oxidized.
[0179] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" is mean to
include, but not be limited to, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0180] The term "aryl" means, unless otherwise stated, a
substituted or unsubstituted polyunsaturated, aromatic, hydrocarbon
substituent which can be a single ring or multiple rings
(preferably from 1 to 3 rings) which are fused together or linked
covalently. The term "heteroaryl" refers to aryl groups (or rings)
that contain from one to four heteroatoms selected from N, O, and
S, wherein the nitrogen, carbon and sulfur atoms are optionally
oxidized, and the nitrogen atom(s) are optionally quaternized. A
heteroaryl group can be attached to the remainder of the molecule
through a heteroatom. Non-limiting examples of aryl and heteroaryl
groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,
1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,
4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,
2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,
5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl,
3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below. "Aryl" and "heteroaryl" also encompass ring
systems in which one or more non-aromatic ring systems are fused,
or otherwise bound, to an aryl or heteroaryl system.
[0181] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0182] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") include both substituted and unsubstituted
forms of the indicated radical. Preferred substituents for each
type of radical are provided below.
[0183] Substituents for the alkyl, and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are
generally referred to as "alkyl substituents" and "heteroalkyl
substituents," respectively, and they can be one or more of a
variety of groups selected from, but not limited to: --OR', .dbd.O,
.dbd.NR', .dbd.N--OR', --NR'R'', --SR', -halogen, --SiR'R''R''',
--OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'', --OC(O)NR'R'',
--NR''C(O)R', --NR'--C(O)NR''R''', --NR''C(O).sub.2R',
--NR--C(NR'R''R''').dbd.NR'''', --NR--C(NR'R'').dbd.NR''',
--S(O)R', --S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --CN
and --NO.sub.2 in a number ranging from zero to (2m'+1), where m'
is the total number of carbon atoms in such radical. R', R'', R'''
and R'''' each preferably independently refer to hydrogen,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, e.g., aryl substituted with 1-3 halogens,
substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or
arylalkyl groups. When a compound of the invention includes more
than one R group, for example, each of the R groups is
independently selected as are each R', R'', R''' and R'''' groups
when more than one of these groups is present. When R' and R'' are
attached to the same nitrogen atom, they can be combined with the
nitrogen atom to form a 5, 6, or 7-membered ring. For example,
--NR'R'' is meant to include, but not be limited to, 1-pyrrolidinyl
and 4-morpholinyl. From the above discussion of substituents, one
of skill in the art will understand that the term "alkyl" is meant
to include groups including carbon atoms bound to groups other than
hydrogen groups, such as haloalkyl (e.g., --CF.sub.3 and
--CH.sub.2CF.sub.3) and acyl (e.g., --C(O)CH.sub.3, --C(O)CF.sub.3,
--C(O)CH.sub.2OCH.sub.3, and the like).
[0184] Similar to the substituents described for the alkyl radical,
the aryl substituents and heteroaryl substituents are generally
referred to as "aryl substituents" and "heteroaryl substituents,"
respectively and are varied and selected from, for example:
halogen, --OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR',
-halogen, --SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R'').dbd.NR''', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and
--NO.sub.2, --R', --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxy, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
the aromatic ring system; and where R', R'', R''' and R'''' are
preferably independently selected from hydrogen,
(C.sub.1-C.sub.8)alkyl and heteroalkyl, unsubstituted aryl and
heteroaryl, (unsubstituted aryl)-(C.sub.1-C.sub.4)alkyl, and
(unsubstituted aryl)oxy-(C.sub.1-C.sub.4)alkyl. When a compound of
the invention includes more than one R group, for example, each of
the R groups is independently selected as are each R', R'', R'''
and R'''' groups when more than one of these groups is present.
[0185] Two of the aryl substituents on adjacent atoms of the aryl
or heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CRR').sub.q--U--, wherein T and U are
independently --NR--, --O--, --CRR'-- or a single bond, and q is an
integer of from 0 to 3. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CRR'--, --O--, --NR--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 4. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CRR').sub.s--X--(CR''R''').sub.d--, where s and d are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituents R, R', R'' and R''' are preferably independently
selected from hydrogen or substituted or unsubstituted
(C.sub.1-C.sub.6) alkyl.
[0186] As used herein, the term "diphosphate" includes but is not
limited to an ester of phosphoric acid containing two phosphate
groups. The term "triphosphate" includes but is not limited to an
ester of phosphoric acid containing three phosphate groups. For
example, particular drugs having a diphosphate or a triphosphate
include:
##STR00001##
[0187] As used herein, the term "heteroatom" includes oxygen (O),
nitrogen (N), sulfur (S) and silicon (Si).
[0188] The symbol "R" is a general abbreviation that represents a
substituent group that is selected from substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, and substituted or unsubstituted heterocyclyl
groups.
[0189] Various aspects of this disclosure are described in further
detail in the following subsections.
Anti-CD22 Antibodies
[0190] The antibodies of this disclosure are characterized by
particular functional features or properties of the antibodies. For
example, the antibodies bind specifically to human CD22.
Preferably, an antibody of the invention binds to CD22 with high
affinity, for example with a K.sub.D of 1.times.10.sup.-7 M or
less.
[0191] The anti-CD22 antibodies of this disclosure preferably
exhibit one or more of the following characteristics:
[0192] (a) internalizing into CD22.sup.+ cells;
[0193] (b) exhibiting antibody dependent cellular cytotoxicity
(ADCC) against CD22.sup.+ cells;
[0194] (c) enhancing cell death of Ramos cells induced by B cell
receptor (BCR) stimulation, and
[0195] (d) inhibits growth of CD22-expressing cells in vivo when
conjugated to a cytotoxin.
[0196] In preferred embodiments, the antibody exhibits at least two
of properties (a), (b), (c) and (d). In yet another embodiment, the
antibody exhibits three of properties (a), (b), (c) and (d). In
another embodiment, the antibody exhibits all four of properties
(a), (b), (c), and (d).
[0197] While the anti-CD22 antibodies of the invention exhibit
certain functional properties, in certain embodiments another
feature of the antibodies is that they do not exhibit other
particular functional properties. For example, in certain
embodiments, the antibody does not have a direct anti-proliferative
effect on Ramos cells. In other embodiments, the antibody does not
induce calcium flux in Ramos cells. In yet other embodiments, the
antibody does not mediate complement dependent cytotoxicity (CDC)
on Ramos cells.
[0198] It is noted that it has been reported that a humanized
anti-CD22 antibody, epratuzumab, lacked a direct anti-proliferative
effect and CDC activity against non-Hodgkin's lymphoma cell lines
yet the antibody did mediate cytotoxic effects against the cell
lines by other means (see Carnahan, J. et al. (2006) Mol. Immunol.
44:1331-1341).
[0199] Preferably, an antibody of this disclosure binds to human
CD22 with a K.sub.D of 1.times.10.sup.-7 M or less, binds to human
CD22 with a K.sub.D of 1.times.10.sup.-8 M or less, binds to human
CD22 with a K.sub.D of 5.times.10.sup.-9 M or less, binds to human
CD22 with a K.sub.D of 3.times.10.sup.-9 M or less, binds to human
CD22 with a K.sub.D of 1.times.10.sup.-9 M or less, or binds to
human CD22 with a K.sub.D of 5.times.10.sup.-10 M or less, or binds
to human CD22 with a K.sub.D of 1.times.10.sup.-10 or binds to
human CD22 with a K.sub.D of 7.times.10.sup.-11 M or less.
[0200] Standard assays to evaluate the binding affinity of the
antibodies toward human CD22 are known in the art, including for
example, ELISA and BIAcore analysis with recombinant CD22 (see
Example 3). The Examples also provide detailed descriptions of
suitable assays for evaluating antibody internalization (Example
4), ADCC activity (Example 5), enhancement of cell death induced by
BCR stimulation (Example 7), direct anti-proliferative effects of
antibodies (Example 8), induction of calcium flux (Example 6), and
CDC activity (Example 9), and anti-proliferative effects of
antibody-drug immunoconjugates on solid tumor cell proliferation in
vivo (Example 10).
Monoclonal Antibodies 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6
and 21F6
[0201] Preferred antibodies of this disclosure are the human
monoclonal antibodies 12C5, 19A3, 16F7, 23C6, 4G6 and 21F6, and the
recombinant human monoclonal antibodies CD22.1, CD22.2, all of
which were isolated and structurally characterized as described in
Examples 1 and 2.
[0202] The V.sub.H amino acid sequences of 12C5, 19A3, CD22.1,
CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs: 31, 32,
61, 33, 34, 81, 82 and 83, respectively, wherein the heavy chains
of 19A3 and CD22.1 are identical and correspond to SEQ ID NO:32 and
the V.sub.H heavy chain of 21F6 correspond to either SEQ ID NO:82
or 83
[0203] The V.sub.L amino acid sequences of 12C5, 19A3, CD22.1,
CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs:35, 36,
37, 38, 39, 40, 84, 85 and 86, respectively, wherein the kappa
light chains of 19A3, CD22.1 and CD22.2 are identical and
correspond to SEQ ID NO:36, the kappa light chain of 16F7
corresponds to either SEQ ID NO:37 or 38, the kappa light chain of
23C6 corresponds to either SEQ ID NO:39 or 40, and the kappa light
chain of 4G6 corresponds to either SEQ ID NO: 84 or 85.
[0204] Given that each of these antibodies can bind to CD22, the
V.sub.H and V.sub.L sequences can be "mixed and matched" to create
other anti-CD22 binding molecules of this disclosure. CD22 binding
of such "mixed and matched" antibodies can be tested using the
binding assays described above and in the Examples (e.g., ELISA or
flow cytometry). Preferably, when V.sub.H and V.sub.L chains are
mixed and matched, a V.sub.H sequence from a particular
V.sub.H/V.sub.L pairing is replaced with a structurally similar
V.sub.H sequence. Likewise, preferably a V.sub.L sequence from a
particular V.sub.H/V.sub.L pairing is replaced with a structurally
similar V.sub.L sequence.
[0205] Accordingly, in one aspect, this disclosure provides an
isolated monoclonal antibody, or antigen binding portion thereof
comprising: [0206] (a) a heavy chain variable region comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs:31-34, 61 and 81-83; and [0207] (b) a light chain variable
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs:35-40 and 84-86; wherein the antibody
specifically binds CD22, preferably human CD22.
[0208] Preferred heavy and light chain combinations include: [0209]
(a) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:31 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:35; or [0210] (b) a
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO:32 or 61, and a light chain variable region comprising
the amino acid sequence of SEQ ID NO:36; or [0211] (c) a heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO:33 and a light chain variable region comprising the amino acid
sequence of SEQ ID NO:37 or 38; or [0212] (d) a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:34
and a light chain variable region comprising the amino acid
sequence of SEQ ID NO:39 or 40; or [0213] (e) a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:81
and a light chain variable region comprising the amino acid
sequence of SEQ ID NO:84 or 85; or [0214] (f) a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:82
or 83 or and a light chain variable region comprising the amino
acid sequence of SEQ ID NO:86.
[0215] In another aspect, this disclosure provides antibodies that
comprise the heavy chain and light chain CDR1s, CDR2s and CDR3s of
12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6, 21F6, or combinations
thereof.
[0216] The amino acid sequences of the V.sub.H CDR1s of 12C5, 19A3,
CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs:
1-4 and 63-65, respectively (wherein the CDR1s of the V.sub.H
sequences of 19A3, CD22.1 and CD22.2 are identical and are shown in
SEQ ID NO:2, and the CDR1s of the V.sub.H1 and V.sub.H2 sequences
of 21F6 are identical and are shown in SEQ ID NOs: 64 and 65,
respectively.
[0217] The amino acid sequences of the V.sub.H CDR2s of 12C5, 19A3,
CD22.1, 16F7, 23C6, CD22.2, 4G6 and 21F6 are shown in SEQ ID NOs:
5-8, 60, and 66-68, respectively (wherein the CDR2s of the V.sub.H
sequences of 19A3 and CD22.1 are identical and are shown in SEQ ID
NO:6, the CDR2 of the V.sub.H sequence of CD22.2 is shown in SEQ ID
NO:60, and the CDR2s of the V.sub.H1 and V.sub.H2 sequences of 21F6
are shown in SEQ ID NOs:67 and 68).
[0218] The amino acid sequences of the V.sub.H CDR3s of 12C5, 19A3,
CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs:
9-12 and 69-71, respectively (wherein the CDR3s of the V.sub.H.
sequences of 19A3, CD22.1 and CD22.2 are identical and are shown in
SEQ ID NO:10, and the CDR3s of the V.sub.H1 and V.sub.H2 sequences
of 21F6 are shown in SEQ ID NOs:70 and 71).
[0219] The amino acid sequences of the V.sub.L CDR1s of 12C5, 19A3,
CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs:
13-18 and 72-74, respectively (wherein the CDR1s of the V.sub.K
sequences of 19A3, CD22.1 and CD22.2 are identical, and are shown
in SEQ ID NO:14, the CDR1s of the V.sub.K.1 and V.sub.K.2 sequences
of 16F7 are shown in SEQ ID NOs: 15 and 16, the CDR1s of the
V.sub.K.1 and V.sub.K.2 sequences of 23C6 are shown in SEQ ID NOs:
17 and 18, and the CDR1s of the V.sub.K.1 and V.sub.K.2 sequences
of 4G6 are shown in SEQ ID NOs 72 and 73).
[0220] The amino acid sequences of the V.sub.L CDR2s of 12C5, 19A3,
CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs:
19-24 and 75-77, respectively (wherein the CDR2s of the V.sub.K
sequences of 19A3, CD22.1 and CD22.2 are identical, and are shown
in SEQ ID NO:20, the CDR2s of the V.sub.K.1 and V.sub.K.2 sequences
of 16F7 are shown in SEQ ID NOs: 21 and 22, the CDR2s of the
V.sub.K.1 and V.sub.K.2 sequences of 23C6 are shown in SEQ ID NOs:
23 and 24, and the CDR2s of the V.sub.K.1 and V.sub.K.2 sequences
of 4G6 are shown in SEQ ID NOs: 75 and 76).
[0221] The amino acid sequences of the V.sub.L CDR1s of 12C5, 19A3,
CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6 are shown in SEQ ID NOs:
25-30 and 78-80, respectively (wherein the CDR3s of the V.sub.K
sequences of 19A3, CD22.1 and CD22.2 are identical, and are shown
in SEQ ID NO:26, the CDR3s of the V.sub.K.1 and V.sub.K.2 sequences
of 16F7 are shown in SEQ ID NOs: 27 and 28, the CDR3s of the
V.sub.K.1 and V.sub.K.2 sequences of 23C6 are shown in SEQ ID NOs:
29 and 30, and the CDR3s of the V.sub.K.1 and V.sub.K.2 sequences
of 4G6 are shown in SEQ ID NOs: 78 and 79).
[0222] The CDR regions are delineated using the Kabat system
(Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242).
[0223] Given that each of these antibodies can bind to CD22 and
that antigen-binding specificity is provided primarily by the CDR1,
CDR2, and CDR3 regions, the V.sub.H CDR1, CDR2, and CDR3 sequences
and V.sub.L CDR1, CDR2, and CDR3 sequences can be "mixed and
matched" (i.e., CDRs from different antibodies can be mixed and
match, although each antibody must contain a V.sub.H CDR1, CDR2,
and CDR3 and a V.sub.L CDR1, CDR2, and CDR3) to create other
anti-CD22 binding molecules of this disclosure. CD22 binding of
such "mixed and matched" antibodies can be tested using the binding
assays described above and in the Examples (e.g., ELISAs,
Biacore.RTM. analysis). Preferably, when V.sub.H CDR sequences are
mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from a
particular V.sub.H sequence is replaced with a structurally similar
CDR sequence(s). Likewise, when V.sub.L CDR sequences are mixed and
matched, the CDR1, CDR2 and/or CDR3 sequence from a particular
V.sub.L sequence preferably is replaced with a structurally similar
CDR sequence(s). It will be readily apparent to the ordinarily
skilled artisan that novel V and V.sub.L sequences can be created
by substituting one or more V.sub.H and/or V.sub.L CDR region
sequences with structurally similar sequences from the CDR
sequences disclosed herein for monoclonal antibodies CDR1s of 12C5,
19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6.
[0224] Accordingly, in another aspect, this disclosure provides an
isolated monoclonal antibody, or antigen binding portion thereof
comprising:
[0225] (a) a heavy chain variable region CDR1 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 1-4
and 63-65;
[0226] (b) a heavy chain variable region CDR2 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:
5-8, 60 and 66-68;
[0227] (c) a heavy chain variable region CDR3 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:
9-12 and 69-71;
[0228] (d) a light chain variable region CDR1 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:
13-18 and 72-74;
[0229] (e) a light chain variable region CDR2 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:
19-24 and 75-77; and
[0230] (f) a light chain variable region CDR3 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:
25-30 and 78-80;
[0231] wherein the antibody specifically binds CD22, preferably
human CD22.
In a preferred embodiment, the antibody comprises:
[0232] (a) a heavy chain variable region CDR1 comprising SEQ ID NO:
1;
[0233] (b) a heavy chain variable region CDR2 comprising SEQ ID
NO:5;
[0234] (c) a heavy chain variable region CDR3 comprising SEQ ID
NO:9;
[0235] (d) a light chain variable region CDR1 comprising SEQ ID
NO:13;
[0236] (e) a light chain variable region CDR2 comprising SEQ ID
NO:19; and
[0237] (f) a light chain variable region CDR3 comprising SEQ ID
NO:25.
In another preferred embodiment, the antibody comprises:
[0238] (a) a heavy chain variable region CDR1 comprising SEQ ID
NO:2;
[0239] (b) a heavy chain variable region CDR2 comprising SEQ ID
NO:6 or 60;
[0240] (c) a heavy chain variable region CDR3 comprising SEQ ID NO:
10;
[0241] (d) a light chain variable region CDR1 comprising SEQ ID NO:
14;
[0242] (e) a light chain variable region CDR2 comprising SEQ ID
NO:20; and
[0243] (f) a light chain variable region CDR3 comprising SEQ ID
NO:26.
In another preferred embodiment, the antibody comprises:
[0244] (a) a heavy chain variable region CDR1 comprising SEQ ID
NO:3;
[0245] (b) a heavy chain variable region CDR2 comprising SEQ ID
NO:7;
[0246] (c) a heavy chain variable region CDR3 comprising SEQ ID NO:
11;
[0247] (d) a light chain variable region CDR1 comprising SEQ ID
NO:15 or 16;
[0248] (e) a light chain variable region CDR2 comprising SEQ ID
NO:21 or 22; and
[0249] (f) a light chain variable region CDR3 comprising SEQ ID
NO:27 or 28.
In another preferred embodiment, the antibody comprises:
[0250] (a) a heavy chain variable region CDR1 comprising SEQ ID
NO:4;
[0251] (b) a heavy chain variable region CDR2 comprising SEQ ID
NO:8;
[0252] (c) a heavy chain variable region CDR3 comprising SEQ ID NO:
12;
[0253] (d) a light chain variable region CDR1 comprising SEQ ID
NO:17 or 18;
[0254] (e) a light chain variable region CDR2 comprising SEQ ID
NO:23 or 24; and
[0255] (f) a light chain variable region CDR3 comprising SEQ ID
NO:29 or 30.
In another preferred embodiment, the antibody comprises:
[0256] (a) a heavy chain variable region CDR1 comprising SEQ ID
NO:63;
[0257] (b) a heavy chain variable region CDR2 comprising SEQ ID NO
66;
[0258] (c) a heavy chain variable region CDR3 comprising SEQ ID
NO:69;
[0259] (d) a light chain variable region CDR1 comprising SEQ ID
NO:72 or 73;
[0260] (e) a light chain variable region CDR2 comprising SEQ ID
NO:75 or 76; and
[0261] (f) a light chain variable region CDR3 comprising SEQ ID
NO:78 or 79.
In another preferred embodiment, the antibody comprises:
[0262] (a) a heavy chain variable region CDR1 comprising SEQ ID
NO:64 or 65;
[0263] (b) a heavy chain variable region CDR2 comprising SEQ ID
NO:67 or 68;
[0264] (c) a heavy chain variable region CDR3 comprising SEQ ID
NO:70 or 71;
[0265] (d) a light chain variable region CDR1 comprising SEQ ID
NO:74;
[0266] (e) a light chain variable region CDR2 comprising SEQ ID
NO:77; and
[0267] (f) a light chain variable region CDR3 comprising SEQ ID
NO:80.
[0268] It is well known in the art that the CDR3 domain,
independently from the CDR1 and/or CDR2 domain(s), alone can
determine the binding specificity of an antibody for a cognate
antigen and that multiple antibodies can predictably be generated
having the same binding specificity based on a common CDR3
sequence. See, for example, Klimka et al., British J. of Cancer
83(2):252-260 (2000) (describing the production of a humanized
anti-CD30 antibody using only the heavy chain variable domain CDR3
of murine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol. Biol.
296:833-849 (2000) (describing recombinant epithelial
glycoprotein-2 (EGP-2) antibodies using only the heavy chain CDR3
sequence of the parental murine MOC-31 anti-EGP-2 antibody); Rader
et al., Proc. Natl. Acad. Sci. U.S.A. 95:8910-8915 (1998)
(describing a panel of humanized anti-integrin
.alpha..sub.v.beta..sub.3 antibodies using a heavy and light chain
variable CDR3 domain of a murine anti-integrin
.alpha..sub.v.beta..sub.3 antibody LM609 wherein each member
antibody comprises a distinct sequence outside the CDR3 domain and
capable of binding the same epitope as the parent muring antibody
with affinities as high or higher than the parent murine antibody);
Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994) (disclosing
that the CDR3 domain provides the most significant contribution to
antigen binding); Barbas et al., Proc. Natl. Acad. Sci. U.S.A.
92:2529-2533 (1995) (describing the grafting of heavy chain CDR3
sequences of three Fabs (SI-1, SI-40, and SI-32) against human
placental DNA onto the heavy chain of an anti-tetanus toxoid Fab
thereby replacing the existing heavy chain CDR3 and demonstrating
that the CDR3 domain alone conferred binding specificity); Ditzel
et al., J. Immunol. 157:739-749 (1996) (describing grafting studies
wherein transfer of only the heavy chain CDR3 of a parent
polyspecific Fab LNA3 to a heavy chain of a monospecific IgG
tetanus toxoid-binding Fab p313 antibody was sufficient to retain
binding specificity of the parent Fab); Berezov et al., BIAjournal
8: Scientific Review 8 (2001) (describing peptide mimetics based on
the CDR3 of an anti-HER2 monoclonal antibody; Igarashi et al., J.
Biochem (Tokyo) 117:452-7 (1995) (describing a 12 amino acid
synthetic polypeptide corresponding to the CDR3 domain of an
anti-phosphatidylserine antibody); Bourgeois et al., J. Virol
72:807-10 (1998) (showing that a single peptide derived form the
heavy chain CDR3 domain of an anti-respiratory syncytial virus
(RSV) antibody was capable of neutralizing the virus in vitro);
Levi et al., Proc. Natl. Acad. Sci. U.S.A. 90:4374-8 (1993)
(describing a peptide based on the heavy chain CDR3 domain of a
murine anti-HIV antibody); Polymenis and Stoller, J. Immunol.
152:5218-5329 (1994) (describing enabling binding of an scFv by
grafting the heavy chain CDR3 region of a Z-DNA-binding antibody)
and Xu and Davis, Immunity 13:37-45 (2000) (describing that
diversity at the heavy chain CDR3 is sufficient to permit otherwise
identical IgM molecules to distinguish between a variety of hapten
and protein antigens). See also, U.S. Pat. Nos. 6,951,646;
6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943;
5,762,905 and 5,760,185, describing patented antibodies defined by
a single CDR domain. Each of these references is hereby
incorporated by reference in its entirety.
[0269] Accordingly, the present disclosure provides monoclonal
antibodies comprising one or more heavy and/or light chain CDR3
domains from an antibody derived from a human or non-human animal,
wherein the monoclonal antibody is capable of specifically binding
to CD22. Within certain aspects, the present disclosure provides
monoclonal antibodies comprising one or more heavy and/or light
chain CDR3 domain from a non-human antibody, such as a mouse or rat
antibody, wherein the monoclonal antibody is capable of
specifically binding to CD22. Within some embodiments, such
inventive antibodies comprising one or more heavy and/or light
chain CDR3 domain from a non-human antibody (a) are capable of
competing for binding with; (b) retain the functional
characteristics; (c) bind to the same epitope; and/or (d) have a
similar binding affinity as the corresponding parental non-human
antibody.
[0270] Within other aspects, the present disclosure provides
monoclonal antibodies comprising one or more heavy and/or light
chain CDR3 domain from a human antibody, such as, for example, a
human antibody obtained from a non-human animal, wherein the human
antibody is capable of specifically binding to CD22. Within other
aspects, the present disclosure provides monoclonal antibodies
comprising one or more heavy and/or light chain CDR3 domain from a
first human antibody, such as, for example, a human antibody
obtained from a non-human animal, wherein the first human antibody
is capable of specifically binding to CD22 and wherein the CDR3
domain from the first human antibody replaces a CDR3 domain in a
human antibody that is lacking binding specificity for CD22 to
generate a second human antibody that is capable of specifically
binding to CD22. Within some embodiments, such inventive antibodies
comprising one or more heavy and/or light chain CDR3 domain from
the first human antibody (a) are capable of competing for binding
with; (b) retain the functional characteristics; (c) bind to the
same epitope; and/or (d) have a similar binding affinity as the
corresponding parental first human antibody.
Antibodies Having Particular Germline Sequences
[0271] In certain embodiments, an antibody of this disclosure
comprises a heavy chain variable region from a particular germline
heavy chain immunoglobulin gene and/or a light chain variable
region from a particular germline light chain immunoglobulin
gene.
[0272] For example, in a preferred embodiment, this disclosure
provides an isolated monoclonal antibody, or an antigen-binding
portion thereof, comprising a heavy chain variable region that is
the product of or derived from a human V.sub.H 7-4.1 gene, a human
V.sub.H 4-34 gene, a human V.sub.H 5-51 gene, or a human V.sub.H
1-69 gene, wherein the antibody specifically binds CD22.
[0273] In another preferred embodiment, this disclosure provides an
isolated monoclonal antibody, or an antigen-binding portion
thereof, comprising a light chain variable region that is the
product of or derived from a human V.sub..lamda. 2b2 gene, a human
V.sub.K L6 gene, a human V.sub.K A27 gene, a human V.sub.K A10
gene, or a human V.sub.K L18 gene, wherein the antibody
specifically binds CD22.
[0274] In yet another preferred embodiment, this disclosure
provides an isolated monoclonal antibody, or antigen-binding
portion thereof, wherein the antibody comprises a heavy chain
variable region that is the product of or derived from a human
V.sub.H 7-4.1 gene and comprises a light chain variable region that
is the product of or derived from a human V.sub..lamda. 2b2 gene,
wherein the antibody specifically binds to CD22, preferably human
CD22.
[0275] In yet another preferred embodiment, this disclosure
provides an isolated monoclonal antibody, or antigen-binding
portion thereof, wherein the antibody comprises a heavy chain
variable region that is the product of or derived from a human
V.sub.H 4-34 gene and comprises a light chain variable region that
is the product of or derived from a human V.sub.K L6 gene, wherein
the antibody specifically binds to CD22, preferably human CD22.
[0276] In yet another preferred embodiment, this disclosure
provides an isolated monoclonal antibody, or antigen-binding
portion thereof, wherein the antibody comprises a heavy chain
variable region that is the product of or derived from a human
V.sub.H 5-51 gene and comprises a light chain variable region that
is the product of or derived from a human V.sub.K A27 or A10 gene,
wherein the antibody specifically binds to CD22, preferably human
CD22.
[0277] In yet another preferred embodiment, this disclosure
provides an isolated monoclonal antibody, or antigen-binding
portion thereof, wherein the antibody comprises a heavy chain
variable region that is the product of or derived from a human
V.sub.H 1-69 gene and comprises a light chain variable region that
is the product of or derived from a human V.sub.K L6 gene, wherein
the antibody specifically binds to CD22, preferably human CD22.
[0278] In yet another preferred embodiment, this disclosure
provides an isolated monoclonal antibody, or antigen-binding
portion thereof, wherein the antibody comprises a heavy chain
variable region that is the product of or derived from a human
V.sub.H 1-69 gene and comprises a light chain variable region that
is the product of or derived from a human V.sub.K A27 or L18 gene,
wherein the antibody specifically binds to CD22, preferably human
CD22.
[0279] Such antibodies also may possess one or more of the
functional characteristics described in detail above, such as
internalization into CD22+ cells, ADCC activity against CD22+ cells
and/or enhancement of cell death of Ramos cells induced by BCR
stimulation cytotoxin.
[0280] An example of an antibody having V.sub.H and V.sub.L of
V.sub.H 7-4.1 and V.sub..lamda. 2b2, respectively, is the 12C5
antibody. An example of an antibody having V.sub.H and V.sub.L of
V.sub.H 4-34 and V.sub.K L6, respectively, is the 19A3 antibody.
Another example of an antibody having V.sub.H and V.sub.L of
V.sub.H 4-34 and V.sub.K L6, respectively, is the CD22.1 antibody.
Another example of an antibody having V.sub.H and V.sub.L of
V.sub.H 4-34 and V.sub.K L6, respectively, wherein the V.sub.H
chain includes an N57Q mutation, is the CD22.2 antibody. Another
example of an antibody having V.sub.H and V.sub.L of V.sub.H 4-34
and V.sub.K L6 germline, respectively, is the 21F6 antibody. An
example of an antibody having V.sub.H and V.sub.L of V.sub.H 5-51
and V.sub.K A27 or A10, respectively, is the 16F7 antibody. An
example of an antibody having V.sub.H and V.sub.L of V.sub.H 1-69
and V.sub.K L6, respectively, is the 23C6 antibody. An example of
an antibody having V.sub.H and V.sub.L of V.sub.H 1-69 and V.sub.K
A27 or L18, respectively, is the 4G6 antibody.
[0281] As used herein, a human antibody comprises heavy or light
chain variable regions that is "the product of" or "derived from" a
particular germline sequence if the variable regions of the
antibody are obtained from a system that uses human germline
immunoglobulin genes. Such systems include immunizing a transgenic
mouse carrying human immunoglobulin genes with the antigen of
interest or screening a human immunoglobulin gene library displayed
on phage with the antigen of interest. A human antibody that is
"the product of" or "derived from" a human germline immunoglobulin
sequence can be identified as such by comparing the amino acid
sequence of the human antibody to the amino acid sequences of human
germline immunoglobulins and selecting the human germline
immunoglobulin sequence that is closest in sequence (i.e., greatest
% identity) to the sequence of the human antibody. A human antibody
that is "the product of" or "derived from" a particular human
germline immunoglobulin sequence may contain amino acid differences
as compared to the germline sequence, due to, for example,
naturally-occurring somatic mutations or intentional introduction
of site-directed mutation. However, a selected human antibody
typically is at least 90% identical in amino acids sequence to an
amino acid sequence encoded by a human germline immunoglobulin gene
and contains amino acid residues that identify the human antibody
as being human when compared to the germline immunoglobulin amino
acid sequences of other species (e.g., murine germline sequences).
In certain cases, a human antibody may be at least 95%, or even at
least 96%, 97%, 98%, or 99% identical in amino acid sequence to the
amino acid sequence encoded by the germline immunoglobulin gene.
Typically, a human antibody derived from a particular human
germline sequence will display no more than 10 amino acid
differences from the amino acid sequence encoded by the human
germline immunoglobulin gene. In certain cases, the human antibody
may display no more than 5, or even no more than 4, 3, 2, or 1
amino acid difference from the amino acid sequence encoded by the
germline immunoglobulin gene.
Homologous Antibodies
[0282] In yet another embodiment, an antibody of this disclosure
comprises heavy and light chain variable regions comprising amino
acid sequences that are homologous to the amino acid sequences of
the preferred antibodies described herein, and wherein the
antibodies retain the desired functional properties of the
anti-CD22 antibodies of this disclosure.
[0283] For example, this disclosure provides an isolated monoclonal
antibody, or antigen binding portion thereof, comprising a heavy
chain variable region and a light chain variable region,
wherein:
[0284] (a) the heavy chain variable region comprises an amino acid
sequence that is at least 80% homologous to an amino acid sequence
selected from the group consisting of SEQ ID NOs:31-34, 61 and
81-83;
[0285] (b) the light chain variable region comprises an amino acid
sequence that is at least 80% homologous to an amino acid sequence
selected from the group consisting of SEQ ID NOs:35-40 and
84-86;
[0286] (c) the antibody specifically binds to human CD22.
[0287] Additionally or alternatively, the antibody may possess one
or more of the following functional properties: (a) binds to human
CD22 with a K.sub.D of 1.times.10.sup.-7 M or less; (b)
internalizes into CD22+ cells; (c) exhibits ADCC activity on CD22+
cells; (d) enhances cell death of Ramos cells induced by, for
example, BCR stimulation; and/or (e) inhibits growth of
CD22-expressing cells in vivo when conjugated to a cytotoxin.
[0288] In various embodiments, the antibody can be, for example, a
human antibody, a humanized antibody or a chimeric antibody.
[0289] In other embodiments, the V.sub.H and/or V.sub.L amino acid
sequences may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to
the sequences set forth above. An antibody having V.sub.H and
V.sub.L regions having high (i.e., 80% or greater) homology to the
V.sub.H and V.sub.L regions of the sequences set forth above, can
be obtained by mutagenesis (e.g., site-directed or PCR-mediated
mutagenesis) of nucleic acid molecules encoding SEQ ID NOs:41-44,
62, or 87-89, or SEQ ID NOs:45-50 or 90-92, followed by testing of
the encoded altered antibody for retained function (i.e., the
functions set forth above) using the functional assays described
herein.
[0290] As used herein, the percent homology between two amino acid
sequences is equivalent to the percent identity between the two
sequences. The percent identity between the 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.
[0291] The percent identity between two amino acid sequences can be
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4: 11-17 (1988)) 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 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.
[0292] Additionally or alternatively, the protein sequences of the
present disclosure can further be used as a "query sequence" to
perform a search against public databases to, for example, to
identify related sequences. Such searches can be performed using
the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10. BLAST protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to the antibody molecules of this disclosure.
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) are useful. See www.ncbi.nlm.nih.gov.
Antibodies with Conservative Modifications
[0293] In certain embodiments, an antibody of this disclosure
comprises a heavy chain variable region comprising CDR1, CDR2 and
CDR3 sequences and a light chain variable region comprising CDR1,
CDR2 and CDR3 sequences, wherein one or more of these CDR sequences
comprise specified amino acid sequences based on the preferred
antibodies described herein (e.g., 12C5, 19A3, CD22.1, CD22.2,
16F7, 23C6, 4G6 and 21F6), or conservative modifications thereof,
and wherein the antibodies retain the desired functional properties
of the anti-CD22 antibodies of this disclosure. It is understood in
the art that certain conservative sequence modification can be made
which do not remove antigen binding. See, for example, Brummell et
al. (1993) Biochem 32:1180-8 (describing mutational analysis in the
CDR3 heavy chain domain of antibodies specific for Salmonella); de
Wildt et al. (1997) Prot. Eng. 10:835-41 (describing mutation
studies in anti-UA1 antibodies); Komissarov et al. (1997) J. Biol.
Chem. 272:26864-26870 (showing that mutations in the middle of
HCDR3 led to either abolished or diminished affinity); Hall et al.
(1992) J. Immunol. 149:1605-12 (describing that a single amino acid
change in the CDR3 region abolished binding activity); Kelley and
O'Connell (1993) Biochem. 32:6862-35 (describing the contribution
of Tyr residues in antigen binding); Adib-Conquy et al. (1998) Int.
Immunol. 10:341-6 (describing the effect of hydrophobicity in
binding) and Beers et al. (2000) Clin. Can. Res. 6:2835-43
(describing HCDR3 amino acid mutants).
[0294] Accordingly, this disclosure provides an isolated monoclonal
antibody, or antigen binding portion thereof, comprising a heavy
chain variable region comprising CDR1, CDR2, and CDR3 sequences and
a light chain variable region comprising CDR1, CDR2, and CDR3
sequences, wherein:
[0295] (a) the heavy chain variable region CDR3 sequence comprises
an amino acid sequence selected from the group consisting of amino
acid sequences of SEQ ID NOs: 9-12, 69-71 and conservative
modifications thereof;
[0296] (b) the light chain variable region CDR3 sequence comprises
an amino acid sequence selected from the group consisting of amino
acid sequence of SEQ ID NOs: 25-30, 79-80, and conservative
modifications thereof; and
[0297] (c) the antibody specifically binds to human CD22.
[0298] Additionally or alternatively, the antibody may possess one
or more of the following functional properties: (a) binds to human
CD22 with a K.sub.D of 1.times.10.sup.-7 M or less; (b)
internalizes into CD22+ cells; (c) exhibits ADCC activity on CD22+
cells; and/or (d) enhances cell death of Ramos cells induced by BCR
stimulation; and/or (e) inhibits growth of CD22-expressing cells in
vivo when conjugated to a cytotoxin.
[0299] In a preferred embodiment, the heavy chain variable region
CDR2 sequence comprises an amino acid sequence selected from the
group consisting of amino acid sequences of SEQ ID NOs:5-8, 60,
66-68, and conservative modifications thereof; and the light chain
variable region CDR2 sequence comprises an amino acid sequence
selected from the group consisting of amino acid sequences of SEQ
ID NOs: 19-24, 75-77, and conservative modifications thereof. In
another preferred embodiment, the heavy chain variable region CDR1
sequence comprises an amino acid sequence selected from the group
consisting of amino acid sequences of SEQ ID NOs: 1-4, 63-65, and
conservative modifications thereof; and the light chain variable
region CDR1 sequence comprises an amino acid sequence selected from
the group consisting of amino acid sequences of SEQ ID NOs: 13-18,
72-74, and conservative modifications thereof.
[0300] In various embodiments, the antibody can be, for example,
human antibodies, humanized antibodies or chimeric antibodies.
As used herein, the term "conservative sequence modifications" is
intended to refer to amino acid modifications that do not
significantly affect or alter the binding characteristics of the
antibody containing the amino acid sequence. Such conservative
modifications include amino acid substitutions, additions and
deletions. Modifications can be introduced into an antibody of this
disclosure by standard techniques known in the art, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions are 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, one
or more amino acid residues within the CDR regions of an antibody
of this disclosure can be replaced with other amino acid residues
from the same side chain family and the altered antibody can be
tested for retained function (i.e., the functions set forth above)
using the functional assays described herein. Antibodies that Bind
to the Same Epitope as Anti-CD22 Antibodies
[0301] In another embodiment, this disclosure provides antibodies
that bind to the same epitope on human CD22 that are recognized by
any of the anti-CD22 monoclonal antibodies of this disclosure
(i.e., antibodies that have the ability to cross-compete for
binding to CD22 with any of the monoclonal antibodies of this
disclosure). In preferred embodiments, the reference antibody for
cross-competition studies can be the monoclonal antibody 12C5
(having V.sub.H and V.sub.L sequences as shown in SEQ ID NOs:31 and
35, respectively), or the monoclonal antibody 19A3 or the
monoclonal antibody CD22.1 or the monoclonal antibody CD22.2
(having V.sub.H and V.sub.L sequences as shown in SEQ ID NOs:32/61
and 36, respectively) or the monoclonal antibody 16F7 (having
V.sub.H and V.sub.L sequences as shown in SEQ ID NOs:33 and 37/38,
respectively) or the monoclonal antibody 23C6 (having V.sub.H and
V.sub.L sequences as shown in SEQ ID NOs:34 and 39/40,
respectively), or the monoclonal antibody 4G6 (having V.sub.H and
V.sub.L sequences as shown in SEQ ID NOs:81 and 84/85,
respectively) or the monoclonal antibody 21F6 (having V.sub.H and
V.sub.L sequences as shown in SEQ ID NOs: 82/83 and 86,
respectively).
[0302] Such cross-competing antibodies can be identified based on
their ability to cross-compete with 12C5, 19A3, CD22.1, CD22.2,
16F7, 23C6, 4G6 and 21F6 in standard CD22 binding assays. For
example, standard ELISA assays can be used in which recombinant
CD22 is immobilized on the plate, one of the antibodies is
fluorescently labeled and the ability of non-labeled antibodies to
compete off the binding of the labeled antibody is evaluated.
Additionally or alternatively, BIAcore analysis can be used to
assess the ability of the antibodies to cross-compete, as described
in Example 3 (regarding the epitope grouping of 12C5, 19A3, CD22.1,
CD22.2, 16F7, 23C6, 4G6 and 21F6). The ability of a test antibody
to inhibit the binding of, for example, 12C5, 19A3, CD22.1, CD22.2,
16F7, 23C6, 4G6 and 21F6 to human CD22 demonstrates that the test
antibody can compete with 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6,
4G6 and 21F6 for binding to human CD22 and thus binds to the same
epitope on human CD22 as is recognized by 12C5, 19A3, CD22.1,
CD22.2, 16F7, 23C6, 4G6 and 21F6. As described in detail in Example
3, the antibodies 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6, 4G6 and
21F6, each bind to a distinct epitope on CD22 and thus belong to
distinct epitope groups. In a preferred embodiment, the antibody
that binds to the same epitope on 12C5, 19A3, CD22.1, CD22.2, 16F7,
23C6, 4G6 and 21F6 is a human monoclonal antibody. Such human
monoclonal antibodies can be prepared and isolated as described in
the Examples.
Engineered and Modified Antibodies
[0303] An antibody of this disclosure further can be prepared using
an antibody having one or more of the V.sub.H and/or V.sub.L
sequences disclosed herein as starting material to engineer a
modified antibody, which modified antibody may have altered
properties from the starting antibody. An antibody can be
engineered by modifying one or more residues within one or both
variable regions (i.e., V.sub.H and/or V.sub.L), for example within
one or more CDR regions and/or within one or more framework
regions. Additionally or alternatively, an antibody can be
engineered by modifying residues within the constant region(s), for
example to alter the effector function(s) of the antibody.
[0304] In certain embodiments, CDR grafting can be used to engineer
variable regions of antibodies. 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;
Queen, C. et al. (1989) Proc. Natl. Acad. See. U.S.A.
86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.)
[0305] Accordingly, another embodiment of this disclosure pertains
to an isolated monoclonal antibody, or antigen binding portion
thereof, comprising a heavy chain variable region comprising CDR1,
CDR2, and CDR3 sequences comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 1-4 and 63-65, SEQ ID
NOs:5-8, 60, and 66-68, and SEQ ID NOs:9-12 and 69-71,
respectively; and a light chain variable region comprising CDR1,
CDR2, and CDR3 sequences comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 13-18 and 72-74, SEQ ID
NOs:19-24 and 75-77, and SEQ ID NOs:25-30 and 78-80, respectively.
Thus, such antibodies contain the V.sub.H and V.sub.L CDR sequences
of monoclonal antibodies 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6,
4G6 and 21F6, yet may contain different framework sequences from
these antibodies.
[0306] Such framework sequences can be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for human heavy
and light chain variable region genes can be found in the "VBase"
human germline sequence database (available on the Internet at
www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; Tomlinson, I. M., et al. (1992) "The
Repertoire of Human Germline V.sub.H Sequences Reveals about Fifty
Groups of V.sub.H Segments with Different Hypervariable Loops" J.
Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) "A
Directory of Human Germ-line V.sub.H Segments Reveals a Strong Bias
in their Usage" Eur. J. Immunol. 24:827-836; the contents of each
of which are expressly incorporated herein by reference. As another
example, the germline DNA sequences for human heavy and light chain
variable region genes can be found in the Genbank database. For
example, the following heavy chain germline sequences found in the
HCo7 HuMAb mouse are available in the accompanying Genbank
accession numbers: 1-69 (NG_0010109, NT_024637 and BC070333), 3-33
(NG_0010109 and NT_024637) and 3-7 (NG_0010109 and NT_024637). As
another example, the following heavy chain germline sequences found
in the HCo12 HuMAb mouse are available in the accompanying Genbank
accession numbers: 1-69 (NG_0010109, NT_024637 and BC070333), 5-51
(NG_0010109 and NT_024637), 4-34 (NG_0010109 and NT_024637), 3-30.3
(CAJ556644) and 3-23 (AJ406678).
[0307] Antibody protein sequences are compared against a compiled
protein sequence database using one of the sequence similarity
searching methods called the Gapped BLAST (Altschul et al. (1997)
Nucleic Acids Research 25:3389-3402), which is well known to those
skilled in the art. BLAST is a heuristic algorithm in that a
statistically significant alignment between the antibody sequence
and the database sequence is likely to contain high-scoring segment
pairs (HSP) of aligned words. Segment pairs whose scores cannot be
improved by extension or trimming is called a hit. Briefly, the
nucleotide sequences of VBASE origin
(http://vbase.mrc-cpe.cam.ac.uk/vbase1/list2.php) are translated
and the region between and including FR1 through FR3 framework
region is retained. The database sequences have an average length
of 98 residues. Duplicate sequences which are exact matches over
the entire length of the protein are removed. A BLAST search for
proteins using the program blastp with default, standard parameters
except the low complexity filter, which is turned off, and the
substitution matrix of BLOSUM62, filters for top 5 hits yielding
sequence matches. The nucleotide sequences are translated in all
six frames and the frame with no stop codons in the matching
segment of the database sequence is considered the potential hit.
This is in turn confirmed using the BLAST program tblastx, which
translates the antibody sequence in all six frames and compares
those translations to the VBASE nucleotide sequences dynamically
translated in all six frames.
[0308] The identities are exact amino acid matches between the
antibody sequence and the protein database over the entire length
of the sequence. The positives (identities+substitution match) are
not identical but amino acid substitutions guided by the BLOSUM62
substitution matrix. If the antibody sequence matches two of the
database sequences with same identity, the hit with most positives
would be decided to be the matching sequence hit.
[0309] Preferred framework sequences for use in the antibodies of
this disclosure are those that are structurally similar to the
framework sequences used by selected antibodies of this disclosure,
e.g., similar to the V.sub.H 7-4.1 (SEQ ID NO:51), V.sub.H 4-34
(SEQ ID NO:52), V.sub.H 5-51 (SEQ ID NO:53), or V.sub.H 1-69 (SEQ
ID NO:54) framework sequences and/or the V.sub..lamda. 2b2 (SEQ ID
NO:55), V.sub.K L6 (SEQ ID NO:56), V.sub.K A27 (SEQ ID NO:57),
V.sub.K A10 (SEQ ID NO:58), or V.sub.K L18 (SEQ ID NO:93) framework
sequences used by preferred monoclonal antibodies of this
disclosure. The V.sub.H CDR1, CDR2, and CDR3 sequences, and the
V.sub.K CDR1, CDR2, and CDR3 sequences, can be grafted onto
framework regions that have the identical sequence as that found in
the germline immunoglobulin gene from which the framework sequence
derive, or the CDR sequences can be grafted onto framework regions
that contain one or more mutations as compared to the germline
sequences. For example, it has been found that in certain instances
it is beneficial to mutate residues within the framework regions to
maintain or enhance the antigen binding ability of the antibody
(see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and
6,180,370 to Queen et al.).
[0310] Another type of variable region modification is to mutate
amino acid residues within the V.sub.H and/or V.sub.L CDR1, CDR2
and/or CDR3 regions to thereby improve one or more binding
properties (e.g., affinity) of the antibody of interest.
Site-directed mutagenesis or PCR-mediated mutagenesis can be
performed to introduce the mutation(s) and the effect on antibody
binding, or other functional property of interest, can be evaluated
in in vitro or in vivo assays as described herein and provided in
the Examples. Preferably conservative modifications (as discussed
above) are introduced. The mutations may be amino acid
substitutions, additions or deletions, but are preferably
substitutions. Moreover, typically no more than one, two, three,
four or five residues within a CDR region are altered.
[0311] Accordingly, in another embodiment, the instant disclosure
provides isolated anti-CD22 monoclonal antibodies, or antigen
binding portions thereof, comprising a heavy chain variable region
comprising: (a) a V.sub.H CDR1 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1-4 or
63-65, or an amino acid sequence having one, two, three, four or
five amino acid substitutions, deletions or additions as compared
to SEQ ID NOs: 1-4 or 63-65; (b) a V.sub.H CDR2 region comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOs:5-8, 60 or 66-68, or an amino acid sequence having one, two,
three, four or five amino acid substitutions, deletions or
additions as compared to SEQ ID NOs:5-8, 60 or 66-68; (c) a V.sub.H
CDR3 region comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs:9-12 or 69-71, or an amino acid
sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID
NOs:9-12 or 69-71; (d) a V.sub.L CDR1 region comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:
13-18 or 72-74, or an amino acid sequence having one, two, three,
four or five amino acid substitutions, deletions or additions as
compared to SEQ ID NOs: 13-18 or 72-74; (e) a V.sub.L CDR2 region
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 19-24 or 75-77, or an amino acid sequence
having one, two, three, four or five amino acid substitutions,
deletions or additions as compared to SEQ ID NOs: 19-24 or 75-77;
and (f) a V.sub.L CDR3 region comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs:25-30 or 78-80, or
an amino acid sequence having one, two, three, four or five amino
acid substitutions, deletions or additions as compared to SEQ ID
NOs:25-30 or 78-80.
[0312] Engineered antibodies of this disclosure include those in
which modifications have been made to framework residues within
V.sub.H and/or V.sub.L, e.g. to improve the properties of the
antibody. Typically such framework modifications are made to
decrease the immunogenicity of the antibody. For example, one
approach is to "backmutate" one or more framework residues to the
corresponding germline sequence. More specifically, an antibody
that has undergone somatic mutation may contain framework residues
that differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived.
[0313] For example, for the 12C5 V.sub..lamda. region, framework
region amino acid positions 40 and 68 (using the Kabat numbering
system) differ from germline. One or both of these positions can be
backmutated to germline sequences by making one or both of the
following substitutions: L40Q and R68K.
[0314] Furthermore, for the 19A3 and the CD22.1 V.sub.H regions,
framework region amino acid position 27 (using the Kabat numbering
system) differs from germline. This position can be backmutated to
the germline sequence by making the following substitution:
R27G.
[0315] Furthermore, for the CD22.2 V.sub.H region, framework region
amino acid positions 27 and 57 (using the Kabat numbering system)
differs from germline. This position can be backmutated to the
germline sequence by making the following substitutions: R27G and
Q57N.
[0316] Furthermore, for the 16F7 V.sub.H region, framework region
amino acid position 28 (using the Kabat numbering system) differs
from germline. This position can be backmutated to the germline
sequence by making the following substitution: N28S.
[0317] Furthermore, for the 16F7 V.sub.K.2 region, framework region
amino acid position 85 (using the Kabat numbering system) differs
from germline. This position can be backmutated to the germline
sequence by making the following substitution: A85T.
[0318] Furthermore, for the 23C6 V.sub.H region, framework region
amino acid positions 14, 79 and 88 (using the Kabat numbering
system) differ from germline. One, two or all three of these
positions can be backmutated to germline sequences by making one,
two or all three of the following substitutions: T14P, V79A and
A88S.
[0319] Furthermore, for the 4G6 V.sub.H region, framework region
amino acid positions P, D, F, D, T, Y and F (using the Kabat
numbering system) differs from germline. This position can be
backmutated to the germline sequence by making one, two, three,
four, five, six or all seven of the following substitution: P?A;
D?G; N?S; F?Y; D?E; T?S; Y?R; F?S. NEED INPUT RE: KABAT
NUMBERING.
[0320] Furthermore, for the 4G6 V.sub.K1 region framework region
amino acid positions T and D (using the Kabat numbering system)
differs from germline. These positions can be backmutated to the
germline sequence by making one or two of the following
substitution: T?K and D?E.
[0321] Furthermore, for the 21F6 V.sub.H1 region, framework region
amino acid position S and I (using the Kabat numbering system)
differs from germline. These positions can be backmutated to the
germline sequence by making the following substitution: S?P and
I?V.
[0322] Furthermore, for the 21F6 V.sub.H2 region framework region
amino acid positions S and M (using the Kabat numbering system)
differs from germline. These positions can be backmutated to the
germline sequence by making the following substitution: S?P and
M?V.
[0323] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Patent Publication No. 2003/0153043 by Carr et al.
[0324] In addition or alternative to modifications made within the
framework or CDR regions, antibodies of this disclosure may be
engineered to include modifications within the Fc region, typically
to alter one or more functional properties of the antibody, such as
serum half-life, complement fixation, Fc receptor binding, and/or
antigen-dependent cellular cytotoxicity. Furthermore, an antibody
of this disclosure may be chemically modified (e.g., one or more
chemical moieties can be attached to the antibody) or be modified
to alter its glycosylation, again to alter one or more functional
properties of the antibody. Each of these embodiments is described
in further detail below. The numbering of residues in the Fc region
is that of the EU index of Kabat.
[0325] In one embodiment, the hinge region of CH1 is modified such
that the number of cysteine residues in the hinge region is
altered, e.g., increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of
cysteine residues in the hinge region of CH1 is altered to, for
example, facilitate assembly of the light and heavy chains or to
increase or decrease the stability of the antibody.
[0326] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half life of the antibody. More
specifically, one or more amino acid mutations are introduced into
the CH.sub.2--CH3 domain interface region of the Fc-hinge fragment
such that the antibody has impaired Staphylococcyl protein A (SpA)
binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Pat. No. 6,165,745
by Ward et al.
[0327] In another embodiment, the antibody is modified to increase
its biological half life. Various approaches are possible. For
example, one or more of the following mutations can be introduced:
T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to
Ward. Alternatively, to increase the biological half life, the
antibody can be altered within the CH1 or CL region to contain a
salvage receptor binding epitope taken from two loops of a CH2
domain of an Fc region of an IgG, as described in U.S. Pat. Nos.
5,869,046 and 6,121,022 by Presta et al.
[0328] In yet other embodiments, the Fc region is altered by
replacing at least one amino acid residue with a different amino
acid residue to alter the effector function(s) of the antibody. For
example, one or more amino acids selected from amino acid residues
234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a
different amino acid residue such that the antibody has an altered
affinity for an effector ligand but retains the antigen-binding
ability of the parent antibody. The effector ligand to which
affinity is altered can be, for example, an Fc receptor or the C1
component of complement. This approach is described in further
detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et
al.
[0329] In another example, one or more amino acids selected from
amino acid residues 329, 331 and 322 can be replaced with a
different amino acid residue such that the antibody has altered C1q
binding and/or reduced or abolished complement dependent
cytotoxicity (CDC). This approach is described in further detail in
U.S. Pat. No. 6,194,551 by Idusogie et al.
[0330] In another example, one or more amino acid residues within
amino acid positions 231 and 239 are altered to thereby alter the
ability of the antibody to fix complement. This approach is
described further in PCT Publication WO 94/29351 by Bodmer et
al.
[0331] In yet another example, the Fc region is modified to
increase the ability of the antibody to mediate antibody dependent
cellular cytotoxicity (ADCC) and/or to increase the affinity of the
antibody for an Fc.gamma. receptor by modifying one or more amino
acids at the following positions: 238, 239, 248, 249, 252, 254,
255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283,
285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305,
307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,
334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398,
414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is
described further in PCT Publication WO 00/42072 by Presta.
Moreover, the binding sites on human IgG for Fc.gamma.R1,
Fc.gamma.RII, Fc.gamma.RIII and FcRn have been mapped and variants
with improved binding have been described (see Shields, R. L. et
al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at
positions 256, 290, 298, 333, 334 and 339 were shown to improve
binding to Fc.gamma.RIII. Additionally, the following combination
mutants were shown to improve Fc.gamma.RIII binding: T256A/S298A,
S298A/E333A, S298A/K224A and S298A/E333A/K334A.
[0332] In still another embodiment, the glycosylation of an
antibody is modified. For example, an aglycoslated antibody can be
made (i.e., the antibody lacks glycosylation). Glycosylation can be
altered to, for example, increase the affinity of the antibody for
antigen. Such carbohydrate modifications can be accomplished by,
for example, altering one or more sites of glycosylation within the
antibody sequence. For example, one or more amino acid
substitutions can be made that result in elimination of one or more
variable region framework glycosylation sites to thereby eliminate
glycosylation at that site. Such aglycosylation may increase the
affinity of the antibody for antigen. Such an approach is described
in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 to Co
et al. Additional approaches for altering glycosylation are
described in further detail in U.S. Pat. No. 7,214,775 to Hanai et
al., U.S. Pat. No. 6,737,056 to Presta, U.S. Pub No. 20070020260 to
Presta, PCT Publication No. WO/2007/084926 to Dickey et al., PCT
Publication No. WO/2006/089294 to Zhu et al., and PCT Publication
No. WO/2007/055916 to Ravetch et al., each of which is hereby
incorporated by reference in its entirety.
[0333] In one exemplary embodiment, a glycosylation site in the
CDR2 region of the V.sub.H chain of the 19A3 antibody was
eliminated by introducing an N57Q mutation (see Example 1), to give
the recombinant antibody CD22.2 having the V.sub.H amino acid
sequence shown in SEQ ID NO:61.
[0334] Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, such as a hypofucosylated
antibody having reduced amounts of fucosyl residues or an antibody
having increased bisecting GlcNac structures. Such altered
glycosylation patterns have been demonstrated to increase the ADCC
ability of antibodies. Such carbohydrate modifications can be
accomplished by, for example, expressing the antibody in a host
cell with altered glycosylation machinery. Cells with altered
glycosylation machinery have been described in the art and can be
used as host cells in which to express recombinant antibodies of
this disclosure to thereby produce an antibody with altered
glycosylation. For example, the cell lines Ms704, Ms705, and Ms709
lack the fucosyltransferase gene, FUT8 (alpha (1,6)
fucosyltransferase), such that antibodies expressed in the Ms704,
Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The
Ms704, Ms705, and Ms709 FUT8.sup.-/- cell lines were created by the
targeted disruption of the FUT8 gene in CHO/DG44 cells using two
replacement vectors (see U.S. Patent Publication No. 20040110704 by
Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng
87:614-22). As another example, EP 1,176,195 by Hanai et al.
describes a cell line with a functionally disrupted FUT8 gene,
which encodes a fucosyl transferase, such that antibodies expressed
in such a cell line exhibit hypofucosylation by reducing or
eliminating the alpha 1,6 bond-related enzyme. Hanai et al. also
describe cell lines which have a low enzyme activity for adding
fucose to the N-acetylglucosamine that binds to the Fc region of
the antibody or does not have the enzyme activity, for example the
rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO
03/035835 by Presta describes a variant CHO cell line, Lec13 cells,
with reduced ability to attach fucose to Asn(297)-linked
carbohydrates, also resulting in hypofucosylation of antibodies
expressed in that host cell (see also Shields, R. L. et al. (2002)
J. Biol. Chem. 277:26733-26740). Antibodies with a modified
glycosylation profile can also be produced in chicken eggs, as
described in US Patent Application No. PCT/US06/05853.
Alternatively, antibodies with a modified glycosylation profile can
be produced in plant cells, such as Lemna. Methods for production
of antibodies in a plant system are disclosed in the U.S. patent
application corresponding to Alston & Bird LLP attorney docket
No. 040989/314911, filed on Aug. 11, 2006. PCT Publication WO
99/54342 by Umana et al. describes cell lines engineered to express
glycoprotein-modifying glycosyl transferases (e.g.,
beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that
antibodies expressed in the engineered cell lines exhibit increased
bisecting GlcNac structures which results in increased ADCC
activity of the antibodies (see also Umana et al. (1999) Nat.
Biotech. 17:176-180). Alternatively, the fucose residues of the
antibody may be cleaved off using a fucosidase enzyme. For example,
the fucosidase alpha-L-fucosidase removes fucosyl residues from
antibodies (Tarentino, A. L. et al. (1975) Biochem.
14:5516-23).
[0335] Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, wherein that alteration
relates to the level of sialyation of the antibody. Such
alterations are described in PCT Publication No. WO/2007/084926 to
Dickey et al, and PCT Publication No. WO/2007/055916 to Ravetch et
al., both of which are incorporated by reference in their entirety.
For example, one may employ an enzymatic reaction with sialidase,
such as, for example, Arthrobacter ureafacens sialidase. The
conditions of such a reaction are generally described in the U.S.
Pat. No. 5,831,077, which is hereby incorporated by reference in
its entirety. Other non-limiting examples of suitable enzymes are
neuraminidase and N-Glycosidase F, as described in Schloemer et
al., J. Virology, 15(4), 882-893 (1975) and in Leibiger et al.,
Biochem J., 338, 529-538 (1999), respectively. Desialylated
antibodies may be further purified by using affinity
chromatography. Alternatively, one may employ methods to increase
the level of sialyation, such as by employing sialyltransferase
enzymes. Conditions of such a reaction are generally described in
Basset et al., Scandinavian Journal of Immunology, 51(3), 307-311
(2000).
[0336] Another modification of the antibodies herein that is
contemplated by this disclosure is pegylation. An antibody can be
pegylated to, for example, increase the biological (e.g., serum)
half life of the antibody. To pegylate an antibody, the antibody,
or fragment thereof, typically is reacted with polyethylene glycol
(PEG), such as a reactive ester or aldehyde derivative of PEG,
under conditions in which one or more PEG groups become attached to
the antibody or antibody fragment. Preferably, the pegylation is
carried out via an acylation reaction or an alkylation reaction
with a reactive PEG molecule (or an analogous reactive
water-soluble polymer). As used herein, the term "polyethylene
glycol" is intended to encompass any of the forms of PEG that have
been used to derivatize other proteins, such as mono (C1-C10)
alkoxy- or aryloxy-polyethylene glycol or polyethylene
glycol-maleimide. In certain embodiments, the antibody to be
pegylated is an aglycosylated antibody. Methods for pegylating
proteins are known in the art and can be applied to the antibodies
of this disclosure. See for example, EP 0 154 316 by Nishimura et
al. and EP 0 401 384 by Ishikawa et al.
Antibody Fragments and Antibody Mimetics
[0337] The instant invention is not limited to traditional
antibodies and may be practiced through the use of antibody
fragments and antibody mimetics. As detailed below, a wide variety
of antibody fragment and antibody mimetic technologies have now
been developed and are widely known in the art. While a number of
these technologies, such as domain antibodies, Nanobodies, and
UniBodies make use of fragments of, or other modifications to,
traditional antibody structures, there are also alternative
technologies, such as Affibodies, DARPins, Anticalins, Avimers, and
Versabodies that employ binding structures that, while they mimic
traditional antibody binding, are generated from and function via
distinct mechanisms.
[0338] Domain Antibodies (dAbs) are the smallest functional binding
units of antibodies, corresponding to the variable regions of
either the heavy (VH) or light (VL) chains of human antibodies.
Domain Antibodies have a molecular weight of approximately 13 kDa.
Domantis has developed a series of large and highly functional
libraries of fully human VH and VL dAbs (more than ten billion
different sequences in each library), and uses these libraries to
select dAbs that are specific to therapeutic targets. In contrast
to many conventional antibodies, Domain Antibodies are well
expressed in bacterial, yeast, and mammalian cell systems. Further
details of domain antibodies and methods of production thereof may
be obtained by reference to U.S. Pat. Nos. 6,291,158; 6,582,915;
6,593,081; 6,172,197; 6,696,245; U.S. Serial No. 2004/0110941;
European patent application No. 1433846 and European Patents
0368684 & 0616640; WO05/035572, WO04/101790, WO04/081026,
WO04/058821, WO04/003019 and WO03/002609, each of which is herein
incorporated by reference in its entirety.
[0339] Nanobodies are antibody-derived therapeutic proteins that
contain the unique structural and functional properties of
naturally-occurring heavy-chain antibodies. These heavy-chain
antibodies contain a single variable domain (VHH) and two constant
domains (CH2 and CH3). Importantly, the cloned and isolated VHH
domain is a perfectly stable polypeptide harbouring the full
antigen-binding capacity of the original heavy-chain antibody.
Nanobodies have a high homology with the VH domains of human
antibodies and can be further humanized without any loss of
activity. Importantly, Nanobodies have a low immunogenic potential,
which has been confirmed in primate studies with Nanobody lead
compounds.
[0340] Nanobodies combine the advantages of conventional antibodies
with important features of small molecule drugs. Like conventional
antibodies, Nanobodies show high target specificity, high affinity
for their target and low inherent toxicity. However, like small
molecule drugs they can inhibit enzymes and readily access receptor
clefts. Furthermore, Nanobodies are extremely stable, can be
administered by means other than injection (see, e.g., WO
04/041867, which is herein incorporated by reference in its
entirety) and are easy to manufacture. Other advantages of
Nanobodies include recognizing uncommon or hidden epitopes as a
result of their small size, binding into cavities or active sites
of protein targets with high affinity and selectivity due to their
unique 3-dimensional, drug format flexibility, tailoring of
half-life and ease and speed of drug discovery.
[0341] Nanobodies are encoded by single genes and are efficiently
produced in almost all prokaryotic and eukaryotic hosts, e.g., E.
coli (see, e.g., U.S. Pat. No. 6,765,087, which is herein
incorporated by reference in its entirety), molds (for example
Aspergillus or Trichoderma) and yeast (for example Saccharomyces,
Kluyveromyces, Hansenula or Pichia) (see, e.g., U.S. Pat. No.
6,838,254, which is herein incorporated by reference in its
entirety). The production process is scalable and multi-kilogram
quantities of Nanobodies have been produced. Because Nanobodies
exhibit a superior stability compared with conventional antibodies,
they can be formulated as a long shelf-life, ready-to-use
solution.
[0342] The Nanoclone method (see, e.g., WO 06/079372, which is
herein incorporated by reference in its entirety) is a proprietary
method for generating Nanobodies against a desired target, based on
automated high-throughout selection of B-cells and could be used in
the context of the instant invention.
[0343] UniBodies are another antibody fragment technology, however
this one is based upon the removal of the hinge region of IgG4
antibodies. The deletion of the hinge region results in a molecule
that is essentially half the size of traditional IgG4 antibodies
and has a univalent binding region rather than the bivalent binding
region of IgG4 antibodies. It is also well known that IgG4
antibodies are inert and thus do not interact with the immune
system, which may be advantageous for the treatment of diseases
where an immune response is not desired, and this advantage is
passed onto UniBodies. For example, UniBodies may function to
inhibit or silence, but not kill, the cells to which they are
bound. Additionally, UniBody binding to cancer cells do not
stimulate them to proliferate. Furthermore, because UniBodies are
about half the size of traditional IgG4 antibodies, they may show
better distribution over larger solid tumors with potentially
advantageous efficacy. UniBodies are cleared from the body at a
similar rate to whole IgG4 antibodies and are able to bind with a
similar affinity for their antigens as whole antibodies. Further
details of UniBodies may be obtained by reference to patent
application WO2007/059782, which is herein incorporated by
reference in its entirety.
[0344] Affibody molecules represent a new class of affinity
proteins based on a 58-amino acid residue protein domain, derived
from one of the IgG-binding domains of staphylococcal protein A.
This three helix bundle domain has been used as a scaffold for the
construction of combinatorial phagemid libraries, from which
Affibody variants that target the desired molecules can be selected
using phage display technology (Nord K, Gunneriusson E, Ringdahl J,
Stahl S, Uhlen M, Nygren P A, Binding proteins selected from
combinatorial libraries of an .alpha.-helical bacterial receptor
domain, Nat Biotechnol 1997; 15:772-7. Ronmark J, Gronlund H, Uhlen
M, Nygren P A, Human immunoglobulin A (IgA)-specific ligands from
combinatorial engineering of protein A, Eur J Biochem 2002;
269:2647-55). The simple, robust structure of Affibody molecules in
combination with their low molecular weight (6 kDa), make them
suitable for a wide variety of applications, for instance, as
detection reagents (Ronmark J, Hansson M, Nguyen T, et al,
Construction and characterization of affibody-Fc chimeras produced
in Escherichia coli, J Immunol Methods 2002; 261:199-211) and to
inhibit receptor interactions (Sandstorm K, Xu Z, Forsberg G,
Nygren P A, Inhibition of the CD28-CD80 co-stimulation signal by a
CD28-binding Affibody ligand developed by combinatorial protein
engineering, Protein Eng 2003; 16:691-7). Further details of
Affibodies and methods of production thereof may be obtained by
reference to U.S. Pat. No. 5,831,012 which is herein incorporated
by reference in its entirety.
[0345] Labelled Affibodies may also be useful in imaging
applications for determining abundance of Isoforms.
[0346] DARPins (Designed Ankyrin Repeat Proteins) are one example
of an antibody mimetic DRP (Designed Repeat Protein) technology
that has been developed to exploit the binding abilities of
non-antibody polypeptides. Repeat proteins such as ankyrin or
leucine-rich repeat proteins, are ubiquitous binding molecules,
which occur, unlike antibodies, intra- and extracellularly. Their
unique modular architecture features repeating structural units
(repeats), which stack together to form elongated repeat domains
displaying variable and modular target-binding surfaces. Based on
this modularity, combinatorial libraries of polypeptides with
highly diversified binding specificities can be generated. This
strategy includes the consensus design of self-compatible repeats
displaying variable surface residues and their random assembly into
repeat domains.
[0347] DARPins can be produced in bacterial expression systems at
very high yields and they belong to the most stable proteins known.
Highly specific, high-affinity DARPins to a broad range of target
proteins, including human receptors, cytokines, kinases, human
proteases, viruses and membrane proteins, have been selected.
DARPins having affinities in the single-digit nanomolar to
picomolar range can be obtained.
[0348] DARPins have been used in a wide range of applications,
including ELISA, sandwich ELISA, flow cytometric analysis (FACS),
immunohistochemistry (IHC), chip applications, affinity
purification or Western blotting. DARPins also proved to be highly
active in the intracellular compartment for example as
intracellular marker proteins fused to green fluorescent protein
(GFP). DARPins were further used to inhibit viral entry with IC50
in the pM range. DARPins are not only ideal to block
protein-protein interactions, but also to inhibit enzymes.
Proteases, kinases and transporters have been successfully
inhibited, most often an allosteric inhibition mode. Very fast and
specific enrichments on the tumor and very favorable tumor to blood
ratios make DARPins well suited for in vivo diagnostics or
therapeutic approaches.
[0349] Additional information regarding DARPins and other DRP
technologies can be found in U.S. Patent Application Publication
No. 2004/0132028 and International Patent Application Publication
No. WO 02/20565, both of which are hereby incorporated by reference
in their entirety.
[0350] Anticalins are an additional antibody mimetic technology,
however in this case the binding specificity is derived from
lipocalins, a family of low molecular weight proteins that are
naturally and abundantly expressed in human tissues and body
fluids. Lipocalins have evolved to perform a range of functions in
vivo associated with the physiological transport and storage of
chemically sensitive or insoluble compounds. Lipocalins have a
robust intrinsic structure comprising a highly conserved
.beta.-barrel which supports four loops at one terminus of the
protein. These loops form the entrance to a binding pocket and
conformational differences in this part of the molecule account for
the variation in binding specificity between individual
lipocalins.
[0351] While the overall structure of hypervariable loops supported
by a conserved .beta.-sheet framework is reminiscent of
immunoglobulins, lipocalins differ considerably from antibodies in
terms of size, being composed of a single polypeptide chain of
160-180 amino acids which is marginally larger than a single
immunoglobulin domain.
[0352] Lipocalins are cloned and their loops are subjected to
engineering in order to create Anticalins. Libraries of
structurally diverse Anticalins have been generated and Anticalin
display allows the selection and screening of binding function,
followed by the expression and production of soluble protein for
further analysis in prokaryotic or eukaryotic systems. Studies have
successfully demonstrated that Anticalins can be developed that are
specific for virtually any human target protein can be isolated and
binding affinities in the nanomolar or higher range can be
obtained.
[0353] Anticalins can also be formatted as dual targeting proteins,
so-called Duocalins. A Duocalin binds two separate therapeutic
targets in one easily produced monomeric protein using standard
manufacturing processes while retaining target specificity and
affinity regardless of the structural orientation of its two
binding domains.
[0354] Modulation of multiple targets through a single molecule is
particularly advantageous in diseases known to involve more than a
single causative factor. Moreover, bi- or multivalent binding
formats such as Duocalins have significant potential in targeting
cell surface molecules in disease, mediating agonistic effects on
signal transduction pathways or inducing enhanced internalization
effects via binding and clustering of cell surface receptors.
Furthermore, the high intrinsic stability of Duocalins is
comparable to monomeric Anticalins, offering flexible formulation
and delivery potential for Duocalins.
[0355] Additional information regarding Anticalins can be found in
U.S. Pat. No. 7,250,297 and International Patent Application
Publication No. WO 99/16873, both of which are hereby incorporated
by reference in their entirety.
[0356] Another antibody mimetic technology useful in the context of
the instant invention are Avimers. Avimers are evolved from a large
family of human extracellular receptor domains by in vitro exon
shuffling and phage display, generating multidomain proteins with
binding and inhibitory properties. Linking multiple independent
binding domains has been shown to create avidity and results in
improved affinity and specificity compared with conventional
single-epitope binding proteins. Other potential advantages include
simple and efficient production of multitarget-specific molecules
in Escherichia coli, improved thermostability and resistance to
proteases. Avimers with sub-nanomolar affinities have been obtained
against a variety of targets.
[0357] Additional information regarding Avimers can be found in
U.S. Patent Application Publication Nos. 2006/0286603,
2006/0234299, 2006/0223114, 2006/0177831, 2006/0008844,
2005/0221384, 2005/0164301, 2005/0089932, 2005/0053973,
2005/0048512, 2004/0175756, all of which are hereby incorporated by
reference in their entirety.
[0358] Versabodies are another antibody mimetic technology that
could be used in the context of the instant invention. Versabodies
are small proteins of 3-5 kDa with >15% cysteines, which form a
high disulfide density scaffold, replacing the hydrophobic core
that typical proteins have. The replacement of a large number of
hydrophobic amino acids, comprising the hydrophobic core, with a
small number of disulfides results in a protein that is smaller,
more hydrophilic (less aggregation and non-specific binding), more
resistant to proteases and heat, and has a lower density of T-cell
epitopes, because the residues that contribute most to MHC
presentation are hydrophobic. All four of these properties are
well-known to affect immunogenicity, and together they are expected
to cause a large decrease in immunogenicity.
[0359] The inspiration for Versabodies comes from the natural
injectable biopharmaceuticals produced by leeches, snakes, spiders,
scorpions, snails, and anemones, which are known to exhibit
unexpectedly low immunogenicity. Starting with selected natural
protein families, by design and by screening the size,
hydrophobicity, proteolytic antigen processing, and epitope density
are minimized to levels far below the average for natural
injectable proteins.
[0360] Given the structure of Versabodies, these antibody mimetics
offer a versatile format that includes multi-valency,
multi-specificity, a diversity of half-life mechanisms, tissue
targeting modules and the absence of the antibody Fc region.
Furthermore, Versabodies are manufactured in E. coli at high
yields, and because of their hydrophilicity and small size,
Versabodies are highly soluble and can be formulated to high
concentrations. Versabodies are exceptionally heat stable (they can
be boiled) and offer extended shelf-life.
[0361] Additional information regarding Versabodies can be found in
U.S. Patent Application Publication No. 2007/0191272 which is
hereby incorporated by reference in its entirety.
[0362] The detailed description of antibody fragment and antibody
mimetic technologies provided above is not intended to be a
comprehensive list of all technologies that could be used in the
context of the instant specification. For example, and also not by
way of limitation, a variety of additional technologies including
alternative polypeptide-based technologies, such as fusions of
complimentary determining regions as outlined in Qui et al., Nature
Biotechnology, 25(8) 921-929 (2007), which is hereby incorporated
by reference in its entirety, as well as nucleic acid-based
technologies, such as the RNA aptamer technologies described in
U.S. Pat. Nos. 5,789,157, 5,864,026, 5,712,375, 5,763,566,
6,013,443, 6,376,474, 6,613,526, 6,114,120, 6,261,774, and
6,387,620, all of which are hereby incorporated by reference, could
be used in the context of the instant invention.
Antibody Physical Properties
[0363] The antibodies of the present disclosure may be further
characterized by the various physical properties of the anti-CD22
antibodies. Various assays may be used to detect and/or
differentiate different classes of antibodies based on these
physical properties.
[0364] In some embodiments, antibodies of the present disclosure
may contain one or more glycosylation sites in either the light or
heavy chain variable region. The presence of one or more
glycosylation sites in the variable region may result in increased
immunogenicity of the antibody or an alteration of the pK of the
antibody due to altered antigen binding (Marshall et al (1972) Annu
Rev Biochem 41:673-702; Gala F A and Morrison S L (2004) J Immunol
172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro R G
(2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature
316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).
Glycosylation has been known to occur at motifs containing an
N-X-S/T sequence. Variable region glycosylation may be tested using
a Glycoblot assay, which cleaves the antibody to produce a Fab, and
then tests for glycosylation using an assay that measures periodate
oxidation and Schiff base formation. Alternatively, variable region
glycosylation may be tested using Dionex light chromatography
(Dionex-LC), which cleaves saccharides from a Fab into
monosaccharides and analyzes the individual saccharide content. In
some instances, it is preferred to have an anti-CD22 antibody that
does not contain variable region glycosylation. This can be
achieved either by selecting antibodies that do not contain the
glycosylation motif in the variable region or by mutating residues
within the glycosylation motif using standard techniques well known
in the art.
[0365] In a preferred embodiment, the antibodies of the present
disclosure do not contain asparagine isomerism sites. A deamidation
or isoaspartic acid effect may occur on N-G or D-G sequences,
respectively. The deamidation or isoaspartic acid effect results in
the creation of isoaspartic acid which decreases the stability of
an antibody by creating a kinked structure off a side chain carboxy
terminus rather than the main chain. The creation of isoaspartic
acid can be measured using an iso-quant assay, which uses a
reverse-phase HPLC to test for isoaspartic acid.
[0366] Each antibody will have a unique isoelectric point (pI), but
generally antibodies will fall in the pH range of between 6 and
9.5. The pI for an IgG1 antibody typically falls within the pH
range of 7-9.5 and the pI for an IgG4 antibody typically falls
within the pH range of 6-8. Antibodies may have a pI that is
outside this range. Although the effects are generally unknown,
there is speculation that antibodies with a pI outside the normal
range may have some unfolding and instability under in vivo
conditions. The isoelectric point may be tested using a capillary
isoelectric focusing assay, which creates a pH gradient and may
utilize laser focusing for increased accuracy (Janini et al (2002)
Electrophoresis 23:1605-11; Ma et al. (2001) Chromatographia
53:S75-89; Hunt et al (1998) J Chromatogr A 800:355-67). In some
instances, it is preferred to have an anti-CD22 antibody that
contains a pI value that falls in the normal range. This can be
achieved either by selecting antibodies with a pI in the normal
range, or by mutating charged surface residues using standard
techniques well known in the art.
[0367] Each antibody will have a melting temperature that is
indicative of thermal stability (Krishnamurthy R and Manning M C
(2002) Curr Pharm Biotechnol 3:361-71). A higher thermal stability
indicates greater overall antibody stability in vivo. The melting
point of an antibody may be measure using techniques such as
differential scanning calorimetry (Chen et al (2003) Pharm Res
20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52). T.sub.M1
indicates the temperature of the initial unfolding of the antibody.
T.sub.M2 indicates the temperature of complete unfolding of the
antibody. Generally, it is preferred that the T.sub.M1 of an
antibody of the present disclosure is greater than 60.degree. C.,
preferably greater than 65.degree. C., even more preferably greater
than 70.degree. C. Alternatively, the thermal stability of an
antibody may be measure using circular dichroism (Murray et al.
(2002) J. Chromatogr Sci 40:343-9).
[0368] In a preferred embodiment, antibodies are selected that do
not rapidly degrade. Fragmentation of an anti-CD22 antibody may be
measured using capillary electrophoresis (CE) and MALDI-MS, as is
well understood in the art (Alexander A J and Hughes D E (1995)
Anal Chem 67:3626-32).
[0369] In another preferred embodiment, antibodies are selected
that have minimal aggregation effects. Aggregation may lead to
triggering of an unwanted immune response and/or altered or
unfavorable pharmacokinetic properties. Generally, antibodies are
acceptable with aggregation of 25% or less, preferably 20% or less,
even more preferably 15% or less, even more preferably 10% or less
and even more preferably 5% or less. Aggregation may be measured by
several techniques well known in the art, including size-exclusion
column (SEC) high performance liquid chromatography (HPLC), and
light scattering to identify monomers, dimers, trimers or
multimers.
Methods of Engineering Antibodies
[0370] As discussed above, the anti-CD22 antibodies having V.sub.H
and V.sub.L sequences disclosed herein can be used to create new
anti-CD22 antibodies by modifying the V.sub.H and/or V.sub.L
sequences, or the constant region(s) attached thereto. Thus, in
another aspect of this disclosure, the structural features of an
anti-CD22 antibody of this disclosure, e.g. 12C5, 19A3, CD22.1,
CD22.2, 16F7, 23C6, 4G6 and 21F6, are used to create structurally
related anti-CD22 antibodies that retain at least one functional
property of the antibodies of this disclosure, such as binding to
human CD22. For example, one or more CDR regions of 12C5, 19A3,
CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6, or mutations thereof, can
be combined recombinantly with known framework regions and/or other
CDRs to create additional, recombinantly-engineered, anti-CD22
antibodies of this disclosure, as discussed above. Other types of
modifications include those described in the previous section. The
starting material for the engineering method is one or more of the
V.sub.H and/or V.sub.L sequences provided herein, or one or more
CDR regions thereof. To create the engineered antibody, it is not
necessary to actually prepare (i.e., express as a protein) an
antibody having one or more of the V.sub.H and/or V.sub.L sequences
provided herein, or one or more CDR regions thereof. Rather, the
information contained in the sequence(s) is used as the starting
material to create a "second generation" sequence(s) derived from
the original sequence(s) and then the "second generation"
sequence(s) is prepared and expressed as a protein.
[0371] Accordingly, in another embodiment, this disclosure provides
a method for preparing an anti-CD22 antibody comprising:
[0372] (a) providing: (i) a heavy chain variable region antibody
sequence comprising a CDR1 sequence selected from the group
consisting of SEQ ID NOs: 1-4 and 63-65; a CDR2 sequence selected
from the group consisting of SEQ ID NOs: 5-8, 60, and 66-68 and/or
a CDR3 sequence selected from the group consisting of SEQ ID NOs:
9-12 and 69-71; and/or (ii) a light chain variable region antibody
sequence comprising a CDR1 sequence selected from the group
consisting of SEQ ID NOs: 13-18 and 72-74; a CDR2 sequence selected
from the group consisting of SEQ ID NOs: 19-24 and 75-77; and/or a
CDR3 sequence selected from the group consisting of SEQ ID NOs:
25-30 and 78-80;
[0373] (b) altering at least one amino acid residue within the
heavy chain variable region antibody sequence and/or the light
chain variable region antibody sequence to create at least one
altered antibody sequence; and
[0374] (c) expressing the altered antibody sequence as a
protein.
[0375] For example, standard molecular biology techniques can be
used to prepare and express the altered antibody sequence.
[0376] Preferably, the antibody encoded by the altered antibody
sequence(s) is one that retains one, some or all of the functional
properties of the anti-CD22 antibodies described herein, which
functional properties include, but are not limited to: [0377] (a)
internalizing into CD22+ cells; [0378] (b) exhibiting ADCC activity
on CD22+ cells; [0379] (c) enhancing cell death of Ramos cells
induced by BCR stimulation; [0380] (d) not having a direct
anti-proliferative effect on Ramos cells; [0381] (d) not inducing
calcium flux in Ramos cells; [0382] (e) not mediating CDC activity
on Ramos cells; and/or [0383] (f) inhibits growth of
CD22-expressing cells in vivo when conjugated to a cytotoxin
[0384] The functional properties of the altered antibodies can be
assessed using standard assays available in the art and/or
described herein, such as those set forth in the Examples.
[0385] In certain embodiments of the methods of engineering
antibodies of this disclosure, mutations can be introduced randomly
or selectively along all or part of an anti-CD22 antibody coding
sequence and the resulting modified anti-CD22 antibodies can be
screened for binding activity and/or other functional properties as
described herein. Mutational methods have been described in the
art. For example, PCT Publication WO 02/092780 by Short describes
methods for creating and screening antibody mutations using
saturation mutagenesis, synthetic ligation assembly, or a
combination thereof. Alternatively, PCT Publication WO 03/074679 by
Lazar et al. describes methods of using computational screening
methods to optimize physiochemical properties of antibodies.
Nucleic Acid Molecules Encoding Antibodies of this Disclosure
[0386] Another aspect of this disclosure pertains to nucleic acid
molecules that encode the antibodies of this disclosure. 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. (1987) Current Protocols
in Molecular Biology, Greene Publishing and Wiley Interscience, New
York. A nucleic acid of this disclosure can be, for example, DNA or
RNA and may or may not contain intronic sequences. In a preferred
embodiment, the nucleic acid is a cDNA molecule.
[0387] Nucleic acids of this disclosure can be obtained using
standard molecular biology techniques. For antibodies expressed by
hybridomas (e.g., hybridomas prepared from transgenic mice carrying
human immunoglobulin genes as described further below), cDNAs
encoding the light and heavy chains of the antibody made by the
hybridoma can be obtained by standard PCR amplification or cDNA
cloning techniques. For antibodies obtained from an immunoglobulin
gene library (e.g., using phage display techniques), a nucleic acid
encoding such antibodies can be recovered from the gene
library.
[0388] Preferred nucleic acids molecules of this disclosure are
those encoding the V.sub.H and V.sub.L sequences of the 12C5, 19A3,
CD22.1, CD22.2, 16F7, 23C6, 4G6 and 21F6 monoclonal antibodies. DNA
sequences encoding the V.sub.H sequences of 12C5, 19A3, CD22.1,
16F7, 23C6, CD22.2, 4G6 and 21F6 are shown in SEQ ID NOs: 41-44, 62
and 87-89, respectively (wherein the heavy chains of 19A3 and
CD22.1 are identical and correspond to SEQ ID NO:42; the heavy
chain of CD22.2 corresponds to SEQ ID NO:62; and the heavy chains
of 21F6 correspond to SEQ ID NOs:82 and 83). DNA sequences encoding
the V.sub.L sequences of 12C5, 19A3, CD22.1, CD22.2, 16F7, 23C6,
4G6 and 21F6 are shown in SEQ ID NOs: 45-50 and 90-92, respectively
(wherein the kappa light chains of 19A3, CD22.1 and CD22.2 are
identical and correspond to SEQ ID NO:46, the kappa light chain of
16F7 corresponds to either SEQ ID NO:47 or 48, the kappa light
chain of 23C6 corresponds to either SEQ ID NO:49 or 50, and the
kappa light chain of 4G6 corresponds to either SEQ ID NO:90 or
91).
[0389] Once DNA fragments encoding V.sub.H and V.sub.L segments are
obtained, these DNA fragments can be further manipulated by
standard recombinant DNA techniques, for example to convert the
variable region genes to full-length antibody chain genes, to Fab
fragment genes or to a scFv gene. In these manipulations, a
V.sub.L- or V.sub.H-encoding DNA fragment is operatively linked to
another DNA fragment encoding another protein, such as an antibody
constant region or a flexible linker. The term "operatively
linked", as used in this context, is intended to mean that the two
DNA fragments are joined such that the amino acid sequences encoded
by the two DNA fragments remain in-frame.
[0390] The isolated DNA encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat,
E. A., el al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgQ1, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG1 or IgG4 constant region. For a Fab fragment
heavy chain gene, the V.sub.H-encoding DNA can be operatively
linked to another DNA molecule encoding only the heavy chain CH1
constant region.
[0391] The isolated DNA encoding the V.sub.L region can be
converted to a full-length light chain gene (as well as a Fab light
chain gene) by operatively linking the V.sub.L-encoding DNA to
another DNA molecule encoding the light chain constant region, CL.
The sequences of human light chain constant region genes are known
in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242) and DNA
fragments encompassing these regions can be obtained by standard
PCR amplification. In preferred embodiments, the light chain
constant region can be a kappa or lambda constant region.
[0392] To create a scFv gene, the V.sub.H- and V.sub.L-encoding DNA
fragments are operatively linked to another fragment encoding a
flexible linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, such that the V.sub.H and V.sub.L sequences
can be expressed as a contiguous single-chain protein, with the
V.sub.L and V.sub.H regions joined by the flexible linker (see
e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990)
Nature 348:552-554).
Production of Monoclonal Antibodies of this Disclosure
[0393] Monoclonal antibodies (mAbs) of the present disclosure can
be produced by a variety of techniques, including conventional
monoclonal antibody methodology e.g., the standard somatic cell
hybridization technique of Kohler and Milstein (1975) Nature 256:
495. Although somatic cell hybridization procedures are preferred,
in principle, other techniques for producing monoclonal antibody
can be employed e.g., viral or oncogenic transformation of B
lymphocytes.
[0394] The preferred animal system for preparing hybridomas is the
murine system. Hybridoma production in the mouse is a very
well-established procedure. Immunization protocols and techniques
for isolation of immunized splenocytes for fusion are known in the
art. Fusion partners (e.g., murine myeloma cells) and fusion
procedures are also known.
[0395] Chimeric or humanized antibodies of the present disclosure
can be prepared based on the sequence of a non-human monoclonal
antibody prepared as described above. DNA encoding the heavy and
light chain immunoglobulins can be obtained from the non-human
hybridoma of interest and engineered to contain non-murine (e.g.,
human) immunoglobulin sequences using standard molecular biology
techniques. For example, to create a chimeric antibody, murine
variable regions can be linked to human constant regions using
methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to
Cabilly et al.). To create a humanized antibody, murine CDR regions
can be inserted into a human framework using methods known in the
art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.).
[0396] In a preferred embodiment, the antibodies of this disclosure
are human monoclonal antibodies. Such human monoclonal antibodies
directed against CD22 can be generated using transgenic or
transchromosomic mice carrying parts of the human immune system
rather than the mouse system. These transgenic and transchromosomic
mice include mice referred to herein as the HuMAb Mouse.RTM. and KM
Mouse.RTM., respectively, and are collectively referred to herein
as "human Ig mice."
[0397] The HuMAb Mouse.RTM. (Medarex.RTM., Inc.) contains human
immunoglobulin gene miniloci that encode 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 (see e.g., Lonberg, 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. 13: 65-93, and Harding, F. and
Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). Preparation
and use of the HuMAb Mouse.RTM., and the genomic modifications
carried by such mice, is further described 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; Taylor, L. et al.
(1994) International Immunology 6: 579-591; and Fishwild, D. et al.
(1996) Nature Biotechnology 14: 845-851, the contents of all of
which are hereby specifically incorporated by reference in their
entirety. 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; U.S. Pat. No.
5,545,807 to Surani et al.; PCT Publication Nos. WO 92/03918, WO
93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962,
all to Lonberg and Kay; and PCT Publication No. WO 01/14424 to
Korman et al.
[0398] In another embodiment, human antibodies of this disclosure
can be raised using a mouse that carries human immunoglobulin
sequences on transgenes and transchomosomes, such as a mouse that
carries a human heavy chain transgene and a human light chain
transchromosome. This mouse is referred to herein as a "KM
Mouse.RTM.," and is described in detail in PCT Publication WO
02/43478 to Ishida et al.
[0399] Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-CD22 antibodies of this disclosure. For
example, an alternative transgenic system referred to as the
Xenomouse (Abgenix, Inc.) can be used; such mice are described in,
for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598;
6,150,584 and 6,162,963 to Kucherlapati et al.
[0400] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-CD22 antibodies of this disclosure. For
example, mice carrying both a human heavy chain transchromosome and
a human light chain tranchromosome, referred to as "TC mice" can be
used; such mice are described in Tomizuka et al. (2000) Proc. Natl.
Acad. Sci. USA 97:722-727. Furthermore, cows carrying human heavy
and light chain transchromosomes have been described in the art
(e.g., Kuroiwa et al. (2002) Nature Biotechnology 20:889-894 and
PCT application No. WO 2002/092812) and can be used to raise
anti-CD22 antibodies of this disclosure.
[0401] Human monoclonal antibodies of this disclosure can also be
prepared using phage display methods for screening libraries of
human immunoglobulin genes. Such phage display methods for
isolating human antibodies are established in the art. See for
example: U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S. Pat. No.
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.
[0402] Human monoclonal antibodies of this disclosure can also be
prepared using SCID mice into which human immune cells have been
reconstituted such that a human antibody response can be generated
upon immunization. Such mice are described in, for example, U.S.
Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.
[0403] In another embodiment, human anti-CD22 antibodies are
prepared using a combination of human Ig mouse and phage display
techniques, as described in U.S. Pat. No. 6,794,132 by Buechler et
al. More specifically, the method first involves raising an
anti-CD22 antibody response in a human Ig mouse (such as a HuMab
mouse or KM mouse as described above) by immunizing the mouse with
a CD22 antigen, followed by isolating nucleic acids encoding human
antibody chains from lymphatic cells of the mouse and introducing
these nucleic acids into a display vector (e.g., phage) to provide
a library of display packages. Thus, each library member comprises
a nucleic acid encoding a human antibody chain and each antibody
chain is displayed from the display package. The library then is
screened with a CD22 antigen to isolate library members that
specifically bind CD22. Nucleic acid inserts of the selected
library members then are isolated and sequenced by standard methods
to determine the light and heavy chain variable sequences of the
selected CD22 binders. The variable regions can be converted to
full-length antibody chains by standard recombinant DNA techniques,
such as cloning of the variable regions into an expression vector
that carries the human heavy and light chain constant regions such
that the V.sub.H region is operatively linked to the C.sub.H region
and the V.sub.L region is operatively linked to the C.sub.L
region.
Immunization of Human Ig Mice
[0404] When human Ig mice are used to raise human antibodies of
this disclosure, such mice can be immunized with a purified or
enriched preparation of CD22 antigen and/or recombinant CD22, or
cells expressing CD22, or a CD22 fusion protein, as described by
Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwild, D.
et al. (1996) Nature Biotechnology 14: 845-851; and PCT Publication
WO 98/24884 and WO 01/14424. Preferably, the mice will be 6-16
weeks of age upon the first infusion. For example, a purified or
recombinant preparation (5-50 .mu.g) of CD22 antigen can be used to
immunize the human Ig mice intraperitoneally. Most preferably, the
immunogen used to raise the antibodies of this disclosure is a
combination of recombinant human CD22 extracellular domain and CHO
cells engineered to express full-length human CD22 on the cell
surface (described further in Example 1).
[0405] Detailed procedures to generate fully human monoclonal
antibodies to CD22 are described in Example 1 below. Cumulative
experience with various antigens has shown that the transgenic mice
respond when initially immunized intraperitoneally (IP) with
antigen in complete Freund's adjuvant, followed by every other week
IP immunizations (up to a total of 6) with antigen in incomplete
Freund's adjuvant. However, adjuvants other than Freund's are also
found to be effective. In addition, whole cells in the absence of
adjuvant are found to be highly immunogenic. 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-CD22 human immunoglobulin can be used for
fusions. Mice can be boosted intravenously with antigen 3 days
before sacrifice and removal of the spleen. It is expected that 2-3
fusions for each immunization may need to be performed. Between 6
and 24 mice are typically immunized for each antigen. Usually both
HCo7 and HCo12 strains are used. In addition, both HCo7 and HCo12
transgene can be bred together into a single mouse having two
different human heavy chain transgenes (HCo7/HCo12). Alternatively
or additionally, the KM Mouse.RTM. and/or KM-.lamda.HAC strains can
be used, as described in Example 1.
Generation of Hybridomas Producing Human Monoclonal Antibodies of
this Disclosure
[0406] To generate hybridomas producing human monoclonal antibodies
of this disclosure, splenocytes and/or 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 be screened for the production of
antigen-specific antibodies. For example, single cell suspensions
of splenic lymphocytes from immunized mice can be fused to
one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma
cells (ATCC, CRL 1580) with 50% PEG. Alternatively, the single cell
suspension of splenic lymphocytes from immunized mice can be fused
using an electric field based electrofusion method, using a
CytoPulse large chamber cell fusion electroporator (CytoPulse
Sciences, Inc., Glen Burnie Md.). Cells are plated at approximately
2.times.10.sup.5 in flat bottom microtiter plate, followed by a two
week incubation in selective medium containing 20% fetal Clone
Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM
L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM
2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin,
50 mg/ml gentamycin and 1.times.HAT (Sigma; the HAT is added 24
hours after the fusion). 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 monoclonal
IgM and IgG antibodies. 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 human IgG, the monoclonal antibodies can be subcloned
at least twice by limiting dilution. The stable subclones can then
be cultured in vitro to generate small amounts of antibody in
tissue culture medium for characterization.
[0407] To purify human monoclonal antibodies, selected hybridomas
can be grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis
and high performance liquid chromatography to ensure purity. The
buffer solution can be exchanged into PBS, and the concentration
can be determined by OD.sub.280 using 1.43 extinction coefficient.
The monoclonal antibodies can be aliquoted and stored at
-80.degree. C.
Generation of Transfectomas Producing Monoclonal Antibodies of this
Disclosure
[0408] Antibodies of this disclosure 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 (e.g., Morrison, S. (1985) Science 229:1202).
[0409] For example, to express the antibodies, or antibody
fragments thereof, DNAs encoding partial or full-length light and
heavy chains, can be obtained by standard molecular biology
techniques (e.g., PCR amplification or cDNA cloning using a
hybridoma that expresses the antibody of interest) and the DNAs can
be inserted into expression vectors such that the genes are
operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is
intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used.
[0410] The antibody light chain gene and the antibody heavy chain
gene can be inserted into separate vector or, more typically, both
genes are inserted into the same expression vector. The antibody
genes are inserted into the expression vector by standard methods
(e.g., ligation of complementary restriction sites on the antibody
gene fragment and vector, or blunt end ligation if no restriction
sites are present). The light and heavy chain variable regions of
the antibodies described herein can be used to create full-length
antibody genes of any antibody isotype by inserting them into
expression vectors already encoding heavy chain constant and light
chain constant regions of the desired isotype such that the V.sub.H
segment is operatively linked to the C.sub.H segment(s) within the
vector and the V.sub.L segment is operatively linked to the C.sub.L
segment within the vector. Additionally or alternatively, the
recombinant expression vector can encode a signal peptide that
facilitates secretion of the antibody chain from a host cell. The
antibody chain gene can be cloned into the vector such that the
signal peptide is linked in-frame to the amino terminus of the
antibody chain gene. The signal peptide can be an immunoglobulin
signal peptide or a heterologous signal peptide (i.e., a signal
peptide from a non-immunoglobulin protein).
[0411] In addition to the antibody chain genes, the recombinant
expression vectors of this disclosure carry regulatory sequences
that control the expression of the antibody chain genes in a host
cell. The term "regulatory sequence" is intended to include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel (Gene Expression Technology.
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990)). It will be appreciated by those skilled in the art that
the design of the expression vector, including the selection of
regulatory sequences, may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV), Simian Virus 40
(SV40), adenovirus, (e.g., the adenovirus major late promoter
(AdMLP) and polyoma. Alternatively, nonviral regulatory sequences
may be used, such as the ubiquitin promoter or .beta.-globin
promoter. Still further, regulatory elements composed of sequences
from different sources, such as the SR.alpha. promoter system,
which contains sequences from the SV40 early promoter and the long
terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.
et al. (1988) Mol. Cell. Biol. 8:466-472).
[0412] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of this disclosure
may carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all to Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0413] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of this disclosure in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Prokaryotic expression
of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss, M. A. and Wood,
C. R. (1985) Immunology Today 6: 12-13).
[0414] Preferred mammalian host cells for expressing the
recombinant antibodies of this disclosure include Chinese Hamster
Ovary (CHO cells) (including dhfr.sup.- CHO cells, described in
Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220,
used with a DHFR selectable marker, e.g., as described in R. J.
Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO
myeloma cells, COS cells and SP2 cells. In particular, for use with
NSO myeloma cells, another preferred expression system is the GS
gene expression system disclosed in WO 87/04462 (to Wilson), WO
89/01036 (to Bebbington) and EP 338,841 (to Bebbington). When
recombinant expression vectors encoding antibody genes are
introduced into mammalian host cells, the antibodies are produced
by culturing the host cells for a period of time sufficient to
allow for expression of the antibody in the host cells or, more
preferably, secretion of the antibody into the culture medium in
which the host cells are grown. Antibodies can be recovered from
the culture medium using standard protein purification methods.
Characterization of Antibody Binding to Antigen
[0415] Antibodies of the invention can be tested for binding to
CD22 by, for example, standard ELISA. Briefly, microtiter plates
are coated with purified and/or recombinant CD22 (e.g., CD22 ECD as
described in Example 1) at 0.25 .mu.g/ml in PBS, and then blocked
with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g.,
dilutions of plasma from CD22-immunized mice) are added to each
well and incubated for 1-2 hours at 37.degree. C. The plates are
washed with PBS/Tween and then incubated with secondary reagent
(e.g., for human antibodies, 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 pNPP
substrate (1 mg/ml), and analyzed at OD of 405-650. Preferably,
mice that develop the highest titers will be used for fusions.
[0416] An ELISA assay as described above can also be used to screen
for hybridomas that show positive reactivity with CD22 immunogen.
Hybridomas that bind with high avidity to CD22 are subcloned and
further characterized. One clone from each hybridoma, which retains
the reactivity of the parent cells (by ELISA), can be chosen for
making a 5-10 vial cell bank stored at -140.degree. C., and for
antibody purification.
[0417] To purify anti-CD22 antibodies, selected hybridomas can be
grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis
and high performance liquid chromatography to ensure purity. The
buffer solution can be exchanged into PBS, and the concentration
can be determined by OD280 using 1.43 extinction coefficient. The
monoclonal antibodies can be aliquoted and stored at -80.degree.
C.
[0418] To determine if the selected anti-CD22 monoclonal antibodies
bind to unique epitopes, each antibody can be biotinylated using
commercially available reagents (Pierce, Rockford, Ill.).
Competition studies using unlabeled monoclonal antibodies and
biotinylated monoclonal antibodies can be performed using CD22
coated-ELISA plates as described above. Biotinylated mAb binding
can be detected with a strep-avidin-alkaline phosphatase probe.
[0419] To determine the isotype of purified antibodies, isotype
ELISAs can be performed using reagents specific for antibodies of a
particular isotype. For example, to determine the isotype of a
human monoclonal antibody, wells of microtiter plates can be coated
with 1 .mu.g/ml of anti-human immunoglobulin overnight at 4.degree.
C. After blocking with 1% BSA, the plates are reacted with 1
.mu.g/ml or less of test monoclonal antibodies or purified isotype
controls, at ambient temperature for one to two hours. The wells
can then be reacted with either human IgG1 or human IgM-specific
alkaline phosphatase-conjugated probes. Plates are developed and
analyzed as described above.
[0420] Anti-CD22 human IgGs can be further tested for reactivity
with CD22 antigen by Western blotting. Briefly, CD22 can be
prepared and subjected to sodium dodecyl sulfate polyacrylamide gel
electrophoresis. After electrophoresis, the separated antigens are
transferred to nitrocellulose membranes, blocked with 10% fetal
calf serum, and probed with the monoclonal antibodies to be tested.
Human IgG binding can be detected using anti-human IgG alkaline
phosphatase and developed with BCIP/NBT substrate tablets (Sigma
Chem. Co., St. Louis, Mo.).
[0421] The binding specificity of an antibody of this disclosure
may also be determined by monitoring binding of the antibody to
cells expressing CD22, for example by flow cytometry. A cell line
that naturally expresses CD22, such as Daudi cells or Raji cells,
may be used or a cell line, such as a CHO cell line, may be
transfected with an expression vector encoding a transmembrane form
of CD22. The transfected protein may comprise a tag, such as a
myc-tag, preferably at the N-terminus, for detection using an
antibody to the tag. Binding of an antibody of this disclosure to
CD22 may be determined by incubating the transfected cells with the
antibody, and detecting bound antibody. Binding of an antibody to
the tag on the transfected protein may be used as a positive
control.
Bispecific Molecules
[0422] In another aspect, the present disclosure features
bispecific molecules comprising an anti-CD22 antibody, or a
fragment thereof, of this disclosure. An antibody of this
disclosure, or antigen-binding portions thereof, can be derivatized
or linked to another functional molecule, e.g., another peptide or
protein (e.g., another antibody or ligand for a receptor) to
generate a bispecific molecule that binds to at least two different
binding sites or target molecules. The antibody of this disclosure
may in fact be derivatized or linked to more than one other
functional molecule to generate multispecific molecules that bind
to more than two different binding sites and/or target molecules;
such multispecific molecules are also intended to be encompassed by
the term "bispecific molecule" as used herein. To create a
bispecific molecule of this disclosure, an antibody of this
disclosure can be functionally linked (e.g., by chemical coupling,
genetic fusion, noncovalent association or otherwise) to one or
more other binding molecules, such as another antibody, antibody
fragment, peptide or binding mimetic, such that a bispecific
molecule results.
[0423] Accordingly, the present disclosure includes bispecific
molecules comprising at least one first binding specificity for
CD22 and a second binding specificity for a second target epitope.
In a particular embodiment of this disclosure, the second target
epitope is an Fc receptor, e.g., human Fc.gamma.RI (CD64) or a
human Fc.alpha. receptor (CD89). Therefore, this disclosure
includes bispecific molecules capable of binding both to Fc.gamma.R
or Fc.alpha.R expressing effector cells (e.g., monocytes,
macrophages or polymorphonuclear cells (PMNs)), and to target cells
expressing CD22. These bispecific molecules target CD22 expressing
cells to effector cell and trigger Fc receptor-mediated effector
cell activities, such as phagocytosis of CD22 expressing cells,
antibody dependent cell-mediated cytotoxicity (ADCC), cytokine
release, or generation of superoxide anion.
[0424] In an embodiment of this disclosure in which the bispecific
molecule is multispecific, the molecule can further include a third
binding specificity, in addition to an anti-Fc binding specificity
and an anti-CD22 binding specificity. In one embodiment, the third
binding specificity is an anti-enhancement factor (EF) portion,
e.g., a molecule which binds to a surface protein involved in
cytotoxic activity and thereby increases the immune response
against the target cell. The "anti-enhancement factor portion" can
be an antibody, functional antibody fragment or a ligand that binds
to a given molecule, e.g., an antigen or a receptor, and thereby
results in an enhancement of the effect of the binding determinants
for the Fc receptor or target cell antigen. The "anti-enhancement
factor portion" can bind an Fc receptor or a target cell antigen.
Alternatively, the anti-enhancement factor portion can bind to an
entity that is different from the entity to which the first and
second binding specificities bind. For example, the
anti-enhancement factor portion can bind a cytotoxic T-cell (e.g.
via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell
that results in an increased immune response against the target
cell).
[0425] In one embodiment, the bispecific molecules of this
disclosure comprise as a binding specificity at least one antibody,
or an antibody fragment thereof, including, e.g., an Fab, Fab',
F(ab').sub.2, Fv, Fd, dAb or a single chain Fv. The antibody may
also be a light chain or heavy chain dimer, or any minimal fragment
thereof such as a Fv or a single chain construct as described in
U.S. Pat. No. 4,946,778 to Ladner et al., the contents of which is
expressly incorporated by reference.
[0426] In one embodiment, the binding specificity for an Fc.gamma.
receptor is provided by a monoclonal antibody, the binding of which
is not blocked by human immunoglobulin G (IgG). As used herein, the
term "IgG receptor" refers to any of the eight .gamma.-chain genes
located on chromosome 1. These genes encode a total of twelve
transmembrane or soluble receptor isoforms which are grouped into
three Fc.gamma. receptor classes: Fc.gamma.RI (CD64),
Fc.gamma.RII(CD32), and Fc.gamma.RIII (CD16). In one preferred
embodiment, the Fc.gamma. receptor a human high affinity
Fc.gamma.RI. The human Fc.gamma.RI is a 72 kDa molecule, which
shows high affinity for monomeric IgG (10.sup.8-10.sup.9
M.sup.-1).
[0427] The production and characterization of certain preferred
anti-Fc.gamma. antibodies are described in PCT Publication WO
88/00052 and in U.S. Pat. No. 4,954,617 to Fanger et al., the
teachings of which are fully incorporated by reference herein.
These antibodies bind to an epitope of Fc.gamma.RI, Fc.gamma.RII or
Fc.gamma.RIII at a site which is distinct from the Fc.gamma.
binding site of the receptor and, thus, their binding is not
blocked substantially by physiological levels of IgG. Specific
anti-Fc.gamma.RI antibodies useful in this disclosure are mAb 22,
mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb 32
is available from the American Type Culture Collection, ATCC
Accession No. HB9469. In other embodiments, the anti-Fc.gamma.
receptor antibody is a humanized form of monoclonal antibody 22
(H22). The production and characterization of the H22 antibody is
described in Graziano, R. F. et al. (1995) J. Immunol 155 (10):
4996-5002 and PCT Publication WO 94/10332 to Tempest et al. The H22
antibody producing cell line was deposited at the American Type
Culture Collection under the designation HA022CL1 and has the
accession no. CRL 11177.
[0428] In still other preferred embodiments, the binding
specificity for an Fc receptor is provided by an antibody that
binds to a human IgA receptor, e.g., an Fc-alpha receptor
(Fc.alpha.RI (CD89)), the binding of which is preferably not
blocked by human immunoglobulin A (IgA). The term "IgA receptor" is
intended to include the gene product of one .alpha.-gene
(Fc.alpha.RI) located on chromosome 19. This gene is known to
encode several alternatively spliced transmembrane isoforms of 55
to 110 kDa. Fc.alpha.RI (CD89) is constitutively expressed on
monocytes/macrophages, eosinophilic and neutrophilic granulocytes,
but not on non-effector cell populations. Fc.alpha.RI has medium
affinity (.apprxeq.5.times.10.sup.7 M.sup.-1) for both IgA1 and
IgA2, which is increased upon exposure to cytokines such as G-CSF
or GM-CSF (Morton, H. C. et al. (1996) Critical Reviews in
Immunology 16:423-440). Four Fc.alpha.RI-specific monoclonal
antibodies, identified as A3, A59, A62 and A77, which bind
Fc.alpha.RI outside the IgA ligand binding domain, have been
described (Monteiro, R. C. et al. (1992) J. Immunol. 148:1764).
[0429] Fc.alpha.RI and Fc.gamma.RI are preferred trigger receptors
for use in the bispecific molecules of this disclosure because they
are (1) expressed primarily on immune effector cells, e.g.,
monocytes, PMNs, macrophages and dendritic cells; (2) expressed at
high levels (e.g., 5,000-100,000 per cell); (3) mediators of
cytotoxic activities (e.g., ADCC, phagocytosis); and (4) mediate
enhanced antigen presentation of antigens, including self-antigens,
targeted to them.
[0430] While human monoclonal antibodies are preferred, other
antibodies which can be employed in the bispecific molecules of
this disclosure are murine, chimeric and humanized monoclonal
antibodies.
[0431] The bispecific molecules of the present disclosure can be
prepared by conjugating the constituent binding specificities,
e.g., the anti-FcR and anti-CD22 binding specificities, using
methods known in the art. For example, each binding specificity of
the bispecific molecule can be generated separately and then
conjugated to one another. When the binding specificities are
proteins or peptides, a variety of coupling or cross-linking agents
can be used for covalent conjugation. Examples of cross-linking
agents include protein A, carbodiimide,
N-succinimidyl-S-acetyl-thioacetate (SATA),
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide
(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate
(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med.
160:1686; Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA
82:8648). Other methods include those described in Paulus (1985)
Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science
229:81-83, and Glennie et al. (1987) J. Immunol. 139: 2367-2375).
Preferred conjugating agents are SATA and sulfo-SMCC, both
available from Pierce Chemical Co. (Rockford, Ill.).
[0432] When the binding specificities are antibodies, they can be
conjugated via sulfhydryl bonding of the C-terminus hinge regions
of the two heavy chains. In a particularly preferred embodiment,
the hinge region is modified to contain an odd number of sulfhydryl
residues, preferably one, prior to conjugation.
[0433] Alternatively, both binding specificities can be encoded in
the same vector and expressed and assembled in the same host cell.
This method is particularly useful where the bispecific molecule is
a mAb.times.mAb, mAb.times.Fab, Fab.times.F(ab').sub.2 or
ligand.times.Fab fusion protein. A bispecific molecule of this
disclosure can be a single chain molecule comprising one single
chain antibody and a binding determinant, or a single chain
bispecific molecule comprising two binding determinants. Bispecific
molecules may comprise at least two single chain molecules. Methods
for preparing bispecific molecules are described for example in
U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405;
5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858, all of
which are expressly incorporated herein by reference.
[0434] Binding of the bispecific molecules to their specific
targets can be confirmed by, for example, enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis,
bioassay (e.g., growth inhibition), or Western Blot assay. Each of
these assays generally detects the presence of protein-antibody
complexes of particular interest by employing a labeled reagent
(e.g., an antibody) specific for the complex of interest. For
example, the FcR-antibody complexes can be detected using e.g., an
enzyme-linked antibody or antibody fragment which recognizes and
specifically binds to the antibody-FcR complexes. Alternatively,
the complexes can be detected using any of a variety of other
immunoassays. For example, the antibody can be radioactively
labeled and used in a radioimmunoassay (RIA) (see, for example,
Weintraub, B., Principles of Radioimmunoassays, Seventh Training
Course on Radioligand Assay Techniques, The Endocrine Society,
March, 1986, which is incorporated by reference herein). The
radioactive isotope can be detected by such means as the use of a
counter or a scintillation counter or by autoradiography.
Linkers
[0435] The present invention provides for antibody-partner
conjugates where the antibody is linked to the partner through a
chemical linker. In some embodiments, the linker is a peptidyl
linker, and is depicted herein as
(L.sup.4).sub.p-F-(L.sup.1).sub.m. Other linkers include hydrazine
and disulfide linkers, and is depicted herein as
(L.sup.4).sub.p-H-(L.sup.1).sub.m or
(L.sup.4).sub.p-J-(L.sup.1).sub.m, respectively. In addition to the
linkers being attached to the partner, the present invention also
provides cleavable linker arms that are appropriate for attachment
to essentially any molecular species. The linker arm aspect of the
invention is exemplified herein by reference to their attachment to
a therapeutic moiety. It will, however, be readily apparent to
those of skill in the art that the linkers can be attached to
diverse species including, but not limited to, diagnostic agents,
analytical agents, biomolecules, targeting agents, detectable
labels and the like.
[0436] The use of peptidyl and other linkers in antibody-partner
conjugates is described in U.S. Provisional Patent Application Ser.
Nos. 60/295,196; 60/295,259; 60/295,342; 60/304,908; 60/572,667;
60/661,174; 60/669,871; 60/720,499; 60/730,804; 60/735,657;
60/891,028; and U.S. patent application Ser. Nos. 10/160,972;
10/161,234; 11/134,685; 11/134,826; and 11/398,854 and U.S. Pat.
No. 6,989,452 and PCT Patent Application No. PCT/US2006/37793, all
of which are incorporated herein by reference.
[0437] Additional linkers are described in U.S. Pat. No. 6,214,345
(Bristol-Myers Squibb), U.S. Pat. Appl. 2003/0096743 and U.S. Pat.
Appl. 2003/0130189 (both to Seattle Genetics), de Groot et al., J.
Med. Chem. 42, 5277 (1999); de Groot et al. J. Org. Chem. 43, 3093
(2000); de Groot et al., J. Med. Chem. 66, 8815, (2001); WO
02/083180 (Syntarga); Carl et al., J. Med. Chem. Lett. 24, 479,
(1981); Dubowchik et al., Bioorg & Med. Chem. Lett. 8, 3347
(1998).
[0438] In one aspect, the present invention relates to linkers that
are useful to attach targeting groups to therapeutic agents and
markers. In another aspect, the invention provides linkers that
impart stability to compounds, reduce their in vivo toxicity, or
otherwise favorably affect their pharmacokinetics, bioavailability
and/or pharmacodynamics. It is generally preferred that in such
embodiments, the linker is cleaved, releasing the active drug, once
the drug is delivered to its site of action. Thus, in one
embodiment of the invention, the linkers of the invention are
traceless, such that once removed from the therapeutic agent or
marker (such as during activation), no trace of the linker's
presence remains.
[0439] In another embodiment of the invention, the linkers are
characterized by their ability to be cleaved at a site in or near
the target cell such as at the site of therapeutic action or marker
activity. Such cleavage can be enzymatic in nature. This feature
aids in reducing systemic activation of the therapeutic agent or
marker, reducing toxicity and systemic side effects. Preferred
cleavable groups for enzymatic cleavage include peptide bonds,
ester linkages, and disulfide linkages. In other embodiments, the
linkers are sensitive to pH and are cleaved through changes in
pH.
[0440] An important aspect of the current invention is the ability
to control the speed with which the linkers cleave. Often a linker
that cleaves quickly is desired. In some embodiments, however, a
linker that cleaves more slowly may be preferred. For example, in a
sustained release formulation or in a formulation with both a quick
release and a slow release component, it may be useful to provide a
linker which cleaves more slowly. WO 02/096910 provides several
specific ligand-drug complexes having a hydrazine linker. However,
there is no way to "tune" the linker composition dependent upon the
rate of cyclization required, and the particular compounds
described cleave the ligand from the drug at a slower rate than is
preferred for many drug-linker conjugates. In contrast, the
hydrazine linkers of the current invention provide for a range of
cyclization rates, from very fast to very slow, thereby allowing
for the selection of a particular hydrazine linker based on the
desired rate of cyclization.
[0441] For example, very fast cyclization can be achieved with
hydrazine linkers that produce a single 5-membered ring upon
cleavage. Preferred cyclization rates for targeted delivery of a
cytotoxic agent to cells are achieved using hydrazine linkers that
produce, upon cleavage, either two 5-membered rings or a single
6-membered ring resulting from a linker having two methyls at the
geminal position. The gem-dimethyl effect has been shown to
accelerate the rate of the cyclization reaction as compared to a
single 6-membered ring without the two methyls at the geminal
position. This results from the strain being relieved in the ring.
Sometimes, however, substitutents may slow down the reaction
instead of making it faster. Often the reasons for the retardation
can be traced to steric hindrance. For example, the gem dimethyl
substitution allows for a much faster cyclization reaction to occur
compared to when the geminal carbon is a CH.sub.2.
[0442] It is important to note, however, that in some embodiments,
a linker that cleaves more slowly may be preferred. For example, in
a sustained release formulation or in a formulation with both a
quick release and a slow release component, it may be useful to
provide a linker which cleaves more slowly. In certain embodiments,
a slow rate of cyclization is achieved using a hydrazine linker
that produces, upon cleavage, either a single 6-membered ring,
without the gem-dimethyl substitution, or a single 7-membered
ring.
[0443] The linkers also serve to stabilize the therapeutic agent or
marker against degradation while in circulation. This feature
provides a significant benefit since such stabilization results in
prolonging the circulation half-life of the attached agent or
marker. The linker also serves to attenuate the activity of the
attached agent or marker so that the conjugate is relatively benign
while in circulation and has the desired effect, for example is
toxic, after activation at the desired site of action. For
therapeutic agent conjugates, this feature of the linker serves to
improve the therapeutic index of the agent.
[0444] The stabilizing groups are preferably selected to limit
clearance and metabolism of the therapeutic agent or marker by
enzymes that may be present in blood or non-target tissue and are
further selected to limit transport of the agent or marker into the
cells. The stabilizing groups serve to block degradation of the
agent or marker and may also act in providing other physical
characteristics of the agent or marker. The stabilizing group may
also improve the agent or marker's stability during storage in
either a formulated or non-formulated form.
[0445] Ideally, the stabilizing group is useful to stabilize a
therapeutic agent or marker if it serves to protect the agent or
marker from degradation when tested by storage of the agent or
marker in human blood at 37.degree. C. for 2 hours and results in
less than 20%, preferably less than 10%, more preferably less than
5% and even more preferably less than 2%, cleavage of the agent or
marker by the enzymes present in the human blood under the given
assay conditions.
[0446] The present invention also relates to conjugates containing
these linkers. More particularly, the invention relates to prodrugs
that may be used for the treatment of disease, especially for
cancer chemotherapy. Specifically, use of the linkers described
herein provide for prodrugs that display a high specificity of
action, a reduced toxicity, and an improved stability in blood
relative to prodrugs of similar structure.
[0447] The linkers of the present invention as described herein may
be present at a variety of positions within the partner
molecule.
[0448] Thus, there is provided a linker that may contain any of a
variety of groups as part of its chain that will cleave in vivo,
e.g., in the blood stream, at a rate which is enhanced relative to
that of constructs that lack such groups. Also provided are
conjugates of the linker arms with therapeutic and diagnostic
agents. The linkers are useful to form prodrug analogs of
therapeutic agents and to reversibly link a therapeutic or
diagnostic agent to a targeting agent, a detectable label, or a
solid support. The linkers may be incorporated into complexes that
include the cytotoxins of the invention.
[0449] In addition to the cleavable peptide, hydrazine, or
disulfide group, one or more self-immolative linker groups L.sup.1
are optionally introduced between the cytoCytotoxin And the
targeting agent. These linker groups may also be described as
spacer groups and contain at least two reactive functional groups.
Typically, one chemical functionality of the spacer group bonds to
a chemical functionality of the therapeutic agent, e.g., cytotoxin,
while the other chemical functionality of the spacer group is used
to bond to a chemical functionality of the targeting agent or the
cleavable linker. Examples of chemical functionalities of spacer
groups include hydroxy, mercapto, carbonyl, carboxy, amino, ketone,
and mercapto groups.
[0450] The self-immolative linkers, represented by L.sup.1, are
generally a substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl or
substituted or unsubstituted heteroalkyl group. In one embodiment,
the alkyl or aryl groups may comprise between 1 and 20 carbon
atoms. They may also comprise a polyethylene glycol moiety.
[0451] Exemplary spacer groups include, for example,
6-aminohexanol, 6-mercaptohexanol, 10-hydroxydecanoic acid, glycine
and other amino acids, 1,6-hexanediol, .beta.-alanine,
2-aminoethanol, cysteamine (2-aminoethanethiol), 5-aminopentanoic
acid, 6-aminohexanoic acid, 3-maleimidobenzoic acid, phthalide,
.alpha.-substituted phthalides, the carbonyl group, aminal esters,
nucleic acids, peptides and the like.
[0452] The spacer can serve to introduce additional molecular mass
and chemical functionality into the cytotoxin-targeting agent
complex. Generally, the additional mass and functionality will
affect the serum half-life and other properties of the complex.
Thus, through careful selection of spacer groups, cytotoxin
complexes with a range of serum half-lives can be produced.
[0453] The spacer(s) located directly adjacent to the drug moiety
is also denoted as (L.sup.1).sub.m, wherein m is an integer
selected from 0, 1, 2, 3, 4, 5, and 6. When multiple L.sup.1
spacers are present, either identical or different spacers may be
used. L.sup.1 may be any self-immolative group.
[0454] L.sup.4 is a linker moiety that preferably imparts increased
solubility or decreased aggregation properties to conjugates
utilizing a linker that contains the moiety or modifies the
hydrolysis rate of the conjugate. The L.sup.4 linker does not have
to be self immolative. In one embodiment, the L.sup.4 moiety is
substituted alkyl, unsubstituted alkyl, substituted aryl,
unsubstituted aryl, substituted heteroalkyl, or unsubstituted
heteroalkyl, any of which may be straight, branched, or cyclic. The
substitutions may be, for example, a lower (C.sup.1-C.sup.6) alkyl,
alkoxy, aklylthio, alkylamino, or dialkylamino. In certain
embodiments, L.sup.4 comprises a non-cyclic moiety. In another
embodiment, L.sup.4 comprises any positively or negatively charged
amino acid polymer, such as polylysine or polyargenine. L.sup.4 can
comprise a polymer such as a polyethylene glycol moiety.
Additionally the L.sup.4 linker can comprise, for example, both a
polymer component and a small chemical moiety.
[0455] In a preferred embodiment, L.sup.4 comprises a polyethylene
glycol (PEG) moiety. The PEG portion of L.sup.4 may be between 1
and 50 units long. Preferably, the PEG will have 1-12 repeat units,
more preferably 3-12 repeat units, more preferably 2-6 repeat
units, or even more preferably 3-5 repeat units and most preferably
4 repeat units. L.sup.4 may consist solely of the PEG moiety, or it
may also contain an additional substituted or unsubstituted alkyl
or heteroalkyl. It is useful to combine PEG as part of the L.sup.4
moiety to enhance the water solubility of the complex.
Additionally, the PEG moiety reduces the degree of aggregation that
may occur during the conjugation of the drug to the antibody.
[0456] In some embodiments, L.sup.4 comprises
##STR00002##
directly attached to the N-terminus of (AA.sup.1).sub.c. R.sup.20
is a member selected from H, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, and acyl. Each R.sup.25,
R.sup.25', R.sup.26, and R.sup.26' is independently selected from
H, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, and substituted or unsubstituted
heterocycloalkyl; and s and t are independently integers from 1 to
6. Preferably, R.sup.20, R.sup.25, R.sup.25', R.sup.26 and
R.sup.26' are hydrophobic. In some embodiments, R.sup.20 is H or
alkyl (preferably, unsubstituted lower alkyl). In some embodiments,
R.sup.25, R.sup.25', R.sup.26 and R.sup.26' are independently H or
alkyl (preferably, unsubstituted C.sup.1 to C.sup.4 alkyl). In some
embodiments, R.sup.25, R.sup.25', R.sup.26 and R.sup.26' are all H.
In some embodiments, t is 1 and s is 1 or 2.
[0457] Peptide Linkers (F)
[0458] As discussed above, the peptidyl linkers of the invention
can be represented by the general formula:
(L.sup.4).sub.p-F-(L.sup.1).sub.m, wherein F represents the linker
portion comprising the peptidyl moiety. In one embodiment, the F
portion comprises an optional additional self-immolative linker(s),
L.sup.2, and a carbonyl group. In another embodiment, the F portion
comprises an amino group and an optional spacer group(s),
L.sup.3.
[0459] Accordingly, in one embodiment, the conjugate comprising the
peptidyl linker comprises a structure of the following formula
(a):
##STR00003##
[0460] In this embodiment, L.sup.1 is a self-immolative linker, as
described above, and L.sup.4 is a moiety that preferably imparts
increased solubility, or decreased aggregation properties, or
modifies the hydrolysis rate, as described above. L.sup.2
represents a self-immolative linker(s). In addition, m is 0, 1, 2,
3, 4, 5, or 6; and o and p are independently 0 or 1. AA.sup.1
represents one or more natural amino acids, and/or unnatural
.alpha.-amino acids; c is an integer from 1 and 20. In some
embodiments, c is in the range of 2 to 5 or c is 2 or 3.
[0461] In the peptide linkers of the invention of the above formula
(a), AA.sup.1 is linked, at its amino terminus, either directly to
L.sup.4 or, when L.sup.4 is absent, directly to the X.sup.4 group
(i.e., the targeting agent, detectable label, protected reactive
functional group or unprotected reactive functional group). In some
embodiments, when L.sup.4 is present, L.sup.4 does not comprise a
carboxylic acyl group directly attached to the N-terminus of
(AA.sup.1).sub.c. Thus, it is not necessary in these embodiments
for there to be a carboxylic acyl unit directly between either
L.sup.4 or X.sup.4 and AA.sup.1, as is necessary in the peptidic
linkers of U.S. Pat. No. 6,214,345.
[0462] In another embodiment, the conjugate comprising the peptidyl
linker comprises a structure of the following formula (b):
##STR00004##
[0463] In this embodiment, L.sup.4 is a moiety that preferably
imparts increased solubility, or decreased aggregation properties,
or modifies the hydrolysis rate, as described above; L.sup.3 is a
spacer group comprising a primary or secondary amine or a carboxyl
functional group, and either the amine of L.sup.3 forms an amide
bond with a pendant carboxyl functional group of D or the carboxyl
of L.sup.3 forms an amide bond with a pendant amine functional
group of D; and o and p are independently 0 or 1. AA.sup.1
represents one or more natural amino acids, and/or unnatural
.alpha.-amino acids; c is an integer from 1 and 20. In this
embodiment, L.sup.1 is absent (i.e., m is 0 in the general
formula).
[0464] In the peptide linkers of the invention of the above formula
(b), AA.sup.1 is linked, at its amino terminus, either directly to
L.sup.4 or, when L.sup.4 is absent, directly to the X.sup.4 group
(i.e., the targeting agent, detectable label, protected reactive
functional group or unprotected reactive functional group). In some
embodiments, when L.sup.4 is present, L.sup.4 does not comprise a
carboxylic acyl group directly attached to the N-terminus of
(AA.sup.1).sub.c. Thus, it is not necessary in these embodiments
for there to be a carboxylic acyl unit directly between either
L.sup.4 or X.sup.4 and AA.sup.1, as is necessary in the peptidic
linkers of U.S. Pat. No. 6,214,345.
[0465] The Self-Immolative Linker L.sup.2
[0466] The self-immolative linker L.sup.2 is a bifunctional
chemical moiety which is capable of covalently linking together two
spaced chemical moieties into a normally stable tripartate
molecule, releasing one of said spaced chemical moieties from the
tripartate molecule by means of enzymatic cleavage; and following
said enzymatic cleavage, spontaneously cleaving from the remainder
of the molecule to release the other of said spaced chemical
moieties. In accordance with the present invention, the
self-immolative spacer is covalently linked at one of its ends to
the peptide moiety and covalently linked at its other end to the
chemically reactive site of the drug moiety whose derivatization
inhibits pharmacological activity, so as to space and covalently
link together the peptide moiety and the drug moiety into a
tripartate molecule which is stable and pharmacologically inactive
in the absence of the target enzyme, but which is enzymatically
cleavable by such target enzyme at the bond covalently linking the
spacer moiety and the peptide moiety to thereby effect release of
the peptide moiety from the tripartate molecule. Such enzymatic
cleavage, in turn, will activate the self-immolating character of
the spacer moiety and initiate spontaneous cleavage of the bond
covalently linking the spacer moiety to the drug moiety, to thereby
effect release of the drug in pharmacologically active form.
[0467] The self-immolative linker L.sup.2 may be any
self-immolative group. Preferably L.sup.2 is a substituted alkyl,
unsubstituted alkyl, substituted heteroalkyl, unsubstituted
heteroalkyl, unsubstituted heterocycloalkyl, substituted
heterocycloalkyl, substituted and unsubstituted aryl, and
substituted and unsubstituted heteroaryl.
[0468] One particularly preferred self-immolative spacer L.sup.2
may be represented by the formula (c):
##STR00005##
[0469] The aromatic ring of the aminobenzyl group may be
substituted with one or more "K" groups. A "K" group is a
substituent on the aromatic ring that replaces a hydrogen otherwise
attached to one of the four non-substituted carbons that are part
of the ring structure. The "K" group may be a single atom, such as
a halogen, or may be a multi-atom group, such as alkyl,
heteroalkyl, amino, nitro, hydroxy, alkoxy, haloalkyl, and cyano.
Each K is independently selected from the group consisting of
substituted alkyl, unsubstituted alkyl, substituted heteroalkyl,
unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl,
substituted heteroaryl, unsubstituted heteroaryl, substituted
heterocycloalkyl, unsubstituted heterocycloalkyl, halogen,
NO.sub.2, NR.sup.21R.sup.22, NR.sup.21COR.sup.22,
OCONR.sup.21R.sup.22, OCOR.sup.21, and OR.sup.21, wherein R.sup.21
and R.sup.22 are independently selected from the group consisting
of H, substituted alkyl, unsubstituted alkyl, substituted
heteroalkyl, unsubstituted heteroalkyl, substituted aryl,
unsubstituted aryl, substituted heteroaryl, unsubstituted
heteroaryl, substituted heterocycloalkyl and unsubstituted
heterocycloalkyl. Exemplary K substituents include, but are not
limited to, F, Cl, Br, I, NO.sub.2, OH, OCH.sub.3, NHCOCH.sub.3,
N(CH.sub.3).sub.2, NHCOCF.sub.3 and methyl. For "K.sub.i", i is an
integer of 0, 1, 2, 3, or 4. In one preferred embodiment, i is
0.
[0470] The ether oxygen atom of the structure shown above is
connected to a carbonyl group. The line from the NR.sup.24
functionality into the aromatic ring indicates that the amine
functionality may be bonded to any of the five carbons that both
form the ring and are not substituted by the --CH.sub.2--O-- group.
Preferably, the NR.sup.24 functionality of X is covalently bound to
the aromatic ring at the para position relative to the
--CH.sub.2--O-- group. R.sup.24 is a member selected from the group
consisting of H, substituted alkyl, unsubstituted alkyl,
substituted heteroalkyl, and unsubstituted heteroalkyl. In a
specific embodiment, R.sup.24 is hydrogen.
[0471] In one embodiment, the invention provides a peptide linker
of formula (a) above, wherein F comprises the structure:
##STR00006##
where R.sup.24 is selected from the group consisting of H,
substituted alkyl, unsubstituted alkyl, substituted heteroalkyl,
and unsubstituted heteroalkyl. Each K is a member independently
selected from the group consisting of substituted alkyl,
unsubstituted alkyl, substituted heteroalkyl, unsubstituted
heteroalkyl, substituted aryl, unsubstituted aryl, substituted
heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl,
unsubstituted heterocycloalkyl, halogen, NO.sub.2,
NR.sup.21R.sup.22, NR.sup.21COR.sup.22, OCONR.sup.21R.sup.22,
OCOR.sup.21, and OR.sup.21 where R.sup.21 and R.sup.22 are
independently selected from the group consisting of H, substituted
alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted
heteroalkyl, substituted aryl, unsubstituted aryl, substituted
heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl,
unsubstituted heterocycloalkyl; and i is an integer of 0, 1, 2, 3,
or 4.
[0472] In another embodiment, the peptide linker of formula (a)
above comprises a --F-(L.sup.1).sub.m- that comprises the
structure:
##STR00007##
where each R.sup.24 is a member independently selected from the
group consisting of H substituted alkyl, unsubstituted alkyl,
substituted heteroalkyl, and unsubstituted heteroalkyl.
[0473] In some embodiments, the self-immolative spacer L.sup.1 or
L.sup.2 includes
##STR00008##
where each R.sup.17, R.sup.18, and R.sup.19 is independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl and substituted or unsubstituted aryl,
and w is an integer from 0 to 4. In some embodiments, R.sup.17 and
R.sup.18 are independently H or alkyl (preferably, unsubstituted
C.sub.1-4 alkyl). Preferably, R.sup.17 and R.sup.18 are C1-4 alkyl,
such as methyl or ethyl. In some embodiments, w is 0. While not
wishing to be bound to any particular theory, it has been found
experimentally that this particular self-immolative spacer cyclizes
relatively quickly.
[0474] In some embodiments, L.sup.1 or L.sup.2 includes
##STR00009##
[0475] The Spacer Group L.sup.3
[0476] The spacer group L.sup.3 is characterized in that it
comprises a primary or secondary amine or a carboxyl functional
group, and either the amine of the L.sup.3 group forms an amide
bond with a pendant carboxyl functional group of D or the carboxyl
of L.sup.3 forms an amide bond with a pendant amine functional
group of D. L.sup.3 can be selected from the group consisting of
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, or substituted or unsubstituted
heterocycloalkyl. In a preferred embodiment, L.sup.3 comprises an
aromatic group. More preferably, L.sup.3 comprises a benzoic acid
group, an aniline group or indole group. Non-limiting examples of
structures that can serve as an -L.sup.3-NH-- spacer include the
following structures:
##STR00010##
where Z is a member selected from O, S and NR.sup.23, and where
R.sup.23 is a member selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, and acyl.
[0477] Upon cleavage of the linker of the invention containing
L.sup.3, the L.sup.3 moiety remains attached to the drug, D.
Accordingly, the L.sup.3 moiety is chosen such that its presence
attached to D does not significantly alter the activity of D. In
another embodiment, a portion of the drug D itself functions as the
L.sup.3 spacer. For example, in one embodiment, the drug, D, is a
duocarmycin derivative in which a portion of the drug functions as
the L.sup.3 spacer. Non-limiting examples of such embodiments
include those in which NH.sub.2-(L.sup.3)-D has a structure
selected from the group consisting of:
##STR00011##
where Z is a member selected from O, S and NR.sup.23, where
R.sup.23 is a member selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, and acyl; and
where the NH.sub.2 group on each structure reacts with
(AA.sup.1).sub.c to form -(AA.sup.1).sub.c-NH--.
[0478] The Peptide Sequence AA.sup.1
[0479] The group AA.sup.1 represents a single amino acid or a
plurality of amino acids that are joined together by amide bonds.
The amino acids may be natural amino acids and/or unnatural
.alpha.-amino acids.
[0480] The peptide sequence (AA.sup.1).sub.c is functionally the
amidification residue of a single amino acid (when c=1) or a
plurality of amino acids joined together by amide bonds. The
peptide of the current invention is selected for directing
enzyme-catalyzed cleavage of the peptide by an enzyme in a location
of interest in a biological system. For example, for conjugates
that are targeted to a cell using a targeting agent, but not
internalized by that cell, a peptide is chosen that is cleaved by
one or more proteases that may exist in the extracellular matrix,
e.g., due to release of the cellular contents of nearby dying
cells, such that the peptide is cleaved extracellularly. The number
of amino acids within the peptide can range from 1 to 20; but more
preferably there will be 1-8 amino acids, 1-6 amino acids or 1, 2,
3 or 4 amino acids comprising (AA.sup.1).sub.c. Peptide sequences
that are susceptible to cleavage by specific enzymes or classes of
enzymes are well known in the art.
[0481] Many peptide sequences that are cleaved by enzymes in the
serum, liver, gut, etc. are known in the art. An exemplary peptide
sequence of the invention includes a peptide sequence that is
cleaved by a protease. The focus of the discussion that follows on
the use of a protease-sensitive sequence is for clarity of
illustration and does not serve to limit the scope of the present
invention.
[0482] When the enzyme that cleaves the peptide is a protease, the
linker generally includes a peptide containing a cleavage
recognition sequence for the protease. A cleavage recognition
sequence for a protease is a specific amino acid sequence
recognized by the protease during proteolytic cleavage. Many
protease cleavage sites are known in the art, and these and other
cleavage sites can be included in the linker moiety. See, e.g.,
Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth.
Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175
(1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al.
Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol. 244:
412 (1994); Bouvier et al. Meth. Enzymol. 248: 614 (1995), Hardy et
al., in Amyloid Protein Precursor in Development, Aging, and
Alzheimer's Disease, ed. Masters et al. pp. 190-198 (1994).
[0483] The amino acids of the peptide sequence (AA.sup.1).sub.c are
chosen based on their suitability for selective enzymatic cleavage
by particular molecules such as tumor-associated protease. The
amino acids used may be natural or unnatural amino acids. They may
be in the L or the D configuration. In one embodiment, at least
three different amino acids are used. In another embodiment, only
two amino acids are used.
[0484] In a preferred embodiment, the peptide sequence
(AA.sup.1).sub.c is chosen based on its ability to be cleaved by a
lysosomal proteases, non-limiting examples of which include
cathepsins B, C, D, H, L and S. Preferably, the peptide sequence
(AA.sup.1).sub.c is capable of being cleaved by cathepsin B in
vitro, which can be tested using in vitro protease cleavage assays
known in the art.
[0485] In another embodiment, the peptide sequence (AA.sup.1).sub.c
is chosen based on its ability to be cleaved by a tumor-associated
protease, such as a protease that is found extracellularly in the
vicinity of tumor cells, non-limiting examples of which include
thimet oligopeptidase (TOP) and CD10. The ability of a peptide to
be cleaved by TOP or CD10 can be tested using in vitro protease
cleavage assays known in the art.
[0486] Suitable, but non-limiting, examples of peptide sequences
suitable for use in the conjugates of the invention include
Val-Cit, Cit-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit,
Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N.sup.9-tosyl-Arg,
Phe-N.sup.9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys,
Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ. ID NO:
94), .beta.-Ala-Leu-Ala-Leu (SEQ. ID NO: 95) and Gly-Phe-Leu-Gly
(SEQ. ID NO: 96), Val-Ala, Leu-Leu-Gly-Leu (SEQ. ID NO: 97),
Leu-Asn-Ala, and Lys-Leu-Val. Preferred peptides sequences are
Val-Cit and Val-Lys.
[0487] In another embodiment, the amino acid located the closest to
the drug moiety is selected from the group consisting of: Ala, Asn,
Asp, Cit, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, and Val. In yet another embodiment, the amino acid
located the closest to the drug moiety is selected from the group
consisting of: Ala, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
[0488] Proteases have been implicated in cancer metastasis.
Increased synthesis of the protease urokinase was correlated with
an increased ability to metastasize in many cancers. Urokinase
activates plasmin from plasminogen, which is ubiquitously located
in the extracellular space and its activation can cause the
degradation of the proteins in the extracellular matrix through
which the metastasizing tumor cells invade. Plasmin can also
activate the collagenases thus promoting the degradation of the
collagen in the basement membrane surrounding the capillaries and
lymph system thereby allowing tumor cells to invade into the target
tissues (Dano, et al. Adv. Cancer. Res., 44:139 (1985)). Thus, it
is within the scope of the present invention to utilize as a linker
a peptide sequence that is cleaved by urokinase.
[0489] The invention also provides the use of peptide sequences
that are sensitive to cleavage by tryptases. Human mast cells
express at least four distinct tryptases, designated
.alpha..beta.I, .beta.II, and .beta.III. These enzymes are not
controlled by blood plasma proteinase inhibitors and only cleave a
few physiological substrates in vitro. The tryptase family of
serine proteases has been implicated in a variety of allergic and
inflammatory diseases involving mast cells because of elevated
tryptase levels found in biological fluids from patients with these
disorders. However, the exact role of tryptase in the
pathophysiology of disease remains to be delineated. The scope of
biological functions and corresponding physiological consequences
of tryptase are substantially defined by their substrate
specificity.
[0490] Tryptase is a potent activator of pro-urokinase plasminogen
activator (uPA), the zymogen form of a protease associated with
tumor metastasis and invasion. Activation of the plasminogen
cascade, resulting in the destruction of extracellular matrix for
cellular extravasation and migration, may be a function of tryptase
activation of pro-urokinase plasminogen activator at the P4-P1
sequence of Pro-Arg-Phe-Lys (SEQ. ID NO: 98) (Stack, et al.,
Journal of Biological Chemistry 269 (13): 9416-9419 (1994)).
Vasoactive intestinal peptide, a neuropeptide that is implicated in
the regulation of vascular permeability, is also cleaved by
tryptase, primarily at the Thr-Arg-Leu-Arg (SEQ. ID NO: 99)
sequence (Tam, et al., Am. J. Respir. Cell Mol. Biol. 3: 27-32
(1990)). The G-protein coupled receptor PAR-2 can be cleaved and
activated by tryptase at the Ser-Lys-Gly-Arg (SEQ. ID NO: 100)
sequence to drive fibroblast proliferation, whereas the thrombin
activated receptor PAR-1 is inactivated by tryptase at the
Pro-Asn-Asp-Lys (SEQ. ID NO: 101) sequence (Molino et al., Journal
of Biological Chemistry 272(7): 4043-4049 (1997)). Taken together,
this evidence suggests a central role for tryptase in tissue
remodeling as a consequence of disease. This is consistent with the
profound changes observed in several mast cell-mediated disorders.
One hallmark of chronic asthma and other long-term respiratory
diseases is fibrosis and thickening of the underlying tissues that
could be the result of tryptase activation of its physiological
targets. Similarly, a series of reports have shown angiogenesis to
be associated with mast cell density, tryptase activity and poor
prognosis in a variety of cancers (Coussens et al., Genes and
Development 13(11): 1382-97 (1999)); Takanami et al., Cancer
88(12): 2686-92 (2000); Toth-Jakatics et al., Human Pathology
31(8): 955-960 (2000); Ribatti et al., International Journal of
Cancer 85(2): 171-5 (2000)).
[0491] Methods are known in the art for evaluating whether a
particular protease cleaves a selected peptide sequence. For
example, the use of 7-amino-4-methyl coumarin (AMC) fluorogenic
peptide substrates is a well-established method for the
determination of protease specificity (Zimmerman, M., et al.,
(1977) Analytical Biochemistry 78:47-51). Specific cleavage of the
anilide bond liberates the fluorogenic AMC leaving group allowing
for the simple determination of cleavage rates for individual
substrates. More recently, arrays (Lee, D., et al., (1999)
Bioorganic and Medicinal Chemistry Letters 9:1667-72) and
positional-scanning libraries (Rano, T. A., et al., (1997)
Chemistry and Biology 4:149-55) of AMC peptide substrate libraries
have been employed to rapidly profile the N-terminal specificity of
proteases by sampling a wide range of substrates in a single
experiment. Thus, one of skill in the art may readily evaluate an
array of peptide sequences to determine their utility in the
present invention without resort to undue experimentation.
[0492] The antibody-partner conjugate of the current invention may
optionally contain two or more linkers. These linkers may be the
same or different. For example, a peptidyl linker may be used to
connect the drug to the ligand and a second peptidyl linker may
attach a diagnostic agent to the complex. Other uses for additional
linkers include linking analytical agents, biomolecules, targeting
agents, and detectable labels to the antibody-partner complex.
[0493] Also within the scope of the present invention are compounds
of the invention that are poly- or multi-valent species, including,
for example, species such as dimers, trimers, tetramers and higher
homologs of the compounds of the invention or reactive analogues
thereof. The poly- and multi-valent species can be assembled from a
single species or more than one species of the invention. For
example, a dimeric construct can be "homo-dimeric" or
"heterodimeric." Moreover, poly- and multi-valent constructs in
which a compound of the invention or a reactive analogue thereof,
is attached to an oligomeric or polymeric framework (e.g.,
polylysine, dextran, hydroxyethyl starch and the like) are within
the scope of the present invention. The framework is preferably
polyfunctional (i.e. having an array of reactive sites for
attaching compounds of the invention). Moreover, the framework can
be derivatized with a single species of the invention or more than
one species of the invention.
[0494] Moreover, the present invention includes compounds that are
functionalized to afford compounds having water-solubility that is
enhanced relative to analogous compounds that are not similarly
functionalized. Thus, any of the substituents set forth herein can
be replaced with analogous radicals that have enhanced water
solubility. For example, it is within the scope of the invention
to, for example, replace a hydroxyl group with a diol, or an amine
with a quaternary amine, hydroxy amine or similar more
water-soluble moiety. In a preferred embodiment, additional water
solubility is imparted by substitution at a site not essential for
the activity towards the ion channel of the compounds set forth
herein with a moiety that enhances the water solubility of the
parent compounds. Methods of enhancing the water-solubility of
organic compounds are known in the art. Such methods include, but
are not limited to, functionalizing an organic nucleus with a
permanently charged moiety, e.g., quaternary ammonium, or a group
that is charged at a physiologically relevant pH, e.g. carboxylic
acid, amine. Other methods include, appending to the organic
nucleus hydroxyl- or amine-containing groups, e.g. alcohols,
polyols, polyethers, and the like. Representative examples include,
but are not limited to, polylysine, polyethyleneimine,
poly(ethyleneglycol) and poly(propyleneglycol). Suitable
functionalization chemistries and strategies for these compounds
are known in the art. See, for example, Dunn, R. L., et al., Eds.
POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series
Vol. 469, American Chemical Society, Washington, D.C. 1991.
[0495] Hydrazine Linkers (H)
[0496] In a second embodiment, the conjugate of the invention
comprises a hydrazine self-immolative linker, wherein the conjugate
has the structure:
X.sup.4-(L.sup.4).sub.p-H-(L.sup.1).sub.m-D
wherein D, L.sup.1, L.sup.4, and X.sup.4 are as defined above and
described further herein, and H is a linker comprising the
structure:
##STR00012##
[0497] wherein n.sub.1 is an integer from 1-10; n.sub.2 is 0, 1, or
2; each R.sup.24 is a member independently selected from the group
consisting of H, substituted alkyl, unsubstituted alkyl,
substituted heteroalkyl, and unsubstituted heteroalkyl; and I is
either a bond (i.e., the bond between the carbon of the backbone
and the adjacent nitrogen) or:
##STR00013##
[0498] wherein n.sub.3 is 0 or 1, with the proviso that when
n.sub.3 is 0, n.sub.2 is not 0; and n.sub.4 is 1, 2, or 3, wherein
when I is a bond, n.sub.1 is 3 and n.sub.2 is 1, D can not be
##STR00014##
[0499] where R is Me or CH.sub.2--CH.sub.2--NMe.sub.2.
[0500] In one embodiment, the substitution on the phenyl ring is a
para substitution. In preferred embodiments, n.sub.1 is 2, 3, or 4
or n.sub.1 is 3. In preferred embodiments, n.sub.2 is 1. In
preferred embodiments, I is a bond (i.e., the bond between the
carbon of the backbone and the adjacent nitrogen). In one aspect,
the hydrazine linker, H, can form a 6-membered self immolative
linker upon cleavage, for example, when n.sub.3 is 0 and n4 is 2.
In another aspect, the hydrazine linker, H, can form two 5-membered
self immolative linkers upon cleavage. In yet other aspects, H
forms a 5-membered self immolative linker, H forms a 7-membered
self immolative linker, or H forms a 5-membered self immolative
linker and a 6-membered self immolative linker, upon cleavage. The
rate of cleavage is affected by the size of the ring formed upon
cleavage. Thus, depending upon the rate of cleavage desired, an
appropriate size ring to be formed upon cleavage can be
selected.
[0501] Five Membered Hydrazine Linkers
[0502] In one embodiment, the hydrazine linker comprises a
5-membered hydrazine linker, wherein H comprises the structure:
##STR00015##
[0503] In a preferred embodiment, n.sub.1 is 2, 3, or 4. In another
preferred embodiment, n.sub.1 is 3.
In the above structure, each R.sup.24 is a member independently
selected from the group consisting of H, substituted alkyl,
unsubstituted alkyl, substituted heteroalkyl, and unsubstituted
heteroalkyl. In one embodiment, each R.sup.24 is independently H or
a C.sub.1-C.sub.6 alkyl. In another embodiment, each R.sup.24 is
independently H or a C.sub.1-C.sub.3 alkyl, more preferably H or
CH.sub.3. In another embodiment, at least one R.sup.24 is a methyl
group. In another embodiment, each R.sub.24 is H. Each R.sup.24 is
selected to tailor the compounds steric effects and for altering
solubility.
[0504] The 5-membered hydrazine linkers can undergo one or more
cyclization reactions that separate the drug from the linker, and
can be described, for example, by:
##STR00016##
[0505] An exemplary synthetic route for preparing a five membered
linker of the invention is:
##STR00017##
The Cbz-protected DMDA b is reacted with 2,2-Dimethyl-malonic acid
a in solution with thionyl chloride to form a
Cbz-DMDA-2,2-dimethylmalonic acid c. Compound c is reacted with
Boc-N-methyl hydrazine d in the presence of EDC to form
DMDA-2,2-dimetylmalonic-Boc-N-methylhydrazine e.
[0506] Six Membered Hydrazine Linkers
[0507] In another embodiment, the hydrazine linker comprises a
6-membered hydrazine linker, wherein H comprises the structure:
##STR00018##
[0508] In a preferred embodiment, n.sub.1 is 3. In the above
structure, each R.sup.24 is a member independently selected from
the group consisting of H, substituted alkyl, unsubstituted alkyl,
substituted heteroalkyl, and unsubstituted heteroalkyl. In one
embodiment, each R.sup.24 is independently H or a C.sub.1-C.sub.6
alkyl. In another embodiment, each R.sup.24 is independently H or a
C.sub.1-C.sub.3 alkyl, more preferably H or CH.sub.3. In another
embodiment, at least one R.sup.24 is a methyl group. In another
embodiment, each R.sub.24 is H. Each R.sup.24 is selected to tailor
the compounds steric effects and for altering solubility. In a
preferred embodiment, H comprises the structure:
##STR00019##
[0509] In one embodiment, H comprises a geminal dimethyl
substitution. In one embodiment of the above structure, each
R.sup.24 is independently an H or a substituted or unsubstituted
alkyl.
[0510] The 6-membered hydrazine linkers will undergo a cyclization
reaction that separates the drug from the linker, and can be
described as:
##STR00020##
[0511] An exemplary synthetic route for preparing a six membered
linker of the invention is:
##STR00021##
[0512] The Cbz-protected dimethyl alanine a in solution with
dichlormethane, was reacted with HOAt, and CPI to form a
Cbz-protected dimethylalanine hydrazine b. The hydrazine b is
deprotected by the action of methanol, forming compound c.
[0513] Other Hydrazine Linkers
[0514] It is contemplated that the invention comprises a linker
having seven members. This linker would likely not cyclize as
quickly as the five or six membered linkers, but this may be
preferred for some antibody-partner conjugates. Similarly, the
hydrazine linker may comprise two six membered rings or a hydrazine
linker having one six and one five membered cyclization products. A
five and seven membered linker as well as a six and seven membered
linker are also contemplated.
[0515] Another hydrazine structure, H, has the formula:
##STR00022##
[0516] where q is 0, 1,2, 3, 4, 5, or 6; and
[0517] each R.sup.24 is a member independently selected from the
group consisting of H, substituted alkyl, unsubstituted alkyl,
substituted heteroalkyl, and unsubstituted heteroalkyl. This
hydrazine structure can also form five-, six-, or seven-membered
rings and additional components can be added to form multiple
rings.
[0518] Disulfide Linkers (J)
[0519] In yet another embodiment, the linker comprises an
enzymatically cleavable disulfide group. In one embodiment, the
invention provides a cytotoxic antibody-partner compound having a
structure according to Formula (d):
##STR00023##
wherein D, L.sup.1, L.sup.4, and X.sup.4 are as defined above and
described further herein, and J is a disulfide linker comprising a
group having the structure:
##STR00024##
[0520] wherein each R.sup.24 is a member independently selected
from the group consisting of H, substituted alkyl, unsubstituted
alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl; each
K is a member independently selected from the group consisting of
substituted alkyl, unsubstituted alkyl, substituted heteroalkyl,
unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl,
substituted heteroaryl, unsubstituted heteroaryl, substituted
heterocycloalkyl, unsubstituted heterocycloalkyl, halogen,
NO.sub.2, NR.sup.21R.sup.22, NR.sup.21COR.sup.22,
OCONR.sup.21R.sup.22, OCOR.sup.21, and OR.sup.21 wherein R.sup.21
and R.sup.22 are independently selected from the group consisting
of H, substituted alkyl, unsubstituted alkyl, substituted
heteroalkyl, unsubstituted heteroalkyl, substituted aryl,
unsubstituted aryl, substituted heteroaryl, unsubstituted
heteroaryl, substituted heterocycloalkyl and unsubstituted
heterocycloalkyl; i is an integer of 0, 1, 2, 3, or 4; and d is an
integer of 0, 1, 2, 3, 4, 5, or 6.
[0521] The aromatic ring of the disulfides linker may be
substituted with one or more "K" groups. A "K" group is a
substituent on the aromatic ring that replaces a hydrogen otherwise
attached to one of the four non-substituted carbons that are part
of the ring structure. The "K" group may be a single atom, such as
a halogen, or may be a multi-atom group, such as alkyl,
heteroalkyl, amino, nitro, hydroxy, alkoxy, haloalkyl, and cyano.
Exemplary K substituents independently include, but are not limited
to, F, Cl, Br, I, NO.sub.2, OH, OCH.sub.3, NHCOCH.sub.3,
N(CH.sub.3).sub.2, NHCOCF.sub.3 and methyl. For "K.sub.i", i is an
integer of 0, 1, 2, 3, or 4. In a specific embodiment, i is 0.
[0522] In a preferred embodiment, the linker comprises an
enzymatically cleavable disulfide group of the following
formula:
##STR00025##
[0523] In this embodiment, the identities of L.sup.4, X.sup.4, p,
and R.sup.24 are as described above, and d is 0, 1, 2, 3, 4, 5, or
6. In a particular embodiment, d is 1 or 2.
[0524] A more specific disulfide linker is shown in the formula
below:
##STR00026##
[0525] A specific example of this embodiment is as follows:
##STR00027##
[0526] Preferably, d is 1 or 2.
[0527] Another disulfide linker is shown in the formula below:
##STR00028##
[0528] A specific example of this embodiment is as follows:
##STR00029##
[0529] Preferably, d is 1 or 2.
[0530] In various embodiments, the disulfides are ortho to the
amine. In another specific embodiment, a is 0. In preferred
embodiments, R.sup.24 is independently selected from H and
CH.sub.3.
[0531] An exemplary synthetic route for preparing a disulfide
linker of the invention is as follows:
##STR00030##
[0532] A solution of 3-mercaptopropionic acid a is reacted with
aldrithiol-2 to form 3-methyl benzothiazolium iodide b.
3-methylbenzothiazolium iodide c is reacted with sodium hydroxide
to form compound d. A solution of compound d with methanol is
further reacted with compound b to form compound e. Compound e
deprotected by the action of acetyl chloride and methanol forming
compound f.
[0533] For further discussion of types of cytotoxins, linkers and
other methods for conjugating therapeutic agents to antibodies, see
also PCT Publication WO 2007/059404 to Gangwar et al. and entitled
"Cytotoxic Compounds And Conjugates," Saito, G. et al. (2003) Adv.
Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003) Cancer
Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell
3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan,
I. and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs
3:1089-1091; Senter, P. D. and Springer, C. J. (2001) Adv. Drug
Deliv. Rev. 53:247-264, each of which are hereby incorporated by
reference in their entirety.
Partner Molecules
[0534] In one aspect, the present invention features an antibody
conjugated to a partner molecule, such as a cytotoxin, a drug
(e.g., an immunosuppressant) or a radiotoxin. Such conjugates are
also referred to herein as "immunoconjugates." Immunoconjugates
that include one or more cytotoxins are referred to as
"immunotoxins." A cytotoxin or cytotoxic agent includes any agent
that is detrimental to (e.g., kills) cells.
[0535] Examples of partner molecules of the present invention
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. Examples of partner molecules also 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).
[0536] Other preferred examples of partner molecules that can be
conjugated to an antibody of the invention include duocarmycins,
calicheamicins, maytansines and auristatins, and derivatives
thereof. An example of a calicheamicin antibody conjugate is
commercially available (Mylotarg.RTM.; American Home Products).
[0537] Preferred examples of partner molecule are CC-1065 and the
duocarmycins. CC-1065 was first isolated from Streptomyces zelensis
in 1981 by the Upjohn Company (Hanka et al., J. Antibiot. 31: 1211
(1978); Martin et al., J. Antibiot. 33: 902 (1980); Martin et al.,
J. Antibiot. 34: 1119 (1981)) and was found to have potent
antitumor and antimicrobial activity both in vitro and in
experimental animals (Li et al., Cancer Res. 42: 999 (1982)).
CC-1065 binds to double-stranded B-DNA within the minor groove
(Swenson et al., Cancer Res. 42: 2821 (1982)) with the sequence
preference of 5'-d(A/GNTTA)-3' and 5'-d(AAAAA)-3' and alkylates the
N3 position of the 3'-adenine by its CPI left-hand unit present in
the molecule (Hurley et al., Science 226: 843 (1984)). Despite its
potent and broad antitumor activity, CC-1065 cannot be used in
humans because it causes delayed death in experimental animals.
[0538] Many analogues and derivatives of CC-1065 and the
duocarmycins are known in the art. The research into the structure,
synthesis and properties of many of the compounds has been
reviewed. See, for example, Boger et al., Angew. Chem. Int. Ed.
Engl. 35: 1438 (1996); and Boger et al., Chem. Rev. 97: 787
(1997).
[0539] A group at Kyowa Hakko Kogya Co., Ltd. has prepared a number
of CC-1065 derivatives. See, for example, U.S. Pat. Nos. 5,101,038;
5,641,780; 5,187,186; 5,070,092; 5,703,080; 5,070,092; 5,641,780;
5,101,038; and 5,084,468; and published PCT application, WO
96/10405 and published European application 0 537 575 A1.
[0540] The Upjohn Company (Pharmacia Upjohn) has also been active
in preparing derivatives of CC-1065. See, for example, U.S. Pat.
Nos. 5,739,350; 4,978,757, 5,332, 837 and 4,912,227.
[0541] A particularly preferred aspect of the current invention
provides a cytotoxic compound having a structure according to the
following formula (e):
##STR00031##
in which ring system A is a member selected from substituted or
unsubstituted aryl substituted or unsubstituted heteroaryl and
substituted or unsubstituted heterocycloalkyl groups. Exemplary
ring systems include phenyl and pyrrole.
[0542] The symbols E and G are independently selected from H,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, a heteroatom, a single bond or E and G are optionally
joined to form a ring system selected from substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl and
substituted or unsubstituted heterocycloalkyl.
[0543] The symbol X represents a member selected from O, S and
NR.sup.23. R.sup.23 is a member selected from H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and
acyl.
[0544] The symbol R.sup.3 represents a member selected from
(.dbd.O), SR.sup.11, NHR.sup.11 and OR.sup.11, in which R.sup.11 is
H, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, monophosphates, diphosphates, triphosphates,
sulfonates, acyl, C(O)R.sup.12R.sup.13, C(O)OR.sup.12,
C(O)NR.sup.12R.sup.13, P(O)(OR.sup.12).sub.2,
C(O)CHR.sup.12R.sup.13, SR.sup.12 or SiR.sup.12R.sup.13R.sup.14.
The symbols R.sup.12, R.sup.13, and R.sup.14 independently
represent H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl and substituted or unsubstituted aryl,
where R.sup.12 and R.sup.13 together with the nitrogen or carbon
atom to which they are attached are optionally joined to form a
substituted or unsubstituted heterocycloalkyl ring system having
from 4 to 6 members, optionally containing two or more heteroatoms.
One or more of R.sup.12, R.sup.13, or R.sup.14 can include a
cleavable group within its structure.
[0545] R.sup.4, R.sup.4', R.sup.5 and R.sup.5' are members
independently selected from H, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocycloalkyl, halogen,
NO.sub.2, NR.sup.15R.sup.16, NC(O)R.sup.15, OC(O)NR.sup.15R.sup.16,
OC(O)OR.sup.15, C(O)R.sup.15, SR.sup.15, OR.sup.15,
CR.sup.15.dbd.NR.sup.16, and O(CH.sub.2).sub.nN(CH.sub.3).sub.2,
where n is an integer from 1 to 20, or any adjacent pair of
R.sup.4, R.sup.4', R.sup.5 and R.sup.5', together with the carbon
atoms to which they are attached, are joined to form a substituted
or unsubstituted cycloalkyl or heterocycloalkyl ring system having
from 4 to 6 members. R.sup.15 and R.sup.16 independently represent
H, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl and substituted or unsubstituted peptidyl, where
R.sup.15 and R.sup.16 together with the nitrogen atom to which they
are attached are optionally joined to form a substituted or
unsubstituted heterocycloalkyl ring system having from 4 to 6
members, optionally containing two or more heteroatoms. One
exemplary structure is aniline.
[0546] R.sup.4, R.sup.4', R.sup.5, R.sup.5', R.sup.11, R.sup.12,
R.sup.13, R.sup.15 and R.sup.16 optionally contain one or more
cleavable groups within their structure, such as a cleavable linker
or cleavable substrate. Exemplary cleavable groups include, but are
not limited to peptides, amino acids, hydrazines, disulfides, and
cephalosporin derivatives.
[0547] In some embodiments, at least one of R.sup.4, R.sup.4',
R.sup.5, R.sup.5', R.sup.11, R.sup.12, R.sup.13, R.sup.15 and
R.sup.16 is used to join the drug to a linker or enzyme cleavable
substrate of the present invention, as described herein, for
example to L.sup.1, if present or to F, H, J, or X.sup.2, or J.
[0548] In a still further exemplary embodiment, at least one of
R.sup.4, R.sup.4', R.sup.5, R.sup.5', R.sup.11, R.sup.12, R.sup.13,
R.sup.15 and R.sup.16 bears a reactive group appropriate for
conjugating the compound. In a further exemplary embodiment,
R.sup.4, R.sup.4', R.sup.5, R.sup.5', R.sup.11, R.sup.12, R.sup.13,
R.sup.15 and R.sup.16 are independently selected from H,
substituted alkyl and substituted heteroalkyl and have a reactive
functional group at the free terminus of the alkyl or heteroalkyl
moiety. One or more of R.sup.4, R.sup.4', R.sup.5, R.sup.5',
R.sup.11, R.sup.12, R.sup.13, R.sup.15 and R.sup.16 may be
conjugated to another species, e.g, targeting agent, detectable
label, solid support, etc.
[0549] R.sup.6 is a single bond which is either present or absent.
When R.sup.6 is present, R.sup.6 and R.sup.7 are joined to form a
cyclopropyl ring. R.sup.7 is CH.sub.2--X.sup.1 or --CH.sub.2--.
When R.sup.7 is --CH.sub.2-- it is a component of the cyclopropane
ring. The symbol X.sup.1 represents a leaving group such as a
halogen, for example Cl, Br or F. The combinations of R.sup.6 and
R.sup.7 are interpreted in a manner that does not violate the
principles of chemical valence.
[0550] X.sup.1 may be any leaving group. Useful leaving groups
include, but are not limited to, halogens, azides, sulfonic esters
(e.g., alkylsulfonyl, arylsulfonyl), oxonium ions, alkyl
perchlorates, ammonioalkanesulfonate esters, alkylfluorosulfonates
and fluorinated compounds (e.g., triflates, nonaflates, tresylates)
and the like. Particular halogens useful as leaving groups are F,
Cl and Br. The choice of these and other leaving groups appropriate
for a particular set of reaction conditions is within the abilities
of those of skill in the art (see, for example, March J, Advanced
Organic Chemistry, 2nd Edition, John Wiley and Sons, 1992; Sandler
S R, Karo W, Organic Functional Group Preparations, 2nd Edition,
Academic Press, Inc., 1983; and Wade L G, Compendium of Organic
Synthetic Methods, John Wiley and Sons, 1980).
[0551] The curved line within the six-membered ring indicates that
the ring may have one or more degrees of unsaturation, and it may
be aromatic. Thus, ring structures such as those set forth below,
and related structures, are within the scope of Formula (f):
##STR00032##
[0552] In some embodiments, at least one of R.sup.4, R.sup.4',
R.sup.5, and R.sup.5' links said drug to L.sup.1, if present, or to
F, H, J, or X.sup.2, and includes
##STR00033##
where v is an integer from 1 to 6; and each R.sup.27, R.sup.27',
R.sup.28, and R.sup.28' is independently selected from H,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, and substituted or unsubstituted
heterocycloalkyl. In some embodiments, R.sup.27, R.sup.27',
R.sup.28, and R.sup.28' are all H. In some embodiments, v is an
integer from 1 to 3 (preferably, 1). This unit can be used to
separate aryl substituents from the drug and thereby resist or
avoid generating compounds that are substrates for multi-drug
resistance.
[0553] In one embodiment, R.sup.11 includes a moiety, X.sup.5, that
does not self-cyclize and links the drug to L, if present, or to F,
H, J, or X.sup.2. The moiety, X.sup.5, is preferably cleavable
using an enzyme and, when cleaved, provides the active drug. As an
example, R.sup.11 can have the following structure (with the right
side coupling to the remainder of the drug):
##STR00034##
[0554] In an exemplary embodiment, ring system A of formula (e) is
a substituted or unsubstituted phenyl ring. Ring system A may be
substituted with one or more aryl group substituents as set forth
in the definitions section herein. In some embodiments, the phenyl
ring is substituted with a CN or methoxy moiety.
[0555] In some embodiments, at least one of R.sup.4, R.sup.4',
R.sup.5, and R.sup.5' links said drug to L.sup.1, if present, or to
F, H, J, or X.sup.2, and R.sup.3 is selected from SR.sup.11,
NHR.sup.11 and OR.sup.11. R.sup.11 is selected from --SO(OH).sub.2,
--PO(OH).sub.2, -AA.sub.n, --Si(CH.sub.3).sub.2C(CH.sub.3).sub.3,
--C(O)OPhNH(AA).sub.m,
##STR00035##
or any other sugar or combination of sugars,
##STR00036##
and pharmaceutically acceptable salts thereof, where n is any
integer in the range of 1 to 10, m is any integer in the range of 1
to 4, p is any integer in the range of 1 to 6, and AA is any
natural or non-natural amino acid. In some embodiments, AA.sub.n or
AA.sub.m is selected from the same amino acid sequences described
above for the peptide linkers (F) and optionally is the same as the
amino acid sequence used in the linker portion of R.sup.4,
R.sup.4', R.sup.5, or R.sup.5'. In at least some embodiments,
R.sup.3 is cleavable in vivo to provide an active drug compound. In
at least some embodiments, R.sup.3 increases in vivo solubility of
the compound. In some embodiments, the rate of decrease of the
concentration of the active drug in the blood is substantially
faster than the rate of cleavage of R.sup.3 to provide the active
drug. This may be particularly useful where the toxicity of the
active drug is substantially higher than that of the prodrug form.
In other embodiments, the rate of cleavage of R.sup.3 to provide
the active drug is faster than the rate of decrease of
concentration of the active drug in the blood.
[0556] In another exemplary embodiment, the invention provides a
compound having a structure according to Formula (g):
##STR00037##
In this embodiment, the identities of the substituents R.sup.3,
R.sup.4, R.sup.4', R.sup.5, R.sup.5', R.sup.6, R.sup.7 and X are
substantially as described above for Formula (a), as well as
preferences for particular embodiments. The symbol Z is a member
independently selected from O, S and NR.sup.23. The symbol R.sup.23
represents a member selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, and acyl. Each
R.sup.23 is independently selected. The symbol R.sup.1 represents
H, substituted or unsubstituted lower alkyl, or C(O)R.sup.8 or
CO.sub.2R.sup.8. R.sup.8 is a member selected from substituted
alkyl, unsubstituted alkyl, NR.sup.9R.sup.10, NR.sup.9NHR.sup.10
and OR.sup.9. R.sup.9 and R.sup.10 are independently selected from
H, substituted or unsubstituted alkyl and substituted or
unsubstituted heteroalkyl. R.sup.2 is H, or substituted or
unsubstituted lower alkyl. It is generally preferred that when
R.sup.2 is substituted alkyl, it is other than a perfluoroalkyl,
e.g., CF.sub.3. In one embodiment, R.sup.2 is a substituted alkyl
wherein the substitution is not a halogen. In another embodiment,
R.sup.2 is an unsubstituted alkyl.
[0557] In some embodiments R.sup.1 is an ester moiety, such as
CO.sub.2CH.sub.3. In some embodiments, R.sup.2 is a lower alkyl
group, which may be substituted or unsubstituted. A presently
preferred lower alkyl group is CH.sub.3. In some preferred
embodiments, R.sup.1 is CO.sub.2CH.sub.3 and R.sup.2 is
CH.sub.3.
[0558] In some embodiments, R.sup.4, R.sup.4', R.sup.5, and
R.sup.5' are members independently selected from H, halogen,
NH.sub.2, OMe, O(CH.sub.2).sub.2N(R.sup.29).sub.2 and NO.sub.2.
Each R.sup.29 is independently H or lower alkyl (e.g., methyl).
[0559] In some embodiments, the drug is selected such that the
leaving group X.sup.1 is a member selected from the group
consisting of halogen, alkylsulfonyl, arylsulfonyl, and azide. In
some embodiments, X.sup.1 is F, Cl, or Br.
[0560] In some embodiments, Z is O or NH. In some embodiments, X is
O.
[0561] In yet another exemplary embodiment, the invention provides
compounds having a structure according to Formula (h) or (i):
##STR00038##
[0562] Another preferred structure of the duocarmycin analog of
Formula (e) is a structure in which the ring system A is an
unsubstituted or substituted phenyl ring. The preferred
substituents on the drug molecule described hereinabove for the
structure of Formula 7 when the ring system A is a pyrrole are also
preferred substituents when the ring system A is an unsubstituted
or substituted phenyl ring.
[0563] For example, in a preferred embodiment, the drug (D)
comprises a structure (j):
##STR00039##
[0564] In this structure, R.sup.3, R.sup.6, R.sup.7, X are as
described above for Formula (e). Furthermore, Z is a member
selected from O, S and NR.sup.23, wherein R.sup.23 is a member
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, and acyl;
[0565] R.sup.1 is H, substituted or unsubstituted lower alkyl,
C(O)R.sup.8, or CO.sub.2R.sup.8, wherein R.sup.8 is a member
selected from NR.sup.9R.sup.10 and OR.sup.9, in which R.sup.9 and
R.sup.10 are members independently selected from H, substituted or
unsubstituted alkyl and substituted or unsubstituted
heteroalkyl;
[0566] R.sup.1' is H, substituted or unsubstituted lower alkyl, or
C(O)R.sup.8, wherein R.sup.8 is a member selected from
NR.sup.9R.sup.10 and OR.sup.9, in which R.sup.9 and R.sup.10 are
members independently selected from H, substituted or unsubstituted
alkyl and substituted or unsubstituted heteroalkyl;
[0567] R.sup.2 is H, or substituted or unsubstituted lower alkyl or
unsubstituted heteroalkyl or cyano or alkoxy; and R.sup.2' is H, or
substituted or unsubstituted lower alkyl or unsubstituted
heteroalkyl.
[0568] At least one of R.sup.4, R.sup.4', R.sup.5, R.sup.5',
R.sup.11, R.sup.12, R.sup.13, R.sup.15 or R.sup.16 links the drug
to L.sup.1, if present, or to F, H, J, or X.sup.2.
[0569] Another embodiment of the drug (D) comprises a structure (k)
where R.sup.4 and R.sup.4' have been joined to from a
heterocycloalkyl:
##STR00040##
[0570] In this structure, R.sup.3, R.sup.5, R.sup.5', R.sup.6,
R.sup.7, X are as described above for Formula (e). Furthermore, Z
is a member selected from O, S and NR.sup.23, wherein R.sup.23 is a
member selected from H, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, and acyl;
[0571] R.sup.32 is selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl, halogen, NO.sub.2, NR.sup.15R.sup.16,
NC(O)R.sup.15, OC(O)NR.sup.15R.sup.16, OC(O)OR.sup.15,
C(O)R.sup.15, SR.sup.15, OR.sup.15, CR.sup.15.dbd.NR.sup.16, and
O(CH.sub.2).sub.nN(CH.sub.3).sub.2, where n is an integer from 1 to
20. R.sup.15 and R.sup.16 independently represent H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocycloalkyl and
substituted or unsubstituted peptidyl, where R.sup.15 and R.sup.16
together with the nitrogen atom to which they are attached are
optionally joined to form a substituted or unsubstituted
heterocycloalkyl ring system having from 4 to 6 members, optionally
containing two or more heteroatoms. R.sup.32 optionally contains
one or more cleavable groups within its structure, such as a
cleavable linker or cleavable substrate. Exemplary cleavable groups
include, but are not limited to, peptides, amino acids, hydrazines,
disulfides, and cephalosporin derivatives. Moreover, any selection
of substituents described herein for R.sup.4, R.sup.4', R.sup.5,
R.sup.5', R.sup.15, and R.sup.16 is also applicable to
R.sup.32.
[0572] At least one of R.sup.5, R.sup.5', R.sup.11, R.sup.12,
R.sup.13, R.sup.15, R.sup.16, or R.sup.32 links the drug to
L.sup.1, if present, or to F, H, J, or X.sup.2. In at least some
embodiments, R.sup.32 links the drug to L.sup.1, if present, or to
F, H, J, or X.sup.2.
[0573] One preferred embodiment of this compound is:
##STR00041##
[0574] R.sup.1 is H, substituted or unsubstituted lower alkyl,
C(O)R.sup.8, or CO.sub.2R.sup.8, wherein R.sup.8 is a member
selected from NR.sup.9R.sup.10 and OR.sup.9, in which R.sup.9 and
R.sup.10 are members independently selected from H, substituted or
unsubstituted alkyl and substituted or unsubstituted
heteroalkyl;
[0575] R.sup.1' is H, substituted or unsubstituted lower alkyl, or
C(O)R.sup.8, wherein R.sup.8 is a member selected from
NR.sup.9R.sup.10 and OR.sup.9, in which R.sup.9 and R.sup.10 are
members independently selected from H, substituted or unsubstituted
alkyl and substituted or unsubstituted heteroalkyl;
[0576] R.sup.2 is H, or substituted or unsubstituted lower alkyl or
unsubstituted heteroalkyl or cyano or alkoxy; and R.sup.2' is H, or
substituted or unsubstituted lower alkyl or unsubstituted
heteroalkyl.
[0577] A further embodiment has the formula:
##STR00042##
[0578] In this structure, A, R.sup.6, R.sup.7, X, R.sup.4,
R.sup.4', R.sup.5, and R.sup.3' are as described above for Formula
(e). Furthermore, Z is a member selected from O, S and NR.sup.23,
where R.sup.23 is a member selected from H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and
acyl;
[0579] R.sup.33 is selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl, halogen, NO.sub.2, NR.sup.15R.sup.16,
NC(O)R.sup.15, OC(O)NR.sup.15R.sup.16, OC(O)OR.sup.15,
C(O)R.sup.15, SR.sup.15, OR.sup.15, CR.sup.15.dbd.NR.sup.16, and
O(CH.sub.2).sub.nN(CH.sub.3).sub.2, where n is an integer from 1 to
20. R.sup.15 and R.sup.16 independently represent H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocycloalkyl and
substituted or unsubstituted peptidyl, where R.sup.15 and R.sup.16
together with the nitrogen atom to which they are attached are
optionally joined to form a substituted or unsubstituted
heterocycloalkyl ring system having from 4 to 6 members, optionally
containing two or more heteroatoms. R.sup.33 links the drug to L,
if present, or to F, H, J, or X.sup.2.
[0580] Preferably, A is substituted or unsubstituted phenyl or
substituted or unsubstituted pyrrole. Moreover, any selection of
substituents described herein for R.sup.11 is also applicable to
R.sup.33.
[0581] Ligands
[0582] X.sup.4 represents a ligand selected from the group
consisting of protected reactive functional groups, unprotected
reactive functional groups, detectable labels, and targeting
agents. Preferred ligands are targeting agents, such as antibodies
and fragments thereof.
[0583] In some embodiments, the group X.sup.4 can be described as a
member selected from R.sup.29, COOR.sup.29, C(O)NR.sup.29, and
C(O)NNR.sup.29 wherein R.sup.29 is a member selected from
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl and substituted or unsubstituted heteroaryl. In yet
another exemplary embodiment, R.sup.29 is a thiol reactive member.
In a further exemplary embodiment, R.sup.29 is a thiol reactive
member selected from haloacetyl and alkyl halide derivatives,
maleimides, aziridines, and acryloyl derivatives. The above thiol
reactive members can act as reactive protective groups that can be
reacted with, for example, a side chain of an amino acid of a
targeting agent, such as an antibody, to thereby link the targeting
agent to the linker-drug moiety.
[0584] Detectable Labels
[0585] The particular label or detectable group used in conjunction
with the compounds and methods of the invention is generally not a
critical aspect of the invention, as long as it does not
significantly interfere with the activity or utility of the
compound of the invention. The detectable group can be any material
having a detectable physical or chemical property. Such detectable
labels have been well developed in the field of immunoassays and,
in general, most any label useful in such methods can be applied to
the present invention. Thus, a label is any composition detectable
by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Useful labels in the present
invention include magnetic beads (e.g., DYNABEADS.TM.), fluorescent
dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and
the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S,
.sup.14C, or .sup.32P), enzymes (e.g., horse radish peroxidase,
alkaline phosphatase and others commonly used in an ELISA), and
colorimetric labels such as colloidal gold or colored glass or
plastic beads (e.g., polystyrene, polypropylene, latex, etc.).
[0586] The label may be coupled directly or indirectly to a
compound of the invention according to methods well known in the
art. As indicated above, a wide variety of labels may be used, with
the choice of label depending on sensitivity required, ease of
conjugation with the compound, stability requirements, available
instrumentation, and disposal provisions.
[0587] When the compound of the invention is conjugated to a
detectable label, the label is preferably a member selected from
the group consisting of radioactive isotopes, fluorescent agents,
fluorescent agent precursors, chromophores, enzymes and
combinations thereof. Methods for conjugating various groups to
antibodies are well known in the art. For example, a detectable
label that is frequently conjugated to an antibody is an enzyme,
such as horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, and glucose oxidase.
[0588] Non-radioactive labels are often attached by indirect means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to
a component of the conjugate. The ligand then binds to another
molecules (e.g., streptavidin) molecule, which is either inherently
detectable or covalently bound to a signal system, such as a
detectable enzyme, a fluorescent compound, or a chemiluminescent
compound.
[0589] Components of the conjugates of the invention can also be
conjugated directly to signal generating compounds, e.g., by
conjugation with an enzyme or fluorophore. Enzymes of interest as
labels will primarily be hydrolases, particularly phosphatases,
esterases and glycosidases, or oxidotases, particularly
peroxidases. Fluorescent compounds include fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone,
etc. Chemiluminescent compounds include luciferin, and
2,3-dihydrophthalazinediones, e.g., luminol. For a review of
various labeling or signal producing systems that may be used, see,
U.S. Pat. No. 4,391,904.
[0590] Means of detecting labels are well known to those of skill
in the art. Thus, for example, where the label is a radioactive
label, means for detection include a scintillation counter or
photographic film as in autoradiography. Where the label is a
fluorescent label, it may be detected by exciting the fluorochrome
with the appropriate wavelength of light and detecting the
resulting fluorescence. The fluorescence may be detected visually,
by means of photographic film, by the use of electronic detectors
such as charge coupled devices (CCDs) or photomultipliers and the
like. Similarly, enzymatic labels may be detected by providing the
appropriate substrates for the enzyme and detecting the resulting
reaction product. Finally simple colorimetric labels may be
detected simply by observing the color associated with the label.
Thus, in various dipstick assays, conjugated gold often appears
pink, while various conjugated beads appear the color of the
bead.
[0591] Fluorescent labels are presently preferred as they have the
advantage of requiring few precautions in handling, and being
amenable to high-throughput visualization techniques (optical
analysis including digitization of the image for analysis in an
integrated system comprising a computer). Preferred labels are
typically characterized by one or more of the following: high
sensitivity, high stability, low background, low environmental
sensitivity and high specificity in labeling. Many fluorescent
labels are commercially available from the SIGMA chemical company
(Saint Louis, Mo.), Molecular Probes (Eugene, Oreg.), R&D
systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology
(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto,
Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,
Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.
(Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka
Chemie AG, Buchs, Switzerland), and Applied Biosystems (Foster
City, Calif.), as well as many other commercial sources known to
one of skill. Furthermore, those of skill in the art will recognize
how to select an appropriate fluorophore for a particular
application and, if it not readily available commercially, will be
able to synthesize the necessary fluorophore de novo or
synthetically modify commercially available fluorescent compounds
to arrive at the desired fluorescent label.
[0592] In addition to small molecule fluorophores, naturally
occurring fluorescent proteins and engineered analogues of such
proteins are useful in the present invention. Such proteins
include, for example, green fluorescent proteins of cnidarians
(Ward et al., Photochem. Photobiol. 35:803-808 (1982); Levine et
al., Comp. Biochem. Physiol., 72B:77-85 (1982)), yellow fluorescent
protein from Vibrio fischeri strain (Baldwin et al., Biochemistry
29:5509-15 (1990)), Peridinin-chlorophyll from the dinoflagellate
Symbiodinium sp. (Morris et al., Plant Molecular Biology 24:673:77
(1994)), phycobiliproteins from marine cyanobacteria, such as
Synechococcus, e.g., phycoerythrin and phycocyanin (Wilbanks et
al., J. Biol. Chem. 268:1226-35 (1993)), and the like.
[0593] Generally, prior to forming the linkage between the
cytoCytotoxin And the targeting (or other) agent, and optionally,
the spacer group, at least one of the chemical functionalities will
be activated. One skilled in the art will appreciate that a variety
of chemical functionalities, including hydroxy, amino, and carboxy
groups, can be activated using a variety of standard methods and
conditions. For example, a hydroxyl group of the cytotoxin or
targeting agent can be activated through treatment with phosgene to
form the corresponding chloroformate, or p-nitrophenylchloroformate
to form the corresponding carbonate.
[0594] In an exemplary embodiment, the invention makes use of a
targeting agent that includes a carboxyl functionality. Carboxyl
groups may be activated by, for example, conversion to the
corresponding acyl halide or active ester. This reaction may be
performed under a variety of conditions as illustrated in March,
supra pp. 388-89. In an exemplary embodiment, the acyl halide is
prepared through the reaction of the carboxyl-containing group with
oxalyl chloride. The activated agent is reacted with a cytotoxin or
cytotoxin-linker arm combination to form a conjugate of the
invention. Those of skill in the art will appreciate that the use
of carboxyl-containing targeting agents is merely illustrative, and
that agents having many other functional groups can be conjugated
to the linkers of the invention.
[0595] Reactive Functional Groups
[0596] For clarity of illustration the succeeding discussion
focuses on the conjugation of a cytotoxin of the invention to a
targeting agent. The focus exemplifies one embodiment of the
invention from which, others are readily inferred by one of skill
in the art. No limitation of the invention is implied, by focusing
the discussion on a single embodiment.
[0597] Exemplary compounds of the invention bear a reactive
functional group, which is generally located on a substituted or
unsubstituted alkyl or heteroalkyl chain, allowing their facile
attachment to another species. A convenient location for the
reactive group is the terminal position of the chain.
[0598] Reactive groups and classes of reactions useful in
practicing the present invention are generally those that are well
known in the art of bioconjugate chemistry. The reactive functional
group may be protected or unprotected, and the protected nature of
the group may be changed by methods known in the art of organic
synthesis. Preferred classes of reactions available with reactive
cytoCytotoxin Analogues are those which proceed under relatively
mild conditions. These include, but are not limited to nucleophilic
substitutions (e.g., reactions of amines and alcohols with acyl
halides, active esters), electrophilic substitutions (e.g., enamine
reactions) and additions to carbon-carbon and carbon-heteroatom
multiple bonds (e.g., Michael reaction, Diels-Alder addition).
These and other useful reactions are discussed in, for example,
March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons,
New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press,
San Diego, 1996; and Feeney et al., Modification of Proteins;
Advances in Chemistry Series, Vol. 198, American Chemical Society,
Washington, D.C., 1982.
[0599] Exemplary reaction types include the reaction of carboxyl
groups and various derivatives thereof including, but not limited
to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid
halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl,
alkenyl, alkynyl and aromatic esters. Hydroxyl groups can be
converted to esters, ethers, aldehydes, etc. Haloalkyl groups are
converted to new species by reaction with, for example, an amine, a
carboxylate anion, thiol anion, carbanion, or an alkoxide ion.
Dienophile (e.g., maleimide) groups participate in Diels-Alder.
Aldehyde or ketone groups can be converted to imines, hydrazones,
semicarbazones or oximes, or via such mechanisms as Grignard
addition or alkyllithium addition. Sulfonyl halides react readily
with amines, for example, to form sulfonamides. Amine or sulfhydryl
groups are, for example, acylated, alkylated or oxidized. Alkenes,
can be converted to an array of new species using cycloadditions,
acylation, Michael addition, etc. Epoxides react readily with
amines and hydroxyl compounds.
[0600] One skilled in the art will readily appreciate that many of
these linkages may be produced in a variety of ways and using a
variety of conditions. For the preparation of esters, see, e.g.,
March supra at 1157; for thioesters, see, March, supra at 362-363,
491, 720-722, 829, 941, and 1172; for carbonates, see, March, supra
at 346-347; for carbamates, see, March, supra at 1156-57; for
amides, see, March supra at 1152; for ureas and thioureas, see,
March supra at 1174; for acetals and ketals, see, Greene et al.
supra 178-210 and March supra at 1146; for acyloxyalkyl
derivatives, see, Prodrugs: Topical and Ocular Drug Delivery, K. B.
Sloan, ed., Marcel Dekker, Inc., New York, 1992; for enol esters,
see, March supra at 1160; for N-sulfonylimidates, see, Bundgaard et
al., J. Med. Chem., 31:2066 (1988); for anhydrides, see, March
supra at 355-56, 636-37, 990-91, and 1154; for N-acylamides, see,
March supra at 379; for N-Mannich bases, see, March supra at
800-02, and 828; for hydroxymethyl ketone esters, see, Petracek et
al. Annals NY Acad. Sci., 507:353-54 (1987); for disulfides, see,
March supra at 1160; and for phosphonate esters and
phosphonamidates.
[0601] The reactive functional groups can be unprotected and chosen
such that they do not participate in, or interfere with, the
reactions. Alternatively, a reactive functional group can be
protected from participating in the reaction by the presence of a
protecting group. Those of skill in the art will understand how to
protect a particular functional group from interfering with a
chosen set of reaction conditions. For examples of useful
protecting groups, See Greene et al., Protective Groups in Organic
Synthesis, John Wiley & Sons, New York, 1991.
[0602] Typically, the targeting agent is linked covalently to a
cytotoxin using standard chemical techniques through their
respective chemical functionalities. Optionally, the linker or
agent is coupled to the agent through one or more spacer groups.
The spacer groups can be equivalent or different when used in
combination.
[0603] Generally, prior to forming the linkage between the
cytoCytotoxin And the reactive functional group, and optionally,
the spacer group, at least one of the chemical functionalities will
be activated. One skilled in the art will appreciate that a variety
of chemical functionalities, including hydroxy, amino, and carboxy
groups, can be activated using a variety of standard methods and
conditions. In an exemplary embodiment, the invention comprises a
carboxyl functionality as a reactive functional group. Carboxyl
groups may be activated as described hereinabove.
[0604] Cleavable Substrate
[0605] The cleavable substrates of the current invention are
depicted as "X.sup.2". Preferably, the cleavable substrate is a
cleavable enzyme substrate that can be cleaved by an enzyme.
Preferably, the enzyme is preferentially associated, directly or
indirectly, with the tumor or other target cells to be treated. The
enzyme may be generated by the tumor or other target cells to be
treated. For example, the cleavable substrate can be a peptide that
is preferentially cleavable by an enzyme found around or in a tumor
or other target cell. Additionally or alternatively, the enzyme can
be attached to a targeting agent that binds specifically to tumor
cells, such as an antibody specific for a tumor antigen.
[0606] As examples of enzyme cleavable substrates suitable for
coupling to the drugs described above, PCT Patent Applications
Publication Nos. WO 00/33888, WO 01/95943, WO 01/95945, WO
02/00263, and WO 02/100353, all of which are incorporated herein by
reference, disclose attachment of a cleavable peptide to a drug.
The peptide is cleavable by an enzyme, such as a trouase (such as
thimet oligopeptidase), CD10 (neprilysin), a matrix metalloprotease
(such as MMP2 or MMP9), a type II transmembrane serine protease
(such as Hepsin, testisin, TMPRSS4, or matriptase/MT-SP1), or a
cathepsin, associated with a tumor. In this embodiment, a prodrug
includes the drug as described above, a peptide, a stabilizing
group, and optionally a linking group between the drug and the
peptide. The stabilizing group is attached to the end of the
peptide to protect the prodrug from degradation before arriving at
the tumor or other target cell. Examples of suitable stabilizing
groups include non-amino acids, such as succinic acid, diglycolic
acid, maleic acid, polyethylene glycol, pyroglutamic acid, acetic
acid, naphthylcarboxylic acid, terephthalic acid, and glutaric acid
derivatives; as well as non-genetically-coded amino acids or
aspartic acid or glutamic acid attached to the N-terminus of the
peptide at the .beta.-carboxy group of aspartic acid or the
.gamma.-carboxyl group of glutamic acid.
[0607] The peptide typically includes 3-12 (or more) amino acids.
The selection of particular amino acids will depend, at least in
part, on the enzyme to be used for cleaving the peptide, as well
as, the stability of the peptide in vivo. One example of a suitable
cleavable peptide is .beta.-AlaLeuAlaLeu. This can be combined with
a stabilizing group to form succinyl-.beta.-AlaLeuAlaLeu. Other
examples of suitable cleavable peptides are provided in the
references cited above.
[0608] As one illustrative example, CD10, also known as neprilysin,
neutral endopeptidase (NEP), and common acute lymphoblastic
leukemia antigen (CALLA), is a type II cell-surface zinc-dependent
metalloprotease. Cleavable substrates suitable for use with CD10
include LeuAlaLeu and IleAlaLeu. Other known substrates for CD10
include peptides of up to 50 amino acids in length, although
catalytic efficiency often declines as the substrate gets
larger.
[0609] Another illustrative example is based on matrix
metalloproteases (MMP). Probably the best characterized proteolytic
enzymes associated with tumors, there is a clear correlation of
activation of MMPs within tumor microenvironments. In particular,
the soluble matrix enzymes MMP2 (gelatinase A) and MMP9 (gelatinase
B), have been intensively studied, and shown to be selectively
activated during tissue remodeling including tumor growth. Peptide
sequences designed to be cleaved by MMP2 and MMP9 have been
designed and tested for conjugates of dextran and methotrexate
(Chau et al., Bioconjugate Chem. 15:931-941 (2004)); PEG
(polyethylene glycol) and doxorubicin (Bae et al., Drugs Exp. Clin.
Res. 29:15-23 (2004)); and albumin and doxorubicin (Kratz et al.,
Bioorg. Med. Chem. Lett. 11:2001-2006 (2001)). Examples of suitable
sequences for use with MMPs include, but are not limited to,
ProValGlyLeuIleGly (SEQ. ID NO. 102), GlyProLeuGlyVal (SEQ. ID NO.
103), GlyProLeuGlyIleAlaGlyGln (SEQ. ID NO. 104), ProLeuGlyLeu
(SEQ. ID NO. 105), GlyProLeuGlyMetLeuSerGln (SEQ. ID NO. 106), and
GlyProLeuGlyLeuTrpAlaGln (SEQ. ID NO. 107). (See, e.g., the
previously cited references as well as Kline et al., Mol.
Pharmaceut. 1:9-22 (2004) and Liu et al., Cancer Res. 60:6061-6067
(2000).) Other cleavable substrates can also be used.
[0610] Yet another example is type II transmembrane serine
proteases. This group of enzymes includes, for example, hepsin,
testisin, and TMPRSS4. GlnAlaArg is one substrate sequence that is
useful with matriptase/MT-SP1 (which is over-expressed in breast
and ovarian cancers) and LeuSerArg is useful with hepsin
(over-expressed in prostate and some other tumor types). (See,
e.g., Lee et. al., J. Biol. Chem. 275:36720-36725 and Kurachi and
Yamamoto, Handbook of Proeolytic Enzymes Vol. 2, 2.sup.nd edition
(Barrett A J, Rawlings N D & Woessner J F, eds) pp. 1699-1702
(2004).) Other cleavable substrates can also be used.
[0611] Another type of cleavable substrate arrangement includes
preparing a separate enzyme capable of cleaving the cleavable
substrate that becomes associated with the tumor or cells. For
example, an enzyme can be coupled to a tumor-specific antibody (or
other entity that is preferentially attracted to the tumor or other
target cell such as a receptor ligand) and then the enzyme-antibody
conjugate can be provided to the patient. The enzyme-antibody
conjugate is directed to, and binds to, antigen associated with the
tumor. Subsequently, the drug-cleavable substrate conjugate is
provided to the patient as a prodrug. The drug is only released in
the vicinity of the tumor when the drug-cleavable substrate
conjugate interacts with the enzyme that has become associated with
the tumor so that the cleavable substrate is cleaved and the drug
is freed. For example, U.S. Pat. Nos. 4,975,278; 5,587,161;
5,660,829; 5,773,435; and 6,132,722, all of which are incorporated
herein by reference, disclose such an arrangement. Examples of
suitable enzymes and substrates include, but are not limited to,
.beta.-lactamase and cephalosporin derivatives, carboxypeptidase G2
and glutamic and aspartic folate derivatives.
[0612] In one embodiment, the enzyme-antibody conjugate includes an
antibody, or antibody fragment, that is selected based on its
specificity for an antigen expressed on a target cell, or at a
target site, of interest. A discussion of antibodies is provided
hereinabove. One example of a suitable cephalosporin-cleavable
substrate is
##STR00043##
[0613] Examples of Conjugates
[0614] The linkers and cleavable substrates of the invention can be
used in conjugates containing a variety of partner molecules.
Examples of conjugates of the invention are described in further
detail below. Unless otherwise indicated, substituents are defined
as set forth above in the sections regarding cytotoxins, linkers,
and cleavable substrates.
[0615] A. Linker Conjugates
[0616] One example of a suitable conjugate is a compound of the
formula:
##STR00044##
wherein L.sup.1 is a self-immolative linker; m is an integer 0, 1,
2, 3, 4, 5, or 6; F is a linker comprising the structure:
##STR00045##
wherein AA.sup.1 is one or more members independently selected from
the group consisting of natural amino acids and unnatural
.alpha.-amino acids; c is an integer from 1 to 20; L.sup.2 is a
self-immolative linker and comprises
##STR00046##
wherein each R.sup.17, R.sup.18, and R.sup.19 is independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl and substituted or unsubstituted aryl,
and w is an integer from 0 to 4; o is 1; L.sup.4 is a linker
member; p is 0 or 1; X.sup.4 is a member selected from the group
consisting of protected reactive functional groups, unprotected
reactive functional groups, detectable labels, and targeting
agents; and D comprises a structure:
##STR00047##
wherein the ring system A is a member selected from substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl and
substituted or unsubstituted heterocycloalkyl groups; E and G are
members independently selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a
single bond, or E and G are joined to form a ring system selected
from substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl and substituted or unsubstituted
heterocycloalkyl; X is a member selected from O, S and NR.sup.23;
R.sup.23 is a member selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, and acyl; R.sup.3
is OR.sup.11, wherein R.sup.11 is a member selected from the group
consisting of H, substituted alkyl, unsubstituted alkyl,
substituted heteroalkyl, unsubstituted heteroalkyl, monophosphates,
diphosphates, triphosphates, sulfonates, acyl,
C(O)R.sup.12R.sup.13, C(O)OR.sup.12, C(O)NR.sup.12R.sup.13,
P(O)(OR.sup.12).sub.2, C(O)CHR.sup.12R.sup.13, SR.sup.12 and
SiR.sup.12R.sup.13R.sup.14, R.sup.4, R.sup.4', R.sup.5 and R.sup.5'
are members independently selected from the group consisting of H,
substituted alkyl, unsubstituted alkyl, substituted aryl,
unsubstituted aryl, substituted heteroaryl, unsubstituted
heteroaryl, substituted heterocycloalkyl, unsubstituted
heterocycloalkyl, halogen, NO.sub.2, NR.sup.15R.sup.16,
NC(O)R.sup.15, OC(O)NR.sup.15R.sup.16, OC(O)OR.sup.15,
C(O)R.sup.15, SR.sup.15, OR.sup.15, CR.sup.15.dbd.NR.sup.16, and
O(CH.sub.2).sub.nN(CH.sub.3).sub.2, or any adjacent pair of
R.sup.4, R.sup.4', R.sup.5 and R.sup.5', together with the carbon
atoms to which they are attached, are joined to form a substituted
or unsubstituted cycloalkyl or heterocycloalkyl ring system having
from 4 to 6 members; wherein n is an integer from 1 to 20; R.sup.15
and R.sup.16 are independently selected from H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocycloalkyl, and
substituted or unsubstituted peptidyl, wherein R.sup.15 and
R.sup.16 together with the nitrogen atom to which they are attached
are optionally joined to form a substituted or unsubstituted
heterocycloalkyl ring system having from 4 to 6 members, optionally
containing two or more heteroatoms; R.sup.6 is a single bond which
is either present or absent and when present R.sup.6 and R.sup.7
are joined to form a cyclopropyl ring; and R.sup.7 is
CH.sub.2--X.sup.1 or --CH.sub.2-- joined in said cyclopropyl ring
with R.sup.6, wherein X.sup.1 is a leaving group, wherein R.sup.11
links said drug to L.sup.1, if present, or to F.
[0617] In some embodiments, the drug has structure (c) or (f)
above. One specific example of a compound suitable for use as a
conjugate is
##STR00048##
[0618] Another example of a type of conjugate is a compound of the
formula
##STR00049##
wherein L.sup.1 is a self-immolative linker; m is an integer 0, 1,
2, 3, 4, 5, or 6; F is a linker comprising the structure:
##STR00050##
wherein AA.sup.1 is one or more members independently selected from
the group consisting of natural amino acids and unnatural
.alpha.-amino acids; c is an integer from 1 to 20; L.sup.2 is a
self-immolative linker; o is 0 or 1; L.sup.4 is a linker member; p
is 0 or 1; X.sup.4 is a member selected from the group consisting
of protected reactive functional groups, unprotected reactive
functional groups, detectable labels, and targeting agents; and D
comprises a structure:
##STR00051##
wherein the ring system A is a member selected from substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl and
substituted or unsubstituted heterocycloalkyl groups; E and G are
members independently selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a
single bond, or E and G are joined to form a ring system selected
from substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl and substituted or unsubstituted
heterocycloalkyl; X is a member selected from O, S and NR.sup.23;
R.sup.23 is a member selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, and acyl; R.sup.3
is a member selected from the group consisting of (.dbd.O),
SR.sup.11, NHR.sup.11 and OR.sup.11, wherein R.sup.11 is a member
selected from the group consisting of H, substituted alkyl,
unsubstituted alkyl, substituted heteroalkyl, unsubstituted
heteroalkyl, monophosphates, diphosphates, triphosphates,
sulfonates, acyl, C(O)R.sup.12R.sup.13, C(O)OR.sup.12,
C(O)NR.sup.12R.sup.13, P(O)(OR.sup.12).sub.2,
C(O)CHR.sup.12R.sup.13, SR.sup.12 and SiR.sup.12R.sup.13R.sup.14,
in which R.sup.12, R.sup.13, and R.sup.14 are members independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl and substituted or unsubstituted aryl,
wherein R.sup.12 and R.sup.13 together with the nitrogen or carbon
atom to which they are attached are optionally joined to form a
substituted or unsubstituted heterocycloalkyl ring system having
from 4 to 6 members, optionally containing two or more heteroatoms;
R.sup.4, R.sup.4', R.sup.5 and R.sup.5' are members independently
selected from the group consisting of H, substituted alkyl,
unsubstituted alkyl, substituted aryl, unsubstituted aryl,
substituted heteroaryl, unsubstituted heteroaryl, substituted
heterocycloalkyl, unsubstituted heterocycloalkyl, halogen,
NO.sub.2, NR.sup.15R.sup.16, NC(O)R.sup.15, OC(O)NR.sup.15R.sup.16,
OC(O)OR.sup.15, C(O)R.sup.15, SR.sup.15, OR.sup.15,
CR.sup.15.dbd.NR.sup.16, and O(CH.sub.2).sub.nN(CH.sub.3).sub.2, or
any adjacent pair of R.sup.4, R.sup.4', R.sup.5 and R.sup.5',
together with the carbon atoms to which they are attached, are
joined to form a substituted or unsubstituted cycloalkyl or
heterocycloalkyl ring system having from 4 to 6 members, wherein n
is an integer from 1 to 20; R.sup.15 and R.sup.16 are independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocycloalkyl, and substituted or unsubstituted
peptidyl, wherein R.sup.15 and R.sup.16 together with the nitrogen
atom to which they are attached are optionally joined to form a
substituted or unsubstituted heterocycloalkyl ring system having
from 4 to 6 members, optionally containing two or more heteroatoms;
wherein at least one of R.sup.4, R.sup.4', R.sup.5 and R.sup.5'
links said drug to L, if present, or to F, and comprises
##STR00052##
wherein v is an integer from 1 to 6; and each R.sup.27, R.sup.27',
R.sup.28, and R28' is independently selected from H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, and substituted or unsubstituted heterocycloalkyl;
R.sup.6 is a single bond which is either present or absent and when
present R.sup.6 and R.sup.7 are joined to form a cyclopropyl ring;
and R.sup.7 is CH.sub.2--X.sup.1 or --CH.sub.2-- joined in said
cyclopropyl ring with R.sup.6, wherein X.sup.1 is a leaving
group.
[0619] In some embodiment, the drug has structure (c) or (f) above.
One specific example of a compound suitable for use as a conjugate
is
##STR00053##
where r is an integer in the range from 0 to 24.
[0620] Another example of a suitable conjugate is a compound of the
formula
##STR00054##
wherein L.sup.1 is a self-immolative linker; m is an integer 0, 1,
2, 3, 4, 5, or 6; F is a linker comprising the structure:
##STR00055##
wherein AA.sup.1 is one or more members independently selected from
the group consisting of natural amino acids and unnatural
.alpha.-amino acids; c is an integer from 1 to 20; L.sup.3 is a
spacer group comprising a primary or secondary amine or a carboxyl
functional group; wherein if L.sup.3 is present, m is 0 and either
the amine of L.sup.3 forms an amide bond with a pendant carboxyl
functional group of D or the carboxyl of L.sup.3 forms an amide
bond with a pendant amine functional group of D; o is 0 or 1;
L.sup.4 is a linker member, wherein L.sup.4 comprises
##STR00056##
directly attached to the N-terminus of (AA.sup.1).sub.c, wherein
R.sup.20 is a member selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, and acyl, each
R.sup.25, R.sup.25', R.sup.26, and R.sup.26' is independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, and substituted or
unsubstituted heterocycloalkyl; and s and t are independently
integers from 1 to 6; p is 1; X.sup.4 is a member selected from the
group consisting of protected reactive functional groups,
unprotected reactive functional groups, detectable labels, and
targeting agents; and D comprises a structure:
##STR00057##
wherein the ring system A is a member selected from substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl and
substituted or unsubstituted heterocycloalkyl groups; E and G are
members independently selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a
single bond, or E and G are joined to form a ring system selected
from substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl and substituted or unsubstituted
heterocycloalkyl; X is a member selected from O, S and NR.sup.23;
R.sup.23 is a member selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, and acyl; R.sup.3
is a member selected from the group consisting of (.dbd.O),
SR.sup.11, NHR.sup.11 and OR.sup.11, wherein R.sup.11 is a member
selected from the group consisting of H, substituted alkyl,
unsubstituted alkyl, substituted heteroalkyl, unsubstituted
heteroalkyl, monophosphates, diphosphates, triphosphates,
sulfonates, acyl, C(O)R.sup.12R.sup.13, C(O)OR.sup.12,
C(O)NR.sup.12R.sup.13, P(O)(OR.sup.12).sub.2,
C(O)CHR.sup.12R.sup.13, SR.sup.12 and SiR.sup.12R.sup.13R.sup.14,
in which R.sup.12, R.sup.13, and R.sup.14 are members independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl and substituted or unsubstituted aryl,
wherein R.sup.12 and R.sup.13 together with the nitrogen or carbon
atom to which they are attached are optionally joined to form a
substituted or unsubstituted heterocycloalkyl ring system having
from 4 to 6 members, optionally containing two or more heteroatoms;
R.sup.4, R.sup.4', R.sup.5 and R.sup.5' are members independently
selected from the group consisting of H, substituted alkyl,
unsubstituted alkyl, substituted aryl, unsubstituted aryl,
substituted heteroaryl, unsubstituted heteroaryl, substituted
heterocycloalkyl, unsubstituted heterocycloalkyl, halogen,
NO.sub.2, NR.sup.15R.sup.16, NC(O)R.sup.15, OC(O)NR.sup.15R.sup.16,
OC(O)OR.sup.15, C(O)R.sup.15, SR.sup.15, OR.sup.15,
CR.sup.15.dbd.NR.sup.16, and O(CH.sub.2).sub.nN(CH.sub.3).sub.2, or
any adjacent pair of R.sup.4, R.sup.4', R.sup.5 and R.sup.5',
together with the carbon atoms to which they are attached, are
joined to form a substituted or unsubstituted cycloalkyl or
heterocycloalkyl ring system having from 4 to 6 members, wherein n
is an integer from 1 to 20; R.sup.15 and R.sup.16 are independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocycloalkyl, and substituted or unsubstituted
peptidyl, wherein R.sup.15 and R.sup.16 together with the nitrogen
atom to which they are attached are optionally joined to form a
substituted or unsubstituted heterocycloalkyl ring system having
from 4 to 6 members, optionally containing two or more heteroatoms;
R.sup.6 is a single bond which is either present or absent and when
present R.sup.6 and R.sup.7 are joined to form a cyclopropyl ring;
and R.sup.7 is CH.sub.2--X.sup.1 or --CH.sub.2-- joined in said
cyclopropyl ring with R.sup.6, wherein X.sup.1 is a leaving group,
wherein at least one of R.sup.4, R.sup.4', R.sup.5, R.sup.5',
R.sup.15 or R.sup.16 links said drug to L, if present, or to F.
[0621] In some embodiment, the drug has structure (c) or (f) above.
One specific example of a compound suitable for use as conjugate
is
##STR00058##
where r is an integer in the range from 0 to 24.
[0622] Other examples of suitable compounds for use as conjugates
include:
##STR00059## ##STR00060## ##STR00061## ##STR00062##
where R is
##STR00063##
and r is an integer in the range from 0 to 24
[0623] Conjugates can also be formed using the drugs having
structure (g), such as the following compounds:
##STR00064## ##STR00065## ##STR00066##
(where r is an integer in the range from 0 to 24.
[0624] Conjugates can also be formed using the drugs having the
following structures:
##STR00067##
Synthesis of such toxins, as well as details regarding their
linkage to antibodies is disclosed in U.S. patent application
having Ser. No. 60/991,300.
[0625] B. Cleavable Linker Conjugates
[0626] One example of a suitable conjugate is a compound having the
following structure:
##STR00068##
wherein L.sup.1 is a self-immolative spacer; m is an integer of 0,
1, 2, 3, 4, 5, or 6; X.sup.2 is a cleavable substrate; and D
comprises a structure:
##STR00069##
wherein the ring system A is a member selected from substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl and
substituted or unsubstituted heterocycloalkyl groups; E and G are
members independently selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a
single bond, or E and G are joined to form a ring system selected
from substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl and substituted or unsubstituted
heterocycloalkyl; X is a member selected from O, S and NR.sup.23;
R.sup.23 is a member selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, and acyl; R.sup.3
is a member selected from the group consisting of (.dbd.O),
SR.sup.11, NHR.sup.11 and OR.sup.11, wherein R.sup.11 is a member
selected from the group consisting of H, substituted alkyl,
unsubstituted alkyl, substituted heteroalkyl, unsubstituted
heteroalkyl, monophosphates, diphosphates, triphosphates,
sulfonates, acyl, C(O)R.sup.12R.sup.13, C(O)OR.sup.12,
C(O)NR.sup.12R.sup.13, P(O)(OR.sup.12).sub.2,
C(O)CHR.sup.12R.sup.13, SR.sup.12 and SiR.sup.12R.sup.13R.sup.14,
in which R.sup.12, R.sup.13, and R.sup.14 are members independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl and substituted or unsubstituted aryl,
wherein R.sup.12 and R.sup.13 together with the nitrogen or carbon
atom to which they are attached are optionally joined to form a
substituted or unsubstituted heterocycloalkyl ring system having
from 4 to 6 members, optionally containing two or more heteroatoms;
R.sup.6 is a single bond which is either present or absent and when
present R.sup.6 and R.sup.7 are joined to form a cyclopropyl ring;
and R.sup.7 is CH.sub.2--X.sup.1 or --CH.sub.2-- joined in said
cyclopropyl ring with R.sup.6, wherein X.sup.1 is a leaving group,
R.sup.4, R.sup.4', R.sup.5 and R.sup.5' are members independently
selected from the group consisting of H, substituted alkyl,
unsubstituted alkyl, substituted aryl, unsubstituted aryl,
substituted heteroaryl, unsubstituted heteroaryl, substituted
heterocycloalkyl, unsubstituted heterocycloalkyl, halogen,
NO.sub.2, NR.sup.15R.sup.16, NC(O)R.sup.15, OC(O)NR.sup.15R.sup.16,
OC(O)OR.sup.15, C(O)R.sup.15, SR.sup.15, OR.sup.15,
CR.sup.15.dbd.NR.sup.16, and O(CH.sub.2).sub.nN(CH.sub.3).sub.2, or
any adjacent pair of R.sup.4, R.sup.4', R.sup.5 and R.sup.5',
together with the carbon atoms to which they are attached, are
joined to form a substituted or unsubstituted cycloalkyl or
heterocycloalkyl ring system having from 4 to 6 members, wherein n
is an integer from 1 to 20; R.sup.15 and R.sup.16 are independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocycloalkyl, and substituted or unsubstituted
peptidyl, wherein R.sup.15 and R.sup.16 together with the nitrogen
atom to which they are attached are optionally joined to form a
substituted or unsubstituted heterocycloalkyl ring system having
from 4 to 6 members, optionally containing two or more heteroatoms;
wherein at least one of members R.sup.4, R.sup.4', R.sup.5 and
R.sup.5' links said drug to L, if present, or to X.sup.2, and is
selected from the group consisting of
##STR00070##
wherein R.sup.30, R.sup.30', R.sup.31, and R.sup.31' are
independently selected from H, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl, and
substituted or unsubstituted heterocycloalkyl; and v is an integer
from 1 to 6.
[0627] Examples of suitable cleavable linkers include
.beta.-AlaLeuAlaLeu and
##STR00071##
Pharmaceutical Compositions
[0628] In another aspect, the present disclosure provides a
composition, e.g., a pharmaceutical composition, containing one or
a combination of monoclonal antibodies, or antigen-binding
portion(s) thereof, of the present disclosure, formulated together
with a pharmaceutically acceptable carrier. Such compositions may
include one or a combination of (e.g., two or more different)
antibodies, or immunoconjugates or bispecific molecules of this
disclosure. For example, a pharmaceutical composition of this
disclosure can comprise a combination of antibodies (or
immunoconjugates or bispecifics) that bind to different epitopes on
the target antigen or that have complementary activities.
[0629] Pharmaceutical compositions of this disclosure also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include an
anti-CD22 antibody of the present disclosure combined with at least
one other anti-cancer, anti-inflammatory or immunosuppressant
agent. Examples of therapeutic agents that can be used in
combination therapy are described in greater detail below in the
section on uses of the antibodies of this disclosure.
[0630] As used herein, "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,
immunoconjugate, or bispecific 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.
[0631] The pharmaceutical compounds of this disclosure may include
one or more pharmaceutically acceptable salts. 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.
[0632] A pharmaceutical composition of this disclosure also may
include a pharmaceutically acceptable anti-oxidant. 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.
[0633] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of this
disclosure 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.
[0634] 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.
[0635] Pharmaceutically acceptable 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 this disclosure is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0636] 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.
[0637] 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.
[0638] 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 percent, this
amount will range from about 0.01 percent to about ninety-nine
percent of active ingredient, preferably from about 0.1 percent to
about 70 percent, most preferably from about 1 percent to about 30
percent of active ingredient in combination with a pharmaceutically
acceptable carrier.
[0639] 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. 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 this disclosure 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.
[0640] For administration of the antibody, the dosage ranges from
about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the
host body weight. For example dosages can be 0.3 mg/kg body weight,
1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10
mg/kg body weight or within the range of 1-10 mg/kg. An exemplary
treatment regime entails administration once per week, once every
two weeks, once every three weeks, once every four weeks, once a
month, once every 3 months or once every three to 6 months.
Preferred dosage regimens for an anti-CD22 antibody of this
disclosure include 1 mg/kg body weight or 3 mg/kg body weight via
intravenous administration, with the antibody being given using one
of the following dosing schedules: (i) every four weeks for six
dosages, then every three months; (ii) every three weeks; (iii) 3
mg/kg body weight once followed by 1 mg/kg body weight every three
weeks.
[0641] In some methods, two or more monoclonal antibodies with
different binding specificities are administered simultaneously, in
which case the dosage of each antibody administered falls within
the ranges indicated. Antibody is usually administered on multiple
occasions. Intervals between single dosages can be, for example,
weekly, monthly, every three months or yearly. Intervals can also
be irregular as indicated by measuring blood levels of antibody to
the target antigen in the patient. In some methods, dosage is
adjusted to achieve a plasma antibody concentration of about 1-1000
.mu.g/ml and in some methods about 25-300 .mu.g/ml.
[0642] Alternatively, antibody can be administered as a sustained
release formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of
the antibody in the patient. In general, human antibodies show the
longest half life, followed by humanized antibodies, chimeric
antibodies, and nonhuman antibodies. The dosage and frequency of
administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some patients continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated, and preferably until the patient shows
partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic
regime.
[0643] For use in the prophylaxis and/or treatment of diseases
related to abnormal cellular proliferation, a circulating
concentration of administered compound of about 0.001 .mu.M to 20
.mu.M is preferred, with about 0.01 .mu.M to 5 .mu.M being
preferred.
[0644] Patient doses for oral administration of the compounds
described herein, typically range from about 1 mg/day to about
10,000 mg/day, more typically from about 10 mg/day to about 1,000
mg/day, and most typically from about 50 mg/day to about 500
mg/day. Stated in terms of patient body weight, typical dosages
range from about 0.01 to about 150 mg/kg/day, more typically from
about 0.1 to about 15 mg/kg/day, and most typically from about 1 to
about 10 mg/kg/day, for example 5 mg/kg/day or 3 mg/kg/day.
[0645] In at least some embodiments, patient doses that retard or
inhibit tumor growth can be 1 .mu.mol/kg/day or less. For example,
the patient doses can be 0.9, 0.6, 0.5, 0.45, 0.3, 0.2, 0.15, or
0.1 .mu.mol/kg/day or less (referring to moles of the drug).
Preferably, the antibody-drug conjugate retards growth of the tumor
when administered in the daily dosage amount over a period of at
least five days. In at least some embodiments, the tumor is a
human-type tumor in a SCID mouse. As an example, the SCID mouse can
be a CB17.SCID mouse (available from Taconic, Germantown,
N.Y.).
[0646] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present disclosure 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 disclosure
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.
[0647] A "therapeutically effective dosage" of an anti-CD22
antibody of this disclosure preferably results in a decrease in
severity of disease symptoms, an increase in frequency and duration
of disease symptom-free periods, or a prevention of impairment or
disability due to the disease affliction. For example, for the
treatment of CD22.sup.+ tumors, a "therapeutically effective
dosage" preferably inhibits cell growth or tumor growth by at least
about 20%, more preferably by at least about 40%, even more
preferably by at least about 60%, and still more preferably by at
least about 80% relative to untreated subjects. The ability of a
compound to inhibit tumor growth 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 cell growth, such inhibition can
be measured 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.
[0648] A composition of the present disclosure can be administered
via one or more routes of administration using one or more of 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. Preferred routes of
administration for antibodies of this disclosure include
intravenous, intramuscular, intradermal, intraperitoneal,
subcutaneous, spinal or other parenteral routes of administration,
for example by injection or infusion. The phrase "parenteral
administration" 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.
[0649] Alternatively, an antibody of this disclosure can be
administered via a non-parenteral route, such as a topical,
epidermal or mucosal route of administration, for example,
intranasally, orally, vaginally, rectally, sublingually or
topically.
[0650] 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.
[0651] Therapeutic compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
therapeutic composition of this disclosure 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 disclosure
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 mendicants 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. These patents are incorporated herein by
reference. Many other such implants, delivery systems, and modules
are known to those skilled in the art.
[0652] In certain embodiments, the human monoclonal antibodies of
this disclosure 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 this disclosure 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); 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.
Uses and Methods of the Invention
[0653] The antibodies, particularly the human antibodies, antibody
compositions and methods of the present disclosure have numerous in
vitro and in vivo diagnostic and therapeutic utilities involving
the diagnosis and treatment of diseases and disorders involving
CD22. For example, these molecules can be administered to cells in
culture, in vitro or ex vivo, or to human subjects, e.g., in vivo,
to treat, prevent and to diagnose a variety of disorders.
[0654] As used herein, the term "subject" is intended to include
human and non-human animals. Non-human animals include all
vertebrates, e.g., mammals and non-mammals, such as non-human
primates, sheep, dogs, cats, cows, horses, chickens, amphibians,
and reptiles. Preferred subjects include human patients having
disorders mediated by or modulated by CD22 activity. When
antibodies to CD22 are administered together with another agent,
the two can be administered in either order or simultaneously.
[0655] Given the specific binding of the antibodies of this
disclosure for CD22, the antibodies of this disclosure can be used
to specifically detect CD22 expression on the surface of cells and,
moreover, can be used to purify CD22 via immunoaffinity
purification.
[0656] Suitable routes of administering the antibody compositions
(e.g., human monoclonal antibodies, multispecific and bispecific
molecules and immunoconjugates) of this disclosure in vivo and in
vitro are well known in the art and can be selected by those of
ordinary skill. For example, the antibody compositions can be
administered by injection (e.g., intravenous or subcutaneous).
Suitable dosages of the molecules used will depend on the age and
weight of the subject and the concentration and/or formulation of
the antibody composition.
[0657] As previously described, human anti-CD22 antibodies of this
disclosure can be co-administered with one or other more
therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent or
an immunosuppressive agent. The antibody can be linked to the agent
(as an immunocomplex) or can be administered separate from the
agent. In the latter case (separate administration), the antibody
can be administered before, after or concurrently with the agent or
can be co-administered with other known therapies, e.g., an
anti-cancer therapy, e.g., radiation. Such therapeutic agents
include, among others, anti-neoplastic agents such as doxorubicin
(adriamycin), cisplatin bleomycin sulfate, carmustine,
chlorambucil, and cyclophosphamide hydroxyurea which, by
themselves, are only effective at levels which are toxic or
subtoxic to a patient. Cisplatin is intravenously administered as a
100 mg/kg dose once every four weeks and adriamycin is
intravenously administered as a 60-75 mg/ml dose once every 21
days. Co-administration of human anti-CD22 antibodies, or antigen
binding fragments thereof, of the present disclosure with
chemotherapeutic agents provides two anti-cancer agents which
operate via different mechanisms which yield a cytotoxic effect to
human tumor cells. Such co-administration can solve problems due to
development of resistance to drugs or a change in the antigenicity
of the tumor cells that would render them unreactive with the
antibody.
[0658] Target-specific effector cells, e.g., effector cells linked
to compositions (e.g., human antibodies, multispecific and
bispecific molecules) of this disclosure can also be used as
therapeutic agents. Effector cells for targeting can be human
leukocytes such as macrophages, neutrophils or monocytes. Other
cells include eosinophils, natural killer cells and other IgG- or
IgA-receptor bearing cells. If desired, effector cells can be
obtained from the subject to be treated. The target-specific
effector cells can be administered as a suspension of cells in a
physiologically acceptable solution. The number of cells
administered can be in the order of 10.sup.8-10.sup.9 but will vary
depending on the therapeutic purpose. In general, the amount will
be sufficient to obtain localization at the target cell, e.g., a
tumor cell expressing CD22, and to effect cell killing by, e.g.,
phagocytosis. Routes of administration can also vary.
[0659] Therapy with target-specific effector cells can be performed
in conjunction with other techniques for removal of targeted cells.
For example, anti-tumor therapy using the compositions (e.g., human
antibodies, multispecific and bispecific molecules) of this
disclosure and/or effector cells armed with these compositions can
be used in conjunction with chemotherapy. Additionally, combination
immunotherapy may be used to direct two distinct cytotoxic effector
populations toward tumor cell rejection. For example, anti-CD22
antibodies linked to anti-Fc-gamma RI or anti-CD3 may be used in
conjunction with IgG- or IgA-receptor specific binding agents.
[0660] Bispecific and multispecific molecules of this disclosure
can also be used to modulate Fc.gamma.R or Fc.gamma.R levels on
effector cells, such as by capping and elimination of receptors on
the cell surface. Mixtures of anti-Fc receptors can also be used
for this purpose.
[0661] The compositions (e.g., human, humanized, or chimeric
antibodies, multispecific and bispecific molecules and
immunoconjugates) of this disclosure which have complement binding
sites, such as portions from IgG1, -2, or -3 or IgM which bind
complement, can also be used in the presence of complement. In one
embodiment, ex vivo treatment of a population of cells comprising
target cells with a binding agent of this disclosure and
appropriate effector cells can be supplemented by the addition of
complement or serum containing complement. Phagocytosis of target
cells coated with a binding agent of this disclosure can be
improved by binding of complement proteins. In another embodiment
target cells coated with the compositions (e.g., human antibodies,
multispecific and bispecific molecules) of this disclosure can also
be lysed by complement. In yet another embodiment, the compositions
of this disclosure do not activate complement.
[0662] The compositions (e.g., human, humanized, or chimeric
antibodies, multispecific and bispecific molecules and
immunoconjugates) of this disclosure can also be administered
together with complement. Accordingly, within the scope of this
disclosure are compositions comprising human antibodies,
multispecific or bispecific molecules and serum or complement.
These compositions are advantageous in that the complement is
located in close proximity to the human antibodies, multispecific
or bispecific molecules. Alternatively, the human antibodies,
multispecific or bispecific molecules of this disclosure and the
complement or serum can be administered separately.
[0663] The antibodies of this disclosure also can be used in
combination with one or more additional therapeutic antibodies or
other binding agents, such as Ig fusion proteins. Non-limiting
examples of other antibodies or binding agents with which an
anti-CD22 antibody of this disclosure can be administered in
combination include antibodies or binding agents to CTLA-4, PSMA,
CD30, IP-10, IFN-.gamma., CD70, PD-1, PD-L1, TNF, TNF-R, VEGF,
VEGF-R, CCR5, IL-1, IL-18, IL-18R, CD19, Campath-1, EGFR, CD33,
CD20, Her-2, CD25, gpIIb/IIIa, IgE, CD11a, .alpha.4 integrin.
[0664] Also within the scope of the present disclosure are kits
comprising antibody compositions of this disclosure (e.g., human
antibodies, bispecific or multispecific molecules, or
immunoconjugates) and instructions for use. The kit can further
contain one ore more additional reagents, such as an
immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent,
or one or more additional human antibodies of this disclosure
(e.g., a human antibody having a complementary activity which binds
to an epitope in the CD22 antigen distinct from the first human
antibody).
[0665] Accordingly, patients treated with antibody compositions of
this disclosure can be additionally administered (prior to,
simultaneously with, or following administration of a human
antibody of this disclosure) with another therapeutic agent, such
as a cytotoxic or radiotoxic agent, which enhances or augments the
therapeutic effect of the human antibodies.
[0666] In other embodiments, the subject can be additionally
treated with an agent that modulates, e.g., enhances or inhibits,
the expression or activity of Fc.gamma. or Fc.gamma. receptors by,
for example, treating the subject with a cytokine. Preferred
cytokines for administration during treatment with the
multispecific molecule include of granulocyte colony-stimulating
factor (G-CSF), granulocyte-macrophage colony-stimulating factor
(GM-CSF), interferon-.gamma. (IFN-.gamma.), and tumor necrosis
factor (TNF).
[0667] The compositions (e.g., human antibodies, multispecific and
bispecific molecules) of this disclosure can also be used to target
cells expressing CD22, for example for labeling such cells. For
such use, the binding agent can be linked to a molecule that can be
detected. Thus, this disclosure provides methods for localizing ex
vivo or in vitro cells expressing CD22. The detectable label can
be, e.g., a radioisotope, a fluorescent compound, an enzyme, or an
enzyme co-factor.
[0668] In a particular embodiment, this disclosure provides methods
for detecting the presence of CD22 antigen in a sample, or
measuring the amount of CD22 antigen, comprising contacting the
sample, and a control sample, with a human monoclonal antibody, or
an antigen binding portion thereof, which specifically binds to
CD22, under conditions that allow for formation of a complex
between the antibody or portion thereof and CD22. The formation of
a complex is then detected, wherein a difference complex formation
between the sample compared to the control sample is indicative the
presence of CD22 antigen in the sample.
[0669] In yet another embodiment, immunoconjugates of the invention
can be used to target compounds (e.g., therapeutic agents, labels,
cytotoxins, radiotoxoins immunosuppressants, etc.) to cells which
express CD22 by linking such compounds to the antibody. For
example, an anti-CD22 antibody can be conjugated to any of the
toxin compounds described in U.S. Pat. Nos. 6,281,354 and
6,548,530, U.S. patent publication Nos. 20030050331, 20030064984,
20030073852, and 20040087497, or published in WO 03/022806. Thus,
the invention also provides methods for localizing ex vivo or in
vivo cells expressing CD22 (e.g., with a detectable label, such as
a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor). Alternatively, the immunoconjugates can be used to kill
cells which have CD22 cell surface receptors by targeting
cytotoxins or radiotoxins to CD22.
[0670] CD22 is known to be expressed on a large percentage of B
cell lymphomas and also is known to be involved in regulating B
cell activity such that autoimmune disorders can be treated via
targeting of CD22. Accordingly, the anti-CD22 antibodies (and
immunoconjugates and bispecific molecules) of this disclosure can
be used to modulate CD22 activity in each of these clinical
situations.
[0671] Accordingly, in one aspect, the invention provides a method
of inhibiting growth of a CD22-expressing tumor cell. The method
comprises contacting the CD22-expressing tumor cell with the
antibody, or antigen-binding portion thereof, of the invention such
that growth of the CD22-expressing tumor cell is inhibited.
Preferably, the CD22-expressing tumor cell is a B cell lymphoma,
such as a non-Hodgkin's lymphoma. Other types of CD22-expressing
tumor cells include Burkitt's lymphomas and B cell chronic
lymphocytic leukemias.
[0672] In one embodiment of the method of inhibiting tumor cell
growth, the antibody, or antigen-binding portion thereof, is
conjugated to a partner molecule, such as a therapeutic agent, such
as a cytotoxin, radioisotope or chemotherapeutic agent. In other
embodiments, the antibody, or antigen-binding portion thereof, in
administered in combination with one or more additional anti-tumor
agents. The antibody can be used in combination other cancer
treatments, such as surgery and/or radiation, and/or with other
anti-neoplastic agents, such as the anti-neoplastic agents
discussed and set forth above, including chemotherapeutic drugs and
other anti-tumor antigen antibodies, including but not limited to
an anti-CD20 antibody (e.g., Rituxan.RTM.).
[0673] In another aspect, the invention provides a method of
treating an inflammatory or autoimmune disorder in a subject. The
method comprises administering to the subject the antibody, or
antigen-binding portion thereof, of the invention such that the
inflammatory or autoimmune disorder in the subject is treated.
Non-limiting examples of preferred autoimmune disorders include
systemic lupus erythematosus and rheumatoid arthritis. Other
examples of autoimmune disorders include inflammatory bowel disease
(including ulcerative colitis and Crohn's disease), Type I
diabetes, multiple sclerosis, Sjogren's syndrome, autoimmune
thyroiditis (including Grave's disease and Hashimoto's
thyroiditis), psoriasis and glomerulonephritis. The antibody can be
used alone or in combination with other anti-inflammatory or
immunsuppresant agents, such as non-steroidal anti-inflammatory
drugs (NSAIDs), corticosteroids (e.g., prednisone, hydrocortisone),
methotrexate, COX-2 inhibitors, TNF antagonists (e.g., etanercept,
infliximab, adalimumab) and immunosuppressants (such as
6-mercaptopurine, azathioprine and cyclosporine A).
[0674] The present disclosure is further illustrated by the
following examples, which should not be construed as further
limiting. The contents of all figures and all references, patents
and published patent applications cited throughout this application
are expressly incorporated herein by reference.
Example 1
Generation of Human Monoclonal Antibodies Against CD22
[0675] Anti-CD22 human monoclonal antibodies were generated using
transgenic mice that express human antibody genes, as follows.
Antigen
[0676] The antigens used to raise anti-CD22 antibodies were the
extracellular domain of human CD22 and the full-length CD22 protein
expressed on CHO cells. To obtain the extracellular domain, a cDNA
encoding human CD22 (commercially available from Open Biosystems,
Inc.) was used to construct an expression vector encoding the
entire CD22.beta. extracellular domain (CD22 ECD) fused to a
C-terminal hexahistidine tag. After transfection of CHO cells and
selection of stable transfectants by standard techniques, CD22 ECD
was purified from the cell culture medium using metal chelate
chromatography. In addition, recombinant CHO cells were created
that expressed full-length CD22 on the cell surface by transfecting
the cells with an expression vector that contained the full-length
CD22 cDNA. After selection of the transfected cells, those cells
expressing high levels of CD22 on the cell surface were isolated by
fluorescent-activated cell sorting, based on reactivity with a
fluorescein-labeled anti-CD22 (commercially available from
Becton-Dickinson-Pharmingen).
Mouse Strains
[0677] Fully human monoclonal antibodies to CD22 were prepared
using HCo7/HCo12 and HCo12/Balbc strains of the transgenic HuMAb
Mouse.RTM., and the KM and KM-.lamda.HAC strains of transgenic
transchromosomic mice, all of which express human antibody
genes.
[0678] In each of these mouse strains, the endogenous mouse kappa
light chain gene has been homozygously disrupted as described in
Chen et al. (1993) EMBO J. 12:811-820 and the endogenous mouse
heavy chain gene has been homozygously disrupted as described in
Example 1 of PCT Publication WO 01/09187. Each of these strains
carries a human kappa light chain transgene, KCo5 (as described in
Fishwild et al. (1996) Nature Biotechnology 14:845-851) and also
contains the SC20 transchromosome, which carries the human Ig heavy
chain locus, as described in PCT Publication WO 02/43478.
[0679] The HCo7 strain carries the HCo7 human heavy chain transgene
as described in U.S. Pat. Nos. 5,545,806; 5,625,825; and 5,545,807.
The HCo12 strain carries the HCo12 human heavy chain transgene as
described in Example 2 of PCT Publication WO 01/09187.
[0680] The KM Mouse.RTM. strain is described in detail in U.S.
Application No. 20020199213.
[0681] The KM-.lamda.HAC strain is very similar to the KM strain in
that the endogenous mouse heavy chain and kappa light chain loci
have been disrupted and the SC20 transchromosome and KCo5 transgene
have bee inserted, but the KM-.lamda.HAC strain also carries a
human artificial chromosome derived from human chromosome 22 that
carries the human lambda light chain locus. Thus, the KM-.lamda.HAC
strain can express human antibodies that utilize either a lambda
light chain or a kappa light chain. The KM-.lamda.HAC mice are also
described in detail in U.S. Application No. 20060015958.
Immunization
[0682] To raise fully human monoclonal antibodies to CD22, animals
of the strains described above were immunized with recombinant
human CD22 ECD and CD22-expressing CHO cells (prepared as described
above for the antigen). General immunization schemes for the
raising human antibodies in mice strains carrying human antibody
genes are described in, for example, Lonberg, N. et al (1994)
Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature
Biotechnology 14: 845-851 and PCT Publication WO 98/24884. Mice
were 10-12 weeks of age when the immunizations were initiated. Mice
were immunized weekly intraperitoneally and subcutaneously with 20
.mu.g of CD22 ECD or 10.sup.7 transfected CHO cells with RIBI as
adjuvant. The first two immunizations were performed with CD22 ECD
in RIBI adjuvant followed by six additional weekly immunizations
alternately using CD22 ECD or transfected cells (up to a total of 8
immunizations). The immune response was monitored in blood
harvested by retroorbital bleeds. The serum was screened by ELISA
and FACS. Mice with adequate titer of anti-CD22 human IgG
immunoglobulin were used for fusions. Mice were boosted once with
CD22 ECD and once with CD22 expressing CHO cells both intravenously
and intraperitoneally on days -4 and -3, respectively, before
sacrifice and removal of the spleen.
Antibody Selection
[0683] To identify mice producing antibodies that bound CD22, sera
from immunized mice were screened by flow cytometry for binding to
CHO cells expressing human CD22 as well as to parental CHO cells.
The sera were also screened by flow cytometry (FACS) on human Daudi
B cells, which express CD22. Sera from all immunized mice were
tested at a dilution of 1:50 in the FACS experiment. After addition
of diluted serum to the cells and incubation for 30 minutes at
37.degree. C., cells were washed and binding was detected with a
PE-labeled anti-human IgG Ab. Flow cytometric analyses were
performed using a FACSCalibur flow cytometry (Becton Dickinson, San
Jose, Calif.). A murine anti-CD22 monoclonal antibody (M anti-CD22)
was used as positive control in the experiment. All three mice
tested exhibited titer to CHO-CD22 and CHO parental cells (CHO-S).
Binding to CHO-S cells reflect the presence of antibodies binding
to molecules other than CD22 on the surface of CHO cells. This
result was expected since mice were immunized with CHO transfected
cells. Titer to human Daudi cells was also detected in the three
mice indicating the potential presence of antibodies specific to
CD22 that could bind CD22 from a non-recombinant source.
[0684] Sera were further tested for binding to human CD22 ECD by
ELISA. Briefly, microtiter plates were coated with purified CD22
ECD protein produced in CHO cells at 1-2.5 .mu.g/ml in PBS (50
.mu.l/well) for 2 hrs at room temperature. The plate was then
blocked with 300 .mu.l/well of 1% BSA in PBS. Dilutions of sera
(100 to 20000) from CD22-immunized mice were added to each well and
incubated for 1-2 hours at ambient temperature. The plates were
washed with PBS/Tween and incubated with a goat-anti-human IgG
polyclonal antibody conjugated with horseradish peroxidase (HRP)
for 1 hour at room temperature. After washing, the plates were
developed with ABTS substrate (Sigma #A9941) in phosphate citrate
buffer with perborate (Sigma#P4922) or Moss ABTS-1000 and analyzed
by spectrophotometry at OD 415-495 nm. The three mice tested had
good titer of anti-CD22 antibodies and were therefore used for
fusions.
Splenocyte Fusions
[0685] Mouse splenocytes were fused to a mouse myeloma cell line
using electric field based electrofusion using a Cyto Pulse large
chamber cell fusion electroporator (Cyto Pulse Sciences, Inc., Glen
Burnie, Md.). Single cell suspensions of splenocytes from immunized
mice were fused to Ag8.653 mouse myeloma cells (ATCC, CRL 1581) at
a ratio of 1:1. Cells were plated at approximately
2.times.10.sup.4/well in flat bottom microtiter plates. Plates were
incubated for one week in DMEM high glucose medium with
L-glutamine, sodium pyruvate (Mediatech, Inc., Herndon, Va.), 10%
fetal Bovine Serum (Hyclone, Logan, Utah), 18% P388DI conditional
media, 5% Origen Hybridoma cloning factor (BioVeris, Gaithersburg,
Va.), 4 mM L-glutamine, 5 mM HEPES, 0.055 mM
.beta.-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml
streptomycin and 1.times. Hypoxanthine-aminopterin-thymidine (HAT).
After one week (day 7), HAT growth media was replaced with medium
containing HT. When extensive hybridoma growth occurred (day
10-11), hybridoma supernatants were tested for the presence of
human IgG antibodies in an HTRF homogeneous assay. Fusions from
KM-.lamda.HAC mice were screened for presence of human IgG bearing
either a human kappa or a human lambda light chain. Positive
hybridomas were then screened by FACS on Daudi cells and by ELISA
for the presence of CD22 specific human IgG antibodies. ELISA and
FACS experiments were performed as described above except that
hybridoma supernatants (50-100 .mu.l/well) were used instead of
serum dilutions. The antigen specific parental hybridoma lines were
transferred to 24 well plates, screened again and, if still
positive for human IgG, subcloned once by limiting dilution. The
stable subclones were then scaled up in vitro and antibodies were
purified for further characterization.
[0686] Eighteen subclones were chosen for expansion for antibody
purification. The isotypes of the expanded subclones included the
following isotypes: IgG1; IgG4; IgG4/IgM; IgG1/IgM;
IgG1/IgG2/.lamda.; and IgG4/.lamda.. Thirteen of the purified
antibodies were titrated by ELISA and FACS and each exhibited
specific binding to human CD22 in both assays. Four subclones,
12C5, 19A3, 16F7, 23C6, were selected for further structural
analysis and sequencing.
Production of Recombinant Antibodies CD22.1 and CD22.2
[0687] The anti-CD22 antibody 19A3 was expressed in CHO cells as a
human IgG1 (f allotype) and the recombinant antibody was designated
CD22.1. In addition, a variant of 19A3 designated CD22.2 was made
in which the mutation N57Q was made to remove the N-glycosylation
site in the CDR2 region of the V.sub.H chain.
[0688] The V.sub.K and V.sub.H regions of 19A3 were amplified by
PCR from cDNA clones and cloned into pCR4Blunt-TOPO (Invitrogen) to
introduce restriction sites for cloning. Site directed mutagenesis
was then performed to introduce an N57Q mutation into the heavy
chain sequence to remove the N-glycosylation site in CDR2. The 19A3
V.sub.K was subcloned into the pICOFSneoK2.hCMV2.1 kb vector to
produce vector pICOFSneoK2.hCMV2.1 kb(CD22.19A3), and the V.sub.H
(both wild type and N57Q mutation) regions were subcloned into the
pICOFSpurG vector to produce vectors pICOFSpurG(CD22.19A3) and
pICOFSpurG(CD22.19A3.VH.N57Q). These constructs for expression of
light and heavy chain were linearized and co-transfected into CHO-S
cells using DMRIE-C (Invitrogen) and stable clones selected using
standard techniques.
[0689] CHO-S clone 8G9 was chosen for CD22.1 expression. An
overgrown culture of this clone produced approximately 75 mg/liter
of antibody. CHO-S clone 17E11 was chosen for CD22.2 expression and
yielded approximately 413 mg/liter in overgrown culture. The
structure and function of the recombinant antibodies CD22.1 and
CD22.2 were then determined (see Example 3 and Example 10,
below).
Example 2
Structural Characterization of Human Anti-CD22 Monoclonal
Antibodies
[0690] The cDNA sequences encoding the heavy and light chain
variable regions of the mAbs expressed by the 12C5, 19A3, 16F7,
23C6, CD22.1, CD22.2, 4G6 and 21F6 clones described in Example 1
were sequenced using standard DNA sequencing techniques and the
expressed proteins were characterized by standard protein chemistry
analysis.
[0691] Characterization of 12C5, 19A3, CD22.1, CD22.2, 16F7 and
23C6
[0692] The 12C5 clone was found to express an antibody comprising
an IgG1 heavy chain and a lambda light chain. The 19A3 clone was
found to express an antibody comprising an IgG1 heavy chain and a
kappa light chain. The heavy and light chains of the recombinant
mAb expressed by the 8G9 clone were identical to those expressed by
the 19A3 clone. The heavy chain of the recombinant mAb expressed by
the 17E11 was identical to that of the 19A3 with the exception of
the introduced N57Q mutation. The light chain of the recombinant
mAb expressed by the 17E11 clone was identical to that expressed by
the 19A3 clone. The 16F7 clone was found to express antibodies
comprising an IgG1 heavy chain and one of two different kappa light
chains (referred to herein as V.sub.K.1 and V.sub.K.2, wherein 43%
of antibody protein comprised V.sub.K.1 and 57% of antibody protein
comprised V.sub.K.2). The 23C6 clone also was found to express
antibodies comprising an IgG1 heavy chain and one of two different
kappa light chains (V.sub.K.1 and V.sub.K.2, wherein 40% of
antibody protein comprised V.sub.K.1 and 60% of antibody protein
comprised V.sub.K.2). The 4G6 clone was found to express antibodies
comprising an IgG1 heavy chain and one of two different kappa light
chains (referred to herein as V.sub.K.1 and V.sub.K.2). The 21F6
clone was found to express antibodies comprising one of two
different IgG1 heavy chains (referred to herein as V.sub.H1 and
V.sub.H2) and a kappa light chain.
[0693] The nucleotide and amino acid sequences of the heavy chain
variable region of 12C5 are shown in FIG. 1A and in SEQ ID NO:41
and 31, respectively.
[0694] The nucleotide and amino acid sequences of the lambda light
chain variable region of 12C5 are shown in FIG. 1B and in SEQ ID
NO:45 and 35, respectively.
[0695] Comparison of the 12C5 heavy chain immunoglobulin sequence
to the known human germline immunoglobulin heavy chain sequences
demonstrated that the 12C5 heavy chain utilizes a V.sub.H segment
from human germline V.sub.H 7-4.1, a D segment from the human
germline 3-3, and a JH segment from human germline JH 6B. Further
analysis of the 12C5 V.sub.H sequence using the Kabat system of CDR
region determination led to the delineation of the heavy chain
CDR1, CDR2 and CDR3 regions as shown in FIG. 1A and in SEQ ID NOs:
1, 5 and 9, respectively.
[0696] Comparison of the 12C5 light chain immunoglobulin sequence
to the known human germline immunoglobulin light chain sequences
demonstrated that the 12C5 lambda light chain utilizes a
V.sub..lamda. segment from human germline V.sub..lamda. 2b2 and a
J.lamda. segment from human germline JL 2. Further analysis of the
12C5 V.sub..lamda. sequence using the Kabat system of CDR region
determination led to the delineation of the light chain CDR1, CDR2
and CDR3 regions as shown in FIG. 1B and in SEQ ID NOs: 13, 19 and
25, respectively.
[0697] The nucleotide and amino acid sequences of the heavy chain
variable region of 19A3 are shown in FIG. 2A and in SEQ ID NOs:42
and 32, respectively. The nucleotide and amino acid sequences of
the heavy chain variable region of CD22.1 are identical to those of
19A3, and correspond to the nucleotide and amino acid sequences
shown in FIG. 2A and SEQ ID NOs:42 and 32, respectively.
[0698] The nucleotide and amino acid sequences of the heavy chain
variable region of CD22.2 are shown in FIG. 2C and in SEQ ID NOs:61
and 60, respectively.
[0699] The nucleotide and amino acid sequences of the light chain
variable region of 19A3 are shown in FIG. 2B and in SEQ ID NO:46
and 36, respectively. The nucleotide and amino acid sequences of
the light chain variable regions of both CD22.1 and CD22.2 are
identical to those of 19A3, and correspond to the nucleotide and
amino acid sequences shown in FIG. 2A and SEQ ID NOs:46 and 36,
respectively.
[0700] Comparison of the 19A3/CD22.1 heavy chain immunoglobulin
sequence to the known human germline immunoglobulin heavy chain
sequences demonstrated that the 19A3 heavy chain utilizes a V.sub.H
segment from human germline V.sub.H 4-34, a D segment from the
human germline 3-9, and a JH segment from human germline JH 4B.
Further analysis of the 19A3/CD22.1 V.sub.H sequence using the
Kabat system of CDR region determination led to the delineation of
the heavy chain CDR1, CDR2 and CDR3 regions as shown in FIG. 2A and
in SEQ ID NOs: 2, 6 and 10, respectively
[0701] Comparison of the CD22.2 heavy chain immunoglobulin sequence
to the known human germline immunoglobulin heavy chain sequences
demonstrated that the CD22.2 heavy chain utilizes a V.sub.H segment
from human germline V.sub.H 4-34, a D segment from the human
germline 3-9, and a JH segment from human germline JH 4B. Further
analysis of the CD22.2 V.sub.H sequence using the Kabat system of
CDR region determination led to the delineation of the heavy chain
CDR1, CDR2 and CDR3 regions as shown in FIG. 2C and in SEQ ID NOs:
2, 60 and 10, respectively.
[0702] Comparison of the 19A3/CD22.1/CD22.2 light chain
immunoglobulin sequence to the known human germline immunoglobulin
light chain sequences demonstrated that the 19A3/CD22.1/CD22.2
kappa light chain utilizes a V.sub.K segment from human germline
V.sub.K L6 and a JK segment from human germline JK 1. Further
analysis of the 19A3/CD22.1/CD22.2 V.sub.K sequence using the Kabat
system of CDR region determination led to the delineation of the
light chain CDR1, CDR2 and CDR3 regions as shown in FIG. 2B and in
SEQ ID NOs: 14, 20 and 26, respectively.
[0703] The nucleotide and amino acid sequences of the heavy chain
variable region of 16F7 are shown in FIG. 3A and in SEQ ID NO:43
and 33, respectively.
[0704] The nucleotide and amino acid sequences of the V.sub.K.1
kappa light chain variable region of 16F7 are shown in FIG. 3B and
in SEQ ID NO:47 and 37, respectively.
[0705] The nucleotide and amino acid sequences of the V.sub.K.2
kappa light chain variable region of 16F7 are shown in FIG. 3C and
in SEQ ID NO:48 and 38, respectively.
[0706] Comparison of the 16F7 heavy chain immunoglobulin sequence
to the known human germline immunoglobulin heavy chain sequences
demonstrated that the 16F7 heavy chain utilizes a V.sub.H segment
from human germline V.sub.H 5-51, a D segment from the human
germline 3-10, and a JH segment from human germline JH 3B. Further
analysis of the 16F7 V.sub.H sequence using the Kabat system of CDR
region determination led to the delineation of the heavy chain
CDR1, CDR2 and CDR3 regions as shown in FIG. 3A and in SEQ ID NOs:
3, 7 and 11, respectively.
[0707] Comparison of the 16F7 V.sub.K.1 kappa light chain
immunoglobulin sequence to the known human germline immunoglobulin
kappa light chain sequences demonstrated that the 16F7 V.sub.K.1
kappa light chain utilizes a V.sub.K segment from human germline
V.sub.K A27 and a JK segment from human germline JK 1. Further
analysis of the 16F7 V.sub.K sequence using the Kabat system of CDR
region determination led to the delineation of the light chain
CDR1, CDR2 and CDR3 regions as shown in FIG. 3B and in SEQ ID NOs:
15, 21 and 27, respectively.
[0708] Comparison of the 16F7 V.sub.K.2 kappa light chain
immunoglobulin sequence to the known human germline immunoglobulin
kappa light chain sequences demonstrated that the 16F7 V.sub.K.2
kappa light chain utilizes a V.sub.K segment from human germline
V.sub.K A10 and a JK segment from human germline JK 2. Further
analysis of the 16F7 V.sub.K sequence using the Kabat system of CDR
region determination led to the delineation of the light chain
CDR1, CDR2 and CDR3 regions as shown in FIG. 3C and in SEQ ID NOs:
16, 22 and 28, respectively.
[0709] The nucleotide and amino acid sequences of the heavy chain
variable region of 23C6 are shown in FIG. 4A and in SEQ ID NO:44
and 34, respectively.
[0710] The nucleotide and amino acid sequences of the V.sub.K.1
kappa light chain variable region of 23C6 are shown in FIG. 4B and
in SEQ ID NO:49 and 39, respectively.
[0711] The nucleotide and amino acid sequences of the V.sub.K.2
kappa light chain variable region of 23C6 are shown in FIG. 4C and
in SEQ ID NO:50 and 40, respectively.
[0712] Comparison of the 23C6 heavy chain immunoglobulin sequence
to the known human germline immunoglobulin heavy chain sequences
demonstrated that the 23C6 heavy chain utilizes a V.sub.H segment
from human germline V.sub.H 1-69, a D segment from the human
germline 2-15, and a JH segment from human germline JH 6B. Further
analysis of the 23C6 V.sub.H sequence using the Kabat system of CDR
region determination led to the delineation of the heavy chain
CDR1, CDR2 and CDR3 regions as shown in FIG. 4A and in SEQ ID
NOs:4, 8 and 12, respectively.
[0713] Comparison of the 23C6 V.sub.K.1 kappa light chain
immunoglobulin sequence to the known human germline immunoglobulin
kappa light chain sequences demonstrated that the V.sub.K.1 kappa
light chain utilizes a V.sub.K segment from human germline V.sub.K
L6 and a JK segment from human germline JK 1. Further analysis of
the 23C6 V.sub.K.1 sequence using the Kabat system of CDR region
determination led to the delineation of the light chain CDR1, CDR2
and CDR3 regions as shown in FIG. 4B and in SEQ ID NOs:17, 23 and
29, respectively.
[0714] Comparison of the 23C6 V.sub.K.2 kappa light chain
immunoglobulin sequence to the known human germline immunoglobulin
kappa light chain sequences demonstrated that the V.sub.K.2 kappa
light chain utilizes a V.sub.K segment from human germline V.sub.K
L6 and a JK segment from human germline JK 1. Further analysis of
the 23C6 V.sub.K.2 sequence using the Kabat system of CDR region
determination led to the delineation of the light chain CDR1, CDR2
and CDR3 regions as shown in FIG. 4B and in SEQ ID NOs:18, 24 and
30, respectively.
[0715] FIG. 5A shows the alignment of the 12C5 heavy chain variable
amino acid sequence (SEQ ID NO:31) with the germline V.sub.H 7-4.1
encoded amino acid sequence (SEQ ID NO:51). The CDR1, CDR2 and CDR3
regions are delineated.
[0716] FIG. 5B shows the alignment of the 12C5 lambda light chain
variable amino acid sequence (SEQ ID NO:35) with the germline
V.sub..lamda. 2b2 encoded amino acid sequence (SEQ ID NO:55). The
CDR1, CDR2 and CDR3 regions are delineated.
[0717] FIG. 6A shows the alignment of the 19A3 heavy chain variable
amino acid sequence and the CD22.1 heavy chain variable amino acid
sequence (SEQ ID NO:32) with the germline V.sub.H 4-34 encoded
amino acid sequence (SEQ ID NO:52). The CDR1, CDR2 and CDR3 regions
are delineated.
[0718] FIG. 6B shows the alignment of the 19A3, CD22.1 and CD22.2
kappa light chain variable amino acid sequences (all of which are
identical to SEQ ID NO:36) with the germline V.sub.K L6 encoded
amino acid sequence (SEQ ID NO:56). The CDR1, CDR2 and CDR3 regions
are delineated.
[0719] FIG. 6C shows the alignment of the CD22.2 heavy chain
variable amino acid sequence (SEQ ID NO:32) with the germline
V.sub.H 4-34 encoded amino acid sequence (SEQ ID NO:52). The CDR1,
CDR2 and regions are delineated.
[0720] FIG. 7A shows the alignment of the 16F7 heavy chain variable
amino acid sequence (SEQ ID NO:33) with the germline V.sub.H 5-51
encoded amino acid sequence (SEQ ID NO:53). The CDR1, CDR2 and CDR3
regions are delineated.
[0721] FIG. 7B shows the alignment of the 16F7 V.sub.K.1 kappa
light chain variable amino acid sequence (SEQ ID NO:37) with the
germline V.sub.K A27 encoded amino acid sequence (SEQ ID NO:57).
The CDR1, CDR2 and CDR3 regions are delineated.
[0722] FIG. 7C shows the alignment of the 16F7 V.sub.K.2 kappa
light chain variable amino acid sequence (SEQ ID NO:38) with the
germline V.sub.K A10 encoded amino acid sequence (SEQ ID NO:58).
The CDR1, CDR2 and CDR3 regions are delineated.
[0723] FIG. 8A shows the alignment of the 23C6 heavy chain variable
amino acid sequence (SEQ ID NO:34) with the germline V.sub.H 1-69
encoded amino acid sequence (SEQ ID NO:54). The CDR1, CDR2 and CDR3
regions are delineated.
[0724] FIG. 8B shows the alignment of the 23C6 V.sub.K.1 kappa
light chain variable amino acid sequence (SEQ ID NO:39) and the
V.sub.K.2 kappa light chain variable amino acid sequence (SEQ ID
NO:40) with the germline V.sub.K L6 encoded amino acid sequence
(SEQ ID NO:56). The CDR1, CDR2 and CDR3 regions are delineated.
Characterization of 4G6 and 21F6
[0725] The nucleotide and amino acid sequences of the heavy chain
variable region of 4G6 are shown in FIG. 17A and in SEQ ID NO:87
and 81, respectively.
[0726] The nucleotide and amino acid sequences of the V.sub.K.1
kappa light chain variable region of 4G6 are shown in FIG. 17B and
in SEQ ID NO:90 and 84, respectively.
[0727] The nucleotide and amino acid sequences of the V.sub.K.2
kappa light chain variable region of 4G6 are shown in FIG. 17C and
in SEQ ID NO:91 and 85, respectively.
[0728] Comparison of the 4G6 heavy chain immunoglobulin sequence to
the known human germline immunoglobulin heavy chain sequences
demonstrated that the 4G6 heavy chain utilizes a V.sub.H segment
from human germline V.sub.H 1-69, a D segment from the human
germline 7-27, and a JH segment from human germline JH 4B. Further
analysis of the 4G6 V.sub.H sequence using the Kabat system of CDR
region determination led to the delineation of the heavy chain
CDR1, CDR2 and CDR3 regions as shown in FIG. 17A and in SEQ ID NOs:
63, 66 and 69, respectively.
[0729] Comparison of the 4G6 V.sub.K.1 kappa light chain
immunoglobulin sequence to the known human germline immunoglobulin
kappa light chain sequences demonstrated that the 16F7 V.sub.K.1
kappa light chain utilizes a V.sub.K segment from human germline
V.sub.K L18 and a JK segment from human germline JK 2. Further
analysis of the 4G6 V.sub.K sequence using the Kabat system of CDR
region determination led to the delineation of the light chain
CDR1, CDR2 and CDR3 regions as shown in FIG. 3B and in SEQ ID NOs:
72, 75 and 78, respectively.
[0730] Comparison of the 4G6 V.sub.K.2 kappa light chain
immunoglobulin sequence to the known human germline immunoglobulin
kappa light chain sequences demonstrated that the 4G5 V.sub.K.2
kappa light chain utilizes a V.sub.K segment from human germline
V.sub.K A27 and a JK segment from human germline JK 4. Further
analysis of the 4G6 V.sub.K sequence using the Kabat system of CDR
region determination led to the delineation of the light chain
CDR1, CDR2 and CDR3 regions as shown in FIG. 17C and in SEQ ID NOs:
73, 76 and 79, respectively.
[0731] The nucleotide and amino acid sequences of the V.sub.H0.1
heavy chain variable region of 21F6 are shown in FIG. 18A and in
SEQ ID NO:88 and 82, respectively.
[0732] The nucleotide and amino acid sequences of the V.sub.H.2
heavy chain variable region of 21F6 are shown in FIG. 18B and in
SEQ ID NO:89 and 83, respectively.
[0733] The nucleotide and amino acid sequences of the kappa light
chain variable region of 21F6 are shown in FIG. 18C and in SEQ ID
NO:92 and 86, respectively.
[0734] Comparison of the 21F6 V.sub.H.1 heavy chain immunoglobulin
sequence to the known human germline immunoglobulin heavy chain
sequences demonstrated that the 21F6 heavy chain utilizes a V
segment from human germline V.sub.H 4-34, a D segment from the
human germline 3-9, and a JH segment from human germline JH 4B.
Further analysis of the 21F6 V.sub.H sequence using the Kabat
system of CDR region determination led to the delineation of the
heavy chain CDR1, CDR2 and CDR3 regions as shown in FIG. 3A and in
SEQ ID NOs: 64, 67 and 70, respectively.
[0735] Comparison of the 21F6 V.sub.H.2 kappa light chain
immunoglobulin sequence to the known human germline immunoglobulin
kappa light chain sequences demonstrated that the 21F6 V.sub.H.2
heavy chain utilizes a V.sub.H segment from human germline V.sub.H
4-34, a D segment from the human germline 3-9, and a JH segment
from human germline JH 4B. Further analysis of the 21F6 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the light chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 18B and in SEQ ID NOs: 65, 68 and 71,
respectively.
[0736] Comparison of the 21F6 kappa light chain immunoglobulin
sequence to the known human germline immunoglobulin kappa light
chain sequences demonstrated that the 21F6 kappa light chain
utilizes a V.sub.K segment from human germline V.sub.K L6 and a JK
segment from human germline JK 4. Further analysis of the 21F6
V.sub.H sequence using the Kabat system of CDR region determination
led to the delineation of the light chain CDR1, CDR2 and CDR3
regions as shown in FIG. 18C and in SEQ ID NOs: 74, 77 and 80,
respectively.
[0737] FIG. 19A shows the alignment of the 4G6 heavy chain variable
amino acid sequence (SEQ ID NO:81) with the germline V.sub.H 1-69
encoded amino acid sequence (SEQ ID NO:54). The CDR1, CDR2 and CDR3
regions are delineated.
[0738] FIG. 19B shows the alignment of the 4G6 V.sub.K.1 kappa
light chain variable amino acid sequence (SEQ ID NO:84) with the
germline V.sub.K1 L18 encoded amino acid sequence (SEQ ID NO:93).
The CDR1, CDR2 and CDR3 regions are delineated.
[0739] FIG. 18C shows the alignment of the 4G6 V.sub.K.2 kappa
light chain variable amino acid sequence (SEQ ID NO:85) with the
germline V.sub.K A27 encoded amino acid sequence (SEQ ID NO:57).
The CDR1, CDR2 and CDR3 regions are delineated.
[0740] FIG. 20A shows the alignment of the 21F6 V.sub.H.1 heavy
chain variable amino acid sequence (SEQ ID NO:82) with the germline
V.sub.H 4-34 encoded amino acid sequence (SEQ ID NO:52). The CDR1,
CDR2 and CDR3 regions are delineated.
[0741] FIG. 20B shows the alignment of the 21F6 V.sub.H.2 kappa
light chain variable amino acid sequence (SEQ ID NO:83) with the
germline V.sub.H 4-34 encoded amino acid sequence (SEQ ID NO:52).
The CDR1, CDR2 and CDR3 regions are delineated.
[0742] FIG. 20C shows the alignment of the 21F6 kappa light chain
variable amino acid sequence (SEQ ID NO:86) with the germline
V.sub.K L6 encoded amino acid sequence (SEQ ID NO:56). The CDR1,
CDR2 and CDR3 regions are delineated.
Recombinant Isotype Conversion
[0743] The 12C5, 19A3, 16F7, 23C6, CD22.1, CD22.2, 4G6 and 21F6
variable regions can be converted to full-length antibodies of any
desired isotype using standard recombinant DNA techniques. For
example, DNA encoding the V.sub.H and V.sub.L regions can be cloned
into an expression vector that carries the heavy and light chain
constant regions such that the variable regions are operatively
linked to the constant regions. Alternatively, separate vectors can
be used for expression of the full-length heavy chain and the
full-length light chain. Non-limiting examples of expression
vectors suitable for use in creating full-length antibodies include
the pIE vectors described in U.S. Patent Application No.
20050153394 by Black.
Example 3
Binding Characteristics of Anti-CD22 Human Monoclonal
Antibodies
[0744] In this example, binding affinities of the anti-CD22
antibodies 12C5, 19A3, 16F7, 23C6 and 4G6 were examined by BIAcore
analysis. Retention of CD22 binding affinity by the 19A32
recombinant derivative antibodies CD22.1 and CD22.2 was confirmed
by means of ELISA analysis and FACS flow cytometry.
[0745] Epitope grouping of the 12C5, 19A3, 16F7, and 23C6
antibodies was performed by BIAcore analysis.
[0746] Finally, the CD22 domains to which the anti-CD22 antibodies
of the present invention specifically bind were mapped using CHO
cells that expressed a fusion protein containing only the amino
terminal domains 1 and 2 of CD22.
Binding Affinity and Kinetics
[0747] For determination of antibody affinity (K.sub.D),
experiments were performed in which the CD22 antigen was captured
on a BIAcore chip using an antibody to the His tag present on the
antigen. Anti-His monoclonal antibody ab15149 (Abcam, Stock conc.
0.5 mg/mL) was coated on a CM5 chip at high density (3500 RUs), as
recommended by the manufacturer. CD22 ECD (6.6 .mu.g/mL) was
captured on this surface for 60 sec at a flow-rate of 6 .mu.L/min.
A single concentration (20 .mu.g/mL) of anti-CD22 purified mAbs was
injected over the captured antigen with an association time of 5
minutes and a dissociation time of 8 minutes, at a flow rate of 25
.mu.g/mL. The chip surface was regenerated after each cycle with 10
.mu.L of 25 mM NaOH. Isotype controls were run on the chip and the
data used to subtract non-specific binding. All experiments were
carried out on a BIAcore 3000 surface plasmon resonance instrument,
using BIAcore Control software v 3.2. Data analysis was carried out
using BiaEvaluation v. 3.2 software.
[0748] Fourteen of the selected anti-CD22 antibodies were tested in
the affinity experiment. The range of obtained affinity values for
the twelve antibodies was 0.07-9.95.times.10.sup.-9 M. The results
for the four antibodies structurally characterized in Example 2 are
summarized below in Table 1:
TABLE-US-00001 TABLE 1 BIAcore Binding Data for Anti-CD22 HuMAbs.
Anti-CD22 BIAcore Affinity (K.sub.D) antibody 10.sup.-9 M Positive
control 1.48 12C5 0.23 19A3 0.15 16F7 1.03 23C6 0.87 4G6 0.07
Retention of CD22 Binding Affinity by Recombinant Derivative
Antibodies CD22.1 and CD22.2
[0749] To determine whether CD22.1 and CD22.2 retained CD22 binding
affinity, ELISA analysis was performed in which binding to CD22 ECD
by CD22.1 and CD22.2 were compared to binding by the
hybridoma-derived parental antibody 19A3.
[0750] Recombinant CD22 extracellular domain (CD22 ECD) was coated
on 96-well ELISA plates at 2 .mu.g/ml, and after washing, blocking
with 5% bovine serum albumin and washing again, the test antibodies
were titrated from 10 .mu.g/ml downwards in 1:3 dilutions. After
incubating for an hour, plates were washed, and goat anti-human IgG
HRP conjugate was added to each well. After a further one hour
incubation plates were washed again and bound HRP conjugate
detected through addition of TMB substrate, incubating until color
developed and stopping with 1M hydrochloric acid. Absorbance was
then read in a plate reader at 450 nm. Results (FIG. 12) clearly
showed that the ability of CD22.1 and CD22.2 to bind to CD22 ECD
was equivalent to the parental antibody 19A3. This revealed that
expression of the antibody in CHO cells was successful, and that
the mutation to remove the N-glycosylation site did not affect
antigen binding.
[0751] The ability of the 4G6 and 21F6 anti-CD22 monoclonal
antibodies to bind CD22 ECD was also investigated, and found to
bind specifically to CD22 ECD.
[0752] FACS Analysis of CD22 Expressed on Cell Surfaces
[0753] The ability of CD22.1 and CD22.2 to bind CD22 was also
confirmed by flow cytometry. Either CHO cells transfected with
full-length CD22 (CHO-CD22) or Raji cells were resuspended in FACS
buffer at 2.times.10.sup.5 cells/well, and after pelleting the
cells, antibody was titrated into the wells starting at 10 .mu.g/ml
and serially diluting 1:3. After mixing and incubating on ice for
45 minutes, FACS buffer was added and the cells washed 4 times.
After washing, goat anti-human IgG PE conjugate was added, and
following a further 30 minute incubation on ice, cells were again
washed 4 times before resuspending in FACS buffer and reading PE
fluorescence on a FACS array machine. Results (FIGS. 13 and 14)
showed that CD22.1 and CD22.2 bound strongly and equivalently to
both the CHO-CD22 transfectants and Raji cells.
[0754] Binding of 4G6 and 21F6 to CD22 expressed on the surfaces of
Raji cells and CHO cells transformed with CD22 was also analyzed by
FACS analysis. The results demonstrated that a high level of cell
binding was obtained. See FIGS. 21, 22A and 22B. Neither antibody
was able to bind to CHO cells absent transfection with CD22.
Epitope Grouping
[0755] Epitope binning was carried out by immobilizing selected
antibodies on the CM5 chip, based on standard immobilization
protocols and flowing antibody-antigen complexes over the surface.
Antibodies that had overlapping epitopes were competed out while
those having non-overlapping epitopes gave rise to simultaneous
binding to the antigen. An increasing signal denotes an epitope
different from the antibody coated on the chip, and the opposite is
true if signal decreases. Antibodies that exhibited faster off-rate
constants were chosen to be coated on the Biacore CM5 chip as they
would facilitate easier regeneration for repeated use of the chip.
Purified anti-CD22 antibodies were coated at high densities on
different surfaces of different CM5 chips. Several rounds of
iterative binning were carried out until the distinct epitope
groups were identified. The concentrations of antibodies varied
between 50-200 .mu.g/mL, which were incubated with 4 nM-50 nM CD22
ECD for 2 hrs at RT. The incubated complexes were passed over the
antibody coated surfaces on each chip for 2-6 min at 5-10
.mu.L/min. Each cycle was regenerated by 15-30 mM NaOH. The signal
obtained after 2-5 minutes of injection was plotted against
antibody concentration to determine the epitope groups. Antibodies
were grouped into various epitopes based on the above
interpretation of the experimental observation.
[0756] The results of the epitope grouping experiment were that
four distinct epitope groups could be identified. Of fourteen
anti-CD22 antibodies examined, five were found to be in Epitope
Group 1, three were found to be in Epitope Group 2, four were found
to be in Epitope Group 3, one was found to be in Epitope Group 4
and one was found to be in Epitope Groups 3 & 4, indicating
that there is some overlap between Epitope Groups 3 and 4. The
results for the four antibodies structurally characterized in
Example 2 are summarized below in Table 2:
TABLE-US-00002 TABLE 2 CD22 Epitope Groups Mapped by BIAcore
Anti-CD22 antibody Epitope Group Positive control 1 12C5 4 19A3 1
16F7 3 23C6 2
Recognition of CD22 Amino Terminal Domains
[0757] The extracellular region of CD22 contains 7
immunoglobulin-type domains, of which the amino terminal 2 Ig-type
domains may be particularly important for CD22 ligand binding. In
order to map which domains the human antibodies bound to, a
recombinant construct was made in which only amino terminal domains
1 and 2 of the ECD were fused to the hinge and Fc regions of a
mouse IgG heavy chain. The resultant fusion protein, designated
CD22 d1d2-mFc, was expressed in CHO cells and purified for use in
binding assays. Human antibodies, previously shown to bind to the
entire ECD of CD22, were then tested for their ability to bind to
CD22 d1d2-mFc.
[0758] Goat anti-mouse IgG was coated on 96-well ELISA plates at 5
.mu.g/ml. After incubating overnight at 4.degree. C., plates were
washed and CD22 d1d2-mFc was added to each well at 2 .mu.g/ml
followed by incubation for 1 hour at room temperature. After plate
washing, blocking with 5% bovine serum albumin and washing again,
the test antibodies were added at 10 .mu.g/ml. After incubating for
an hour, plates were washed, and goat anti-human IgG HRP conjugate
was added to each well. After a further one hour incubation plates
were washed again and bound HRP conjugate detected through addition
of TMB substrate, incubating until color developed and stopping
with 1M hydrochloric acid. Absorbance was then read in a plate
reader at 450 nm.
[0759] Results (FIG. 15) showed that antibodies fell into two
groups. Group 1 represented by 23C6, 19A3, and the recombinant
derivatives of 19A3, CD22.1 and CD22.2 bound to CD22 d1d2-mFc
whereas group 2 represented by 12C5 and 16F7 did not. This suggests
that 12C5 and 16F7 recognize epitopes on the CD22 ECD outside of
the amino-terminal domains.
[0760] FACS Analysis of CD22 Expressed on Cell Surfaces
[0761] Binding of CD22.1 and CD22.2 to CD22 was also confirmed by
flow cytometry. Either CHO cells transfected with full-length CD22
(CHO-CD22) or Raji cells were resuspended in FACS buffer at
2.times.10.sup.5 cells/well, and after pelleting the cells,
antibody was titrated into the wells starting at 10 .mu.g/ml and
serially diluting 1:3. After mixing and incubating on ice for 45
minutes, FACS buffer was added and the cells washed 4 times. After
washing, goat anti-human IgG PE conjugate was added, and following
a further 30 minute incubation on ice, cells were again washed 4
times before resuspending in FACS buffer and reading PE
fluorescence on a FACS array machine. Results (FIGS. 13 and 14)
showed that CD22.1 and CD22.2 bound strongly and equivalently to
both the CHO-CD22 transfectants and Raji cells.
[0762] Binding of 4G6 and 21F6 to CD22 expressed on the surfaces of
Raji cells and CHO cells transformed with CD22 was analyzed by FACS
analysis. The results demonstrated that a high level of cell
binding was obtained. See FIGS. 21, 22A and 22B. Neither antibody
was able to bind to CHO cells absent transfection with CD22.
Example 4
Internalization of Anti-CD22 Antibodies
[0763] To determine the ability of the anti-CD22 human antibodies
to internalize into CD22-expressing cells, a Hum-ZAP
internalization assay was used with the Burkitt's lymphoma cell
line Raji, which expresses CD22. The Hum-ZAP assay tests for
internalization of a primary antibody through binding of a
secondary antibody with affinity for human IgG conjugated to the
toxin saporin.
[0764] The CD22-expressing Raji cells were seeded at
2.0.times.10.sup.4 cells/well (35 .mu.l/well). The anti-CD22
antibodies were added to the wells at 1.5 .mu.g/ml (35 .mu.l/well).
Media alone was used as negative control. The Hum-ZAP reagent
(Advanced Targeting Systems, San Diego, Calif., IT-22-25) was then
added at a concentration of 3.0 .mu.g/mL (35 .mu.l/well) to half of
the wells while the other half of the wells received media only.
The plates were incubated for 72 hours at 37.degree. C. The cell
viability was determined using CellTiter-Glo Luminescent Cell
Viability Assay (Promega, Madison, Wis., #G7571). CellTiter-Glo
buffer was mixed with CellTiter-Glo substrate and 100 .mu.L of the
mixture was added to each well. Luminescence was detected using
Veritas Microplate Luminometer and Veritas software (Turner
BioSystems, Sunnyvale, Calif.) per manufacturer directions.
[0765] Twelve different anti-CD22 antibodies were tested and all
exhibited the ability to internalize. The results for the four
antibodies that were structurally characterized in Example 2 are
shown in the bar graph of FIG. 9. As illustrated in FIG. 9, a
marked decrease in Raji cell viability was observed in all wells
containing anti-CD22 antibodies and HumZAP reagent, including wells
with positive control, while cell viability was not affected in
wells containing only the anti-CD22 antibodies with no HumZAP
reagent, demonstrating that the anti-CD22 antibodies do not trigger
cell killing on their own. As expected, in absence of anti-CD22
antibodies, the negative control (referred to as media) did not
show any cell killing in presence or absence of HumZAP reagent.
These data demonstrate that the anti-CD22 antibodies internalize
efficiently and release of saporin inside the cells is responsible
for the killing of CD22-expressing Raji cells in presence of HumZAP
reagent.
Example 5
Assessment of ADCC Activity of Anti-CD22 Antibodies
[0766] To determine the ability of the anti-CD22 human antibodies
kill CD22+ cell lines in the presence of effector cells via
antibody dependent cellular cytotoxicity (ADCC), a fluorescence
cytotoxicity assay was used.
[0767] Human effector cells were prepared from whole blood as
follows. Human peripheral blood mononuclear cells were purified
from heparinized whole blood by standard Ficoll-paque separation.
The cells were resuspended in RPMI1640 media containing 10% FBS
(heat-inactivated) and 200 U/ml of human IL-2 and incubated
overnight at 37.degree. C. The following day, the cells were
collected and washed four times in culture media and resuspended at
1.times.10.sup.7 cells/ml. Target CD22+ cells were incubated with
BATDA reagent (Perkin Elmer, Wellesley, Mass.) at 2.5 .mu.l BATDA
per 1.times.10.sup.6 target cells/mL for 20 minutes at 37.degree.
C. The target cells were washed four times, spun down and brought
to a final volume of 1.times.10.sup.5 cells/ml.
[0768] The CD22+ cell lines Raji (human B lymphocyte Burkitt's
lymphoma; ATCC Accession No. CCL-86) and Daudi (human B lymphocyte
Burkitt's lymphoma; ATCC Accession No. CCL-213) were tested for
antibody specific ADCC to the human anti-CD22 monoclonal antibodies
using the Delfia fluorescence emission analysis as follows. Each
target cell line (100 .mu.l of labeled target cells, 10.sup.4
cells/well) was incubated with 50 .mu.l of effector cells (10.sup.6
cells/well) and 50 .mu.l of antibody (10 ug/ml final
concentration). A target to effector ratio of 1:50 was used
throughout the experiments. In all studies, a human IgG1 isotype
control was used as a negative control. Cells were spun down at
2000 rpm and incubated for one hour incubation at 37.degree. C. The
supernatants were then collected, submitted to centrifugation and
20 .mu.l of supernatant was transferred to a flat bottom plate, to
which 180 .mu.l of Eu solution (Perkin Elmer, Wellesley, Mass.) was
added and read in a RubyStar reader (BMG Labtech). The % lysis was
calculated as follows: (sample release-spontaneous
release*100)/(maximum release-spontaneous release), where the
spontaneous release is the fluorescence from wells which contain
target cells plus effector cells and maximum release is the
fluorescence from wells containing target cells and have been
treated with 2% Triton-X.
[0769] For the Raji and Daudi cell ADCC assays, thirteen different
anti-CD33 antibodies were tested along with negative and positive
control antibodies (hIgG1 and CD20, respectively). In each assay,
ten of the thirteen anti-CD22 antibodies exhibited levels of ADCC
activity equal to or greater than the positive control antibody.
The results for the four antibodies that were structurally
characterized in Example 2 are shown in the graphs of FIGS. 10A
(Daudi cells) and 10B (Raji cells), which show % cell lysis. The
data demonstrate that human anti-CD22 antibodies that exhibit ADCC
activity can be selected, although the degree of cytotoxicity of
each antibody against CD22+ cells may differ depending on which
cell line is used as the target cell.
Example 6
Stimulation of Calcium Flux by Anti-CD22 Antibodies
[0770] To assess the ability of the anti-CD22 human antibodies to
stimulate calcium flux in CD22+ cells, the following calcium flux
assay was used. Viable Ramos cells (ATCC Accession No. CRL-1596)
were counted by trypan blue exclusion microscopy and diluted to
2.times.10.sup.6 cells/ml in RPMI+10% FBS culture media. From this
cell suspension, 2.times.10.sup.5 cells (100 .mu.l/well) was
dispensed into to all the wells of a Poly D-Lysine surface black
with clear bottom 96 well plate (Corning #3667). Loading dye
(Molecular Devices catalog # R7181) was added to the cell
suspension, 100 .mu.l/well. The plate was centrifuges at 1100 rpm
for 4 minutes and then incubated at 37.degree. C. for 30 minutes.
Five-fold dilution series of anti-CD22 antibodies and human IgG1
isotype control, from 50 .mu.g/ml to 40 ng/ml, were prepared in
Component B (Molecular Devices # R7181)+0.1% BSA buffer. Antibodies
were dispensed in triplicate into rows A-F of the previously
prepared 96-well plate. Component B+0.1% BSA was dispensed into all
the wells of rows G&H on the assay plate. Calcium flux was
assayed using a Flex Station (Molecular Devices), adding 22 .mu.l
reagent per well to the assay plate at 17 seconds. Data was
analyzed as FI Max-Min and plotted vs. antibody concentration using
GraphPad.TM. PRISM, non-linear regression, sigmoidal dose response,
variable slope.
[0771] Seventeen different anti-CD22 human antibodies were
evaluated in the assay. The results showed that none of the
seventeen anti-CD22 antibodies tested stimulated significant
calcium flux, as compared to a human IgG1 isotype control or buffer
alone. Ramos cells were previously demonstrated to flux calcium in
response to BCR stimulation with goat anti-human IgM
F(ab').sub.2.
Example 7
Modulation of BCR Stimulation-Induced Effects by Anti-CD22
Antibodies
[0772] In this example, the ability of immobilized anti-CD22
antibody to modulate B Cell Receptor (BCR) stimulation-induced
effects was examined. In the assay, anti-CD22 human antibodies and
human IgG1 isotype control were diluted to 5 .mu.g/ml in RPMI+10%
FBS and dispensed 100 .mu.l/well in triplicate into Microlite 1
Flat Bottom plates (Corning #7416). Following overnight incubation
at 4.degree. C., the plates were washed once with cold PBS, then
once with RPMI 1640 (Mediatech)+10% FBS (GIBCO). Viable Ramos cells
(ATCC Accession No. CRL-1596) were counted by trypan blue exclusion
microscopy and diluted to 2.times.10.sup.5 cells/ml in RPMI+10%
FBS. 20,000 cells (50 .mu.l/well) were dispensed into the antibody
coated 96-well plates. Anti-human IgM F(ab').sub.2 (Jackson catalog
#109-006-129) was diluted to 5 .mu.g/ml in RPMI+10% FBS and 100
.mu.l/well was dispensed for a final concentration of 2.5 .mu.g/ml.
The assay plates were incubated 72 hours. Cell viability was
assayed with the addition of CellTiter-Glo reagent (Promega G7571),
100 .mu.l/well, for 10 minutes. Luminescence was measured using
Luminescence Test 1 plate Nunc96 on Pherastar GMB Labtech. Data was
analyzed as % cell death relative to the human IgG1 isotype
control, which represented 100% cell viability.
[0773] Seventeen different anti-CD22 antibodies were tested in the
assay and this panel exhibited a broad range of activity, ranging
from an observed % cell death value of approximately 80% to an
observed % cell death value approximately equal to the isotype
control, depending on the particular antibody. The results for the
four antibodies that were structurally characterized in Example 2
are shown in the bar graph of FIG. 11, which shows that immobilized
19A3, 23C6 and 12C5 each potently enhanced the anti-proliferative
effects of BCR stimulation cell death whereas 16F7 did not differ
significantly from the isotype control
Example 8
Effects of Anti-CD22 Antibodies on Cell Proliferation
[0774] In this example, the direct effects of soluble anti-CD22
antibodies on cell proliferation, with or without antibody
cross-linking, was examined. In the assay used, viable Ramos cells
(ATCC Accession No. CRL-1596) were counted by trypan blue exclusion
microscopy and diluted to 2.times.10.sup.5 cells/ml in RPMI+10%
FBS. 20,000 cells (50 .mu.l/well) were dispensed into 96-well
culture treated plates. Cross-linking antibody goat anti-human IgG
Fc (Rockland catalog #709-1117) was diluted to 80 .mu.g/ml in
RPMI+10% FBS and 25 .mu.l/well was dispensed into half of the Ramos
cell assay plate. Diluent alone was dispensed (25 .mu.l/well) into
the remainder of the plate. Anti-CD22 human antibodies and human
IgG1 isotype control were diluted to 20 .mu.g/ml in RPMI+10% FBS
and 25 .mu.l/well was dispensed in triplicate to both Ramos plus
cross-linker and Ramos plus diluent containing wells such that the
final antibody concentrations were 5 .mu.g/ml anti-CD22+/-20
.mu.g/ml cross-linking antibody. Assay plates were incubated at
37.degree. C. for 72 hours. Cell viability was assayed with the
addition of CellTiter-Glo reagent (Promega G7571), 100 .mu.l/well,
for 10 minutes. Luminescence was measured using Luminescence Test 1
plate Nunc96 on Pherastar GMB Labtech. Data was analyzed as %
growth inhibition relative to the human IgG1 isotype control, which
represented 0% inhibition.
[0775] Seventeen different anti-CD22 human antibodies were
evaluated in the assay. The results showed that none of the
seventeen anti-CD22 antibodies tested significantly altered the
rate of Ramos cell proliferation, either when the antibodies were
not cross-linked or when they were cross-linked. These results
indicate that none of the anti-CD22 antibodies have a direct
anti-proliferative effect on Ramos cells, even if the antibody is
cross-linked.
Example 9
Assessment of CDC Activity of Anti-CD22 Antibodies
[0776] In this example, the ability of soluble anti-CD22 human
antibodies to mediate complement dependent cytotoxicity (CDC) was
examined. In the assay used, viable Ramos cells (ATCC Accession No.
CRL-1596) were counted by trypan blue exclusion microscopy and
diluted to 1.times.10.sup.6 cells/ml in CDC buffer (RPMI 1640+0.1%
BSA+20 mM HEPES+1% Pen/strep). The cell suspension was dispensed as
50,000 cells (50 .mu.l/well) in a 96-well flat-bottomed tissue
culture treated plate. Human complement (Quidel catalog # A113) was
heat-inactivated by incubating 1 hr at 56.degree. C. Active and
heat-inactivated complement were each diluted 1:3 in CDC buffer and
were dispensed 50 .mu.l/well into the Ramos assay plates. Anti-CD22
human antibodies, human IgG isotype control and an anti-CD20
positive control antibody were each diluted to 40 .mu.g/ml in CDC
buffer. Diluted antibodies were dispensed 50 .mu.l/well in
duplicate into the Ramos assay plates with both active and
heat-inactivated complement such that the final concentration of
antibody was 10 .mu.g/ml. The assay plates were incubated 2 hrs at
37.degree. C. To analyze cell viability, alamar blue reagent
(BioSource catalog # DAL1100) was added 50 .mu.l/well and the
plates were incubated a further 21 hrs at 37.degree. C. Cell
viability was assayed as being proportional to fluorescence measure
using the SPECTROMAX GEMINI fluorescence plate reader (Molecular
Devices S/N G 02243).
[0777] Eighteen different anti-CD22 antibodies were tested, along
with, as a positive control, an anti-CD20 antibody known to exhibit
robust cytotoxicity in the presence of active but not heat
inactivated complement. The results showed that none of the
anti-CD22 antibodies tested exhibited significant CDC activity as
compared to the human IgG isotype control.
Example 10
Inhibition of Solid Tumor Cell Proliferation In Vivo by Anti-CD22
Antibody-Drug Conjugates
[0778] To determine whether drug conjugates of CD22.1 and CD22.2
could be made which could effectively inhibit proliferation of an
established solid tumor in vivo, the anti-CD22 recombinant
antibodies CD22.1 and CD22.2 were conjugated to the cytotoxic drug
Cytotoxin A and the efficacy of the resulting ADC compounds were
examined using a Ramos subcutaneous tumor cell model.
Conjugation of CD22.1 and CD22.2 to Cytotoxic Compound Cytotoxin
A
[0779] CD22.1 and CD22.2 were concentrated to approximately 5
mg/ml, buffer exchanged into 20 mM phosphate buffer, 50 mM NaCl, 2
mM DTPA, 3% Glycerol, pH 7.5 and thiolated with a 14-fold molar
excess of 2-Imminothiolane for 60 minutes at room temperature.
Following thiolation, the antibody was buffer exchanged into 50 mM
HEPES buffer, containing 5 mM glycine, 2 mM DTPA, and 0.5% Povidone
(10 K) pH 5.5. Thiolation was quantified with 4,
4''-dithiodipyridine by measuring thiopyridine release at 324 nM.
Conjugation was achieved by addition of Cytotoxin A at a 3:1 molar
ratio of Cytotoxin A to thiols. Incubation was at room temperature
for 60 minutes followed by blocking of any residual thiols by the
addition of a 10:1 molar ratio of N-ethylmaleimide to thiols to the
reaction mix.
[0780] The resulting conjugates were purified by ion-exchange
chromatography. Each reaction mix was filtered and loaded onto an
SP-Sepharose High Performance column equilibrated with Buffer A (50
mM HEPES, 5 mM Glycine, 0.5% Povidone (10K), pH 5.5). Antibody
conjugates were eluted with 24% Buffer B (50 mM HEPES, 5 mM
Glycine, 1M NaCl, 0.5% Povidone (10K), pH 5.5). Fractions
containing monomeric antibody-Cytotoxin A conjugate were pooled and
dialyzed against 50 mM HEPES, 5 mM glycine, 100 mM NaCl, 0.5%
Povidone (10K), pH 6.0. Substitution ratios were determined by
measuring absorbance at 280 and 340 nm, and the conjugates analyzed
by SEC-HPLC.
[0781] CD22.1-Cytotoxin A conjugate was made with a substitution
ratio of 1.7, and CD22.2 conjugate was made with a substitution
ratio of 1.6.
In Vivo Efficacy of Anti-CD22 Antibody-Drug Conjugates
CD22.1-Cytotoxin A and CD22.2-Cytotoxin A
[0782] SCID mice were implanted subcutaneously with Raji cells at
10 million cells per mouse in matrigel, and tumors allowed to grow
until well established with a median size of approx. 190 mm.sup.3.
Groups of 8 mice were then treated with a single dose of either
CD22.1-Cytotoxin A antibody conjugate, CD22.2-Cytotoxin A
conjugate, a control human IgG-Cytotoxin A conjugate which did not
bind to Raji cells, or with vehicle alone. Tumor size was monitored
for 63 days post dosing, or until animals were euthanized due to
tumor growth beyond 1500 mm.sup.3. CD22.1-Cytotoxin A was
administered at 0.18 .mu.mol/kg drug equivalent, and
CD22.2-Cytotoxin A and control ab-Cytotoxin A were administered at
0.3 .mu.mol/kg drug equivalent. Results demonstrated good
anti-tumor efficacy for both CD22.1 and CD22.2 conjugates (FIG.
16).
SUMMARY OF SEQUENCE LISTING
TABLE-US-00003 [0783] SEQ ID NO: SEQUENCE SEQ ID NO: SEQUENCE 1
V.sub.H CDR1 a.a. 12C5 31 V.sub.H a.a. 12C5 2 V.sub.H CDR1 a.a.
19A3 32 V.sub.H a.a. 19A3 3 V.sub.H CDR1 a.a. 16F7 33 V.sub.H a.a.
16F7 4 V.sub.H CDR1 a.a. 23C6 34 V.sub.H a.a. 23C6 5 V.sub.H CDR2
a.a. 12C5 35 V.sub.K a.a. 12C5 6 V.sub.H CDR2 a.a. 19A3 36 V.sub.K
a.a. 19A3 7 V.sub.H CDR2 a.a. 16F7 37 V.sub.K.1 a.a. 16F7 8 V.sub.H
CDR2 a.a. 23C6 38 V.sub.K.2 a.a. 16F7 39 V.sub.K.1 a.a. 23C6 9
V.sub.H CDR3 a.a. 12C5 40 V.sub.K.2 a.a. 23C6 10 V.sub.H CDR3 a.a.
19A3 11 V.sub.H CDR3 a.a. 16F7 41 V.sub.H n.t. 12C5 12 V.sub.H CDR3
a.a. 23C6 42 V.sub.H n.t. 19A3 43 V.sub.H n.t. 16F7 13 V.sub.K CDR1
a.a. 12C5 44 V.sub.H n.t. 23C6 14 V.sub.K CDR1 a.a. 19A3 15
V.sub.K.1 CDR1 a.a. 16F7 45 V.sub.K n.t. 12C5 16 V.sub.K.2 CDR1
a.a. 16F7 46 V.sub.K n.t. 19A3 17 V.sub.K.1 CDR1 a.a. 23C6 47
V.sub.K.1 n.t. 16F7 18 V.sub.K.2 CDR1 a.a. 23C6 48 V.sub.K.2 n.t.
16F7 49 V.sub.K.1 n.t. 23C6 19 V.sub.K CDR2 a.a. 12C5 50 V.sub.K.2
n.t. 23C6 20 V.sub.K CDR2 a.a. 19A3 21 V.sub.K.1 CDR2 a.a. 16F7 51
V.sub.H 7-4.1 germline a.a. 22 V.sub.K.2 CDR2 a.a. 16F7 52 V.sub.H
4-34 germline a.a. 23 V.sub.K.1 CDR2 a.a. 23C6 53 V.sub.H 5-51
germline a.a. 24 V.sub.K.2 CDR2 a.a. 23C6 54 V.sub.H 1-69 germline
a.a. 25 V.sub.K CDR3 a.a. 12C5 55 V.sub..lamda. 2b2 germline a.a.
26 V.sub.K CDR3 a.a. 19A3 56 V.sub.K L6 germline a.a. 27 V.sub.K.1
CDR3 a.a. 16F7 57 V.sub.K A27 germline a.a. 28 V.sub.K.2 CDR3 a.a.
16F7 58 V.sub.K A10 germline a.a. 29 V.sub.K.1 CDR3 a.a. 23C6 30
V.sub.K.2 CDR3 a.a. 23C6 59 human CD22 (NP_001762) 60 V.sub.H CDR2
a.a. CD22.2 61 V.sub.H a.a. CD22.2 62 V.sub.H n.t. CD22.2 63
V.sub.H CDR1 a.a. 4G6 81 V.sub.H a.a. 4G6 64 V.sub.H1 CDR1 a.a.21F6
82 V.sub.H1 a.a. 21F6 65 V.sub.H2 CDR1 a.a. 21F6 83 V.sub.H2 a.a.
21F6 66 V.sub.H CDR2 a.a. 4G6 84 V.sub.K1 a.a. 4G6 67 V.sub.H1CDR2
a.a. 21F6 85 V.sub.K2 a.a. 4G6 68 V.sub.H2 CDR2 a.a. 21F6 86
V.sub.K a.a. 21F6 69 V.sub.H CDR3 a.a. 4G6 70 V.sub.H1 CDR3 a.a.
21F6 87 V.sub.H n.t. 4G6 71 V.sub.H2 CDR3 a.a. 21F6 88 V.sub.H1
n.t. 21F6 89 V.sub.H2 n.t. 21F6 72 V.sub.K1 CDR1 a.a. 4G6 73
V.sub.K2 CDR1 a.a. 4G6 90 V.sub.K1 n.t. 4G6 74 V.sub.K CDR1 a.a.
21F6 91 V.sub.K2 n.t. 4F6 92 V.sub.K2 n.t. 21F6 75 V.sub.K1 CDR2
a.a. 4G6 76 V.sub.K2 CDR2 a.a. 4G6 93 V.sub.K1 L18 germline a.a. 77
V.sub.K CDR2 a.a. 21F6 94 Peptide Linker 78 V.sub.K1 CDR3 a.a. 4G6
95 Peptide Linker 79 V.sub.K2 CDR3 a.a. 4G6 96 Peptide Linker 80
V.sub.K CDR3 a.a. 21F6 97 Peptide Linker 98 Peptide Linker 99
Peptide Linker 100 Peptide Linker 101 Peptide Linker 102 Peptide
Linker 103 Peptide Linker 104 Peptide Linker 105 Peptide Linker 106
Peptide Linker 107 Peptide Linker 108 12C5 JH6b germline 118 21F6
4-34 germline VH1 109 JL2 germline 119 21F6 JH4b germline VH1 110
JK1 germline 120 21F6 4-34 germline VH2 111 JK4b germline 121 21F6
JH4b germline VH2 112 JK3b germline 122 21F6 VK L6 germline 113 JK1
germline 123 21F6 VK JK4 germline 114 JK2 germline 124 4G6 VH 1-69
germline 115 2-15 germline 125 4G6 VH JH4b germline 116 JK1
germline 126 4G6 VK1 JK2 germline 117 JH4b germline 127 4G6 VK2 A27
germline 128 4G6 VK2 JK4 germline
Sequence CWU 1
1
11415PRTHomo sapiens 1Ser Tyr Ala Met Asn 1 5 25PRTHomo sapiens
2Ser Tyr Tyr Trp Ser 1 5 35PRTHomo sapiens 3Ser Tyr Trp Ile Gly 1 5
45PRTHomo sapiens 4Ser Tyr Gly Ile Asn 1 5 517PRTHomo sapiens 5Trp
Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe Thr 1 5 10
15 Gly 616PRTHomo sapiens 6Asp Ile Asn His Ser Gly Ser Thr Asn Tyr
Asn Pro Ser Leu Lys Ser 1 5 10 15 717PRTHomo sapiens 7Ile Ile Tyr
Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln 1 5 10 15 Gly
817PRTHomo sapiens 8Glu Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala
Gln Lys Phe Gln 1 5 10 15 Gly 910PRTHomo sapiens 9Leu Phe Tyr Tyr
Tyr Phe Gly Met Asp Val 1 5 10 1014PRTHomo sapiens 10Thr Phe Tyr
Asp Ile Leu Thr Gly Tyr Tyr Pro Leu Gly Tyr 1 5 10 1112PRTHomo
sapiens 11Pro Thr Tyr Tyr Phe Gly Ser Val Ala Phe Asp Ile 1 5 10
1219PRTHomo sapiens 12Asp Gln Gly Val Val Val Val Ala Ala Thr His
Tyr Tyr Tyr Tyr Gly 1 5 10 15 Met Asp Val 1314PRTHomo sapiens 13Thr
Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn Leu Val Ser 1 5 10
1411PRTHomo sapiens 14Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1
5 10 1512PRTHomo sapiens 15Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr
Leu Ala 1 5 10 1611PRTHomo sapiens 16Arg Ala Ser Gln Ser Ile Gly
Ser Ser Leu His 1 5 10 1711PRTHomo sapiens 17Arg Ala Ser Gln Ser
Val Ser Ser Tyr Leu Ala 1 5 10 1811PRTHomo sapiens 18Arg Ala Ser
Gln Ser Val Ser Asn Phe Leu Ala 1 5 10 197PRTHomo sapiens 19Glu Val
Ser Lys Arg Pro Ser 1 5 207PRTHomo sapiens 20Asp Ala Ser Asn Arg
Ala Thr 1 5 217PRTHomo sapiens 21Gly Ala Ser Ser Arg Ala Thr 1 5
227PRTHomo sapiens 22Tyr Ala Ser Gln Ser Phe Ser 1 5 237PRTHomo
sapiens 23Asp Ala Ser Asn Arg Ala Thr 1 5 247PRTHomo sapiens 24Asp
Ala Ser Asn Arg Ala Thr 1 5 2510PRTHomo sapiens 25Cys Ser Tyr Ala
Asn Ser Ser Thr Leu Val 1 5 10 268PRTHomo sapiens 26Gln Gln Arg Ser
Asn Trp Pro Thr 1 5 279PRTHomo sapiens 27Gln Gln Tyr Gly Ser Ser
Pro Pro Thr 1 5 289PRTHomo sapiens 28His Gln Ser Ser Ser Leu Pro
Tyr Thr 1 5 299PRTHomo sapiens 29Gln Gln Arg Ser Asn Trp Pro Trp
Thr 1 5 309PRTHomo sapiens 30Gln Gln Arg Ser Asn Trp Pro Pro Thr 1
5 31119PRTHomo sapiens 31Gln Val Gln Leu Val Gln Ser Gly Ser Glu
Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn
Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe 50 55 60 Thr Gly
Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Leu Phe Tyr Tyr Tyr Phe Gly Met Asp Val Trp Gly Gln
Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 32122PRTHomo
sapiens 32Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Arg Ser
Phe Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn His Ser Gly Ser
Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Gly Thr
Phe Tyr Asp Ile Leu Thr Gly Tyr Tyr Pro Leu Gly Tyr Trp 100 105 110
Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 33121PRTHomo
sapiens 33Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Asn
Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser
Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile
Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser
Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr
Pro Thr Tyr Tyr Phe Gly Ser Val Ala Phe Asp Ile Trp Gly 100 105 110
Gln Gly Thr Met Val Thr Val Ser Ser 115 120 34128PRTHomo sapiens
34Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Thr Gly Ser 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30 Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Glu Ile Ile Pro Ile Phe Gly Thr Ala Asn
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp
Glu Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Gly
Val Val Val Val Ala Ala Thr His Tyr Tyr Tyr 100 105 110 Tyr Gly Met
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125
35110PRTHomo sapiens 35Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Leu Val Ser Trp Tyr Gln
Leu His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val
Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser
Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Asn Ser 85 90
95 Ser Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
110 36106PRTHomo sapiens 36Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser
Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr 85
90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 37108PRTHomo
sapiens 37Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Pro Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 38107PRTHomo
sapiens 38Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr
Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Gly Ser Ser 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln
Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala
Ala Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Tyr 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 39107PRTHomo sapiens
39Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Arg Ser Asn Trp Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 40107PRTHomo sapiens 40Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asn Phe 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 41357DNAHomo sapiens 41caggtgcagc tggtgcaatc
tgggtctgag ttgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg cttctggata
caccttcact agttatgcta tgaattgggt gcgacaggcc 120cctggacaag
ggcttgagtg gatgggatgg atcaacacca acactgggaa cccaacgtat
180gcccagggct tcacaggacg gtttgtcttc tccttggaca cctctgtcag
cacggcatat 240ctgcagatca gcagcctaaa ggctgaggac actgccgtgt
attactgtgc taggttattc 300tactactact tcggtatgga cgtctggggc
caagggacca cggtcaccgt ctcctca 35742366DNAHomo sapiens 42caggtgcagc
tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60acctgcgctg
tctatggtag gtccttcagt agttactact ggagctggat ccgccagccc
120ccagggaagg ggctggagtg gattggggac atcaatcata gtggaagcac
caactacaac 180ccgtccctca agagtcgagt caccatatca gtagacacgt
ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc cgcggacacg
gctgtgtatt actgtgcggg aacgttttac 300gatattttga ctggttatta
tccccttggg tactggggcc cgggaaccct ggtcaccgtc 360tcctca
36643363DNAHomo sapiens 43gaggtgcagc tggtgcagtc tggagcagag
gtgaaaaagc ccggggagtc tctgaagatc 60tcctgtaagg gttctggata caactttacc
agctactgga tcggctgggt gcgccagatg 120cccgggaaag gcctggagtg
gatggggatc atctatcctg gtgactctga taccagatac 180agcccgtcct
tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac
240ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc
gaccccgacg 300tattactttg gttcggtggc ttttgatatc tggggccaag
ggacaatggt caccgtctct 360tca 36344384DNAHomo sapiens 44caggtccagc
tggtgcagtc tggggctgag gtgaaaaaga ctgggtcctc ggtgaaggtc 60tcctgcaagg
cttctggagg caccttcagc agctatggta tcaactgggt gcgacaggcc
120cctggacaag ggcttgaatg gatgggagag atcatcccta tctttggtac
agcaaactac 180gcacagaagt tccagggcag agtcacgatt accgcggacg
aatccacgag cacagtctac 240atggagctga gcagcctgag agctgaggac
acggccgtgt attactgtgc gagagatcag 300ggtgtagtgg tggtagctgc
aacccactac tactactacg gtatggacgt ctggggccaa 360gggaccacgg
tcaccgtctc ctca 38445330DNAHomo sapiens 45cagtctgccc tgactcagcc
tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag
tgatgttggg agttataacc ttgtctcctg gtaccaactg 120cacccaggca
aagcccccaa actcatgatt tatgaggtca gtaagcggcc ctcaggggtt
180tctaatcgct tctctggctc caggtctggc aacacggcct ccctgacaat
ctctgggctc 240caggctgagg acgaggctga ttattactgc tgctcatatg
caaatagtag cactttggta 300ttcggcggag ggaccaagct gaccgtccta
33046318DNAHomo sapiens 46gaaattgtgt tgacacagtc tccagccacc
ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc
agctacttag cctggtacca acagaaacct 120ggccaggctc ccaggctcct
catctatgat gcatccaaca gggccactgg catcccagcc 180aggttcagtg
gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct
240gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctacgtt
cggccaaggg 300accaaggtgg aaatcaaa 31847324DNAHomo sapiens
47gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag
cagtatggta gctcacctcc gacgttcggc 300caagggacca aggtggaaat caaa
32448321DNAHomo sapiens 48gaaattgtgc tgactcagtc tccagacttt
cagtctgtga ctccaaagga gaaagtcacc 60atcacctgcc gggccagtca gagcattggt
agtagcttac actggtacca gcagaaacca 120gatcagtctc caaagctcct
catcaagtat gcttcccagt ccttctcagg ggtcccctcg 180aggttcagtg
gcagtggatc tgggacagat ttcaccctca ccatcaatag cctggaagct
240gaagatgctg cagcgtatta ctgtcatcag agtagtagtt taccgtacac
ttttggccag 300gggaccaagc tggagatcaa a 32149321DNAHomo sapiens
49gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact ggccgtggac gttcggccaa 300gggaccaagg tggaaatcaa a
32150321DNAHomo sapiens 50gaaattgtgt tgacacagtc tccagccacc
ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc
aacttcttag cctggtacca acagaaacct 120ggccaggctc ccaggctcct
catctatgat gcatccaaca gggccactgg catcccagcc 180aggttcagtg
gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct
240gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctccgac
gttcggccaa 300gggaccaagg tggaaatcaa a 3215198PRTHomo sapiens 51Gln
Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr
Ala Gln Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Leu Asp Thr
Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 5297PRTHomo
sapiens 52Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile
35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser
Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Arg 5398PRTHomo sapiens 53Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20
25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp
Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser
Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser
Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser
Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg 5498PRTHomo sapiens
54Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp
Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 5598PRTHomo
sapiens 55Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro
Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Ser Tyr 20 25 30 Asn Leu Val Ser Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg
Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly
Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser 85 90 95 Ser Thr
5695PRTHomo sapiens 56Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro 85 90 95
5797PRTHomo sapiens 57Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90
95 Pro 5895PRTHomo sapiens 58Glu Ile Val Leu Thr Gln Ser Pro Asp
Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Gly Ser Ser 20 25 30 Leu His Trp Tyr Gln
Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala
Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70
75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro
85 90 95 594PRTHomo sapiens 59Gln Leu Val Gln 1 6016PRTHomo sapiens
60Asp Ile Gln His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1
5 10 15 61122PRTHomo sapiens 61Gln Val Gln Leu Gln Gln Trp Gly Ala
Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala
Val Tyr Gly Arg Ser Phe Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile
Gln His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70
75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95 Gly Thr Phe Tyr Asp Ile Leu Thr Gly Tyr Tyr Pro Leu
Gly Tyr Trp 100 105 110 Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115
120 62366DNAHomo sapiens 62caggtgcagc tacagcagtg gggcgcagga
ctgttgaagc cttcggagac cctgtccctc 60acctgcgctg tctatggtag gtccttcagt
agttactact ggagctggat ccgccagccc 120ccagggaagg ggctggagtg
gattggggac atccaacata gtggaagcac caactacaac 180ccgtccctca
agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg
240aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcggg
aacgttttac 300gatattttga ctggttatta tccccttggg tactggggcc
cgggaaccct ggtcaccgtc 360tcctca 3666319PRTHomo sapiens 63Tyr Tyr
Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 1 5 10 15
Val Ser Ser 6412PRTHomo sapiens 64Val Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 1 5 10 6511PRTHomo sapiens 65Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 1 5 10 6613PRTHomo sapiens 66Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 6715PRTHomo sapiens
67Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5
10 15 6811PRTHomo sapiens 68Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 1 5 10 6912PRTHomo sapiens 69Tyr Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 1 5 10 709PRTHomo sapiens 70Asp Ile Val Val Val Val
Ala Ala Thr 1 5 7112PRTHomo sapiens 71Trp Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 1 5 10 7213PRTHomo sapiens 72Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 73366DNAHomo sapiens
73caggtccagt tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60tcctgcaagc cttctggaga caccttcagc aactatgcta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggaagg atcatcccta tccttggtat
ggctatctac 180gcaccgaagt tccagggcag agttacgatt accgcggaca
aatccacgaa cacagccttc 240atggatctta ccagcctgta ttttgaggac
acggccgtgt attactgtgc gagagcccca 300acttactggg gatcgaagga
ctactttgac tactggggcc agggaaccct ggtcaccgtc 360tcctca
36674121PRTHomo sapiens 74Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro
Ser Gly Asp Thr Phe Ser Asn Tyr 20 25 30 Ala Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile
Pro Ile Leu Gly Met Ala Ile Tyr Ala Pro Lys Phe 50 55 60 Gln Gly
Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Phe 65 70 75 80
Met Asp Leu Thr Ser Leu Tyr Phe Glu Asp Ala Val Tyr Tyr Cys Ala 85
90 95 Arg Ala Pro Thr Tyr Trp Gly Ser Lys Asp Tyr Phe Asp Tyr Trp
Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
755PRTHomo sapiens 75Asn Tyr Ala Ile Ser 1 5 7617PRTHomo sapiens
76Arg Ile Ile Pro Ile Leu Gly Met Ala Ile Tyr Ala Pro Lys Phe Gln 1
5 10 15 Gly 7713PRTHomo sapiens 77Ala Pro Thr Tyr Trp Gly Ser Lys
Asp Tyr Phe Asp Tyr 1 5 10 78321DNAHomo sapiens 78gccatccagt
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca ggacattagc agtggtttag cctggtatca gcagaaacca
120gggacagctc ctaagctcct gatctatgat gcctccagtt tggaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 240gacgattttg caacttatta ctgtcaacag
tttaatagtt tcccgtacac ttttggccag 300gggaccaagc tggagatcaa a
32179107PRTHomo sapiens 79Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Ile Ser Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Thr Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser
Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Phe Pro Tyr 85
90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
8011PRTHomo sapiens 80Arg Ala Ser Gln Asp Ile Ser Ser Gly Leu Ala 1
5 10 817PRTHomo sapiens 81Asp Ala Ser Ser Leu Glu Ser 1 5
829PRTHomo sapiens 82Gln Gln Phe Asn Ser Phe Pro Tyr Thr 1 5
83321DNAHomo sapiens 83gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact
tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat
ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg
gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagtatggta gctcacccac tttcggcgga
300gggaccaagg tggagatcaa a 32184107PRTHomo sapiens 84Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Ser Ser Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 8512PRTHomo sapiens 85Arg Ala Ser Gln Ser Val
Ser Ser Ser Tyr Leu Ala 1 5 10 867PRTHomo sapiens 86Gly Ala Ser Ser
Arg Ala Thr 1 5 878PRTHomo sapiens 87Gln Gln Tyr Gly Ser Ser Pro
Thr 1 5 88366DNAHomo sapiens 88caggtgcagc tacagcagtg gggcgcagga
ctgttgaagc cttcggagac cctgtccctc 60acctgcgctg tctatggtgg gtccttcagt
ggtcactact ggagctggat ccgccagtcc 120ccagggaagg ggctggagtg
gattggggaa accgatcata gtggaagcac caactacaat 180ccgtccctca
agagtcgagt caccatatca atagacacgt ccaagaatca gttctccctg
240aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag
gacgtattac 300gatattttga ctgattatta cccctttgac tcctggggcc
agggaaccct ggtcaccgtc 360tcctca 36689122PRTHomo sapiens 89Gln Val
Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly His 20
25 30 Tyr Trp Ser Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45 Gly Glu Thr Asp His Ser Gly Ser Thr Asn Tyr Asn Pro
Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Ile Asp Thr Ser Lys
Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Tyr Tyr Asp Ile Leu
Thr Asp Tyr Tyr Pro Phe Asp Ser Trp 100 105 110 Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 905PRTHomo sapiens 90Gly His Tyr Trp
Ser 1 5 9116PRTHomo sapiens 91Glu Thr Asp His Ser Gly Ser Thr Asn
Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 9214PRTHomo sapiens 92Thr Tyr
Tyr Asp Ile Leu Thr Asp Tyr Tyr Pro Phe Asp Ser 1 5 10 93366DNAHomo
sapiens 93caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac
cctgtccctc 60acctgcgctg tctatggtgg gtccttcagt ggtcactact ggagctggat
ccgccagtcc 120ccagggaagg gactggagtg gattggggaa atcgatcata
gtggaagcac caactacaat 180ccgtccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc
cgcggacacg gctatgtatt actgtgcgag gacgtattac 300gatattttga
ctgattatta cccctttgac tcctggggcc agggaaccct ggtcaccgtc 360tcctca
36694122PRTHomo sapiens 94Gln Val Gln Leu Gln Gln Trp Gly Ala Gly
Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val
Tyr Gly Gly Ser Phe Ser Gly His 20 25 30 Tyr Trp Ser Trp Ile Arg
Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp
His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Met Tyr Tyr Cys Ala 85
90 95 Arg Thr Tyr Tyr Asp Ile Leu Thr Asp Tyr Tyr Pro Phe Asp Ser
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
955PRTHomo sapiens 95Gly His Tyr Trp Ser 1 5 9616PRTHomo sapiens
96Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1
5 10 15 9725PRTHomo sapiens 97Thr Tyr Tyr Asp Ile Leu Thr Asp Tyr
Tyr Pro Phe Asp Ser Trp Gly 1 5 10 15 Gln Gly Thr Leu Val Thr Val
Ser Ser 20 25 98321DNAHomo sapiens 98gaaattgtgt tgacacagtc
tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca
gagtgttagc ggctacttag cctggtacca acagaaacct 120ggccaggctc
ccaggctcct catctatgat gtatcctaca gggccactgg catcctagtc
180aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag
cctagagcct 240gaagattttg cagtttatta ctgtcagcag cgtagcaact
ggcccatcac tttcggcgga 300gggaccaagg tggagatcaa a 32199107PRTHomo
sapiens 99Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Gly Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Val Ser Tyr Arg Ala Thr
Gly Ile Leu Val Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg
Ser Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105 10011PRTHomo sapiens 100Arg Ala Ser Gln Ser Val Ser
Gly Tyr Leu Ala 1 5 10 1017PRTHomo sapiens 101Asp Val Ser Tyr Arg
Ala Thr 1 5 1029PRTHomo sapiens 102Gln Gln Arg Ser Asn Trp Pro Ile
Thr 1 5 10397PRTHomo sapiens 103Gln Val Gln Leu Gln Gln Trp Gly Ala
Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala
Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile
Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70
75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95 Arg 10414PRTHomo sapiens 104Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 1 5 10 10597PRTHomo sapiens 105Gln
Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10
15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr
20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn
Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser
Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg 10614PRTHomo sapiens
106Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10
10795PRTHomo sapiens 107Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro 85 90 95
10811PRTHomo sapiens 108Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
1 5 10 10998PRTHomo sapiens 109Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile
Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg 11015PRTHomo sapiens 110Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 15 11193PRTHomo
sapiens 111Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Phe Asn Ser 85 90 11212PRTHomo sapiens
112Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10
11396PRTHomo sapiens 113Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90
95 11411PRTHomo sapiens 114Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 1 5 10
* * * * *
References