U.S. patent application number 12/888312 was filed with the patent office on 2011-03-10 for anti-robo4 antibodies and uses therefor.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Richard Carano, Alexander W. Koch, Franklin V. Peale, JR., Scott Stawicki, Ryan J. Watts, Yan Wu.
Application Number | 20110059112 12/888312 |
Document ID | / |
Family ID | 39591442 |
Filed Date | 2011-03-10 |
United States Patent
Application |
20110059112 |
Kind Code |
A1 |
Koch; Alexander W. ; et
al. |
March 10, 2011 |
ANTI-ROBO4 ANTIBODIES AND USES THEREFOR
Abstract
The invention provides anti-Robo4 antibodies, and compositions
comprising the antibodies and methods of using these antibodies,
including diagnostic and therapeutic methods.
Inventors: |
Koch; Alexander W.;
(Millbrae, CA) ; Stawicki; Scott; (San Francisco,
CA) ; Wu; Yan; (Foster City, CA) ; Carano;
Richard; (San Ramon, CA) ; Peale, JR.; Franklin
V.; (San Carlos, CA) ; Watts; Ryan J.; (San
Mateo, CA) |
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
39591442 |
Appl. No.: |
12/888312 |
Filed: |
September 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12028124 |
Feb 8, 2008 |
7834154 |
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12888312 |
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60891475 |
Feb 23, 2007 |
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60889214 |
Feb 9, 2007 |
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Current U.S.
Class: |
424/172.1 ;
530/387.3; 530/389.1; 530/391.5 |
Current CPC
Class: |
A61P 37/06 20180101;
C07K 2317/55 20130101; A61P 9/10 20180101; A61P 17/06 20180101;
A61K 2039/505 20130101; A61P 27/02 20180101; C07K 2317/77 20130101;
A61P 29/00 20180101; C07K 2317/76 20130101; A61P 35/00 20180101;
C07K 16/2803 20130101; A61P 19/02 20180101; C07K 16/30 20130101;
A61P 35/04 20180101; C07K 2317/565 20130101; C07K 2317/92
20130101 |
Class at
Publication: |
424/172.1 ;
530/389.1; 530/387.3; 530/391.5 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; A61P 35/00 20060101
A61P035/00 |
Claims
1. An isolated anti-Robo4 antibody comprising: (i) HVR-L1
comprising SEQ ID NO:1 (ii) HVR-L2 comprising SEQ ID NO:2 (iii)
HVR-L3 comprising SEQ ID NO:3 (iv) HVR-H1 comprising SEQ ID NO:4
(v) HVR-H2 comprising SEQ ID NO:5; (vi) HVR-H3 comprising SEQ ID
NO:6.
2. The antibody of claim 1, comprising a light chain variable
domain comprising an amino acid sequence selected from the group
consisting of: SEQ ID NOS:72-97 as shown in FIGS. 1A and 2A.
3. The antibody of claim 1, comprising a heavy chain variable
domain comprising an amino acid sequence selected from the group
consisting of: SEQ ID NOS:140-165 as shown in FIGS. 1B and 2B.
4-5. (canceled)
6. The antibody of claim 1, comprising (i) a light chain variable
domain comprising an HVR-L1 comprising SEQ ID NO:1, an HVR-L2
comprising SEQ ID NO:2, and an HVR-L3 comprising SEQ ID NO:3; and
(ii) a heavy chain variable domain comprising an HVR-H1 comprising
SEQ ID NO:4, an HVR-H2 comprising SEQ ID NO:5, and an HVR-H3
comprising SEQ ID NO:6.
7. The antibody of claim 1, wherein the antibody is humanized.
8. The antibody of claim 1, wherein the antibody is selected from
the group consisting of a Fab, a Fab', and a (Fab').sub.2.
9. The antibody of claim 1, further comprising a cytotoxic
agent.
10. The antibody of claim 9, wherein the cytotoxic agent is
selected from the group consisting of:
N.sup.2'-deacetyl-N-.sup.2'(3-mercapto-1-oxopropyl)-maytansine
(DM1), monomethyl auristatin E (MMAE), monomethyl auristatin F
(MMAF), and combinations thereof.
11. The antibody of claim 10, further comprising a detectable
label.
12. The antibody of claim 11, wherein the detectable label is
selected from the group consisting of: biotin, a fluorescent dye, a
radionuclide, chelating agent 1, 4, 7,
10-tetraazacyclododecane-N,N', N'', N'''-tetraacetic acid (DOTA), a
microbubble, a perfluorocarbon nanoparticle emulsion, a metallic
particle, and combinations thereof.
13. The antibody of claim 11, wherein the detectable label is
covalently attached to the antibody at a cysteine residue.
14. The antibody of claim 13, wherein the cysteine residue is at
position 118 of the heavy chain Fc region according to EU
numbering.
15-27. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/028,124, filed Feb. 8, 2008, which claims
the benefit of U.S. Provisional Patent Applications Nos.
60/889,214, filed Feb. 9, 2007 and 60/891,475, filed Feb. 23, 2007,
the disclosures of which are hereby incorporated by reference in
their entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the fields of
angiogenesis, and endothelial cell proliferation and migration.
More specifically, the invention concerns modulators of Robo4, and
uses of such modulators.
BACKGROUND OF THE INVENTION
[0003] The Roundabout family of receptors are molecular guidance
molecules which regulate axon guidance, neuronal migration and
leukocyte chemotaxis in response to interaction with Slit proteins
(Suchting et al., FASEB J. 19:121-123 (2005)). Roundabout receptor
molecules contain five immunoglobulin and three fibronectin domains
in their extracellular region. Magic Roundabout (i.e., Robo4 or
endothelial cell-specific molecule 4 (ESCSM4)) is structurally
distinct from other roundabout family members. Human Magic
Roundabout (Robo4) comprises two immunoglobulin and major
histocompatibility complex domains (amino acids 46-116 and
151-209), two fibronectin type III domains (amino acids 252-335 and
347-432), a transmembrane region (468-490), and a proline rich
region (amino acids 715-772) (see Huminiecki et al., Genomics
79(4):547-552 (2002), and FIG. 1 therein). Mouse and human Robo4
show 75% nucleotide sequence identity (Huminiecki et al., supra
(2002)).
[0004] Robo4 expression analysis indicated that Robo4 expression is
highly restricted, with strong expression in placenta and tumors
including brain, bladder, and colonic metastasis to the liver,
where tumor expression is restricted to tumor vasculature
(Huminiecki et al., supra (2002)). In addition, Robo4 expression is
associated with sites of active angiogenesis, but is not detected
in neuronal tissue (Huminiecki, L. et al., supra (2002)).
[0005] As a membrane associated receptor with extracellular
domains, Robo4 is a useful target for delivering cytotoxic
therapeutics for inhibition of vascular endothelial cell
proliferation during angiogenesis. Robo4 is also a useful target to
delivery of detectable markers to proliferating vascular
endothelial cells. Antibody conjugates targeted to Robo4 (also
referred to as ECSM4) have also been reported as potential
therapeutic compounds where the conjugate comprises a cytotoxin and
as potential diagnostic markers where the conjugate comprises a
detectable label (see, for example, WO 2002036771).
[0006] The use of antibody-drug conjugates for the local delivery
of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos,
Anticancer Res. 19:605-614 (1999); Niculescu-Duvaz and Springer,
Adv. Drg Del. Rev. 26:151-172 (1997); U.S. Pat. No. 4,975,278)
allows targeted delivery of the drug moiety to tumors, and
intracellular accumulation of the drugs.
[0007] A number of antibody-drug conjugates that target other
molecules have been or are being developed. For example,
ZEVALIN.RTM. (ibritumomab tiuxetan, Biogen/Idec) is an
antibody-radioisotope conjugate composed of a murine IgG1 kappa
monoclonal antibody directed against the CD20 antigen found on the
surface of normal and malignant B lymphocytes and .sup.111In or
.sup.90Y radioisotope bound by a thiourea linker-chelator (Wiseman
et al., Eur. Jour. Nucl. Med. 27(7):766-77 (2000); Wiseman et al.
Blood 99(12):4336-42 (2002); Witzig et al., J. Clin. Oncol.
20(10):2453-63 (2002); Witzig et al., J. Clin. Oncol.
20(15):3262-69 (2002)). Although ZEVALIN has activity against
B-cell non-Hodgkin's Lymphoma (NHL), administration results in
severe and prolonged cytopenias in most patients. MYLOTARG.TM.
(gemtuzumab ozogamicin, Wyeth Pharmaceuticals), composed of a
huCD33 antibody linked to calicheamicin, was approved in 2000 for
the treatment of acute myeloid leukemia by injection (Drugs of the
Future (2000) 25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233;
5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285; 5,773,001).
Cantuzumab mertansine (Immunogen, Inc.), composed of a huC242
antibody linked via the disulfide linker SPP to the maytansinoid
drug moiety, DM1, is being developed for the treatment of cancers
that express CanAg antigen, such as colon, pancreatic, gastric, and
others. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen
Inc.), composed of an anti-prostate specific membrane antigen
(PSMA) monoclonal antibody linked to the maytansinoid drug moiety,
DM1, is being developed for the potential treatment of prostate
tumors. The same maytansinoid drug moiety, DM1, was linked through
a non-disulfide noncleavable linker, SMCC, to a mouse murine
monoclonal antibody, TA.1 (Chari et al., Cancer Res. 52:127-131
(1992)). This conjugate was reported to be 200-fold less potent
than the corresponding disulfide linker conjugate.
[0008] The auristatin peptides, auristain E (AE) and
monomethylauristatin (MMAE), synthetic analogs of dolastatin, have
been conjugated to: (i) cBR96, a chimeric monoclonal antibody
specific for Lewis Y on carcinomas; (ii) cAC10 which is specific to
CD30 on hematological malignancies (Klussman et al., Bioconjugate
Chemistry 15(4):765-773 (2004); Doronina et al. Nature Biotech.
21(7):778-784 (2003); Francisco et al. Blood 102(4):1458-1465
(2003); U.S. Patent Publication No. 2004/0018194; (iii) anti-CD20
antibodies such as Rituxan.RTM. (rituximab) (WO 04/032828) for the
treatment of CD20-expressing cancers and immune disorders; (iv)
anti-EphB2 antibodies 2H9 and anti-IL-8 for treatment of colorectal
cancer (Mao et al. Cancer Res. 64(3):781-788 (2004)); (v)
E-selectin antibody (Bhaskar et al. Cancer Res. 63:6387-6394
(2003)); and (vi) other anti-CD30 antibodies (WO 03/043583).
Monomethylauristatin (MMAE) has also been conjugated to 2H9, an
antibody against EphB2R which is a type 1 TM tyrosine kinase
receptor with close homology between mouse and human, and is
over-expressed in colorectal cancer cells (Mao et al., Cancer Res.
64:781-788 (2004)).
[0009] Monomethylauristatin MMAF, a variant of auristatin E (MMAE)
with a phenylalanine at the C-terminus (U.S. Pat. Nos. 5,767,237
and 6,124,431), has been reported to be less potent than MMAE, but
more potent when conjugated to monoclonal antibodies (Senter et al,
Proceedings of the American Association for Cancer Research, Volume
45, Abstract Number 623, presented Mar. 28, 2004). Auristatin F
phenylene diamine (AFP); a phenylalanine variant of MMAE was linked
to an anti-CD70 mAb, 1F6, through the C-terminus of 1F6 via a
phenylene diamine spacer (Law et al, Proceedings of the American
Association for Cancer Research, Volume 45, Abstract Number 625,
presented Mar. 28, 2004).
[0010] There exists a need in the art for additional drugs to treat
diseases and disorders associated angiogenesis including, e.g.,
aberrant angiogenesis associated with cancers dependent on the
growth and proliferation of vasculature of endothelial origin.
There also exists a need in the art for additional endothelial
cell-targeted anti-Robo4 antibody-drug conjugates for the detection
and visualization of blood vessel growth and proliferation in, for
example, cancers or ocular disorders supported or caused by excess
proliferation of blood vessels, as well as in disorders or other
physiological states in which monitoring blood vessel growth is
useful in understanding or treating the physiological state. The
present invention meets these and other needs.
SUMMARY OF THE INVENTION
[0011] The invention provides antibodies that specifically bind to
Robo4 (including, e.g., primate and/or rodent Robo4, such as human
and/or mouse Robo4) and diagnostic and therapeutic methods using
such antibodies. In some embodiments, the antibodies are humanized
or human. In some embodiments, the antibody is selected from the
group consisting of: an intact antibody, an antibody variant, and
antibody derivative, a Fab, a Fab', a (Fab').sub.2, and an Fv. In
one embodiment, the invention provides an anti-Robo4 antibody
having affinity and specificity for human Robo4 and, optionally,
murine Robo4, the antibody comprising, consisting of or consisting
essentially of hypervariable regions (HVRs) of a light chain and
heavy chain variable domain sequence as depicted in FIGS. 1A, 1B,
2A, and 2B (SEQ ID NOS:1-8, 17-71). In another embodiment, the
invention provides an anti-Robo4 antibody having affinity and
specificity for human Robo4 and, optionally, murine Robo4, the
antibody comprising, consisting of or consisting essentially of
framework regions (FRs) of a light chain and heavy chain variable
domain sequence as depicted in FIGS. 1A, 1B, 2A, 2B, 3A, 3B, 4A,
and 4B (SEQ ID NOS:9-16, SEQ ID NO:12, where V is replaced by M,
and SEQ ID NOS:99-139). In another embodiment, the invention
provides an anti-Robo4 antibody having affinity and specificity for
human Robo4 and, optionally, murine Robo4, the antibody comprising,
consisting of or consisting essentially of the light chain and
heavy chain variable domain sequences as depicted in FIGS. 1A and
1B (SEQ ID NOS:72-97) and FIGS. 2A and 2B (SEQ ID NOS:
140-165).
[0012] In one embodiment the invention provides an anti-Robo4
antibody comprising: at least one, two, three, four, five, or six
hypervariable region (HVR) sequence(s) selected from the group
consisting of:
[0013] (i) HVR-L1 comprising sequence A1-A11, wherein A1-A11 is
RASQDVSTAVA (SEQ ID NO:1)
[0014] (ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is
SASFLYS (SEQ ID NO:2)
[0015] (iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is
QQSYTTPPT (SEQ ID NO:3)
[0016] (iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is
GFTINGYYIH (SEQ ID NO:17)
[0017] (v) HVR-H2 comprising sequence E1-E18, wherein E1-E18 is
GFIYPAGGDTDYADSVKG (SEQ ID NO:18);
[0018] (vi) HVR-H3 comprising sequence F1-F17 wherein F1-F17 is
ARLIGNKFGWSSYGMDY (SEQ ID NO:19); and
[0019] (vii) at least one variant HVR, wherein the variant HVR
comprises an insertion, deletion, or substitution of at least one
amino acid residue of the sequence depicted in SEQ ID NO: 1, 2, 3,
17, 18, or 19.
[0020] In another embodiment, the invention provides an anti-Robo4
antibody comprising:
[0021] at least one, two, three, four, five or six hypervariable
region (HVR) sequences selected from the group consisting of:
[0022] (i) HVR-L1 comprising sequence A1-A11, wherein A1-A11 is
RASQDVSTAVA (SEQ ID NO:1)
[0023] (ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is
SASFLYS (SEQ ID NO:2)
[0024] (iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is
QQSYTTPPT (SEQ ID NO:3)
[0025] (iv) HVR-H1 comprising sequence D1-D10, wherein D1-D11 is
GFTISGSWIH (SEQ ID NO:4)
[0026] (v) HVR-H2 comprising sequence E1-E18, wherein E1-E18 is
AVITPAGGYTYYADSVKG (SEQ ID NO:5) and
[0027] (vi) HVR-H3 comprising sequence F1-F16, wherein F1-F16 is
SNRYSGQFVPAYAMDY (SEQ ID NO:6).
[0028] In one embodiment, HVR-L1 of an antibody of the invention
comprises the sequence of SEQ ID NO:1. In one embodiment, HVR-L2 of
an antibody of the invention comprises the sequence of SEQ ID NO:2.
In one embodiment, HVR-L3 of an antibody of the invention comprises
the sequence of SEQ ID NO:3. In one embodiment, HVR-H1 of an
antibody of the invention comprises the sequence of SEQ ID NO:4. In
one embodiment, HVR-H2 of an antibody of the invention comprises
the sequence of SEQ ID NO:5. In one embodiment, HVR-H3 of an
antibody of the invention comprises the sequence of SEQ ID NO:6. In
one embodiment, HVR-L1 comprises RASQSISSYLA (SEQ ID NO:7) or
RASQDGARSLA (SEQ ID NO:39) or RASQDGAIYLA (SEQ ID NO:40). In one
embodiment, HVR-L2 comprises GASSRAS (SEQ ID NO:8) or SASFLAS (SEQ
ID NO:41) or SASLES (SEQ ID NO:42) or SATLAS (SEQ ID NO:43) or
SASFLAS (SEQ ID NO:44) or SASNLAS (SEQ ID NO:45) or SASTLAS (SEQ ID
NO:46). In one embodiment, an antibody of the invention comprising
these sequences (in combination as described herein) is humanized
or human.
[0029] In one aspect, the invention provides an antibody comprising
one, two, three, four, five or six HVRs, wherein each HVR
comprises, consists or consists essentially of a sequence selected
from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, and
8, and wherein SEQ ID NO: 1 or 7 corresponds to an HVR-L1, SEQ ID
NO:2 or 8 corresponds to an HVR-L2, SEQ ID NO:3 corresponds to an
HVR-L3, SEQ ID NO:4 corresponds to an HVR-H1, SEQ ID NO:5
corresponds to an HVR-H2, and SEQ ID NO:6 corresponds to an HVR-H3.
In one embodiment, an antibody of the invention comprises an HVR-L1
comprising SEQ ID NO:7, an HVR-L2 comprising SEQ ID NO:2, an HVR-L3
comprising SEQ ID NO:3, an HVR-H1 comprising SEQ ID NO:4, an HVR-H2
comprising SEQ ID NO:5, and an HVR-H3 comprising SEQ ID NO:6. In
another embodiment, an antibody of the invention comprises an
HVR-L1 comprising SEQ ID NO:1, an HVR-L2 comprising SEQ ID NO:8, an
HVR-L3 comprising SEQ ID NO:3, an HVR-H1 comprising SEQ ID NO:4, an
HVR-H2 comprising SEQ ID NO:5, and an HVR-H3 comprising SEQ ID
NO:6. In one embodiment, an antibody of the invention comprises an
HVR-L1 comprising SEQ ID NO:1, an HVR-L2 comprising SEQ ID NO:2, an
HVR-L3 comprising SEQ ID NO:3, an HVR-H1 comprising SEQ ID NO:4, an
HVR-H2 comprising SEQ ID NO:5, and an HVR-H3 comprising SEQ ID
NO:6. In another embodiment, an antibody of the invention comprises
an HVR-L1 comprising SEQ ID NO:1, an HVR-L2 comprising SEQ ID NO:2,
an HVR-L3 comprising SEQ ID NO:20, an HVR-H1 comprising SEQ ID
NO:17, an HVR-H2 comprising SEQ ID NO:18, and an HVR-H3 comprising
SEQ ID NO:19.
[0030] Variant HVRs in an antibody of the invention can have
modifications of one or more residues within the HVR. In one
embodiment, a HVR-L1 variant comprises SEQ ID NO:1 wherein A1-11
comprises 1-6 (1, 2, 3, 4, 5, or 6) substitutions in any
combination of the following positions: A5 (S), A6 (I or G), A7
(A), A8 (S, R, or I), A9 (Y or S), and A10 (L). In one embodiment,
a HVR-L2 variant comprises SEQ ID NO:2 wherein B1-7 comprises 1-4
(1, 2, 3, 4, 5, or 6) substitutions in any combination of the
following positions: B1 (G), B3 (T), B4 (S, L, N, or T), B5 (R, E,
or A), B6 (A or S) and B7 (Y). In one embodiment, a HVR-L3 variant
comprises SEQ ID NO:3 wherein C1-9 comprises 1-6 (1, 2, 3, 4, 5, or
6) substitutions in any combination of the following positions: C3
(Y, P, T, F or G), C4 (W, F, R or N), C5 (S, N, A, D, F, H, V or
G), C6 (Y, S, A, D, N, L, I, M, Y or G), C7 (L, H, or T) and C8 (L,
F, A, M or S). In one embodiment, a HVR-H1 variant comprises SEQ ID
NO:4 wherein D1-10 comprises 1-9 (1, 2, 3, 4, 5, 6, 7, 8 or 9)
substitutions in any combination of the following positions: D2
(Y), D3 (S), D4 (F or L), D5 (N, T, Y, D, or K), D6 (N or S), D7
(Y, N or R), D8 (Y or A), D9 (M, F, L or N) and D10 (S, E or Q). In
one embodiment, a HVR-H2 variant comprises SEQ ID NO:5 wherein
E1-18 comprises 1-10 (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)
substitutions in any combination of the following positions: E1 (G
or S), E2 (F, G, I, T, or R), E4 (Y or S), E5 (G or S), E6 (T, Y,
or M), E7 (D or L), E8 (S), E9 (D, S, T, H, K, A or V), E10 (I),
and E11 (D, N, A, E, or 1). In one embodiment, a HVR-H3 variant
comprises SEQ ID NO:6 wherein F1-18 comprises 1-17 (1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) substitutions in any
combination of the following positions: F2 (S), F3 (L, G, D, M or
W), F4 (I, G, V, or S), F5 (G, Y, or N), F6 (N, S, or V), F7 (K, Y,
W, or M), F8 (F, S, P, or a deletion), F9 (G, A, S, Y, or a
deletion), F10 (W, R, P, E, G, or a deletion), F11 (S, W, G, H, or
a deletion), F12 (S, W, H, or a deletion), F13 (Y, D, G, V, or a
deletion), F14 (G or a deletion), F15 (V or a deletion), F16 (L or
F), F17 (a), and F18 (V). Letter(s) in parenthesis following each
position indicates an illustrative substitution (i.e., replacement)
amino acid or, where indicated, an amino acid deletion. In one
embodiment, a HVR-L1 comprises the sequence of SEQ ID NO:1; a
HVR-L2 comprises SEQ ID NO:2; a HVR-L3 variant comprises SEQ ID
NO:3 wherein C4, C5, C6, and C6 are R, S, D, and H, respectively; a
HVR-H1 comprises SEQ ID NO:17; a HVR-H2 comprises SEQ ID NO:18; and
a HVR-H3 comprises SEQ ID NO:19 or a HVR-H3 variant comprising SEQ
ID NO:19. In one embodiment of these antibodies, these antibodies
further comprise a human .kappa.light chain framework consensus
sequence. In one embodiment, position 104 in framework region 4 of
the light chain (LC-FR4) (Kabat numbering) is V. In one embodiment,
position 104 of LC-FR4 is M.
[0031] In one embodiment, an antibody of the invention comprises a
variant HVR-H1 SEQ ID NO:4 numbered D1-D10, wherein from 1 to 6
substitutions are selected from the following: D2 (Y), D3 (S), D4
(F or L), D5 (S, T, Y, D, or K), D6 (N or S), D7 (S, N, or R), D8
(W or A), D9 (M, F, L or N), and D10 (S, E, or Q). In one
embodiment, an antibody of the invention comprises a variant HVR-H1
comprising SEQ ID NO:24, 25, 26, 27 or 28. In one embodiment, an
antibody of the invention comprises a variant HVR-H2 SEQ ID NO:5
numbered E1-E18, wherein from 1 to 7 substitutions are selected
from the following: E1 (A or S), E2 (V, G, I, T or R), E4 (T or S),
E5 (G or S), E6 (T, Y, or M), E7 (D or L), E8 (S), E9 (Y, S, T, H,
K, A or V), E10 (I) and E11 (Y, N, A, E, or 1). In one embodiment,
an antibody of the invention comprises a variant HVR-H2 comprising
SEQ ID NO:29, 30, 31, 32, or 33. In one embodiment, an antibody of
the invention comprises a variant HVR-H3 comprising SEQ ID NO:6
numbered F1-F16, wherein from 1-15 substitutions are selected from
the following: F1 (S, G, D, M, or W), F2 (N, G, V, or S), F3 (R, Y
or N), F4 (Y, S, or V), F5 (S, Y, W or M), F6 (G, S, P or a
deletion), F7 (Q, A, S, Y, or a deletion), F8 (F, R, P, E, G, or a
deletion), F9 (V, W, G, H, or a deletion), F10 (P, W, H, or a
deletion), F11 (A, D, G, V, or a deletion), F12 (Y or a deletion),
F13 (A or V), F14 (L or F), and F15 (V). According to the
invention, an amino acid substitution may encompass an amino acid
deletion in a given sequence. In one embodiment of the invention,
an antibody of the invention comprises a variant HVR-H3 comprising
SEQ ID NO:34, 35, 36, 37, or 38. In one embodiment of these
antibodies, the framework consensus sequence comprises substitution
at position 71, 73 and/or 78. In some embodiments of these
antibodies, position 71 is A, 73 is T and/or 78 is A. In one
embodiment of these antibodies, these antibodies further comprise a
human subgroup III heavy chain framework consensus sequence.
[0032] In one aspect, the invention provides an anti-Robo4 antibody
comprising one, two, three, four, five six, or more of the HVR
sequences depicted in FIGS. 1, 2, 3, and (SEQ ID NOS:1-8,
17-71).
[0033] In one embodiment, the invention provides a humanized
anti-Robo4 antibody. A humanized anti-Robo4 antibody of the
invention may comprise one or more human and/or human consensus
non-hypervariable region (e.g., framework) sequences in its heavy
and/or light chain variable domain. In some embodiments, one or
more additional modifications are present within the human and/or
human consensus non-hypervariable region sequences. In one
embodiment, the heavy chain variable domain of an antibody of the
invention comprises a human consensus framework sequence, which in
one embodiment is the subgroup III consensus framework sequence. In
one embodiment, an antibody of the invention comprises a variant
subgroup III consensus framework sequence modified at least one
amino acid position. For example, in one embodiment, a variant
subgroup III consensus framework sequence may comprise a
substitution at one or more of positions 71, 73 and/or 78. In one
embodiment, said substitution is R71A, N73T and/or N78A and/or
L/78A, in any combination thereof. In one embodiment, the light
chain variable domain of an antibody of the invention comprises a
human consensus framework sequence, which in one embodiment is the
subgroup .kappa.I consensus framework sequence. In one embodiment,
the light chain consensus framework sequence is modified at one or
more positions. In one embodiment, the light chain consensus
sequence has at position 104 the amino acid V. In one embodiment,
the light chain consensus sequence is modified at position 104
according to the Kabat numbering system, wherein position 104 is
M.
[0034] In one aspect, the invention provides an antibody comprising
1 to 4 or all of the light chain FR sequences LC-FR1 (SEQ ID NO:9),
LC-FR2 (SEQ ID NO: 10, LC-FR3 (SEQ ID NO:11), and LC-FR4 (SEQ ID
NO:12). In one embodiment, LC-FR4 (SEQ ID NO:12) is a variant
wherein position 104 is M.
[0035] In one aspect, the invention provides an antibody comprising
1 to 4 or all of the heavy chain FR sequences HC-FR1 (SEQ ID
NO:13), HC-FR2 (SEQ ID NO:14), HC-FR3 (SEQ ID NO:15), and HC-FR4
(SEQ ID NO:16).
[0036] In one embodiment, the invention provides a therapeutic
agent for use in a host subject that elicits little to no
immunogenic response against the agent in said subject. For
example, in one embodiment, the invention provides a humanized
antibody that elicits and/or is expected to elicit a human
anti-mouse antibody response (HAMA) at a substantially reduced
level in a host subject compared to a murine antibody raised
against human Robo4. In another example, the invention provides a
humanized antibody that elicits and/or is expected to elicit
minimal or no human anti-mouse antibody response (HAMA). In one
example, an antibody of the invention elicits an anti-mouse
antibody response that is at or below a clinically-acceptable
level.
[0037] The invention also provides antibodies comprising
modifications in hybrid hypervariable positions as described herein
below. In one embodiment, an antibody of the invention comprises a
variant human subgroup consensus framework sequence modified at one
or more hybrid hypervariable positions. In one embodiment, an
antibody of the invention comprises a heavy chain variable domain
comprising a variant human subgroup III consensus framework
sequence modified at one or more of positions 26-35, 49-65, 95-102,
and 94. In one embodiment, an antibody of the invention comprises a
light chain variable domain comprising a variant human kappa
subgroup I consensus framework sequence modified at one or more of
positions 24-34, 50-56, and 39-97.
[0038] In one embodiment, an antibody of the invention comprises at
least a portion (or all) of the framework sequence of human .kappa.
light chain. In one embodiment, an antibody of the invention
comprises at least a portion (or all) of human .kappa. subgroup I
framework consensus sequence. In one embodiment, an antibody of the
invention comprises the framework sequences shown in FIGS. 3A and
3B.
[0039] An antibody of the invention can comprise any suitable human
or human consensus heavy chain framework sequences, provided the
antibody exhibits the desired biological characteristics (e.g., a
desired binding affinity). In one embodiment, an antibody of the
invention comprises at least a portion (or all) of the framework
sequence of human subgroup III heavy chain. In one embodiment, an
antibody of the invention comprises the framework sequences shown
in FIGS. 4A and 4B.
[0040] In one embodiment, an antibody of the invention comprises a
heavy and/or light chain variable domain comprising framework
sequence depicted in SEQ ID NOS:72-137 (FIGS. 1A, 1B, 2A, 2B, 3A,
3B, 4A and 4B).
[0041] In one aspect, an antibody of the invention is a humanized
anti-Robo4 antibody that binds human Robo4 vascular endothelial
cell marker. For example, in one embodiment, an antibody of the
invention binds human Robo4 with an IC50 value that is, less than
1000 nM, less than 500 nM, less than 200 nM, less than 100 nM, less
than 50 nM, less than 30 nM, less than 20 nM less than 10 nM, less
than 5 nM, less than 3 nM, less than 1 nM, less than 0.1 nM, or
less than 0.01 nM. Comparison of abilities to inhibit ligand
binding to its receptor can be performed according to various
methods known in the art, including as described in the Examples
below.
[0042] In one aspect, an antibody of the invention is an anti-Robo4
antibody conjugated to a cytotoxic moiety (an anti-Robo4 antibody
drug conjugate, also referred to as an anti-Robo4 ADC), wherein the
anti-Robo4ADC inhibits Robo4-dependent cell proliferation in a cell
expressing Robo4. Robo4 expressing cells include, but are not
limited to, endothelial cells such as vascular endothelial cells.
Exemplary anti-Robo4 ADCs are disclosed herein. In one embodiment,
the anti-Robo4 antibody is humanized. In one embodiment, the
anti-Robo4 antibody is a human antibody. In one embodiment, the
anti-Robo4 antibody is derived from phage display mutagenesis and
selection.
[0043] In one aspect, an antibody of the invention is an anti-Robo4
antibody ADC that inhibits Robo4-dependent cell proliferation
better than a reference anti-Robo4 antibody that is not conjugated
to a cytotoxic agent. For example, in one embodiment, an anti-Robo4
ADC antibody of the invention inhibits cell proliferation with an
IC50 value that is less than about half that of the reference
anti-Robo4 antibody that is not conjugated to a cytotoxic agent. In
one embodiment, the IC50 value of an ADC antibody of the invention
is about 0.001, 0.01 0.1, 0.2, 0.3 or 0.4 that of the non-ADC
reference antibody. Comparison of abilities to inhibit cell
proliferation can be performed according to various methods known
in the art, including as described in the Examples below. In one
embodiment, IC50 values are determined across an antibody
concentration range from about 0.01 nM to about 100 nM.
[0044] In one embodiment, both the humanized antibody and chimeric
antibody are monovalent. In one embodiment, both the humanized
antibody and chimeric antibody comprise a single Fab region linked
to an Fc region. In one embodiment, the reference chimeric antibody
comprises variable domain sequences depicted in FIG. 7 (SEQ ID NO:
9 and 10) linked to a human Fc region. In one embodiment, the human
Fc region is that of an IgG (e.g., IgG1, 2, 3 or 4).
[0045] In one aspect, the antibody of the invention comprises an Fc
region. In an embodiment, the Fc region is an IgG1, 2, 3 or 4. In
one embodiment the IgG is a native IgG. In one embodiment, the Fc
region exhibits enhance antibody dependent cellular cytotoxicity
(ADCC) relative to wild type ADCC activity of the native IgG1, 2,
3, or 4. In one embodiment, the IgG is an Fc region altered from
the native IgG such that the altered Fc region exhibits enhanced
ADCC activity relative to the native Fc region.
[0046] In one aspect, an antibody of the invention is an anti-Robo4
antibody conjugated to a detectable moiety, such as an imaging
agent. In one aspect, antibodies of the invention may be conjugated
with any label moiety which can be covalently attached to the
antibody through a reactive moiety, an activated moiety, or a
reactive cysteine thiol group (Singh et al., Anal. Biochem.
304:147-15 (2002); Harlow E. and Lane, D. (1999) Using Antibodies:
A Laboratory Manual, Cold Springs Harbor Laboratory Press, Cold
Spring Harbor, N.Y.; Lundblad R. L. (1991) Chemical Reagents for
Protein Modification, 2nd ed. CRC Press, Boca Raton, Fla.). The
attached label may function to: (i) provide a detectable signal;
(ii) interact with a second label to modify the detectable signal
provided by the first or second label, e.g. to give FRET
(fluorescence resonance energy transfer); (iii) stabilize
interactions or increase affinity of binding, with antigen or
ligand; (iv) affect mobility, e.g. electrophoretic mobility or
cell-permeability, by charge, hydrophobicity, shape, or other
physical parameters, or (v) provide a capture moiety, to modulate
ligand affinity, antibody/antigen binding, or ionic complexation.
Labeled antibodies of the invention are further disclosed
herein.
[0047] In an embodiment, the detectable label is an aqueous
insoluble particle that is detectable in vitro or in vivo. In an
embodiment, the particle is a magnetic or metallic particle.
Metallic particles are detectable by such methods as magnetic
resonance imaging (MRI), single-particle or single-molecule
tracking, immunocytochemistry, plasmon frequency with dark-field
illumination, differential interference contrast and video
enhancement, total internal reflection, and photothermal
interference contrast (PIC) technique (see, for example PNAS USA
100(20): 11350-11355 (2003); Sheetz et al., Nature 340:284-288
(1989); Baschong et al., Histochemistry 83:409-411 (1985); Slot and
Geuze, Eur. J. Cell Biol. 38:87-93 (1935); Frey and Frey, J.
Struct. Biol. 127:94-100 (1999); Hainfeld and Powell, J. Histochem.
Cytochem. 48:471-480 (2000); Schultz et al., PNAS USA 97:996-1001
(2000); Gelles et al., Nature 331:450-453 (1988); Sonnichsen et
al., Appl. Phys. Lett. 77:2949-2951 (2000); Boyer et al., Science
297:1160-1163 (2002)). Nanoparticulate agents of the invention may
also include, but are not limited to, microbubbles (see Ellegala et
al., Circulation 108:336-341 (2003)), also referred to as
acoustically active lipospheres (AALs) (see Tartis et al.,
Ultrasound Med. Biol. 32(11): 1771-80 (2006)), superparamagnetic
agents, liposomes, perfluorocarbon nanoparticle emulsions (WO
2005104051), and dendrimers (see Caruthers et al., Methods in
Molecular Medicine, 124:387-400 (2006) and references cited
therein, all of which references are hereby incorporated by
reference in their entirety).
[0048] In one aspect, the invention provides an antibody comprising
a heavy chain variable domain comprising the HVR-H1, HVR-H2 and/or
HVR-H3 sequence depicted in FIGS. 1B and 2B. In one embodiment, the
variable domain comprises HC-FR1, HC-FR2, HC-FR3 and/or HC-FR4
sequence depicted in FIGS. 1B, 2B, 4A and 4B. In one embodiment,
the antibody comprises a CH1 and/or Fc sequence. In one embodiment,
an antibody of the invention comprises a heavy chain variable
domain comprising the HVR-H1, HVR-H2 and/or HVR-H3 sequence, and
the HC-FR1, HC-FR2, HC-FR3 and/or HC-FR4 sequence depicted in FIGS.
1B, 2B, 4A, and 4B. In one embodiment, an antibody of the invention
comprises a heavy chain variable domain comprising the HVR1-HC,
HVR2-HC and/or HVR3-HC sequence depicted in FIGS. 1B, 2B, 4A and
4B, and a CH1 and/or Fc sequence.
[0049] In one aspect, the invention provides an antibody comprising
a light chain variable domain comprising HVR1-LC, HVR2-LC and/or
HVR3-LC sequence depicted in FIG. 1A or 2A. In one embodiment, the
variable domain comprises a FR1-LC, FR2-LC, FR3-LC and/or FR4-LC
sequence depicted in FIGS. 3A and 3B.
[0050] In one embodiment, an antibody of the invention comprises
light and heavy chain variable domains as described in the
preceding two paragraphs. In one embodiment, the antibody is
monovalent and comprises an Fc region. In one embodiment, the Fc
region comprises at least one protuberance (knob) and at least one
cavity (hole), wherein presence of the protuberance and cavity
enhances formation of a complex between an Fc polypeptide
comprising the protuberance and an Fc polypeptide comprising the
cavity, for example as described in WO 2005/063816. In one
embodiment, the Fc region of an antibody of the invention comprises
a first and a second Fc polypeptide, wherein the first and second
polypeptide each comprises one or more mutations with respect to
wild type human Fc. In one embodiment, a cavity mutation is T366S,
L368A and/or Y407V. In one embodiment, a protuberance mutation is
T366W. In one embodiment, the first polypeptide comprises the Fc
sequence depicted in FIG. 13 and the second polypeptide comprises
the Fc sequence depicted in FIG. 14.
[0051] In one embodiment, the invention provides methods of
modulating one or more aspects of Robo4-associated effects (e.g.,
Robo4 activation, downstream molecular signaling (e.g., mitogen
activated protein kinase (MAPK) phosphorylation), cell
proliferation, cell migration, cell survival, cell morphogenesis
and angiogenesis) by administering an anti-Robo4 antibody to a
subject (e.g., a mammalian subject such as a human). In some
embodiments, the anti-Robo4 antibody disrupts ligand binding to
Robo4 (e.g., by binding to a sequence within the Robo4
extracellular domain, and thereby inhibiting interaction of said
bound domain with its binding partner (such as a ligand molecule).
In other embodiments, the Robo4 antibody binds to a sequence that
is not within the Robo4 ligand binding site, wherein said binding
results in disruption of the ability of the Robo4 to interact with
its binding partner.
[0052] In one embodiment of the invention, binding of the antibody
to Robo4 inhibits Robo4 associated endothelial cell proliferation.
In another embodiment of a Robo4 antibody of the invention, binding
of the antibody to Robo4 in a cell inhibits proliferation,
survival, scattering, morphogenesis and/or motility of the cell. In
another embodiment, the cell is an endothelial cell, such as,
without limitation, a vascular endothelial cell.
[0053] In one embodiment, a Robo4 antibody of the invention
specifically binds at least a portion of Robo4 shown in FIG. 5 (SEQ
ID NO:138) or variant thereof. In one embodiment, an antibody of
the invention specifically binds within the full length Robo4 amino
acid sequence lacking the leader sequence (i.e., lacking the region
indicated by dotted underlining in FIG. 5). In one embodiment, an
antibody of the invention specifically binds within the
extracellular domain sequence shown in FIG. 5 (indicated by solid
underlining in FIG. 5). In one embodiment, an antibody of the
invention specifically binds a conformational epitope formed by
part or all of the Robo4 extracellular domain. In one embodiment,
an antibody of the invention specifically binds an amino acid
sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98% sequence
identity or similarity with the sequence shown in FIG. 5 (SEQ ID
NO:138). In one embodiment, an antibody of the invention
specifically binds an amino acid sequence having at least 50%, 60%,
70%, 80%, 90%, 95%, 98% sequence identity or similarity with the
sequence of the Robo4 full length amino acid sequence lacking the
leader sequence (i.e., lacking the region indicated by dotted
underlining in FIG. 5). In one embodiment, an antibody of the
invention specifically binds an amino acid sequence having at least
50%, 60%, 70%, 80%, 90%, 95%, 98% sequence identity or similarity
with the sequence of the Robo4 extracellular domain (the region
indicated by solid underlining in FIG. 5).
[0054] In one embodiment, an antibody of the invention specifically
binds to Robo4 of a first animal species, and does not specifically
bind to Robo4 of a second animal species. In one embodiment, the
first animal species is human and/or primate (e.g., cynomolgus
monkey), and the second animal species is ratus (e.g., rat), murine
(e.g., mouse) and/or canine. In one embodiment, the first animal
species is human. In one embodiment, the first animal species is
primate, for example cynomolgus monkey. In one embodiment, the
second animal species is murine, for example mouse. In one
embodiment, the second animal species is canine. In one embodiment,
an antibody of the invention specifically binds to Robo4 of at
least two species. In one embodiment, the first animal species is
human and/or primate (e.g., cynomolgus monkey), and the second
animal species is murine (e.g., mouse). Antibodies of the invention
that bind more than one species find use in animal modeling studies
for therapeutic or diagnostic agents.
[0055] In one aspect, the invention provides a method of detecting
Robo4 in serum of a mammal, where a concentration of Robo4 in the
serum that is higher than normal levels of Robo4 indicate the
presence of angiogenesis in the mammal. According to the method of
the invention, a detectable or detectably labeled anti-Robo4
antibody of the invention is contacted with a serum sample from a
test mammal, including but not limited to a human. The anti-Robo4
antibody is detected directly via the detectable label or
indirectly by, for example, contact with a detectably labeled or
detectable secondary antibody. A serum concentration (level) of
anti-Robo4 in the test mammal that is higher than anti-Robo4 serum
concentration from a normal mammal indicates angiogenesis in the
test mammal. A normal mammal refers to a mammal known not to be
experiencing angiogenesis or a population of mammals not
experiencing angiogenesis. In one embodiment the test and normal
mammal or population of mammals is the same species, including but
not limited to humans. Serum levels of Robo4 protein have been
detected in human patients with advanced non-small cell lung cancer
(Gom et al., Lung Cancer 49:71-76 (2005)). Serum anti-Robo4 levels
are useful for diagnosis and prognosis of angiogenesis associated
with cancer.
[0056] In one aspect, the invention provides compositions
comprising one or more antibodies of the invention and a carrier.
In one embodiment, the carrier is pharmaceutically acceptable.
[0057] In one aspect, the invention provides nucleic acids encoding
a Robo4 antibody of the invention, vectors comprising the nucleic
acids, and host cells (e.g., E. coli or Chinese Hamster Ovary (CHO)
cells), comprising the nucleic acids and/or vectors.cells
comprising a nucleic acid or a vector of the invention. A vector
can be of any type, for example a recombinant vector such as an
expression vector. Any of a variety of host cells can be used. In
one embodiment, a host cell is a prokaryotic cell, for example, E.
coli. In one embodiment, a host cell is a eukaryotic cell, for
example a mammalian cell such as Chinese Hamster Ovary (CHO)
cell.
[0058] In one aspect, the invention provides methods for making an
anti-Robo4. For example, the invention provides a method of making
a Robo4 antibody, the method comprising expressing in a suitable
host cell a recombinant vector of the invention encoding said
antibody (or fragment thereof), and recovering said antibody.
[0059] In one aspect, the invention provides an article of
manufacture comprising a container; and a composition contained
within the container, wherein the composition comprises one or more
anti-Robo4 antibodies of the invention. In one embodiment, the
composition comprises a nucleic acid of the invention. In one
embodiment, a composition comprising an antibody further comprises
a carrier, which in some embodiments is pharmaceutically
acceptable. In one embodiment, an article of manufacture of the
invention further comprises instructions for administering the
composition (e.g., the antibody) to a subject.
[0060] In one aspect, the invention provides a kit comprising a
first container comprising a composition comprising one or more
anti-Robo4 antibodies of the invention; and a second container
comprising a buffer. In one embodiment, the buffer is
pharmaceutically acceptable. In one embodiment, a composition
comprising an antibody further comprises a carrier, which in some
embodiments is pharmaceutically acceptable. In one embodiment, a
kit further comprises instructions for administering the
composition (e.g., the antibody) to a subject.
[0061] In one aspect, the invention provides use of a Robo4
antibody of the invention in the preparation of a medicament for
the therapeutic and/or prophylactic treatment of a disease, such as
a cancer, a tumor, a cell proliferative disorder, an immune (such
as autoimmune) disorder and/or an angiogenesis-related
disorder.
[0062] In one aspect, the invention provides use of a nucleic acid
of the invention in the preparation of a medicament for the
therapeutic and/or prophylactic treatment of a disease, such as a
cancer, a tumor, a cell proliferative disorder, an immune (such as
autoimmune) disorder and/or an angiogenesis-related disorder.
[0063] In one aspect, the invention provides use of an expression
vector of the invention in the preparation of a medicament for the
therapeutic and/or prophylactic treatment of a disease, such as a
cancer, a tumor, a cell proliferative disorder, an immune (such as
autoimmune) disorder and/or an angiogenesis-related disorder.
[0064] In one aspect, the invention provides use of a host cell of
the invention in the preparation of a medicament for the
therapeutic and/or prophylactic treatment of a disease, such as a
cancer, a tumor, a cell proliferative disorder, an immune disorder
(e.g., an autoimmune disorder such as, for example rheumatoid
arthritis) and/or an angiogenesis-related disorder.
[0065] In one aspect, the invention provides use of an article of
manufacture of the invention in the preparation of a medicament for
the therapeutic and/or prophylactic treatment of a disease, such as
a cancer, a tumor, a cell proliferative disorder, an immune
disorder (e.g., an autoimmune disorder such as, for example
rheumatoid arthritis) and/or an angiogenesis-related disorder.
[0066] In one aspect, the invention provides use of a kit of the
invention in the preparation of a medicament for the therapeutic
and/or prophylactic treatment of a disease, such as a cancer, a
tumor, a cell proliferative disorder, an immune (such as
autoimmune) disorder and/or an angiogenesis-related disorder.
[0067] The invention provides methods and compositions useful for
modulating disease states associated with dysregulation of the
Robo4 signaling axis. The Robo4 signaling pathway is involved in
multiple biological and physiological functions, including, e.g.,
cell proliferation and angiogenesis. Thus, in one aspect, the
invention provides a method comprising administering to a subject
an antibody of the invention.
[0068] In one aspect, the invention provides a method of inhibiting
Robo4 activated cell proliferation, said method comprising
contacting a cell or tissue with an effective amount of a antibody
of the invention, whereby cell proliferation associated with Robo4
activation is inhibited.
[0069] In one aspect, the invention provides a method of treating a
pathological condition associated with dysregulation of Robo4
activation in a subject, said method comprising administering to
the subject an effective amount of an antibody of the invention,
whereby said condition is treated.
[0070] In one aspect, the invention provides a method of inhibiting
the growth of a cell that expresses Robo4, said method comprising
contacting said cell with an antibody of the invention or an
antibody drug conjugate of the invention, thereby causing an
inhibition of growth of said cell.
[0071] In one aspect, the invention provides a method of
therapeutically treating a mammal having a cancerous tumor
comprising a cell that expresses Robo4, said method comprising
administering to said mammal an effective amount of an antibody of
the invention or an antibody drug conjugate of the invention,
thereby effectively treating said mammal.
[0072] In one aspect, the invention provides a method for treating
or preventing a cell proliferative disorder associated with
increased expression or activity of Robo4, said method comprising
administering to a subject in need of such treatment an effective
amount of an antibody of the invention or an antibody drug
conjugate of the invention, thereby effectively treating or
preventing said cell proliferative disorder. In one embodiment,
said proliferative disorder is a pathological condition associated
with abnormal or unwanted endothelial cell proliferation, such as
abnormal or unwanted vascular cell proliferation in disorders
including, but not limited to, cancer, angiogenesis and disorders
associated with (e.g., augmented by endothelial cell proliferation
within the tissue experiencing the disorder) solid tumors and
metastasis, atherosclerosis, retrolental fibroplasia, hemangiomas,
chronic inflammation, intraocular neovascular diseases such as
proliferative retinopathies, e.g., diabetic retinopathy,
age-related macular degeneration (AMD), neovascular glaucoma,
immune rejection of transplanted corneal tissue and other tissues,
rheumatoid arthritis, and psoriasis.
[0073] In another aspect, the invention provides a method of
therapeutically treating a tumor in a mammal, wherein the growth of
said tumor is at least in part dependent upon a growth potentiating
effect of Robo4 expressed in endothelial cells, including without
limitation vascular endothelial cells, the method comprising
contacting said cell with an effective amount of an antibody of the
invention or an antibody drug conjugate of the invention, thereby
inhibiting the growth of endothelial cells in, for example,
angiogenesis associated with the tumor, and thereby effectively
treating said tumor.
[0074] Yet another embodiment of the invention provides methods of
modulating angiogenesis, by contacting a cell or tissue with an
effective amount of an anti-Robo4 antibody described herein. In
some embodiments, the antibody further comprises a cytotoxic agent.
In some embodiments, the angiogenesis is inhibited. In some
embodiments, the angiogenesis is enhanced. In some embodiments, the
angiogenesis is associated with a disorder selected from: cancer,
atherosclerosis, retrolental fibroplasia, hemangiomas, chronic
inflammation, intraocular neovascular diseases, proliferative
retinopathies, diabetic retinopathy, age-related macular
degeneration (AMD), neovascular glaucoma, immune rejection of
transplanted corneal tissue and other tissues, rheumatoid
arthritis, psoriasis, and combinations thereof. In some
embodiments, the angiogenesis is associated with cancer. In some
embodiments, the cancer is selected from the group consisting of:
sarcomas including osteogenic sarcomas and angiosarcomas, squamous
cell cancer, lung cancer, cancer of the peritoneum, hepatocellular
cancer, gastric or stomach cancer including gastrointestinal
cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, cancer of the urinary tract,
hepatoma, breast cancer, colon cancer, rectal cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney or renal cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,
melanoma, multiple myeloma and B-cell lymphoma, brain cancer, head
and neck cancer, and associated metastases and combinations
thereof. In some embodiments, the cell or tissue is in a mammal. In
some embodiments, the mammal is a human. In some embodiments, the
methods further comprise contacting the cell with an agent selected
from: an anti-neoplastic agent, chemotherapeutic agent, a growth
inhibitory agent, a cytotoxic agent, an anti-angiogenic agent, and
combinations thereof. In some embodiments, the agent is an
anti-VEGF antibody. In some embodiments, the methods further
comprise contacting the cell with a second, third, or fourth agent
selected from: an anti-neoplastic agent, chemotherapeutic agent, a
growth inhibitory agent, a cytotoxic agent, an anti-angiogenic
agent, and combinations thereof. In some embodiments, the second,
third, or fourth agent is an anti-VEGF antibody.
[0075] Methods of the invention can be used to affect any suitable
pathological state, for example, cells and/or tissues associated
with upregulation of the Robo4 expression. In one embodiment, a
cell that is targeted in a method of the invention is an
endothelial cell or vascular endothelial cell proliferating within
a tumor (including, e.g., a breast cancer, a colorectal cancer, a
lung cancer, a papillary carcinoma (e.g., of the thyroid gland), a
colon cancer, a pancreatic cancer, an ovarian cancer, a cervical
cancer, a central nervous system cancer, an osteogenic sarcoma, a
renal carcinoma, a hepatocellular carcinoma, a bladder cancer, a
gastric carcinoma, a head and neck squamous carcinoma, a melanoma
and a leukemia, or, a sarcomas (e.g., an osteogenic sarcoma or an
angiosarcoma), or any cancer the growth of which is supported by
endothelial cell proliferation in angiogenesis). In one embodiment,
the pathological conditions are associated with abnormal or
unwanted endothelial cell proliferation. In some embodiments, the
target of an anti-Robo4 antibody or antibody drug conjugate of the
invention, includes abnormal or unwanted vascular cell
proliferation in disorders including, but not limited to, cancer,
(including, e.g., solid tumors and metastasis), atherosclerosis,
retrolental fibroplasia, hemangiomas, chronic inflammation,
intraocular neovascular diseases such as proliferative
retinopathies, e.g., diabetic retinopathy, age-related macular
degeneration (AMD), neovascular glaucoma, immune rejection of
transplanted corneal tissue and other tissues, rheumatoid
arthritis, psoriasis, and disorders associated with angiogenesis
(including, e.g., disorders augmented by endothelial cell
proliferation within the tissue experiencing the disorder).
[0076] Methods of the invention can further comprise additional
treatment steps. For example, in one embodiment, a method further
comprises a step wherein a targeted cell and/or tissue (e.g., a
cancer cell) is exposed to radiation treatment or a
chemotherapeutic agent.
[0077] In one embodiment of methods of the invention, a cell that
is targeted (e.g., an endothelial cell of a tumor tissue or a
retina) is one in which Robo4 expression is increased as compared
to an endothelial cell in normal tissue of the same tissue origin.
In one embodiment, a method of the invention causes the death of a
targeted cell. For example, contact with an anti-Robo4 antibody of
the invention, including contact with an antibody drug conjugate of
the invention, results in uptake of the cytotoxic moiety, which
causes cell death. Alternatively, contact with an anti-Robo4
antibody of the invention conjugated to a cytotoxic compound may
result in internalization of the cytotoxic compound, which results
in cell death. In another alternative embodiment, contact with an
anti-Robo4 antibody of the invention conjugated to a detectable
moiety causes the endothelial cells of, for example, vasculature of
a tissue (e.g. a tumor tissue or retina) to be detected, such as by
imaging techniques well known in the relevant art.
[0078] Another embodiment of the invention provides methods of in
vivo imaging, by administering an anti-Robo4 antibody described
herein to a mammal. In some embodiments the mammal is a human. In
some embodiments, the mammal is suspected of having a disease or
disorder selected from: cancer, atherosclerosis, retrolental
fibroplasia, hemangiomas, chronic inflammation, intraocular
neovascular diseases, proliferative retinopathies, diabetic
retinopathy, age-related macular degeneration (AMD), neovascular
glaucoma, immune rejection of transplanted corneal tissue,
rheumatoid arthritis, psoriasis, and combinations thereof.
[0079] Another embodiment of the invention provides methods of
detecting a cell expressing Robo4, by contacting the cell with an
anti-Robo4 antibody described herein. In some embodiments, the cell
is a vascular endothelial cell. In some embodiments, the cell is in
a mammal. In some embodiments, the mammal is suspected of having a
disease or disorder selected from: cancer, atherosclerosis,
retrolental fibroplasia, hemangiomas, chronic inflammation,
intraocular neovascular diseases, proliferative retinopathies,
diabetic retinopathy, age-related macular degeneration (AMD),
neovascular glaucoma, immune rejection of transplanted corneal
tissue, rheumatoid arthritis, psoriasis, and combinations thereof.
In some embodiments, the mammal is suspected of having a cancer
selected from: squamous cell cancer, lung cancer, cancer of the
peritoneum, hepatocellular cancer, gastric or stomach cancer
including gastrointestinal cancer, pancreatic cancer, glioblastoma,
cervical cancer, ovarian cancer, liver cancer, bladder cancer,
cancer of the urinary tract, hepatoma, breast cancer, colon cancer,
rectal cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney or renal cancer, prostate cancer,
vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma,
penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma,
brain cancer, head and neck cancer, and associated metastases and
combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0080] FIGS. 1A and 1B depict alignment of amino acid sequences of
the variable regions of the light and heavy chains for various
anti-Robo4 clones of the invention (FIGS. 1A and 1B, respectively).
The hypervariable regions (HVRs) of the light and heavy chains are
indicated under the boxed complementary determining regions (CDRs).
In FIG. 1A, the light chain HVRs-L1, L2, and L3 are Kabat positions
24-34, 50-56, and 89-102, respectively. In FIG. 1B, the heavy chain
HVRs-H1, H2, and H3 are Kabat positions 26-35, 49-65, and 93-102,
respectively. Clones were prepared by phage display as disclosed in
the Examples and sequenced.
[0081] FIGS. 2A and 2B depict amino acid sequences of the variable
regions of the light and heavy chains (FIGS. 2A and 2B,
respectively) of selected affinity-matured antibodies from
libraries with individually-randomized HVR. The HVRs in FIGS. 2A
and 2B are the same positions as depicted in FIGS. 1A and 1B.
[0082] FIGS. 3A, 3B, 4A and 4B depict exemplary acceptor human
consensus framework sequences for use in practicing the instant
invention with sequence identifiers as follows:
[0083] Variable light (VL) consensus framework regions minus Kabat
HVRs (FIGS. 3A and 3B) human VL kappa subgroup I consensus
frameworks 1-4 (SEQ ID NOS:9, 10, 99, 100) human VL kappa subgroup
I' consensus frameworks 1-4 (SEQ ID NOS:9, 101, 99, 100) human VL
kappa subgroup II consensus frameworks 1-4 (SEQ ID NOS:102, 103,
104, 100) human VL kappa subgroup III consensus frameworks 1-4 (SEQ
ID NOS:105, 106, 107, 100) human VL kappa subgroup IV consensus
frameworks 1-4 (SEQ ID NOS:108, 109, 110, 100) The locations of
Kabat HVRs L1, L2, and L3 within the framework sequences are
depicted as shaded boxes.
[0084] Variable heavy (VH) consensus framework regions minus Kabat
HVRs (FIGS. 4A and B) human VH subgroup I consensus frameworks 1-4
minus Kabat HVRs (Subgroup IA, SEQ ID NOS:111, 112, 113, 16) and
(subgroup IB, SEQ ID NOS:114, 115, 113, 16) human VH subgroup I
consensus framework 1-4 minus extended HVRs, (subgroup IC, SEQ ID
NOS:114, 115, 116, 16) and (subgroup ID, SEQ ID NOS:114, 115, 117,
16) human VH subgroup II consensus framework 1-4 minus Kabat HVRs
(subgroup IIA, SEQ ID NOS:118, 119, 120, 16), (subgroup IB, SEQ ID
NOS:121, 122, 120, 16) human VH subgroup II consensus framework 1-4
minus extended HVRs (subgroup IIC, SEQ ID NOS:121, 122, 123, 16),
(subgroup IID, SEQ ID NOS:121, 122, 124, 16) human VH subgroup III
consensus frameworks 1-4 minus Kabat HVRs (subgroup IIIA, SEQ ID
NO:125, 126, 127, 16), (subgroup IIIB, SEQ ID NOS:128, 129, 127,
16) human VH subgroup III consensus frameworks 1-4 minus extended
HVRs (subgroup IIIC, SEQ ID NOS:128, 129, 130, 16), (subgroup IIID,
SEQ ID NOS:128, 129, 131, 16) human VH acceptor 1 frameworks 1-4
minus Kabat HVRs (subgroup Acceptor 1A, SEQ ID NOS:132, 126, 133,
16) and (subgroup Acceptor 1B, SEQ ID NOS:128, 129, 133, 16) human
VH acceptor 1 frameworks 1-4 minus extended HVRs (subgroup Acceptor
1C, SEQ ID NOS:128, 129, 134, 16) human VH acceptor 2 frameworks
1-4 minus Kabat HVRs (subgroup Acceptor 2A, SEQ ID NOS:132, 126,
135, 16) and (subgroup Acceptor 2B, SEQ ID NOS:128, 129, 135, 16)
human VH acceptor 2 frameworks 1-4 minus extended HVRs (subgroup
Acceptor 2C, SEQ ID NOS:128, 129, 136, 16) and (subgroup Acceptor
2D, SEQ ID NOS:128, 129, 137, 16).
[0085] FIG. 5A depicts the full length amino acid sequence of the
Robo4 polypeptide. A potential signal sequence is indicated by
dotted underlining. A portion of the extracellular domain is
indicated by solid underlining. FIG. 5B depicts the sequence of the
His-tagged soluble Robo4 extracellular domain fragment (SEQ ID
NO:171) used in, for example, anti-Robo4 Fab phage display
experiments disclosed herein. The Robo4 extracellular domain was
linked to a histidine tag for ease of recovery or detection.
[0086] FIG. 6A depicts the full length sequence of anti-Robo4
antibody YW71.22 heavy chain (SEQ ID NO:169). The alanine shown in
bold text and underlined is the position at which a cysteine was
substituted to generate the thioMAb. FIG. 6B depicts the full
length sequence of anti-Robo4 antibody YW71.22 light chain (SEQ ID
NO:170).
[0087] FIG. 7 depicts the amino acid sequence of murine Robo4
extracellular domain comprising a potential signal sequence and
having the histidine tag RRA(H).sub.5 attached at position H232
(SEQ ID NO:172). A dotted underline indicates the potential signal
sequence which is cleaved during expression in mammalian cells.
[0088] FIG. 8 is a bar graph of the results of a HUVEC migration
assay using Robo4 constructs.
[0089] FIG. 9 shows the results of a BiaCore assay indicating that
Robo4 does not bind Slit2.
[0090] FIG. 10 shows the results of a BiaCore assay indicating that
Robo4 interacts with UNC5B.
[0091] FIG. 11 is a photograph of an ISH assay showing that Robo4
is expressed in fetal mouse endothelium.
[0092] FIGS. 12A and 12B show expression of Robo4 in human HM-7
colon cancer mouse xenograft tumor model. FIGS. 12C and 12D show
Robo4 expression in human MDA-MB-175 breast cancer tumor mouse
xenograft model.
[0093] FIGS. 13A-D show expression of Robo4 in human malignant
melanoma.
[0094] FIGS. 14A-D show expression of Robo4 in small cell lung
cancer. FIGS. 14E-H show Robo4 expression in colon cancer.
[0095] FIGS. 15A-15C depict internalization of anti-Robo4 antibody
71.22 by mouse MS1 cells (FIGS. 15A and 15B), and internalization
of affinity matured anti-Robo4 antibody 71.22.S1.16 by mouse MS1
cells (FIG. 15C).
[0096] FIG. 16 is a bar graph showing the results of endothelial
cell migration assay. Anti-Robo4 antibody did not block VEGF
induced EC migration.
[0097] FIG. 17 is a graph of changes in median tumor volume as a
function of time in xenograft mouse models dosed with control,
anti-VEGF, anti-Robo4 (YW71.22), and anti-Robo4 plus anti-VEGF
antibodies. Naked Robo4 antibody did not inhibit tumor growth in
these experiments.
[0098] FIGS. 18A and 18B show the results of FACS plots indicating
that affinity matured anti-Robo4 clones 71.22.S1.16 and 71.22.S1.21
bind Robo4 endogenously expressed on HUVEC cells. FIGS. 18C and 18D
show the results of FACS plots indicating that the same affinity
matured anti-Robo4 antibody clones bind murine Robo4 on the
endogenously expressed on murine MS1 cells. These plots also show
that the parent antibody, 71.22, also cross-reacts with human and
murine Robo4.
[0099] FIGS. 19A and 19B show that anti-Robo4 antibody 71.22
associates with vasculature following injection into mice as
described in Example 12.
[0100] FIGS. 20A and 20B depict the results of a bead outgrowth
assay indicating that anti-Robo4 antibody 71.22 inhibits HUVEC tube
elongation. FIG. 20A shows that both total number and length of
tubes are reduced upon treatment of HUVECs with 71.22 as compared
to an irrelevant control antibody (anti-ragweed, E25). Anti-VEGF
was used as positive control antibody. FIG. 20B shows
representative examples with concentric circles drawn at 100, 200
and 300 .mu.m.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0101] The present invention provides compositions that bind Robo4
or a portion thereof, kits and articles of manufacture comprising
such compositions, and methods of using such compositions,
including, e.g., methods for modulating ligand binding to the Robo4
receptor and for modulating biological/physiological activities
associated with ligand binding to the Robo4 receptor. The invention
is based in part on the identification of a variety of anti-Robo4
antibodies that bind the Robo4 receptor, a receptor useful as a
therapeutic and diagnostic (e.g., in vivo, in vitro, or ex vivo
imaging) target. The anti-Robo4 antibodies of the invention can
conveniently be used as therapeutic and diagnostic agents for use
in targeting pathological conditions associated with abnormal or
unwanted endothelial cell proliferation, such as abnormal or
unwanted vascular cell proliferation in disorders including, but
not limited to, cancer, dysregulated angiogenesis and disorders
associated with (e.g., augmented by) endothelial cell proliferation
within the tissue experiencing the disorder, solid tumors and
metastasis, atherosclerosis, retrolental fibroplasia, hemangiomas,
chronic inflammation, intraocular neovascular diseases such as
proliferative retinopathies, e.g., diabetic retinopathy,
age-related macular degeneration (AMD), neovascular glaucoma,
immune rejection of transplanted corneal tissue and other tissues,
rheumatoid arthritis, and psoriasis. The anti-Robo4 antibodies of
the invention can be used alone or in combination with additional
agents (including anti-neoplastic compositions, chemotherapeutic
agents, cytotoxic agents, growth-inhibitory agents. In some
embodiments, the invention provides methods for identifying and/or
using Robo4-targeted antibodies to block Robo4 function, deliver
cytotoxic agents to cells expressing Robo4 (e.g., on the cell
surface), and/or deliver imaging agents to cells expressing Robo4
on the cell surface. For example, in some embodiments, detectably
labeled anti-Robo4 antibodies can conveniently be used as an
imaging agent for disease states in which increased vascularization
is harmful (e.g. cancer and macular degeneration) or for
physiological conditions in which endothelial cell proliferation,
vascular cell proliferation, and/or angiogenesis is a desirable
condition (such as in, for example, wound healing).
II. Definitions
[0102] As used herein, the following terms have the meanings
ascribed to them below unless otherwise specified.
[0103] The term "Robo4" or "Magic Roundabout," "Endothelial
Cell-Specific Molecule 4," or "ECSM4" as used herein refers to any
native or variant (whether native or synthetic) Robo4 polypeptide
comprising the amino acid sequence shown in FIG. 5, a variant or a
subsequence thereof to which the antibody of the present invention
specifically binds, including, e.g., the extracellular domain of
Robo4 or any subsequence thereof. The term "wild type Robo4"
generally refers to a polypeptide comprising the amino acid
sequence of a naturally occurring Robo4 protein. The term "wild
type Robo4 sequence" generally refers to an amino acid sequence
found in a naturally occurring Robo4. The Robo4 polypeptide of the
invention is a mammalian Robo4 polypeptide, such as, without
limitation human, equine, bovine, porcine, canine, feline, rodent
Robo4. "Robo4," "Magic Roundabout," "Endothelial Cell-Specific
Molecule 4," or "ECSM4" also refer to nucleic acids and polypeptide
polymorphic variants, alleles, mutants, and interspecies homologs
that: (1) have an amino acid sequence that has greater than about
60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%,
preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater
amino acid sequence identity, preferably over a region of at least
about 25, 50, 100, 200, 500, 1000, or more amino acids, to an amino
acid sequence encoded by a Robo4 nucleic acid (for a human Robo4
polypeptide sequence, see, e.g., FIGS. 5 and 7 and SEQ ID NOS:138
and 171; for a murine Robo4 polypeptide sequence, see, e.g., SEQ ID
NO:172); (2) bind to antibodies, e.g., polyclonal antibodies and/or
monoclonal antibodies, raised against an immunogen comprising an
amino acid sequence of a Robo4 protein, and conservatively modified
variants thereof; (3) specifically hybridize under stringent
hybridization conditions to an anti-sense strand corresponding to a
nucleic acid sequence encoding a Robo4 protein, and conservatively
modified variants thereof, (4) have a nucleic acid sequence that
has greater than about 95%, preferably greater than about 96%, 97%,
98%, 99%, or higher nucleotide sequence identity, preferably over a
region of at least about 25, 50, 100, 200, 500, 1000, or more
nucleotides, to a Robo4 nucleic acid (e.g., a nucleic acid encoding
the polypeptide set forth in FIGS. 5 and 7 and SEQ ID NOS:138, 171,
or 172. Preferably the Robo4 nucleic acid has greater than 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid
sequence encoding SEQ ID NOS:138, 171, or 172, preferably over a
region of at least about 25, 50, 100, 150, 200, 250, 300, 350, 400,
450, or 500, 600, 700, 800, 900, or 1000 or more nucleotides.
[0104] The term "anti-Robo4 antibody" or "an antibody that binds to
Robo4" refers to an antibody that is capable of binding Robo4 with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting Robo4. Preferably,
the extent of binding of an anti-Robo4 antibody to an unrelated,
non-Robo4 protein is less than about 10% of the binding of the
antibody to Robo4 as measured, e.g., by a radioimmunoassay (RIA).
In certain embodiments, an antibody that binds to Robo4 has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM. In certain
embodiments, an anti-Robo4 antibody binds to an epitope of Robo4
that is conserved among Robo4 from different species. In other
embodiments, an ant-Robo4 antibody binds to an epitope of Robo4
that is not conserved among Robo4 from different species.
[0105] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g. bispecific antibodies) formed from
at least two intact antibodies, and antibody fragments so long as
they exhibit the desired biological activity.
[0106] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with research, diagnostic or
therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or nonproteinaceous solutes. In
some embodiments, an antibody is purified (1) to greater than 95%
by weight of antibody as determined by, for example, the Lowry
method, and in some embodiments, to greater than 99% by weight; (2)
to a degree sufficient to obtain at least 15 residues of N-terminal
or internal amino acid sequence by use of, for example, a spinning
cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using, for example, Coomassie blue or silver
stain. Isolated antibody includes the antibody in situ within
recombinant cells since at least one component of the antibody's
natural environment will not be present. Ordinarily, however,
isolated antibody will be prepared by at least one purification
step.
[0107] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end;
the constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light chain and heavy chain variable domains.
[0108] The "variable region" or "variable domain" of an antibody
refers to the amino-terminal domains of the heavy or light chain of
the antibody. The variable domain of the heavy chain may be
referred to as "VH." The variable domain of the light chain may be
referred to as "VL." These domains are generally the most variable
parts of an antibody and contain the antigen-binding sites.
[0109] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions (HVRs) both in the light-chain and the
heavy-chain variable domains. The more highly conserved portions of
variable domains are called the framework regions (FR). The
variable domains of native heavy and light chains each comprise
four FR regions, largely adopting a beta-sheet configuration,
connected by three HVRs, which form loops connecting, and in some
cases forming part of, the beta-sheet structure. The HVRs in each
chain are held together in close proximity by the FR regions and,
with the HVRs from the other chain, contribute to the formation of
the antigen-binding site of antibodies (see Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, National
Institute of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in the binding of an antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody in antibody-dependent cellular
toxicity.
[0110] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0111] Depending on the amino acid sequences of the constant
domains of their heavy chains, antibodies (immunoglobulins) can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and IgA.sub.2. The
heavy chain constant domains that correspond to the different
classes of immunoglobulins are called .alpha., .delta., .epsilon.,
.gamma., and .mu., respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known and described generally in, for
example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B.
Saunders, Co., 2000). An antibody may be part of a larger fusion
molecule, formed by covalent or non-covalent association of the
antibody with one or more other proteins or peptides.
[0112] The terms "full length antibody," "intact antibody" and
"whole antibody" are used herein interchangeably to refer to an
antibody in its substantially intact form, not antibody fragments
as defined below. The terms particularly refer to an antibody with
heavy chains that contain an Fc region.
[0113] A "naked antibody" for the purposes herein is an antibody
that is not conjugated to a cytotoxic moiety or radiolabel.
[0114] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen binding region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0115] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0116] "Fv" is the minimum antibody fragment which contains a
complete antigen-binding site. In one embodiment, a two-chain Fv
species consists of a dimer of one heavy- and one light-chain
variable domain in tight, non-covalent association. In a
single-chain Fv (scFv) species, one heavy- and one light-chain
variable domain can be covalently linked by a flexible peptide
linker such that the light and heavy chains can associate in a
"dimeric" structure analogous to that in a two-chain Fv species. It
is in this configuration that the three HVRs of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. Collectively, the six HVRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three HVRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0117] The Fab fragment contains the heavy- and light-chain
variable domains and also contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0118] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see, e.g., Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York, 1994), pp. 269-315.
[0119] The term "diabodies" refers to antibody fragments with two
antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies may be bivalent or bispecific. Diabodies are described
more fully in, for example, EP 404,097; WO 1993/01161; Hudson et
al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90:
6444-6448 (1993). Triabodies and tetrabodies are also described in
Hudson et al., Nat. Med. 9:129-134 (2003).
[0120] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible mutations, e.g.,
naturally occurring mutations, that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies. In
certain embodiments, such a monoclonal antibody typically includes
an antibody comprising a polypeptide sequence that binds a target,
wherein the target-binding polypeptide sequence was obtained by a
process that includes the selection of a single target binding
polypeptide sequence from a plurality of polypeptide sequences. For
example, the selection process can be the selection of a unique
clone from a plurality of clones, such as a pool of hybridoma
clones, phage clones, or recombinant DNA clones. It should be
understood that a selected target binding sequence can be further
altered, for example, to improve affinity for the target, to
humanize the target binding sequence, to improve its production in
cell culture, to reduce its immunogenicity in vivo, to create a
multispecific antibody, etc., and that an antibody comprising the
altered target binding sequence is also a monoclonal antibody of
this invention. In contrast to polyclonal antibody preparations,
which typically include different antibodies directed against
different determinants (epitopes), each monoclonal antibody of a
monoclonal antibody preparation is directed against a single
determinant on an antigen. In addition to their specificity,
monoclonal antibody preparations are advantageous in that they are
typically uncontaminated by other immunoglobulins.
[0121] The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler and Milstein, Nature,
256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995),
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)),
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),
phage-display technologies (see, e.g., Clackson et al., Nature,
352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597
(1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et
al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, PNAS USA
101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods
284(1-2): 119-132 (2004), and technologies for producing human or
human-like antibodies in animals that have parts or all of the
human immunoglobulin loci or genes encoding human immunoglobulin
sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735;
WO 1991/10741; Jakobovits et al., PNAS USA 90: 2551 (1993);
Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al.,
Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al,
Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368:
856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et
al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature
Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.
Immunol. 13: 65-93 (1995).
[0122] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al., PNAS USA
81:6851-6855 (1984)). Chimeric antibodies include PRIMATIZED.RTM.
antibodies wherein the antigen-binding region of the antibody is
derived from an antibody produced by, e.g., immunizing macaque
monkeys with the antigen of interest.
[0123] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the
following review articles and references cited therein: Vaswani and
Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);
Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and
Gross, Curr. Op. Biotech. 5:428-433 (1994).
[0124] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues. Human antibodies can be
produced using various techniques known in the art, including
phage-display libraries. Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also
available for the preparation of human monoclonal antibodies are
methods described in Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,
147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr.
Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be
prepared by administering the antigen to a transgenic animal that
has been modified to produce such antibodies in response to
antigenic challenge, but whose endogenous loci have been disabled,
e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and
6,150,584 regarding XENOMOUSE.TM. technology). See also, for
example, Li et al., PNAS USA, 103:3557-3562 (2006) regarding human
antibodies generated via a human B-cell hybridoma technology.
[0125] An "antigen" is a predetermined antigen (e.g. a Robo4
sequence) to which an antibody can selectively bind. The target
antigen may be polypeptide, carbohydrate, nucleic acid, lipid,
hapten or other naturally occurring or synthetic compound.
Preferably, the target antigen is a polypeptide.
[0126] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a VL or VH
framework derived from a human immunoglobulin framework, or from a
human consensus framework. An acceptor human framework "derived
from" a human immunoglobulin framework or human consensus framework
may comprise the same amino acid sequence thereof, or may contain
pre-existing amino acid sequence changes. In some embodiments, the
number of pre-existing amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. Where pre-existing amino acid changes are
present in a VH, preferably those changes occur at only three, two,
or one of positions 71H, 73H and 78H; for instance, the amino acid
residues at those positions may be 71A, 73T and/or 78A. In one
embodiment, the VL acceptor human framework is identical in
sequence to the VL human immunoglobulin framework sequence or human
consensus framework sequence. Where pre-existing amino acid changes
are present in a VH, preferably those changes are only at three,
two or one of positions 71H, 73H and 78H; for instance, the amino
acid residues at those positions may be 71A, 73T and/or 78A. In one
embodiment, the VL acceptor human framework is identical in
sequence to the VL human immunoglobulin framework sequence or human
consensus framework sequence.
[0127] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residue in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al.
[0128] A "VH subgroup III consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable heavy subgroup III of Kabat et al. In one embodiment, the
VH subgroup III consensus framework amino acid sequence comprises
at least a portion or all of each of the following sequences:
EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:128)-H1-WVRQAPGKGLEWV (SEQ ID
NO:129)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID
NO:131)-H3-WGQGTLVTVSS (SEQ ID NO:16). In another embodiment, the
VH subgroup III consensus framework amino acid sequence comprises
at least a portion or all of each of the following sequences:
EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:128)-H1-WVRQAPGKGLEWV (SEQ ID
NO:129)-H2-RFTISADTSKNTAYLQMNSLRLRAEDTAVYYC (SEQ ID
NO:137)-H3-WGQGTLVTVSS (SEQ ID NO:16)
[0129] A "VL subgroup I consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable light kappa subgroup I of Kabat et al. In one embodiment,
the VH subgroup I consensus framework amino acid sequence comprises
at least a portion or all of each of the following sequences:
TABLE-US-00001 (SEQ ID NO: 9) DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:
10) -L1-WYQQKPGKAPKLLIY (SEQ ID NO: 99)
-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 100)
-L3-FGQGTKVEIK.
[0130] An "unmodified human framework" is a human framework which
has the same amino acid sequence as the acceptor human framework,
e.g. lacking human to non-human amino acid substitution(s) in the
acceptor human framework.
[0131] The term "hypervariable region", "HVR", or "HV", when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six hypervariable regions;
three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A
number of hypervariable region delineations are in use and are
encompassed herein. The Kabat Complementarity Determining Regions
(CDRs) are HVRs that are based on sequence variability and are the
most commonly used (Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead
to the location of the structural loops (Chothia and Lesk J. Mol.
Biol. 196:901-917 (1987)). The AbM hypervariable regions represent
a compromise between the Kabat CDRs and Chothia structural loops,
and are used by Oxford Molecular's AbM antibody modeling software.
The "contact" hypervariable regions are based on an analysis of the
available complex crystal structures. The residues from each of
these hypervariable regions are noted below.
TABLE-US-00002 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0132] The amino acid position/boundary delineating a hypervariable
region of an antibody can vary, depending on the context and the
various definitions known in the art (as described below). Some
positions within a variable domain may be viewed as hybrid
hypervariable positions in that these positions can be deemed to be
within a hypervariable region under one set of criteria while being
deemed to be outside a hypervariable region under a different set
of criteria. One or more of these positions can also be found in
extended hypervariable regions. In one embodiment, these hybrid
hypervariable positions include one or more of positions 26-30,
26-35 33-35B, 47-49, 49-65, 57-65, 95-102, 93, 94 and 102 in a
heavy chain variable domain. In one embodiment, these hybrid
hypervariable positions include one or more of positions 24-29,
24-34, 35-36, 46-49, 50-56, 89-97, 56 and 97 in a light chain
variable domain.
[0133] As used herein, the HVRs of the light chain are referred to
interchangeably as HVR-L1, -L2, or -L3, or HVR1-LC, HVR2-LC or
HVR3-LC or other similar designation that indicates that a light
chain HVR is referenced. As used herein, the HVRs of the heavy
chain are referred to interchangeably as HVR-H1, -H2, or -H3, or
HVR1-HC, HVR2-HC, or HVR3-HC, or other similar designation that
indicates that a heavy chain HVR is referenced.
[0134] Hypervariable regions may comprise "extended hypervariable
regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and
89-97 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and
93-102, 94-102 or 95-102 (H3) in the VH. The variable domain
residues are numbered according to Kabat et al., supra for each of
these definitions.
[0135] An "altered hypervariable region" for the purposes herein is
a hypervariable region comprising one or more (e.g. one to about
16) amino acid substitution(s) therein.
[0136] An "un-modified hypervariable region" for the purposes
herein is a hypervariable region having the same amino acid
sequence as a non-human antibody from which it was derived, i.e.
one which lacks one or more amino acid substitutions therein.
[0137] "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined. As used herein, LC-FR1-4 or FR1-4-LC or similar
designation is used interchangeably and refers to framework regions
of the light chain. As used herein, HC-FR1-4 or FR1-4-HC or similar
designation is used interchangeably and refers to framework region
of the heavy chain.
[0138] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s).
Preferred affinity matured antibodies will have nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies are produced by procedures known in the art. Marks et
al., Bio/Technology 10:779-783 (1992) describes affinity maturation
by VH and VL domain shuffling. Random mutagenesis of CDR and/or
framework residues is described by: Barbas et al., PNAS USA
91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton
et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J.
Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.
226:889-896 (1992).
[0139] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces biological activity of the antigen it
binds. For example blocking antibodies or antagonist anti-Robo4
antibodies substantially or completely inhibit the angiogenesis by
binding Robo4.
[0140] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat", and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). Using this numbering
system, the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or CDR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0141] The phrase "substantially similar," or "substantially the
same", as used herein, denotes a sufficiently high degree of
similarity between two numeric values (generally one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody) such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values). The difference between said two values is
preferably less than about 50%, preferably less than about 40%,
preferably less than about 30%, preferably less than about 20%,
preferably less than about 10% as a function of the value for the
reference/comparator antibody.
[0142] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (K.sub.d).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present invention. Specific illustrative
embodiments are described in the following.
[0143] In one embodiment, the "K.sub.d," "K.sub.D," or "K.sub.d
value," is measured by a radiolabeled antigen binding assay (RIA)
performed with the Fab version of an antibody of interest and its
antigen as described by the following assay that measures solution
binding affinity of Fabs for antigen by equilibrating Fab with a
minimal concentration of (.sup.125I)-labeled antigen in the
presence of a titration series of unlabeled antigen, then capturing
bound antigen with an anti-Fab antibody-coated plate (Chen, et al.,
(1999) J. Mol Biol 293:865-881). To establish conditions for the
assay, microtiter plates (Dynex) are coated overnight with 5 ug/ml
of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium
carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine
serum albumin in PBS for two to five hours at room temperature
(approximately 23.degree. C.). In a non-adsorbant plate (Nunc
#269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed with serial
dilutions of a Fab of interest (e.g., Fab-12 as described in Presta
et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is
then incubated overnight; however, the incubation may continue for
a longer period (e.g., 65 hours) to insure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
Tween.RTM. 20 in PBS. When the plates have dried, 150 ul/well of
scintillant (MicroScint-20; Packard) is added, and the plates are
counted on a Topcount gamma counter (Packard) for ten minutes.
Concentrations of each Fab that give less than or equal to 20% of
maximal binding are chosen for use in competitive binding assays.
According to another embodiment the Kd or Kd value is measured by
using surface plasmon resonance assays using a BIAcore.TM.-2000 or
a BIAcore.TM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized antigen CM5 chips at .about.10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
into 5 ug/ml (.about.0.2 uM) before injection at a flow rate of 5
ul/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% Tween.RTM. 20 (PBST) at 25.degree.
C. at a flow rate of approximately 25 ul/min. Association rates
(k.sub.on) and dissociation rates (k.sub.off) are calculated using
a simple one-to-one Langmuir binding model (BIAcore Evaluation
Software version 3.2) by simultaneous fitting the association and
dissociation sensorgram. The equilibrium dissociation constant
(K.sub.d) is calculated as the ratio k.sub.off/k.sub.on. See, e.g.,
Chen et al., J. Mol. Biol 293:865-881 (1999). If the on-rate
exceeds 10.sup.6 M.sup.-1S.sup.-1 by the surface plasmon resonance
assay above, then the on-rate can be determined by using a
fluorescent quenching technique that measures the increase or
decrease in fluorescence emission intensity (excitation=295 nm;
emission=340 nm, 16 nm band-pass) at 25.degree. C. of a 20 nM
anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of
increasing concentrations of antigen as measured in a spectrometer,
such as a stop-flow equipped spectrophometer (Aviv Instruments) or
a 8000-series SLM-Aminco.TM. spectrophotometer (ThermoSpectronic)
with a stirred cuvette.
[0144] The phrase "substantially reduced," or "substantially
different", as used herein, denotes a sufficiently high degree of
difference between two numeric values (generally one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody) such that one of skill in the art
would consider the difference between the two values to be of
statistical significance within the context of the biological
characteristic measured by said values (e.g., K.sub.d values, HAMA
response). The difference between said two values is preferably
greater than about 10%, preferably greater than about 20%,
preferably greater than about 30%, preferably greater than about
40%, preferably greater than about 50% as a function of the value
for the reference/comparator antibody.
[0145] "Percent (%) amino acid sequence identity" with respect to a
peptide or polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the specific peptide or polypeptide
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent
amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN or
Megalign (DNASTAR.TM.) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the full
length of the sequences being compared. For purposes herein,
however, % amino acid sequence identity values are generated using
the sequence comparison computer program ALIGN-2. The ALIGN-2
sequence comparison computer program was authored by Genentech,
Inc. and the source code shown has been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559,
where it is registered under U.S. Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through
Genentech, Inc., South San Francisco, Calif. The ALIGN-2 program
should be compiled for use on a UNIX operating system, preferably
digital UNIX V4.0D. All sequence comparison parameters are set by
the ALIGN-2 program and do not vary.
[0146] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A.
[0147] Unless specifically stated otherwise, all % amino acid
sequence identity values used herein are obtained as described in
the immediately preceding paragraph using the ALIGN-2 computer
program.
[0148] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
phage vector. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "recombinant vectors"). In general, expression vectors
of utility in recombinant DNA techniques are often in the form of
plasmids. In the present specification, "plasmid" and "vector" may
be used interchangeably as the plasmid is the most commonly used
form of vector.
[0149] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase, or by a synthetic reaction. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after synthesis, such as by conjugation with a
label. Other types of modifications include, for example, "caps,"
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as, for example,
those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.) and with
charged linkages (e.g., phosphorothioates, phosphorodithioates,
etc.), those containing pendant moieties, such as, for example,
proteins (e.g., nucleases, toxins, antibodies, signal peptides,
poly-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid or semi-solid supports.
The 5' and 3' terminal OH can be phosphorylated or substituted with
amines or organic capping group moieties of from 1 to 20 carbon
atoms. Other hydroxyls may also be derivatized to standard
protecting groups. Polynucleotides can also contain analogous forms
of ribose or deoxyribose sugars that are generally known in the
art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-
or 2'-azido-ribose, carbocyclic sugar analogs, .alpha.-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by P(O)S
("thioate"), P(S)S ("dithioate"), "(O)NR.sub.2 ("amidate"), P(O)R,
P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or R' is
independently H or substituted or unsubstituted alkyl (1-20 C.)
optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0150] "Oligonucleotide," as used herein, generally refers to
short, generally single stranded, generally synthetic
polynucleotides that are generally, but not necessarily, less than
about 200 nucleotides in length. The terms "oligonucleotide" and
"polynucleotide" are not mutually exclusive. The description above
for polynucleotides is equally and fully applicable to
oligonucleotides.
[0151] A "disorder" or "disease" is any condition that would
benefit from treatment with a substance/molecule or method of the
invention. This includes chronic and acute disorders or diseases
including those pathological conditions which predispose the mammal
to the disorder in question. Non-limiting examples of disorders to
be treated herein include malignant and benign tumors;
non-leukemias and lymphoid malignancies; neuronal, glial,
astrocytal, hypothalamic and other glandular, macrophagal,
epithelial, stromal and blastocoelic disorders; and inflammatory,
immunologic and other angiogenesis-related disorders.
[0152] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer. In one embodiment, the cell
proliferative disorder is angiogenesis.
[0153] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer",
"cancerous", "cell proliferative disorder", "proliferative
disorder" and "tumor" are not mutually exclusive as referred to
herein.
[0154] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell proliferation. Examples of cancer include but
are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and
leukemia. More particular examples of such cancers include squamous
cell cancer, lung cancer (including small-cell lung cancer,
non-small cell lung cancer, adenocarcinoma of the lung, and
squamous carcinoma of the lung), cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer (including
gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
breast cancer, colon cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, liver cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma and various types of head and neck
cancer, as well as B-cell lymphoma (including low grade/follicular
non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL;
intermediate grade/follicular NHL; intermediate grade diffuse NHL;
high grade immunoblastic NHL; high grade lymphoblastic NHL; high
grade small non-cleaved cell NHL; bulky disease NHL; mantle cell
lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), and Meigs' syndrome.
[0155] The term "anti-neoplastic composition" or "anti-cancer
composition" or "anti-cancer agent" refers to a composition useful
in treating cancer comprising at least one active therapeutic
agent, e.g., "anti-cancer agent." Examples of therapeutic agents
(anti-cancer agents) include, but are limited to, e.g.,
chemotherapeutic agents, growth inhibitory agents, cytotoxic
agents, agents used in radiation therapy, anti-angiogenesis agents,
apoptotic agents, anti-tubulin agents, and other-agents to treat
cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, an
epidermal growth factor receptor (EGFR) antagonist (e.g., a
tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib
(Tarceva.TM.), platelet derived growth factor inhibitors (e.g.,
Gleevec.TM. (Imatinib Mesylate)), a COX-2 inhibitor (e.g.,
celecoxib), interferons, cytokines, antagonists (e.g., neutralizing
antibodies) that bind to one or more of the following targets
ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA VEGF, or VEGF
receptor(s), TRAIL/Apo2, and other bioactive and organic chemical
agents, etc. Combinations thereof are also included in the
invention.
[0156] An "angiogenic factor or agent" is a growth factor which
stimulates the development of blood vessels, e.g., promote
angiogenesis, endothelial cell growth, stability of blood vessels,
and/or vasculogenesis, etc. For example, angiogenic factors,
include, but are not limited to, e.g., VEGF and members of the VEGF
family, PlGF, PDGF family, fibroblast growth factor family (FGFs),
TIE ligands (Angiopoietins), ephrins, Del-1, fibroblast growth
factors: acidic (aFGF) and basic (bFGF), Follistatin, Granulocyte
colony-stimulating factor (G-CSF), Hepatocyte growth factor
(HGF)/scatter factor (SF), Interleukin-8 (IL-8), Leptin, Midkine,
Placental growth factor, Platelet-derived endothelial cell growth
factor (PD-ECGF), Platelet-derived growth factor, especially
PDGF-BB or PDGFR-beta, Pleiotrophin (PTN), Progranulin, Proliferin,
Transforming growth factor-alpha (TGF-alpha), Transforming growth
factor-beta (TGF-beta), Tumor necrosis factor-alpha (TNF-alpha),
Vascular endothelial growth factor (VEGF)/vascular permeability
factor (VPF), etc. It would also include factors that accelerate
wound healing, such as growth hormone, insulin-like growth factor-I
(IGF-I), VIGF, epidermal growth factor (EGF), CTGF and members of
its family, and TGF-alpha and TGF-beta. See, e.g., Klagsbrun and
D'Amore, Annu. Rev. Physiol., 53:217-39 (1991); Streit and Detmar,
Oncogene, 22:3172-3179 (2003); Ferrara & Alitalo, Nature
Medicine 5(12):1359-1364 (1999); Tonini et al., Oncogene,
22:6549-6556 (2003) (e.g., Table 1 listing known angiogenic
factors); and, Sato Int. J. Clin. Oncol., 8:200-206 (2003).
[0157] The term "VEGF" as used herein refers to the 165-amino acid
human vascular endothelial cell growth factor and related 121-,
189-, and 206-amino acid human vascular endothelial cell growth
factors, as described by Leung et al. Science, 246:1306 (1989), and
Houck et al. Mol. Endocrin., 5:1806 (1991), together with the
naturally occurring allelic and processed forms thereof. The term
"VEGF" also refers to VEGFs from non-human species such as mouse,
rat or primate. Sometimes the VEGF from a specific species are
indicated by terms such as hVEGF for human VEGF, mVEGF for murine
VEGF, and etc. The term "VEGF" is also used to refer to truncated
forms of the polypeptide comprising amino acids 8 to 109 or 1 to
109 of the 165-amino acid human vascular endothelial cell growth
factor. Reference to any such forms of VEGF may be identified in
the present application, e.g., by "VEGF (8-109)," "VEGF (1-109)" or
"VEGF.sub.165." The amino acid positions for a "truncated" native
VEGF are numbered as indicated in the native VEGF sequence. For
example, amino acid position 17 (methionine) in truncated native
VEGF is also position 17 (methionine) in native VEGF. The truncated
native VEGF has binding affinity for the KDR and Flt-1 receptors
comparable to native VEGF. According to a preferred embodiment, the
VEGF is a human VEGF.
[0158] A "VEGF antagonist" refers to a molecule capable of
neutralizing, blocking, inhibiting, abrogating, reducing or
interfering with VEGF activities including its binding to VEGF or
one or more VEGF receptors or the nucleic acid encoding them.
Preferrably, the VEGF antagonist binds VEGF or a VEGF receptor.
VEGF antagonists include anti-VEGF antibodies and antigen-binding
fragments thereof, polypeptides that bind VEGF and VEGF receptors
and block ligand-receptor interaction (e.g., immunoadhesins,
peptibodies), anti-VEGF receptor antibodies and VEGF receptor
antagonists such as small molecule inhibitors of the VEGFR tyrosine
kinases, aptamers that bind VEGF and nucleic acids that hybridize
under stringent conditions to nucleic acid sequences that encode
VEGF or VEGF receptor (e.g., RNAi). According to one preferred
embodiment, the VEGF antagonist binds to VEGF and inhibits
VEGF-induced endothelial cell proliferation in vitro. According to
one preferred embodiment, the VEGF antagonist binds to VEGF or a
VEGF receptor with greater affinity than a non-VEGF or non-VEGF
receptor. According to one preferred embodiment, the VEG antagonist
binds to VEGF or a VEGF receptor with a Kd of between 1 uM and 1
pM. According to another preferred embodiment, the VEGF antagonist
binds to VEGF or a VEGF receptor between 500 nM and 1 pM.
[0159] According a preferred embodiment, the VEGF antagonist is
selected from the group consisting of a polypeptide such as an
antibody, a peptibody, an immunoadhesin, a small molecule or an
aptamer. In a preferred embodiment, the antibody is an anti-VEGF
antibody such as the AVASTIN.RTM. antibody or an anti-VEGF receptor
antibody such as an anti-VEGFR2 or an anti-VEGFR3 antibody. Other
examples of VEGF antagonists include: VEGF-Trap, Mucagen, PTK787,
SU11248, AG-013736, Bay 439006 (sorafenib), ZD-6474, CP632,
CP-547632, AZD-2171, CDP-171, SU-14813, CHIR-258, AEE-788,
SB786034, BAY579352, CDP-791, EG-3306, GW-786034, RWJ-417975/CT6758
and KRN-633.
[0160] An "anti-VEGF antibody" is an antibody that binds to VEGF
with sufficient affinity and specificity. Preferably, the anti-VEGF
antibody of the invention can be used as a therapeutic agent in
targeting and interfering with diseases or conditions wherein the
VEGF activity is involved. An anti-VEGF antibody will usually not
bind to other VEGF homologues such as VEGF-B or VEGF-C, nor other
growth factors such as PlGF, PDGF or bFGF. A preferred anti-VEGF
antibody is a monoclonal antibody that binds to the same epitope as
the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC
HB 10709. More preferably the anti-VEGF antibody is a recombinant
humanized anti-VEGF monoclonal antibody generated according to
Presta et al. (1997) Cancer Res. 57:4593-4599, including but not
limited to the antibody known as bevacizumab (BV; Avastin.RTM.).
According to another embodiment, anti-VEGF antibodies that can be
used include, but are not limited to the antibodies disclosed in WO
2005/012359. According to one embodiment, the anti-VEGF antibody
comprises the variable heavy and variable light region of any one
of the antibodies disclosed in FIGS. 24, 25, 26, 27 and 29 of WO
2005/012359 (e.g., G6, G6-23, G6-31, G6-23.1, G6-23.2, B20, B20-4
and B20.4.1). In another preferred embodiment, the anti-VEGF
antibody known as ranibizumab is the VEGF antagonist administered
for ocular disease such as diabetic neuropathy and AMD.
[0161] The anti-VEGF antibody "Bevacizumab (BV)", also known as
"rhuMAb VEGF" or "Avastin.RTM.", is a recombinant humanized
anti-VEGF monoclonal antibody generated according to Presta et al.
(1997) Cancer Res. 57:4593-4599. It comprises mutated human IgG1
framework regions and antigen-binding complementarity-determining
regions from the murine anti-hVEGF monoclonal antibody A.4.6.1 that
blocks binding of human VEGF to its receptors. Approximately 93% of
the amino acid sequence of Bevacizumab, including most of the
framework regions, is derived from human IgG1, and about 7% of the
sequence is derived from the murine antibody A4.6.1. Bevacizumab
has a molecular mass of about 149,000 daltons and is glycosylated.
Other anti-VEGF antibodies include the antibodies described in U.S.
Pat. No. 6,884,879 and WO 2005/044853.
[0162] The anti-VEGF antibody Ranibizumab or the LUCENTIS.RTM.
antibody or rhuFab V2 is a humanized, affinity-matured anti-human
VEGF Fab fragment. Ranibizumab is produced by standard recombinant
technology methods in Escherichia coli expression vector and
bacterial fermentation. Ranibizumab is not glycosylated and has a
molecular mass of .about.48,000 daltons. See WO98/45331 and
US20030190317.
[0163] Dysregulation of angiogenesis can lead to abnormal
angiogenesis, i.e., when excessive or inappropriate growth of new
blood vessels (e.g., the location, timing or onset of the
angiogenesis being undesired from a medical standpoint) in a
diseased state or such that it causes a diseased state. Excessive,
inappropriate or uncontrolled angiogenesis occurs when there is new
blood vessel growth that contributes to the worsening of the
diseased state or causes a diseased state. The new blood vessels
can feed the diseased tissues, destroy normal tissues, and in the
case of cancer, the new vessels can allow tumor cells to escape
into the circulation and lodge in other organs (tumor metastases).
Disease states involving abnormal angiogenesis include both
non-neoplastic and neoplastic conditions including, e.g., cancer,
especially vascularized solid tumors and metastatic tumors
(including colon cancer, breast cancer, lung cancer (especially
small-cell lung cancer), or prostate cancer), undesired or aberrant
hypertrophy, arthritis, rheumatoid arthritis (RA), inflammatory
bowel disease or IBD (Crohn's disease and ulcerative colitis),
psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis,
atherosclerotic plaques, diabetic and other proliferative
retinopathies including retinopathy of prematurity, retrolental
fibroplasia, neovascular glaucoma, age-related macular
degeneration, diabetic macular edema, corneal neovascularization,
corneal graft neovascularization, corneal graft rejection,
retinal/choroidal neovascularization, neovascularization of the
anterior surface of the iris (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), chronic inflammation, lung
inflammation, acute lung injury/ARDS, sepsis, primary pulmonary
hypertension, malignant pulmonary effusions, cerebral edema (e.g.,
associated with acute stroke/closed head injury/trauma), synovial
inflammation, myositis ossificans, hypertropic bone formation,
osteoarthritis (OA), refractory ascites, polycystic ovarian
disease, endometriosis, 3rd spacing of fluid diseases
(pancreatitis, compartment syndrome, burns, bowel disease), uterine
fibroids, premature labor, chronic inflammation such as IBD, renal
allograft rejection, inflammatory bowel disease, nephrotic
syndrome, undesired or aberrant tissue mass growth (non-cancer),
hemophilic joints, hypertrophic scars, inhibition of hair growth,
Osler-Weber syndrome, pyogenic granuloma retrolental fibroplasias,
scleroderma, trachoma, vascular adhesions, synovitis, dermatitis,
preeclampsia, ascites, pericardial effusion (such as that
associated with pericarditis), and pleural effusion.
[0164] As used herein, "treatment" refers to clinical intervention
in an attempt to alter the natural course of the individual or cell
being treated, and can be performed either for prophylaxis or
during the course of clinical pathology. Desirable effects of
treatment include preventing occurrence or recurrence of disease,
alleviation of symptoms, diminishment of any direct or indirect
pathological consequences of the disease, preventing metastasis,
decreasing the rate of disease progression, amelioration or
palliation of the disease state, and remission or improved
prognosis. In some embodiments, antibodies of the invention are
used to delay development of a disease or disorder.
[0165] An "effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result.
[0166] A "therapeutically effective amount" of a substance/molecule
of the invention, agonist or antagonist may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the substance/molecule, agonist or
antagonist to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the substance/molecule, agonist or
antagonist are outweighed by the therapeutically beneficial
effects. The term "therapeutically effective amount" refers to an
amount of an antibody, polypeptide or antagonist of this invention
effective to "treat" a disease or disorder in a mammal (aka
patient). In the case of cancer, the therapeutically effective
amount of the drug can reduce the number of cancer cells; reduce
the tumor size or weight; inhibit (i.e., slow to some extent and
preferably stop) cancer cell infiltration into peripheral organs;
inhibit (i.e., slow to some extent and preferably stop) tumor
metastasis; inhibit, to some extent, tumor growth; and/or relieve
to some extent one or more of the symptoms associated with the
cancer. To the extent the drug can prevent growth and/or kill
existing cancer cells, it can be cytostatic and/or cytotoxic. In
one embodiment, the therapeutically effective amount is a growth
inhibitory amount. In another embodiment, the therapeutically
effective amount is an amount that extends the survival of a
patient. In another embodiment, the therapeutically effective
amount is an amount that improves progression free survival of a
patient.
[0167] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically but not necessarily,
since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease, the prophylactically effective amount is
less than the therapeutically effective amount.
[0168] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents e.g.
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described below.
A tumoricidal agent causes destruction of tumor cells.
[0169] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem Intl. Ed.
Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antiobiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel
(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM.
Cremophor-free, albumin-engineered nanoparticle formulation of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. doxetaxel (Rhone-Poulenc Rorer, Antony, France);
chloranbucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.); novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone, and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin. Additional
chemotherapeutic agents include the cytotoxic agents useful as
antibody drug conjugates, such as maytansinoids (DM1, for example)
and the auristatins MMAE and MMAF, for example.
[0170] "Chemotherapeutic agents" also include "anti-hormonal
agents" that act to regulate, reduce, block, or inhibit the effects
of hormones that can promote the growth of cancer, and are often in
the form of systemic, or whole-body treatment. They may be hormones
themselves. Examples include anti-estrogens and selective estrogen
receptor modulators (SERMs), including, for example, tamoxifen
(including NOLVADEX.RTM. tamoxifen), EVISTA.RTM. raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. toremifene; anti-progesterones;
estrogen receptor down-regulators (ERDs); agents that function to
suppress or shut down the ovaries, for example, leutinizing
hormone-releasing hormone (LHRH) agonists such as LUPRON.RTM. and
ELIGARD.RTM. leuprolide acetate, goserelin acetate, buserelin
acetate and tripterelin; other anti-androgens such as flutamide,
nilutamide and bicalutamide; and aromatase inhibitors that inhibit
the enzyme aromatase, which regulates estrogen production in the
adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, MEGASE.RTM. megestrol acetate, AROMASIN.RTM.
exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole,
FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole. In addition,
such definition of chemotherapeutic agents includes bisphosphonates
such as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.),
DIDROCAL.RTM. etidronate, NE-58095, ZOMETA.RTM. zoledronic
acid/zoledronate, FOSAMAX.RTM. alendronate, AREDIA.RTM.
pamidronate, SKELID.RTM. tiludronate, or ACTONEL.RTM. risedronate;
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine
analog); antisense oligonucleotides, particularly those that
inhibit expression of genes in signaling pathways implicated in
abherant cell proliferation, such as, for example, PKC-alpha, Raf,
H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such
as THERATOPE.RTM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase
small-molecule inhibitor also known as GW572016); and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0171] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth and/or proliferation
of a cell (e.g., a cell expressing Robo4) either in vitro or in
vivo. Thus, the growth inhibitory agent may be one which
significantly reduces the percentage of Robo4-expressing cells in S
phase. Examples of growth inhibitory agents include agents that
block cell cycle progression (at a place other than S phase), such
as agents that induce G1 arrest and M-phase arrest. Classical
M-phase blockers include the vincas (vincristine and vinblastine),
taxanes, and topoisomerase II inhibitors such as doxorubicin,
epirubicin, daunorubicin, etoposide, and bleomycin. Those agents
that arrest G1 also spill over into S-phase arrest, for example,
DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,
mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and
ara-C. Further information can be found in The Molecular Basis of
Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell
cycle regulation, oncogenes, and antineoplastic drugs" by Murakami
et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The
taxanes (paclitaxel and docetaxel) are anticancer drugs both
derived from the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc
Rorer), derived from the European yew, is a semisynthetic analogue
of paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0172] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexapyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione.
III. Anti-Robo4 Antibodies
[0173] In one aspect, the invention provides antibodies that bind
to Robo4. In one embodiment, an anti-Robo4 antibody is a monoclonal
antibody. In one embodiment, an anti-Robo4 antibody is an antibody
fragment, e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab').sub.2 fragment.
In one embodiment, an anti-Robo4 antibody is a chimeric, humanized,
or human antibody. In one embodiment, an anti-Robo4 antibody is
purified.
[0174] In another aspect of the invention, polynucleotides encoding
anti-Robo4 antibodies are provided. In certain embodiments, vectors
comprising polynucleotides encoding anti-Robo4 antibodies are
provided. In certain embodiments, host cells comprising such
vectors are provided. In another aspect of the invention,
compositions comprising anti-Robo4 antibodies or polynucleotides
encoding anti-Robo4 antibodies are provided. In certain
embodiments, a composition is a pharmaceutical formulation for the
treatment of diseases and disorders including, e.g., cancer,
atherosclerosis, retrolental fibroplasia, hemangiomas, chronic
inflammation, intraocular neovascular diseases, proliferative
retinopathies, diabetic retinopathy, age-related macular
degeneration (AMD), neovascular glaucoma, immune rejection of
transplanted corneal tissue and other tissues, rheumatoid
arthritis, psoriasis, and combinations thereof.
[0175] Exemplary monoclonal antibodies derived from a phage library
are provided herein and described in the Examples below. Those
antibodies are designated YW71.6, YW71.1, YW71.22, YW71.89, YW79.1,
YW79.8, and YW79.11. Those antibodies were affinity matured to
generate YW71.22.S1.2, YW71.22S1.8, YW71.22S1.16, YW71.22S1.23,
YW71.22S1.24, YW71.22S1.27, YW71.22S1.31, YW71.22S1.38,
YW71.22S1.77 YW71.22.S2.21, YW71.22.S2.79, YW71.22.H1.2,
YW71.22.H1.9, YW71.22.H1.46, YW71.22.H1.77, YW71.22.H1.91, and
YW71.22.H2.31. The sequences of the heavy and light chain variable
domains of the antibodies are set forth in FIGS. 1 and 2.
[0176] An antibody of the invention can comprise any suitable human
or human consensus light chain framework sequences, provided the
antibody exhibits the desired biological characteristics (e.g., a
desired binding affinity).
[0177] In one embodiment, human consensus frameworks herein are
from, or derived from, VH subgroup III (see FIGS. 4A and 4B) and/or
VL kappa subgroup I (see FIGS. 3A and 3B) consensus framework
sequences.
[0178] Thus, the VH acceptor human framework may comprise one, two,
three or all four of the following framework sequences:
TABLE-US-00003 FR1 comprising EVQLVESGGGLVQPGGSLRLSCAAS, (SEQ ID
NO: 13) FR2 comprising WVRQAPGKGLEWV, (SEQ ID NO: 14) FR3
comprising FR3 comprises RFTISADTSKNTAYLQMNSLRAEDTAVYYC, (SEQ ID
NO: 15) FR4 comprising WGQGTLVTVSS. (SEQ ID NO: 16)
[0179] In other embodiments, the VH consensus frameworks
include:
human VH subgroup I consensus frameworks 1-4 minus Kabat CDRs (SEQ
ID NOS:111, 112, 113, 16); human VH subgroup I consensus frameworks
1-4 minus extended hypervariable regions (SEQ ID NOS:114, 115, 113,
16 or SEQ ID NOS:114, 115, 116, 16 or SEQ ID NOS:114, 115, 117,
16); human VH subgroup II consensus frameworks 1-4 minus Kabat CDRs
(SEQ ID NOS: 118, 119, 120, 16); human VH subgroup II consensus
frameworks 1-4 minus extended hypervariable regions (SEQ ID
NOS:121, 122, 120, 16 or SEQ ID NOS:121, 122, 123, 16 or SEQ ID
NOS:121, 122, 124, 16); human VH subgroup III consensus frameworks
1-4 minus Kabat CDRs (SEQ ID NO:125, 126, 127, 16); human VH
subgroup III consensus frameworks 1-4 minus extended hypervariable
regions (SEQ ID NOS:128, 129, 127, 16 or SEQ ID NOS:128, 129, 130,
16 or SEQ ID NOS:128, 129, 131, 16); human VH acceptor 1 frameworks
1-4 minus Kabat CDRs (SEQ ID NOS:132, 126, 133, 16); human VH
acceptor 1 frameworks 1-4 minus extended hypervariable regions (SEQ
ID NOS:128, 129, 133, 16 or SEQ ID NOS:128, 129, 134, 16); human VH
acceptor 2 frameworks 1-4 minus Kabat CDRs (SEQ ID NO:132, 126,
135, 16); or human VH acceptor 2 frameworks 1-4 minus extended
hypervariable regions (SEQ ID NOS:128, 126, 135, 16 or SEQ ID
NOS:128, 126, 136, 16 or SEQ ID NOS:128, 126, 137, 16).
[0180] In one embodiment, the VH acceptor human framework region 4
(H-FR4) comprises WGQGTLVTVSS (SEQ ID NO:45).
[0181] The VL acceptor human framework may comprise one, two, three
or four of the following framework sequences:
TABLE-US-00004 FR1 comprising DIQMTQSPSSLSASVGDRVTITC, (SEQ ID NO:
9) FR2 comprising WYQQKPGKAPKLLIY, (SEQ ID NO: 10) FR3 comprising
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC, (SEQ ID NO: 11) FR4 comprising
FGQGTKVEIKR (SEQ ID NO: 173) or FGQGTKMEIKR. (SEQ ID NO: 139)
In other embodiment, the VL consensus frameworks include: human VL
kappa subgroup I consensus frameworks 1-4 (SEQ ID NOS:9, 10, 99,
100); human VL kappa subgroup I consensus frameworks 1-4 (SEQ ID
NOS:9, 101, 99, 100); human VL kappa subgroup II consensus
frameworks 1-4 (SEQ ID NOS:102, 103, 104, 100); human VL kappa
subgroup III consensus frameworks 1-4 (SEQ ID NOS:105, 106, 107,
100); or human VL kappa subgroup IV consensus frameworks 1-4 (SEQ
ID NOS:108, 109, 110, 100)
[0182] While the acceptor may be identical in sequence to the human
framework sequence selected, whether that be from a human
immunoglobulin or a human consensus framework, the present
invention contemplates that the acceptor sequence may comprise
pre-existing amino acid substitutions relative to the human
immunoglobulin sequence or human consensus framework sequence.
These pre-existing substitutions are preferably minimal; usually
four, three, two or one amino acid differences only relative to the
human immunoglobulin sequence or consensus framework sequence.
[0183] Hypervariable region residues of the non-human antibody are
incorporated into the VL and/or VH acceptor human frameworks. For
example, one may incorporate residues corresponding to the Kabat
CDR residues, the Chothia hypervariable loop residues, the Abm
residues, and/or contact residues. Optionally, the extended
hypervariable region residues as follows are incorporated: 24-34
(L1), 50-56 (L2) and 89-97 (L3), 26-35 (H1), 50-65 or 49-65 (H2)
and 93-102, 94-102, or 95-102 (H3).
[0184] While "incorporation" of hypervariable region residues is
discussed herein, it will be appreciated that this can be achieved
in various ways, for example, nucleic acid encoding the desired
amino acid sequence can be generated by mutating nucleic acid
encoding the mouse variable domain sequence so that the framework
residues thereof are changed to acceptor human framework residues,
or by mutating nucleic acid encoding the human variable domain
sequence so that the hypervariable domain residues are changed to
non-human residues, or by synthesizing nucleic acid encoding the
desired sequence, etc.
[0185] In the examples herein, hypervariable region-grafted
variants were generated by Kunkel mutagenesis of nucleic acid
encoding the human acceptor sequences, using a separate
oligonucleotide for each hypervariable region. Kunkel et al.,
Methods Enzymol. 154:367-382 (1987). Appropriate changes can be
introduced within the framework and/or hypervariable region, using
routine techniques, to correct and re-establish proper
hypervariable region-antigen interactions.
[0186] A. Antibody Fragments
[0187] The present invention encompasses antibody fragments.
Antibody fragments may be generated by traditional means, such as
enzymatic digestion, or by recombinant techniques. In certain
circumstances there are advantages of using antibody fragments,
rather than whole antibodies. The smaller size of the fragments
allows for rapid clearance, and may lead to improved access to
solid tumors. For a review of certain antibody fragments, see
Hudson et al. (2003) Nat. Med. 9:129-134.
[0188] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10: 163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Fab and F(ab').sub.2 fragment with increased in
vivo half-life comprising salvage receptor binding epitope residues
are described in U.S. Pat. No. 5,869,046. Other techniques for the
production of antibody fragments will be apparent to the skilled
practitioner. In certain embodiments, an antibody is a single chain
Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and
5,587,458. Fv and scFv are the only species with intact combining
sites that are devoid of constant regions; thus, they may be
suitable for reduced nonspecific binding during in vivo use. scFv
fusion proteins may be constructed to yield fusion of an effector
protein at either the amino or the carboxy terminus of an scFv. See
Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment
may also be a "linear antibody", e.g., as described in U.S. Pat.
No. 5,641,870, for example. Such linear antibodies may be
monospecific or bispecific.
[0189] B. Humanized Antibodies
[0190] The invention encompasses humanized antibodies. Various
methods for humanizing non-human antibodies are known in the art.
For example, a humanized antibody can have one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al. (1986) Nature
321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen
et al. (1988) Science 239:1534-1536), by substituting hypervariable
region sequences for the corresponding sequences of a human
antibody. Accordingly, such "humanized" antibodies are chimeric
antibodies (U.S. Pat. No. 4,816,567) wherein substantially less
than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[0191] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies can be important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework for the humanized
antibody. See, e.g., Sims et al. (1993) J. Immunol. 151:2296;
Chothia et al. (1987) J. Mol. Biol. 196:901. Another method uses a
particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies. See, e.g., Carter et al (1992) PNAS USA, 89:4285;
Presta et al. (1993) J. Immunol., 151:2623.
[0192] It is further generally desirable that antibodies be
humanized with retention of high affinity for the antigen and other
favorable biological properties. To achieve this goal, according to
one method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0193] In some embodiments, the invention provides antibodies that
are humanized such that HAMA response is reduced or eliminated.
Reduction or elimination of a HAMA response is a significant aspect
of clinical development of suitable therapeutic agents. See, e.g.,
Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et
al., Transplantation (1986), 41:572; Shawler et al., J. Immunol.
(1985), 135:1530; Sears et al., J. Biol. Response Mod. (1984),
3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J.
Immunol. (1991), 147:1352; Reichmann et al., Nature (1988),
332:323; Junghans et al., Cancer Res. (1990), 50:1495. Variants of
these antibodies can further be obtained using routine methods
known in the art, some of which are further described below.
[0194] For example, an amino acid sequence from an antibody as
described herein can serve as a starting (parent) sequence for
diversification of the framework and/or hypervariable sequence(s).
A selected framework sequence to which a starting hypervariable
sequence is linked is referred to herein as an acceptor human
framework. While the acceptor human frameworks may be from, or
derived from, a human immunoglobulin (the VL and/or VH regions
thereof), preferably the acceptor human frameworks are from, or
derived from, a human consensus framework sequence as such
frameworks have been demonstrated to have minimal, or no,
immunogenicity in human patients.
[0195] Where the acceptor is derived from a human immunoglobulin,
one may optionally select a human framework sequence that is
selected based on its homology to the donor framework sequence by
aligning the donor framework sequence with various human framework
sequences in a collection of human framework sequences, and select
the most homologous framework sequence as the acceptor.
[0196] C. Human Antibodies
[0197] Human antibodies of the invention can be constructed by
combining Fv clone variable domain sequence(s) selected from
human-derived phage display libraries with known human constant
domain sequences(s) as described above. Alternatively, human
monoclonal antibodies of the invention can be made by the hybridoma
method. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described,
for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et
al., Monoclonal Antibody Production Techniques and Applications,
pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et
al., J. Immunol., 147: 86 (1991).
[0198] It is now possible to produce transgenic animals (e.g. mice)
that are capable, upon immunization, of producing a full repertoire
of human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (JH) gene in
chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge. See,
e.g., Jakobovits et al., PNAS USA, 90: 2551 (1993); Jakobovits et
al., Nature, 362: 255 (1993); Bruggermann et al., Year in Immunol.,
7: 33 (1993).
[0199] Gene shuffling can also be used to derive human antibodies
from non-human, e.g. rodent, antibodies, where the human antibody
has similar affinities and specificities to the starting non-human
antibody. According to this method, which is also called "epitope
imprinting", either the heavy or light chain variable region of a
non-human antibody fragment obtained by phage display techniques as
described herein is replaced with a repertoire of human V domain
genes, creating a population of non-human chain/human chain scFv or
Fab chimeras. Selection with antigen results in isolation of a
non-human chain/human chain chimeric scFv or Fab wherein the human
chain restores the antigen binding site destroyed upon removal of
the corresponding non-human chain in the primary phage display
clone, i.e. the epitope governs (imprints) the choice of the human
chain partner. When the process is repeated in order to replace the
remaining non-human chain, a human antibody is obtained (see PCT WO
93/06213 published Apr. 1, 1993). Unlike traditional humanization
of non-human antibodies by CDR grafting, this technique provides
completely human antibodies, which have no FR or CDR residues of
non-human origin.
[0200] D. Antibody Variants
[0201] In some embodiments, amino acid sequence modification(s) of
the antibodies described herein are contemplated. For example, it
may be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the antibody may be prepared by introducing appropriate changes
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of, residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. The amino acid alterations may be introduced in
the subject antibody amino acid sequence at the time that sequence
is made.
[0202] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (e.g., alanine or
polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
immunoglobulins are screened for the desired activity.
[0203] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0204] In certain embodiments, an antibody of the invention is
altered to increase or decrease the extent to which the antibody is
glycosylated. Glycosylation of polypeptides is typically either
N-linked or O-linked. N-linked refers to the attachment of a
carbohydrate moiety to the side chain of an asparagine residue. The
tripeptide sequences asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars N-aceylgalactosamine,
galactose, or xylose to a hydroxyamino acid, most commonly serine
or threonine, although 5-hydroxyproline or 5-hydroxylysine may also
be used.
[0205] Addition or deletion of glycosylation sites to the antibody
is conveniently accomplished by altering the amino acid sequence
such that one or more of the above-described tripeptide sequences
(for N-linked glycosylation sites) is created or removed. The
alteration may also be made by the addition, deletion, or
substitution of one or more serine or threonine residues to the
sequence of the original antibody (for O-linked glycosylation
sites).
[0206] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
(1997) TIBTECH 15:26-32. The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody of the invention may be made in order to create antibody
variants with certain improved properties.
[0207] For example, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. Such variants may have improved ADCC
function. See, e.g., US Patent Publication Nos. US 2003/0157108
(Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
Examples of publications related to "defucosylated" or
"fucose-deficient" antibody variants include: US 2003/0157108; WO
2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US
2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US
2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO
2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol.
Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng.
87: 614 (2004). Examples of cell lines capable of producing
defucosylated antibodies include Lec 13 CHO cells deficient in
protein fucosylation (Ripka et al. Arch. Biochem. Biophys.
249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L;
and WO 2004/056312 A1, Adams et al., especially at Example 11), and
knockout cell lines, such as alpha-1,6-fucosyltransferase gene,
FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech.
Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng.,
94(4):680-688 (2006); and WO2003/085107).
[0208] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0209] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which further
improve ADCC, for example, substitutions at positions 298, 333,
and/or 334 of the Fc region (Eu numbering of residues). Such
substitutions may occur in combination with any of the variations
described above.
[0210] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for many
applications in which the half life of the antibody in vivo is
important yet certain effector functions (such as complement and
ADCC) are unnecessary or deleterious. In certain embodiments, the
Fc activities of the antibody are measured to ensure that only the
desired properties are maintained. In vitro and/or in vivo
cytotoxicity assays can be conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc
receptor (FcR) binding assays can be conducted to ensure that the
antibody lacks Fc.gamma.R binding (hence likely lacking ADCC
activity), but retains FcRn binding ability. The primary cells for
mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas
monocytes express Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR
expression on hematopoietic cells is summarized in Table 3 on page
464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991).
Non-limiting examples of in vitro assays to assess ADCC activity of
a molecule of interest is described in U.S. Pat. No. 5,500,362
(see, e.g. Hellstrom, I., et al. PNAS USA 83:7059-7063 (1986)) and
Hellstrom, I et al., PNAS USA 82:1499-1502 (1985); U.S. Pat. No.
5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361
(1987)). Alternatively, non-radioactive assays methods may be
employed (see, for example, ACTI.TM. non-radioactive cytotoxicity
assay for flow cytometry (CellTechnology, Inc. Mountain View,
Calif.; and CytoTox 96.RTM. non-radioactive cytotoxicity assay
(Promega, Madison, Wis.). Useful effector cells for such assays
include peripheral blood mononuclear cells (PBMC) and Natural
Killer (NK) cells. Alternatively, or additionally, ADCC activity of
the molecule of interest may be assessed in vivo, e.g., in a animal
model such as that disclosed in Clynes et al. PNAS USA 95:652-656
(1998). C1q binding assays may also be carried out to confirm that
the antibody is unable to bind C1q and hence lacks CDC activity. To
assess complement activation, a CDC assay may be performed (see,
for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163
(1996); Cragg, M. S. et al., Blood 101: 1045-1052 (2003); and
Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn
binding and in vivo clearance/half life determinations can also be
performed using methods known in the art (see, for example,
Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769
(2006)).
[0211] Other antibody variants having one or more amino acid
substitutions are provided. Sites of interest for substitutional
mutagenesis include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are shown in
Table 1 under the heading of "preferred substitutions." More
substantial changes, denominated "exemplary substitutions" are
provided in Table 1, or as further described below in reference to
amino acid classes. Amino acid substitutions may be introduced into
an antibody of interest and the products screened, e.g., for a
desired activity, such as improved antigen binding, decreased
immunogenicity, improved ADCC or CDC, etc.
TABLE-US-00005 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; Ile; Val; Ile
Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine
[0212] Modifications in the biological properties of an antibody
may be accomplished by selecting substitutions that affect (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Amino acids may be grouped
according to similarities in the properties of their side chains
(in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth
Publishers, New York (1975)):
[0213] (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P),
Phe (F), Trp (W), Met (M)
[0214] (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr
(Y), Asn (N), Gln (O)
[0215] (3) acidic: Asp (D), Glu (E)
[0216] (4) basic: Lys (K), Arg (R), H is(H)
[0217] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties:
[0218] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0219] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0220] (3) acidic: Asp, Glu;
[0221] (4) basic: His, Lys, Arg;
[0222] (5) residues that influence chain orientation: Gly, Pro;
[0223] (6) aromatic: Trp, Tyr, Phe.
[0224] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, into the remaining (non-conserved) sites.
[0225] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further development will have modified (e.g.,
improved) biological properties relative to the parent antibody
from which they are generated. An exemplary substitutional variant
is an affinity matured antibody, which may be conveniently
generated using phage display-based affinity maturation techniques.
Briefly, several hypervariable region sites (e.g. 6-7 sites) are
mutated to generate all possible amino acid substitutions at each
site. The antibodies thus generated are displayed from filamentous
phage particles as fusions to at least part of a phage coat protein
(e.g., the gene III product of M13) packaged within each particle.
The phage-displayed variants are then screened for their biological
activity (e.g. binding affinity). In order to identify candidate
hypervariable region sites for modification, scanning mutagenesis
(e.g., alanine scanning) can be performed to identify hypervariable
region residues contributing significantly to antigen binding.
Alternatively, or additionally, it may be beneficial to analyze a
crystal structure of the antigen-antibody complex to identify
contact points between the antibody and antigen. Such contact
residues and neighboring residues are candidates for substitution
according to techniques known in the art, including those
elaborated herein. Once such variants are generated, the panel of
variants is subjected to screening using techniques known in the
art, including those described herein, and variants with superior
properties in one or more relevant assays may be selected for
further development.
[0226] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
[0227] It may be desirable to introduce one or more amino acid
modifications in an Fc region of antibodies of the invention,
thereby generating an Fc region variant. The Fc region variant may
comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3
or IgG4 Fc region) comprising an amino acid modification (e.g. a
substitution) at one or more amino acid positions including that of
a hinge cysteine.
[0228] In accordance with this description and the teachings of the
art, it is contemplated that in some embodiments, an antibody of
the invention may comprise one or more alterations as compared to
the wild type counterpart antibody, e.g. in the Fc region. These
antibodies would nonetheless retain substantially the same
characteristics required for therapeutic utility as compared to
their wild type counterpart. For example, it is thought that
certain alterations can be made in the Fc region that would result
in altered (i.e., either improved or diminished) C1q binding and/or
Complement Dependent Cytotoxicity (CDC), e.g., as described in
WO99/51642. See also Duncan & Winter, Nature 322:738-40 (1988);
U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO94/29351
concerning other examples of Fc region variants. WO00/42072
(Presta) and WO 2004/056312 (Lowman) describe antibody variants
with improved or diminished binding to FcRs. The content of these
patent publications are specifically incorporated herein by
reference. See, also, Shields et al. J. Biol. Chem. 9(2): 6591-6604
(2001). Antibodies with increased half lives and improved binding
to the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). These antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Polypeptide variants with
altered Fc region amino acid sequences and increased or decreased
C1q binding capability are described in U.S. Pat. No. 6,194,551B1,
WO99/51642. The contents of those patent publications are
specifically incorporated herein by reference. See, also, Idusogie
et al. J. Immunol. 164: 4178-4184 (2000).
[0229] In another aspect, the invention provides antibodies
comprising modifications in the interface of Fc polypeptides
comprising the Fc region, wherein the modifications facilitate
and/or promote heterodimerization. These modifications comprise
introduction of a protuberance into a first Fc polypeptide and a
cavity into a second Fc polypeptide, wherein the protuberance is
positionable in the cavity so as to promote complexing of the first
and second Fc polypeptides. Methods of generating antibodies with
these modifications are known in the art, e.g., as described in
U.S. Pat. No. 5,731,168.
[0230] In yet another aspect, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," and "thioFabs" in
which one or more residues of an antibody are substituted with
cysteine residues. In particular embodiments, the substituted
residues occur at accessible sites of the antibody. By substituting
those residues with cysteine, reactive thiol groups are thereby
positioned at accessible sites of the antibody and may be used to
conjugate the antibody to other moieties, such as drug moieties or
linker-drug moieties, as described further herein. In certain
embodiments, any one or more of the following residues may be
substituted with cysteine: V205 (Kabat numbering) of the light
chain; A118 (EU numbering) of the heavy chain; and S400 (EU
numbering) of the heavy chain Fc region. In a preferred embodiment,
A118 (EU numbering) of the heavy chain is substituted for cysteine.
Cysteine engineered thioMabs and thioFabs are described in further
detail herein below.
[0231] E. Antibody Derivatives
[0232] The antibodies of the present invention can be further
modified to contain additional nonproteinaceous moieties that are
known in the art and readily available. Preferably, the moieties
suitable for derivatization of the antibody are water soluble
polymers. Non-limiting examples of water soluble polymers include,
but are not limited to, polyethylene glycol (PEG), copolymers of
ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,
polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
propropylene glycol homopolymers, polypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have advantages in manufacturing due to its
stability in water. The polymer may be of any molecular weight, and
may be branched or unbranched. The number of polymers attached to
the antibody may vary, and if more than one polymer are attached,
they can be the same or different molecules. In general, the number
and/or type of polymers used for derivatization can be determined
based on considerations including, but not limited to, the
particular properties or functions of the antibody to be improved,
whether the antibody derivative will be used in a therapy under
defined conditions, etc.
[0233] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., PNAS USA 102: 11600-11605
(2005)). The radiation may be of any wavelength, and includes, but
is not limited to, wavelengths that do not harm ordinary cells, but
which heat the nonproteinaceous moiety to a temperature at which
cells proximal to the antibody-nonproteinaceous moiety are
killed.
IV. Methods of Making Antibodies
[0234] A. Generating Phage-Derived Antibodies
[0235] Antibodies of the invention may be prepared by phage(mid)
display techniques as disclosed in Lee et al., J. Mol. Biol.
(2004), 340(5): 1073-93). Phage(mid) display allows the generation
of large libraries of protein variants which can be rapidly sorted
for those sequences that bind to a target molecule with high
affinity. Nucleic acids encoding variant polypeptides are generally
fused to a nucleic acid sequence encoding a viral coat protein,
such as the gene III protein or the gene VIII protein. Monovalent
phagemid display systems where the nucleic acid sequence encoding
the protein or polypeptide is fused to a nucleic acid sequence
encoding a portion of the gene III protein have been developed.
(Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: A
Companion to Methods in Enzymology, 3:205 (1991)). In a monovalent
phagemid display system, the gene fusion is expressed at low levels
and wild type gene III proteins are also expressed so that
infectivity of the particles is retained. Methods of generating
peptide libraries and screening those libraries have been disclosed
in many patents (e.g. U.S. Pat. No. 5,723,286, U.S. Pat. No.
5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908 and
U.S. Pat. No. 5,498,530).
[0236] Libraries of antibodies or antigen binding polypeptides have
been prepared in a number of ways including by altering a single
gene by inserting random DNA sequences or by cloning a family of
related genes. Methods for displaying antibodies or antigen binding
fragments using phage(mid) display have been described in U.S. Pat.
Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197,
5,580,717, and 5,658,727. The library is then screened for
expression of antibodies or antigen binding proteins with the
desired characteristics.
[0237] Methods of substituting an amino acid of choice into a
template nucleic acid are well established in the art, some of
which are described herein. For example, hypervariable region
residues can be substituted using the Kunkel method. See, e.g.,
Kunkel et al., Methods Enzymol. 154:367-382 (1987).
[0238] The sequence of oligonucleotides includes one or more of the
designed codon sets for the hypervariable region residues to be
altered. A codon set is a set of different nucleotide triplet
sequences used to encode desired variant amino acids. Codon sets
can be represented using symbols to designate particular
nucleotides or equimolar mixtures of nucleotides as shown in below
according to the IUB code.
[0239] IUB CODES
[0240] G Guanine
[0241] A Adenine
[0242] T Thymine
[0243] C Cytosine
[0244] R (A or G)
[0245] Y (C or T)
[0246] M (A or C)
[0247] K (G or T)
[0248] S(C or G)
[0249] W (A or T)
[0250] H (A or C or T)
[0251] B (C or G or T)
[0252] V (A or C or G)
[0253] D (A or G or T) H
[0254] N (A or C or G or T)
[0255] For example, in the codon set DVK, D can be nucleotides A or
G or T; V can be A or G or C; and K can be G or T. This codon set
can present 18 different codons and can encode amino acids Ala,
Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.
[0256] Oligonucleotide or primer sets can be synthesized using
standard methods. A set of oligonucleotides can be synthesized, for
example, by solid phase synthesis, containing sequences that
represent all possible combinations of nucleotide triplets provided
by the codon set and that will encode the desired group of amino
acids. Synthesis of oligonucleotides with selected nucleotide
"degeneracy" at certain positions is well known in that art. Such
sets of nucleotides having certain codon sets can be synthesized
using commercial nucleic acid synthesizers (available from, for
example, Applied Biosystems, Foster City, Calif.), or can be
obtained commercially (for example, from Life Technologies,
Rockville, Md.). Therefore, a set of oligonucleotides synthesized
having a particular codon set will typically include a plurality of
oligonucleotides with different sequences, the differences
established by the codon set within the overall sequence.
Oligonucleotides, as used according to the invention, have
sequences that allow for hybridization to a variable domain nucleic
acid template and also can include restriction enzyme sites for
cloning purposes.
[0257] In one method, nucleic acid sequences encoding variant amino
acids can be created by oligonucleotide-mediated mutagenesis. This
technique is well known in the art as described by Zoller et al.,
Nucleic Acids Res. 10:6487-6504 (1987). Briefly, nucleic acid
sequences encoding variant amino acids are created by hybridizing
an oligonucleotide set encoding the desired codon sets to a DNA
template, where the template is the single-stranded form of the
plasmid containing a variable region nucleic acid template
sequence. After hybridization, DNA polymerase is used to synthesize
an entire second complementary strand of the template that will
thus incorporate the oligonucleotide primer, and will contain the
codon sets as provided by the oligonucleotide set.
[0258] Generally, oligonucleotides of at least 25 nucleotides in
length are used. An optimal oligonucleotide will have 12 to 15
nucleotides that are completely complementary to the template on
either side of the nucleotide(s) coding for the mutation(s). This
ensures that the oligonucleotide will hybridize properly to the
single-stranded DNA template molecule. The oligonucleotides are
readily synthesized using techniques known in the art such as that
described by Crea et al., PNAS USA, 75:5765 (1978).
[0259] The DNA template is generated by those vectors that are
either derived from bacteriophage M13 vectors (the commercially
available M13 mp18 and M13 mp19 vectors are suitable), or those
vectors that contain a single-stranded phage origin of replication
as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus,
the DNA that is to be mutated can be inserted into one of these
vectors in order to generate single-stranded template. Production
of the single-stranded template is described in sections 4.21-4.41
of Sambrook et al., above.
[0260] To alter the native DNA sequence, the oligonucleotide is
hybridized to the single stranded template under suitable
hybridization conditions. A DNA polymerizing enzyme, usually T7 DNA
polymerase or the Klenow fragment of DNA polymerase I, is then
added to synthesize the complementary strand of the template using
the oligonucleotide as a primer for synthesis. A heteroduplex
molecule is thus formed such that one strand of DNA encodes the
mutated form of gene 1, and the other strand (the original
template) encodes the native, unaltered sequence of gene 1. This
heteroduplex molecule is then transformed into a suitable host
cell, usually a prokaryote such as E. coli JM101. After growing the
cells, they are plated onto agarose plates and screened using the
oligonucleotide primer radiolabeled with a 32-Phosphate to identify
the bacterial colonies that contain the mutated DNA.
[0261] The method described immediately above may be modified such
that a homoduplex molecule is created wherein both strands of the
plasmid contain the mutation(s). The modifications are as follows:
The single stranded oligonucleotide is annealed to the
single-stranded template as described above. A mixture of three
deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine
(dGTP), and deoxyribothymidine (dTT), is combined with a modified
thiodeoxyribocytosine called dCTP-(aS) (which can be obtained from
Amersham). This mixture is added to the template-oligonucleotide
complex. Upon addition of DNA polymerase to this mixture, a strand
of DNA identical to the template except for the mutated bases is
generated. In addition, this new strand of DNA will contain
dCTP-(aS) instead of dCTP, which serves to protect it from
restriction endonuclease digestion. After the template strand of
the double-stranded heteroduplex is nicked with an appropriate
restriction enzyme, the template strand can be digested with ExoIII
nuclease or another appropriate nuclease past the region that
contains the site(s) to be mutagenized. The reaction is then
stopped to leave a molecule that is only partially single-stranded.
A complete double-stranded DNA homoduplex is then formed using DNA
polymerase in the presence of all four deoxyribonucleotide
triphosphates, ATP, and DNA ligase. This homoduplex molecule can
then be transformed into a suitable host cell.
[0262] As indicated previously the sequence of the oligonucleotide
set is of sufficient length to hybridize to the template nucleic
acid and may also, but does not necessarily, contain restriction
sites. The DNA template can be generated by those vectors that are
either derived from bacteriophage M13 vectors or vectors that
contain a single-stranded phage origin of replication as described
by Viera et al., Meth. Enzymol., 153:3 (1987). Thus, the DNA that
is to be mutated must be inserted into one of these vectors in
order to generate single-stranded template. Production of the
single-stranded template is described in sections 4.21-4.41 of
Sambrook et al., supra.
[0263] According to another method, a library can be generated by
providing upstream and downstream oligonucleotide sets, each set
having a plurality of oligonucleotides with different sequences,
the different sequences established by the codon sets provided
within the sequence of the oligonucleotides. The upstream and
downstream oligonucleotide sets, along with a variable domain
template nucleic acid sequence, can be used in a polymerase chain
reaction to generate a "library" of PCR products. The PCR products
can be referred to as "nucleic acid cassettes", as they can be
fused with other related or unrelated nucleic acid sequences, for
example, viral coat proteins and dimerization domains, using
established molecular biology techniques.
[0264] The sequence of the PCR primers includes one or more of the
designed codon sets for the solvent accessible and highly diverse
positions in a hypervariable region. As described above, a codon
set is a set of different nucleotide triplet sequences used to
encode desired variant amino acids.
[0265] Antibody selectants that meet the desired criteria, as
selected through appropriate screening/selection steps can be
isolated and cloned using standard recombinant techniques.
[0266] In one aspect, the HVR-H1, HVR-H2, HVR-H3 sequences are
varied by holding amino acids at particular positions constant and
varying the amino acids at other positions.
[0267] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three of the following:
[0268] (i) a HVR-L1 sequence comprising the sequence of SEQ ID
NO:1;
[0269] (ii) a HVR-L2 sequence comprising the sequence of SEQ ID
NO:2;
[0270] (iii) a HVR-L3 sequence comprising the sequence of SEQ ID
NO:3.
[0271] The amino acid sequences of SEQ ID NOS:1, 2, and 3 are
numbered with respect to individual HVR (i.e., L1, L2, or L3) as
indicated in by in FIGS. 1A (positions 24-34, 50-56, and 89-97,
respectively) and 2A (positions 24-34, 50-56, and 89-97,
respectively), the numbering being consistent with the Kabat
numbering system as described below.
[0272] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three of the following:
[0273] (i) a HVR-H1 sequence comprising the sequence of SEQ ID NO:
4;
[0274] (ii) a HVR-H2 sequence comprising the sequence of SEQ ID
NO:5;
[0275] (iii) a HVR-H3 sequence comprising the sequence of SEQ ID
NO:6.
[0276] The amino acid sequences of SEQ ID NOS:4, 5, and 6 are
numbered with respect to individual HVR (i.e., H1, H2 or H3) as
indicated in by in FIGS. 1B (positions 26-35, 49-65, and 93-102,
respectively) and 2B (positions 26-35, 49-65, and 93-102,
respectively), the numbering being consistent with the Kabat
numbering system as described below.
[0277] In one aspect, the invention provides antibodies comprising
light chain HVR sequences as depicted in FIGS. 1A and 2A.
[0278] In one aspect, the invention provides antibodies comprising
heavy chain HVR sequences as depicted in FIGS. 1B and 2B.
[0279] Some embodiments of antibodies of the invention comprise a
light chain variable domain of humanized 4D5 antibody (huMAb4D5-8)
(HERCEPTIN.RTM. anti-HER2 antibody, Genentech, Inc., South San
Francisco, Calif., USA) (also referred to in U.S. Pat. No.
6,407,213 and Lee et al., J. Mol. Biol. (2004), 340(5):1073-93) as
depicted in SEQ ID NO:98 below.
TABLE-US-00006 (SEQ ID NO: 98) 1 Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Val Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr Thr Pro Pro
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107
[0280] (HVR Residues are Underlined)
[0281] In one embodiment, the huMAb4D5-8 light chain variable
domain sequence is modified at one or more of positions 30, 66 and
91 (Asn, Arg and His as indicated in bold/italics above,
respectively). In one embodiment, the modified huMAb4D5-8 sequence
comprises Ser in position 30, Gly in position 66 and/or Ser in
position 91. Accordingly, in one embodiment, an antibody of the
invention comprises a light chain variable domain comprising the
sequence depicted in SEQ ID NO:167 below:
TABLE-US-00007 (SEQ ID NO: 167) 1 Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Val Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr Thr Pro Pro
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107
[0282] (HVR Residues are Underlined)
Substituted residues with respect to huMAb4D5-8 are indicated in
bold/italics above.
[0283] Antibodies of the invention can comprise any suitable
framework variable domain sequence, provided binding activity to
Robo4 is substantially retained, such as at least 1%, at least 10%,
at least 20%, at least 30%, at least 40%, at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 100% or
greater binding activity relative to an antibody of the invention
disclosed herein. For example, in some embodiments, antibodies of
the invention comprise a human subgroup III heavy chain framework
consensus sequence. In one embodiment of these antibodies, the
framework consensus sequence comprises substitution at position 71,
73 and/or 78. In some embodiments of these antibodies, position 71
is A, 73 is T and/or 78 is A. In one embodiment, these antibodies
comprise heavy chain variable domain framework sequences of
huMAb4D5-8 (HERCEPTIN.RTM., Genentech, Inc., South San Francisco,
Calif., USA) (also referred to in U.S. Pat. Nos. 6,407,213 and
5,821,337, and Lee et al., J. Mol. Biol. 340(5): 1073-93 (2004)).
In one embodiment, these antibodies further comprise a human
.kappa.I light chain framework consensus sequence. In one
embodiment, these antibodies comprise light chain HVR sequences of
huMAb4D5-8 as described in U.S. Pat. Nos. 6,407,213 and 5,821,337.)
In one embodiment, these antibodies comprise light chain variable
domain sequences of huMAb4D5-8 (SEQ ID NOS: 98 or 167)
(HERCEPTIN.RTM. anti-HER2 antibody, Genentech, Inc., South San
Francisco, Calif., USA) (also referred to in U.S. Pat. Nos.
6,407,213 & 5,821,337, and Lee et al., J. Mol. Biol.
340(5):1073-93 (2004)).
[0284] In one embodiment, antibodies of the invention may comprise
framework region sequences of huMAb4D5-8 light and heavy chains as
provided below (SEQ ID NOS:9, 10, 168, 12 (light chain) and SEQ ID
NOS:13, 14, 15, 16 (heavy chain)). Numbers in superscript/bold
indicate amino acid positions according to Kabat.
Framework Sequences of huMAb4D5-8 Light Chain
TABLE-US-00008 (SEQ ID NO: 9) LC-FR1 .sup.1Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys.sup.23 (SEQ ID NO: 10) LC-FR2 .sup.35Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr.sup.49 (SEQ ID NO: 168) LC-FR3
.sup.57G1y Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys.sup.88 (SEQ ID NO: 12) LC-FR4 .sup.98Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys.sup.107
Framework Sequences of huMAb4D5-8 Heavy Chain
TABLE-US-00009 (SEQ ID NO: 13) HC-FR1 .sup.1Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser.sup.25 (SEQ ID NO: 14) HC-FR2 .sup.36Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Va1.sup.48 (SEQ ID NO: 15) HC-FR3
.sup.66Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu
Gln Met Asn.sup.82a Ser.sup.82b Leu.sup.82c Arg.sup.83 Ala Glu Asp
Thr Ala Val Tyr Tyr Cys.sup.92 (SEQ ID NO: 16) HC-FR4 .sup.103Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser.sup.113
[0285] In one embodiment, antibodies of the invention may comprise
modified/variant_framework region sequences of huMAb4D5-8 light and
heavy chains as provided below (SEQ ID NOS:9, 10, 11, 12 (light
chain) and SEQ ID NOS: 13, 14, 15, 16 (heavy chain)). Numbers in
superscript/bold indicate amino acid positions according to
Kabat.
Framework sequences of huMAb4D5-8 Light Chain Modified at Position
66 (Underlined)
TABLE-US-00010 (SEQ ID NO: 9) LC-FR1 .sup.1Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys.sup.23 (SEQ ID NO: 10) LC-FR2 .sup.35Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr.sup.49 (SEQ ID NO: 11) LC-FR3
.sup.57Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys.sup.88 (SEQ ID NO: 12) LC-FR4 .sup.98Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys.sup.107
Framework Sequences of huMAb4D5-8 Heavy Chain Modified at Positions
71, 73 and 78 (Underlined)
TABLE-US-00011 (SEQ ID NO: 13) HC-FR1 .sup.1Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser.sup.25 (SEQ ID NO: 14) HC-FR2 .sup.36Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val.sup.48 (SEQ ID NO: 15) HC-FR3
.sup.66Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
Gln Met Asn.sup.82a Ser.sup.82b Leu.sup.82c Arg.sup.83 Ala Glu Asp
Thr Ala Val Tyr Tyr Cys.sup.92 (SEQ ID NO: 16) HC-FR4 .sup.103Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser.sup.113
[0286] In one embodiment, an antibody of the invention is affinity
matured to obtain the target binding affinity desired. In one
example, an affinity matured antibody of the invention comprises
substitution at amino acid positions of the light chain as follows.
In one embodiment, a variant HVR-L1 A1-A11 (Kabat positions 24-34)
is RASQDVSTAVA (SEQ ID NO:1) having 1, 2, 3, 4, or 5 substitutions
in any combination of the following positions: A6 (G), A7 (A), A8
(R or I), A9 (S or Y), and A10 (L). In one embodiment, a variant
HVR-L2 B1-B7 (Kabat positions 50-56) is SASFLYS (SEQ ID NO:2)
having 1, 2, 3, 4, or 5 substitutions in any combination of the
following positions: B3 (T), B4 (L, N or T), B5 (E or A), B6 (A or
S), and B7 (Y or a deletion). In one embodiment, a variant HVR-L3
C1-C9 (Kabat positions 89-97) is QQSYTTPPT (SEQ ID NO:3) having 1,
2, 3, 4, 5 or 6 substitutions in any combination of the following
positions: C3 (P, T, F, or G), C4 (F, R, or N), CS (A, S, D, F, H,
N, V, or G), C6 (A, D, N, L, I, M, Y, or G), C7 (L, H, or T), and
C8 (A, M, F, or S).
[0287] In one embodiment, an antibody of the affinity matured
antibody of the invention comprises substitution at amino acid
positions of the heavy chain as follows. In one embodiment, a
variant HVR-H1 D1-D10 (Kabat positions 26-35) is GFTINGYYIH (SEQ ID
NO:17) having 1, 2, 3, 4, or 5 substitutions in any combination of
the following positions: D3 (S), D4 (L), D5 (Y, D, or K), D9 (F, L
or N), and D10 (E or Q). In one embodiment, a variant HVR-H2 E1-E18
(Kabat positions 49-65) is GFIYPAGGDTDYADSVKG (SEQ ID NO:18) having
1, 2, 3, 4, or 5 substitutions in any combination of the following
positions: E2 (R), E5 (S), E7 (L), E9 (H, K, A, or V), and E11 (A,
E or I). In one embodiment, HVR-H3 F1-F18 (Kabat positions 93-102)
is ARLIGNKFGWSSYG*MDY (SEQ ID NO:19), wherein "*" in the amino acid
sequence indicates a deletion at position F15, Kabat position
100).
[0288] In one embodiment, the affinity matured antibody of the
invention comprises one, two or three HVRs (L1, L2, and/or L3)
depicted in FIG. 2A. In one embodiment, the affinity matured
antibody of the invention comprises one, two, or three HVRs (H1,
H2, and/or H3) depicted in FIG. 2B. In one embodiment, the affinity
matured antibody of the invention comprises one, two, three, four,
five, or all six HVRs selected from the HVRs depicted in FIGS. 2A
and 2B.
[0289] In one embodiment, the affinity matured antibody of the
invention comprises the light chain variable region sequence of any
of the sequences depicted in FIG. 2A. In one embodiment, the
affinity matured antibody of the invention comprises the heavy
chain variable region sequence of any of the sequences depicted in
FIG. 2B.
[0290] In one embodiment, the affinity matured antibody of the
invention comprises a light chain variable region sequence
(comprising framework sequences and HVR sequences) shown in FIG. 2A
and the heavy chain variable region sequences (comprising framework
sequences and HVR sequences) of the corresponding antibody shown in
FIG. 2B.
[0291] In one aspect, the invention provides an antibody that
competes with any of the above-mentioned antibodies for binding to
Robo4. In one aspect, the invention provides an antibody that binds
to the same epitope on Robo4 as any of the above-mentioned
antibodies.
[0292] B. Hybridoma-Based Methods
[0293] Monoclonal antibodies of the invention can also be made
using the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975), and further described, e.g., in Hongo et
al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies:
A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell
Hybridomas 563-681 (Elsevier, N.Y., 1981), and Ni, Xiandai
Mianyixue, 26(4):265-268 (2006) regarding human-human hybridomas.
Additional methods include those described, for example, in U.S.
Pat. No. 7,189,826 regarding production of monoclonal human natural
IgM antibodies from hybridoma cell lines. Human hybridoma
technology (Trioma technology) is described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3): 185-91 (2005). Other hybridoma
techniques, see, e.g., US 2006/258841; US 2006/183887 (fully human
antibodies), US 2006/059575; US 2005/287149; US 2005/100546; US
2005/026229; and U.S. Pat. Nos. 7,078,492 and 7,153,507.
[0294] C. Vectors, Host Cells, and Recombinant Methods
[0295] Antibodies may also be produced using recombinant methods.
For recombinant production of an anti-Robo4 antibody, nucleic acid
encoding the antibody is isolated and inserted into a replicable
vector for further cloning (amplification of the DNA) or for
expression. DNA encoding the antibody may be readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the antibody). Many
vectors are available. The vector components generally include, but
are not limited to, one or more of the following: a signal
sequence, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence.
[0296] 1. Signal Sequence Component
[0297] An antibody of the invention may be produced recombinantly
not only directly, but also as a fusion polypeptide with a
heterologous polypeptide, which is preferably a signal sequence or
other polypeptide having a specific cleavage site at the N-terminus
of the mature protein or polypeptide. The heterologous signal
sequence selected preferably is one that is recognized and
processed (i.e., cleaved by a signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process a
native antibody signal sequence, the signal sequence is substituted
by a prokaryotic signal sequence selected, for example, from the
group of the alkaline phosphatase, penicillinase, lpp, or
heat-stable enterotoxin II leaders. For yeast secretion the native
signal sequence may be substituted by, e.g., the yeast invertase
leader, .alpha. factor leader (including Saccharomyces and
Kluyveromyces .alpha.-factor leaders), or acid phosphatase leader,
the C. albicans glucoamylase leader, or the signal described in WO
90/13646. In mammalian cell expression, mammalian signal sequences
as well as viral secretory leaders, for example, the herpes simplex
gD signal, are available.
[0298] 2. Origin of Replication
[0299] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2.mu. plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. Generally,
the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter).
[0300] 3. Selection Gene Component
[0301] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli.
[0302] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0303] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up antibody-encoding nucleic acid, such as DHFR, glutamine
synthetase (GS), thymidine kinase, metallothionein-I and -II,
preferably primate metallothionein genes, adenosine deaminase,
ornithine decarboxylase, etc.
[0304] For example, cells transformed with the DHFR gene are
identified by culturing the transformants in a culture medium
containing methotrexate (Mtx), a competitive antagonist of DHFR.
Under these conditions, the DHFR gene is amplified along with any
other co-transformed nucleic acid. A Chinese hamster ovary (CHO)
cell line deficient in endogenous DHFR activity (e.g., ATCC
CRL-9096) may be used.
[0305] Alternatively, cells transformed with the GS gene are
identified by culturing the transformants in a culture medium
containing L-methionine sulfoximine (Msx), an inhibitor of GS.
Under these conditions, the GS gene is amplified along with any
other co-transformed nucleic acid. The GS selection/amplification
system may be used in combination with the DHFR
selection/amplification system described above.
[0306] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody of interest, wild-type DHFR gene,
and another selectable marker such as aminoglycoside
3'-phosphotransferase (APH) can be selected by cell proliferation
in medium containing a selection agent for the selectable marker
such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,
or G418. See U.S. Pat. No. 4,965,199.
[0307] A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 (Stinchcomb et al., Nature,
282:39 (1979)). The trp1 gene provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12
(1977). The presence of the trp1 lesion in the yeast host cell
genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
[0308] In addition, vectors derived from the 1.6 .mu.m circular
plasmid pKD1 can be used for transformation of Kluyveromyces
yeasts. Alternatively, an expression system for large-scale
production of recombinant calf chymosin was reported for K. lactis.
Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy
expression vectors for secretion of mature recombinant human serum
albumin by industrial strains of Kluyveromyces have also been
disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
[0309] 4. Promoter Component
[0310] Expression and cloning vectors generally contain a promoter
that is recognized by the host organism and is operably linked to
nucleic acid encoding an antibody. Promoters suitable for use with
prokaryotic hosts include the phoA promoter, .beta.-lactamase and
lactose promoter systems, alkaline phosphatase promoter, a
tryptophan (trp) promoter system, and hybrid promoters such as the
tac promoter. However, other known bacterial promoters are
suitable. Promoters for use in bacterial systems also will contain
a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding an antibody.
[0311] Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
[0312] Examples of suitable promoter sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase or other
glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phospho-fructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0313] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657. Yeast enhancers also are advantageously used with yeast
promoters.
[0314] Antibody transcription from vectors in mammalian host cells
can be controlled, for example, by promoters obtained from the
genomes of viruses such as polyoma virus, fowlpox virus, adenovirus
(such as Adenovirus 2), bovine papilloma virus, avian sarcoma
virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian
Virus 40 (SV40), or from heterologous mammalian promoters, e.g.,
the actin promoter or an immunoglobulin promoter, from heat-shock
promoters, provided such promoters are compatible with the host
cell systems.
[0315] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the Rous Sarcoma
Virus long terminal repeat can be used as the promoter.
[0316] 5. Enhancer Element Component
[0317] Transcription of a DNA encoding an antibody of this
invention by higher eukaryotes is often increased by inserting an
enhancer sequence into the vector. Many enhancer sequences are now
known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the
antibody-encoding sequence, but is preferably located at a site 5'
from the promoter.
[0318] 6. Transcription Termination Component
[0319] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding
antibody. One useful transcription termination component is the
bovine growth hormone polyadenylation region. See WO94/11026 and
the expression vector disclosed therein.
[0320] 7. Selection and Transformation of Host Cells
[0321] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0322] Full length antibody, antibody fusion proteins, and antibody
fragments can be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) that by itself shows effectiveness in tumor cell
destruction. Full length antibodies have greater half life in
circulation. Production in E. coli is faster and more cost
efficient. For expression of antibody fragments and polypeptides in
bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S.
Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523 (Simmons
et al.), which describes translation initiation region (TIR) and
signal sequences for optimizing expression and secretion. See also
Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed.,
Humana Press, Totowa, N.J., 2003), pp. 245-254, describing
expression of antibody fragments in E. coli. After expression, the
antibody may be isolated from the E. coli cell paste in a soluble
fraction and can be purified through, e.g., a protein A or G column
depending on the isotype. Final purification can be carried out
similar to the process for purifying antibody expressed e.g., in
CHO cells.
[0323] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors. Saccharomyces cerevisiae, or common
baker's yeast, is the most commonly used among lower eukaryotic
host microorganisms. However, a number of other genera, species,
and strains are commonly available and useful herein, such as
Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger. For a review discussing the use of
yeasts and filamentous fungi for the production of therapeutic
proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414
(2004).
[0324] Certain fungi and yeast strains may be selected in which
glycosylation pathways have been "humanized," resulting in the
production of an antibody with a partially or fully human
glycosylation pattern. See, e.g., Li et al., Nat. Biotech.
24:210-215 (2006) (describing humanization of the glycosylation
pathway in Pichia pastoris); and Gerngross et al., supra.
[0325] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains and
variants and corresponding permissive insect host cells from hosts
such as Spodoptera frugiperda (caterpillar), Aedes aegypti
(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster
(fruitfly), and Bombyx mori have been identified. A variety of
viral strains for transfection are publicly available, e.g., the
L-1 variant of Autographa californica NPV and the Bm-5 strain of
Bombyx mori NPV, and such viruses may be used as the virus herein
according to the present invention, particularly for transfection
of Spodoptera frugiperda cells.
[0326] Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, duckweed (Lemnaceae), alfalfa (M. truncatula), and
tobacco can also be utilized as hosts. See, e.g., U.S. Pat. Nos.
5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429
(describing PLANTIBODIES.TM. technology for producing antibodies in
transgenic plants).
[0327] Vertebrate cells may be used as hosts, and propagation of
vertebrate cells in culture (tissue culture) has become a routine
procedure. Examples of useful mammalian host cell lines are monkey
kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney line (293 or 293 cells subcloned for growth in
suspension culture, Graham et al., J. Gen Virol. 36:59 (1977));
baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells
(TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells
(CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC
CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);
canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells
(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75);
human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT
060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.
Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human
hepatoma line (Hep G2). Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells
(Urlaub et al., PNAS USA 77:4216 (1980)); and myeloma cell lines
such as NSO and Sp2/0. For a review of certain mammalian host cell
lines suitable for antibody production, see, e.g., Yazaki and Wu,
Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana
Press, Totowa, N.J., 2003), pp. 255-268.
[0328] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0329] 8. Culturing the Host Cells
[0330] The host cells used to produce an antibody of this invention
may be cultured in a variety of media. Commercially available media
such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),
(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
((DMEM), Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0331] 9. Purification of Antibodies
[0332] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, are removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10: 163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an Amicon or Millipore
Pellicon ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0333] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography,
hydrophobic interaction chromatography, gel electrophoresis,
dialysis, and affinity chromatography, with affinity chromatography
being among one of the typically preferred purification steps. The
suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a C.sub.H3 domain, the Bakerbond
ABX.TM.resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSE.TM. chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available
depending on the antibody to be recovered.
[0334] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
[0335] In general, various methodologies for preparing antibodies
for use in research, testing, and clinical are well-established in
the art, consistent with the above-described methodologies and/or
as deemed appropriate by one skilled in the art for a particular
antibody of interest.
V. Immunoconjugates
[0336] The invention also provides immunoconjugates
(interchangeably referred to as "antibody-drug conjugates," or
"ADCs") comprising any of the anti-Robo4 antibodies of the
invention conjugated to one or more cytotoxic agents, such as a
chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin
(e.g., an enzymatically active toxin of bacterial, fungal, plant,
or animal origin, or fragments thereof), or a radioactive isotope
(i.e., a radioconjugate). Conjugates of an antibody and one or more
small molecule toxins, such as a calicheamicin, maytansinoids, a
trichothecene, and CC1065, and the derivatives of these toxins that
have toxin activity, are also contemplated herein.
[0337] In certain embodiments, an immunoconjugate comprises an
anti-Robo4 antibody and a chemotherapeutic agent or other toxin.
Chemotherapeutic agents useful in the generation of
immunoconjugates are described herein (e.g., above). Enzymatically
active toxins and fragments thereof can also be used and are
described herein.
[0338] In certain embodiments, an immunoconjugate comprises an
anti-Robo4 antibody and one or more small molecule toxins,
including, but not limited to, small molecule drugs such as a
calicheamicin, maytansinoid, dolastatin, auristatin, trichothecene,
and CC1065, and the derivatives of these drugs that have cytotoxic
activity. Examples of such immunoconjugates are discussed in
further detail below.
[0339] A. Exemplary Immunoconjugates
[0340] An immunoconjugate (or "antibody-drug conjugate" ("ADC")) of
the invention may be of Formula I, below, wherein an anti-Robo4
antibody is conjugated (i.e., covalently attached) to one or more
drug moieties (D) through an optional linker (L).
Ab-(L-D).sub.p Formula I
[0341] Accordingly, the anti-Robo4 antibody may be conjugated to
the drug either directly or via a linker. In Formula I, p is the
average number of drug moieties per antibody, which can range,
e.g., from about 1 to about 20 drug moieties per antibody, and in
certain embodiments, from 1 to about 8 drug moieties per
antibody.
[0342] B. Exemplary Linkers
[0343] Exemplary linkers and drug moieties are disclosed herein and
in U.S. Patent Publication Nos. 20050238649 A1; 20050276812 A1; and
20070092940 A1. A linker may comprise one or more linker
components. Exemplary linker components include 6-maleimidocaproyl
("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or
"vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl
(a "PAB"), N-Succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"),
N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate
("SMCC"), and N-Succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"),
N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (see, e.g.,
Carlsson et al., Biochem. J., 173, 723-737 (1978)), N-succinimidyl
4-(2-pyridyldithio) butanoate (SPDB) (see, e.g., U.S. Pat. No.
4,563,304), N-succinimidyl
4-methyl-4-[2-(5-nitro-pyridyl)-dithio]pentanoate (SMNP), and the
polyethylene glycol derivative, methoxy-polyethylene oxide (mPEO).
Various linker components are known in the art, some of which are
described below.
[0344] A linker may be a "cleavable linker," facilitating release
of a drug in the cell. For example, an acid-labile linker (e.g.,
hydrazone), protease-sensitive (e.g., peptidase-sensitive) linker,
photolabile linker, dimethyl linker or disulfide-containing linker
(Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0345] In some embodiments, a linker component may comprise a
"stretcher unit" that links an antibody to another linker component
or to a drug moiety. Exemplary stretcher units are shown below
(wherein the wavy line indicates sites of covalent attachment to an
antibody, another linker component or a drug moiety):
##STR00001##
[0346] In some embodiments, a linker component may comprise an
amino acid unit. In one such embodiment, the amino acid unit allows
for cleavage of the linker by a protease, thereby facilitating
release of the drug from the immunoconjugate upon exposure to
intracellular proteases, such as lysosomal enzymes. See, e.g.,
Doronina et al. (2003). Nat. Biotechnol. 21:778-784. Exemplary
amino acid units include, but are not limited to, a dipeptide, a
tripeptide, a tetrapeptide, and a pentapeptide. Exemplary
dipeptides include: valine-citrulline (vc or val-cit),
alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or
phe-lys); or N-methyl-valine-citrulline (Me-val-cit). Exemplary
tripeptides include: glycine-valine-citrulline (gly-val-cit) and
glycine-glycine-glycine (gly-gly-gly). An amino acid unit may
comprise amino acid residues that occur naturally, as well as minor
amino acids and non-naturally occurring amino acid analogs, such as
citrulline. Amino acid units can be designed and optimized in their
selectivity for enzymatic cleavage by a particular enzyme, for
example, a tumor-associated protease, cathepsin B, C and D, or a
plasmin protease.
[0347] In some embodiments, a linker component may comprise a
"spacer" unit that links the antibody to a drug moiety, either
directly or by way of a stretcher unit and/or an amino acid unit. A
spacer unit may be "self-immolative" or a "non-self-immolative." A
"non-self-immolative" spacer unit is one in which part or all of
the spacer unit remains bound to the drug moiety upon enzymatic
(e.g., proteolytic) cleavage of the ADC. Examples of
non-self-immolative spacer units include, but are not limited to, a
glycine spacer unit and a glycine-glycine spacer unit. Other
combinations of peptidic spacers susceptible to sequence-specific
enzymatic cleavage are also contemplated. For example, enzymatic
cleavage of an ADC containing a glycine-glycine spacer unit by a
tumor-cell associated protease would result in release of a
glycine-glycine-drug moiety from the remainder of the ADC. In one
such embodiment, the glycine-glycine-drug moiety is then subjected
to a separate hydrolysis step in the tumor cell, thus cleaving the
glycine-glycine spacer unit from the drug moiety.
[0348] A "self-immolative" spacer unit allows for release of the
drug moiety without a separate hydrolysis step. In certain
embodiments, a spacer unit of a linker comprises a p-aminobenzyl
unit. In one such embodiment, a p-aminobenzyl alcohol is attached
to an amino acid unit via an amide bond, and a carbamate,
methylcarbamate, or carbonate is made between the benzyl alcohol
and a cytotoxic agent. See, e.g., Hamann et al. (2005) Expert Opin.
Ther. Patents (2005) 15:1087-1103. In one embodiment, the spacer
unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments, the
phenylene portion of a p-amino benzyl unit is substituted with Qm,
wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8 alkyl),
-halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
Examples of self-immolative spacer units further include, but are
not limited to, aromatic compounds that are electronically similar
to p-aminobenzyl alcohol (see, e.g., US 2005/0256030 A1), such as
2-aminoimidazol-5-methanol derivatives (Hay et al. (1999) Bioorg.
Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzylacetals.
Spacers can be used that undergo cyclization upon amide bond
hydrolysis, such as substituted and unsubstituted 4-aminobutyric
acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223);
appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring
systems (Storm, et al., J. Amer. Chem. Soc., 1972, 94, 5815); and
2-aminophenylpropionic acid amides (Amsberry, et al., J. Org.
Chem., 1990, 55, 5867). Elimination of amine-containing drugs that
are substituted at the a-position of glycine (Kingsbury, et al., J.
Med. Chem., 1984, 27, 1447) are also examples of self-immolative
spacers useful in ADCs.
[0349] In one embodiment, a spacer unit is a branched
bis(hydroxymethyl)styrene (BHMS) unit as depicted below, which can
be used to incorporate and release multiple drugs.
##STR00002##
[0350] wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -halogen, -nitro or -cyano; m is an integer ranging from
0-4; n is 0 or 1; and p ranges raging from 1 to about 20.
[0351] A linker may comprise any one or more of the above linker
components. In certain embodiments, a linker is as shown in
brackets in the following ADC Formula II
Ab-([Aa-Ww-Yy]-D).sub.p Formula II
[0352] wherein A is a stretcher unit, and a is an integer from 0 to
1; W is an amino acid unit, and w is an integer from 0 to 12; Y is
a spacer unit, and y is 0, 1, or 2; and Ab, D, and p are defined as
above for Formula I. Exemplary embodiments of such linkers are
described in US 20050238649 A1, which is expressly incorporated
herein by reference.
[0353] Exemplary linker components and combinations thereof are
shown below in the context of ADCs of Formula II:
##STR00003##
[0354] Linkers components, including stretcher, spacer, and amino
acid units, may be synthesized by methods known in the art, such as
those described in US 2005-0238649 A1.
[0355] C. Exemplary Drug Moieties
[0356] 1. Maytansine and Maytansinoids
[0357] In some embodiments, an immunoconjugate comprises an
antibody of the invention conjugated to one or more maytansinoid
molecules. Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the
disclosures of which are hereby expressly incorporated by
reference.
[0358] Maytansine compounds inhibit cell proliferation by
inhibiting the formation of microtubules during mitosis through
inhibition of polymerization of the microtubulin protein, tubulin
(Remillard et al. (1975) Science 189:1002-1005; U.S. Pat. No.
5,208,020). Maytansine and maytansinoids are highly cytotoxic but
their clinical use in cancer therapy has been greatly limited by
their severe systemic side-effects primarily attributed to their
poor selectivity for tumors. Clinical trials with maytansine had
been discontinued due to serious adverse effects on the central
nervous system and gastrointestinal system (Issel et al., (1978)
Can. Treatment. Rev. 5:199-207).
[0359] Maytansinoid drug moieties are attractive drug moieties in
antibody-drug conjugates because they are: (i) relatively
accessible to prepare by fermentation or chemical modification or
derivatization of fermentation products, (ii) amenable to
derivatization with functional groups suitable for conjugation
through non-disulfide linkers to antibodies, (iii) stable in
plasma, and (iv) effective against a variety of tumor cell
lines.
[0360] Maytansine compounds suitable for use as maytansinoid drug
moieties are well known in the art and can be isolated from natural
sources according to known methods or produced using genetic
engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973).
Maytansinol and maytansinol analogues may also be prepared
synthetically according to known methods. Exemplary embodiments of
maytansinoid drug moieties include: DM1; DM3; and DM4, as disclosed
herein below.
[0361] As with other drug moieties, all stereoisomers of the
maytansinoid drug moiety are contemplated for the compounds of the
invention, i.e. any combination of R and S configurations at the
chiral carbons of D. In one embodiment, the maytansinoid drug
moiety (D) will have the following stereochemistry:
##STR00004##
[0362] Exemplary embodiments of maytansinoid drug moieties include:
DM1, (CR.sub.2).sub.m.dbd.CH.sub.2CH.sub.2; DM3,
(CR.sub.2).sub.m.dbd.CH.sub.2CH.sub.2CH(CH.sub.3); and DM4,
(CR.sub.2).sub.m.dbd.CH.sub.2CH.sub.2C(CH.sub.3).sub.2, having the
structures:
##STR00005##
[0363] In an attempt to improve the therapeutic index of targeted
antibodies, maytansine and maytansinoids have been conjugated to
antibodies specifically binding to tumor cell antigens.
Immunoconjugates containing maytansinoids and their therapeutic use
are disclosed, for example, in U.S. Pat. Nos. 5,208,020, 5,416,064
and European Patent EP 0 425 235 B1, the disclosures of which are
hereby expressly incorporated by reference. Liu et al., PNAS USA
93:8618-8623 (1996) described immunoconjugates comprising a
maytansinoid designated DM1 linked to the monoclonal antibody C242
directed against human colorectal cancer. The conjugate was found
to be highly cytotoxic towards cultured colon cancer cells, and
showed antitumor activity in an in vivo tumor growth assay. Chari
et al., Cancer Research 52:127-131 (1992) describe immunoconjugates
in which a maytansinoid was conjugated via a disulfide linker to
the murine antibody A7 binding to an antigen on human colon cancer
cell lines, or to another murine monoclonal antibody TA.1 that
binds the HER-2/neu oncogene. The cytotoxicity of the
TA.1-maytansonoid conjugate was tested in vitro on the human breast
cancer cell line SK-BR-3, which expresses 3.times.10.sup.5 HER-2
surface antigens per cell. The drug conjugate achieved a degree of
cytotoxicity similar to the free maytansinoid drug, which could be
increased by increasing the number of maytansinoid molecules per
antibody molecule. The A7-maytansinoid conjugate showed low
systemic cytotoxicity in mice.
[0364] Antibody-maytansinoid conjugates are prepared by chemically
linking an antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. An average of 3-4
maytansinoid molecules conjugated per antibody molecule has shown
efficacy in enhancing cytotoxicity of target cells without
negatively affecting the function or solubility of the antibody,
although even one molecule of toxin/antibody would be expected to
enhance cytotoxicity over the use of naked antibody. Maytansinoids
are well known in the art and can be synthesized by known
techniques or isolated from natural sources. Suitable maytansinoids
are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the
other patents and nonpatent publications referred to hereinabove.
Preferred maytansinoids are maytansinol and maytansinol analogues
modified in the aromatic ring or at other positions of the
maytansinol molecule, such as various maytansinol esters.
[0365] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and
Chari et al., Cancer Research 52:127-131 (1992). The linking groups
include disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred.
[0366] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al, Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0367] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
[0368] 2. Auristatins, Dolastatins
[0369] In some embodiments, an immunoconjugate comprises an
antibody of the invention conjugated to dolastatin or a dolastatin
peptidic analog or derivative, e.g., an auristatin (U.S. Pat. Nos.
5,635,483; 5,780,588). Dolastatins and auristatins have been shown
to interfere with microtubule dynamics, GTP hydrolysis, and nuclear
and cellular division (Woyke et al. (2001) Antimicrob. Agents and
Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No.
5,663,149) and antifungal activity (Pettit et al. (1998)
Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or
auristatin drug moiety may be attached to the antibody through the
N (amino) terminus or the C (carboxyl) terminus of the peptidic
drug moiety (WO 02/088172).
[0370] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, disclosed in
Senter et al, Proceedings of the American Association for Cancer
Research, Volume 45, Abstract Number 623, presented Mar. 28, 2004,
the disclosure of which is expressly incorporated by reference in
its entirety.
[0371] A peptidic drug moiety may be selected from Formulas D.sub.E
and D.sub.F below:
##STR00006## [0372] wherein the wavy line of D.sub.E and D.sub.F
indicates the covalent attachment site to an antibody or
antibody-linker component, and independently at each location:
[0373] R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl; [0374]
R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); [0375] R.sup.4
is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); [0376] R.sup.5
is selected from H and methyl; [0377] or R.sup.4 and R.sup.5
jointly form a carbocyclic ring and have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from H, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and 6;
[0378] R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl; [0379]
R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); [0380] each
R.sup.8 is independently selected from H, OH, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle and O---(C.sub.1-C.sub.8 alkyl);
[0381] R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl; [0382]
R.sup.10 is selected from aryl or C.sub.3-C.sub.8 heterocycle;
[0383] Z is O, S, NH, or NR, wherein R.sup.12 is C.sub.1-C.sub.8
alkyl; [0384] R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl,
aryl, C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14,
or --(R.sup.13O).sub.m--CH(R.sup.15).sub.2; [0385] m is an integer
ranging from 1-1000; [0386] R.sup.13 is C.sub.2-C.sub.8 alkyl;
[0387] R.sup.14 is H or C.sub.1-C.sub.8 alkyl; [0388] each
occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl; [0389] each
occurrence of R.sup.16 is independently H, C.sub.1-C.sub.8 alkyl,
or --(CH.sub.2).sub.n--COOH; [0390] R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and [0391] n is an integer ranging from 0 to 6.
[0392] In one embodiment, R.sup.3, R.sup.4 and R.sup.7 are
independently isopropyl or sec-butyl and R.sup.5 is --H or methyl.
In an exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.5 is --H, and R.sup.7 is sec-butyl.
[0393] In yet another embodiment, R.sup.2 and R.sup.6 are each
methyl, and R.sup.9 is --H.
[0394] In still another embodiment, each occurrence of R.sup.8 is
--OCH.sub.3.
[0395] In an exemplary embodiment, R.sup.3 and R.sup.4 are each
isopropyl, R.sup.2 and R.sup.6 are each methyl, R.sup.5 is --H,
R.sup.7 is sec-butyl, each occurrence of R.sup.8 is --OCH.sub.3,
and R.sup.9 is --H.
[0396] In one embodiment, Z is --O-- or --NH--.
[0397] In one embodiment, R.sup.10 is aryl.
[0398] In an exemplary embodiment, R.sup.10 is -phenyl.
[0399] In an exemplary embodiment, when Z is --O--, R.sup.11 is
--H, methyl or t-butyl.
[0400] In one embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--N(R.sup.16).sub.2, and R.sup.16 is
--C.sub.1-C.sub.8 alkyl or --(CH.sub.2).sub.n--COOH.
[0401] In another embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--SO.sub.3H.
[0402] An exemplary auristatin embodiment of formula D.sub.E is
MMAE, wherein the wavy line indicates the covalent attachment to a
linker (L) of an antibody-drug conjugate:
##STR00007##
[0403] An exemplary auristatin embodiment of formula D.sub.F is
MMAF, wherein the wavy line indicates the covalent attachment to a
linker (L) of an antibody-drug conjugate (see US 2005/0238649 and
Doronina et al. (2006) Bioconjugate Chem. 17:114-124):
##STR00008##
[0404] Other drug moieties include the following MMAF derivatives,
wherein the wavy line indicates the covalent attachment to a linker
(L) of an antibody-drug conjugate:
##STR00009## ##STR00010##
[0405] In one aspect, hydrophilic groups including but not limited
to, triethylene glycol esters (TEG), as shown above, can be
attached to the drug moiety at R.sup.11. Without being bound by any
particular theory, the hydrophilic groups assist in the
internalization and non-agglomeration of the drug moiety.
[0406] Exemplary embodiments of ADCs of Formula I comprising an
auristatin/dolastatin or derivative thereof are described in US
2005-0238649 A1 and Doronina et al. (2006) Bioconjugate Chem.
17:114-124, which is expressly incorporated herein by reference.
Exemplary embodiments of ADCs of Formula I comprising MMAE or MMAF
and various linker components have the following structures and
abbreviations (wherein "Ab" is an antibody; p is 1 to about 8,
"Val-Cit" is a valine-citrulline dipeptide; and "S" is a sulfur
atom:
##STR00011## ##STR00012##
[0407] Exemplary embodiments of ADCs of Formula I comprising MMAF
and various linker components further include Ab-MC-PAB-MMAF and
Ab-PAB-MMAF. Interestingly, immunoconjugates comprising MMAF
attached to an antibody by a linker that is not proteolytically
cleavable have been shown to possess activity comparable to
immunoconjugates comprising MMAF attached to an antibody by a
proteolytically cleavable linker. See, Doronina et al. (2006)
Bioconjugate Chem. 17:114-124. In such instances, drug release is
believed to be effected by antibody degradation in the cell.
Id.
[0408] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry.
Auristatin/dolastatin drug moieties may be prepared according to
the methods of: US 2005-0238649 A1; U.S. Pat. No. 5,635,483; U.S.
Pat. No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc.
111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design
13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit
et al. (1996) J. Chem. Soc. Perkin Trans. 15:859-863; and Doronina
(2003) Nat. Biotechnol. 21(7):778-784.
[0409] In particular, auristatin/dolastatin drug moieties of
formula D.sub.F, such as MMAF and derivatives thereof, may be
prepared using methods described in US 2005-0238649 A1 and Doronina
et al. (2006) Bioconjugate Chem. 17:114-124. Auristatin/dolastatin
drug moieties of formula D.sub.E, such as MMAE and derivatives
thereof, may be prepared using methods described in Doronina et al.
(2003) Nat. Biotech. 21:778-784. Drug-linker moieties MC-MMAF,
MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE may be conveniently
synthesized by routine methods, e.g., as described in Doronina et
al. (2003) Nat. Biotech. 21:778-784, and U.S. Patent Publication
No. 20050238649 A1, and then conjugated to an antibody of
interest.
[0410] 3. Calicheamicin
[0411] Another immunoconjugate of interest comprises an antibody
conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics is capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma..sub.1.sup.I, .alpha..sub.2.sup.I,
.alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I, PSAG and
.theta..sup.I.sub.1 (Hinman et al., Cancer Research 53:3336-3342
(1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the
aforementioned U.S. patents to American Cyanamid). Another
anti-tumor drug that the antibody can be conjugated is QFA which is
an antifolate. Both calicheamicin and QFA have intracellular sites
of action and do not readily cross the plasma membrane. Therefore,
cellular uptake of these agents through antibody mediated
internalization greatly enhances their cytotoxic effects.
[0412] 4. Other Cytotoxic Agents
[0413] Other antitumor agents that can be conjugated to the
antibodies of the invention include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known
collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0414] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0415] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0416] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
antibodies. Examples include At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu.
[0417] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
Tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al.
(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods in detail.
[0418] Anti-Robo4 antibodies of the invention may be conjugated to
nanoparticulate agents which may in turn be conjugated to cytotoxic
moieties. Suitable nanoparticular agents include, but are not
limited to, microbubbles (see Ellegala et al., Circulation
108:336-341 (2003))), also referred to as acoustically active
lipospheres (AALs) (see Tartis et al., Ultrasound Med. Biol.
32(11):1771-80 (2006)), superparamagnetic agents, liposomes,
perfluorocarbon nanoparticle emulsions (WO2005014051), and
dendrimers (see Caruthers et al., Methods in Molecular Medicine,
124:387-400 (2006) and references cited therein, all of which
references are hereby incorporated by reference in their
entirety).
[0419] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat.
No. 5,208,020) may be used.
[0420] The compounds of the invention expressly contemplate, but
are not limited to, ADC prepared with cross-linker reagents: BMPS,
EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB,
SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,
U.S.A). See pages 467-498, 2003-2004 Applications Handbook and
Catalog.
[0421] 5. Drug Loading
[0422] Drug loading is represented by p and is the average number
of drug moieties per antibody in a molecule of Formula I, Ia, Ia'
or II. Drug loading may range from 1 to 20 drug moieties (D) per
antibody. ADCs of Formula I include collections of antibodies
conjugated with a range of drug moieties, from 1 to 20. The average
number of drug moieties per antibody in preparations of ADC from
conjugation reactions may be characterized by conventional means
such as mass spectroscopy, ELISA assay, and HPLC. The quantitative
distribution of ADC in terms of p may also be determined. In some
instances, separation, purification, and characterization of
homogeneous ADC where p is a certain value from ADC with other drug
loadings may be achieved by means such as reverse phase HPLC or
electrophoresis.
[0423] For some antibody-drug conjugates, p may be limited by the
number of attachment sites on the antibody. For example, where the
attachment is a cysteine thiol, as in the exemplary embodiments
above, an antibody may have only one or several cysteine thiol
groups, or may have only one or several sufficiently reactive thiol
groups through which a linker may be attached. In certain
embodiments, higher drug loading, e.g. p>5, may cause
aggregation, insolubility, toxicity, or loss of cellular
permeability of certain antibody-drug conjugates. In certain
embodiments, the drug loading for an ADC of the invention ranges
from 1 to about 8; from about 2 to about 6; from about 3 to about
5; from about 3 to about 4; from about 3.1 to about 3.9; from about
3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to
about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about
3.7. Indeed, it has been shown that for certain ADCs, the optimal
ratio of drug moieties per antibody may be less than 8, and may be
about 2 to about 5. See US 2005-0238649 A1 (herein incorporated by
reference in its entirety).
[0424] In certain embodiments, fewer than the theoretical maximum
of drug moieties are conjugated to an antibody during a conjugation
reaction. An antibody may contain, for example, lysine residues
that do not react with the drug-linker intermediate or linker
reagent, as discussed below. Generally, antibodies do not contain
many free and reactive cysteine thiol groups which may be linked to
a drug moiety; indeed most cysteine thiol residues in antibodies
exist as disulfide bridges. In certain embodiments, an antibody may
be reduced with a reducing agent such as dithiothreitol (DTT) or
tricarbonylethylphosphine (TCEP), under partial or total reducing
conditions, to generate reactive cysteine thiol groups. In certain
embodiments, an antibody is subjected to denaturing conditions to
reveal reactive nucleophilic groups such as lysine or cysteine.
[0425] The loading (drug/antibody ratio) of an ADC may be
controlled in different ways, e.g., by: (i) limiting the molar
excess of drug-linker intermediate or linker reagent relative to
antibody, (ii) limiting the conjugation reaction time or
temperature, (iii) partial or limiting reductive conditions for
cysteine thiol modification, (iv) engineering by recombinant
techniques the amino acid sequence of the antibody such that the
number and position of cysteine residues is modified for control of
the number and/or position of linker-drug attachements (such as
thioMab or thioFab prepared as disclosed herein and in
WO2006/034488 (herein incorporated by reference in its entirety)).
In an embodiment of the invention, an amino acid residue of the
antibody of the invention is substituted with a cysteine residue.
In an embodiment, the amino acid at position 118 of the heavy chain
Fc region of the antibody is a cysteine or is substituted with a
cysteine residue (where 118 refers to the amino acid position in
the Fc region of the antibody numbered according to EU numbering,
see Kabat, E. A. et al., Sequences of Proteins of Immunological
Interest (U.S. Dept. of Health and Hum. Serv., Bethesda (1991)). In
an embodiment, the cysteine residue at position 118 of the heavy
chain Fc region (EU numbering) is covalently attached to a drug
moiety or a detectable label, such as the drug moieties or
detectable labels disclosed herein. In FIG. 6A, the alanine of the
heavy chain Fc region shown in bold text and underlined is the
position at which a cysteine was substituted to generate the
thioMAb.
[0426] It is to be understood that where more than one nucleophilic
group reacts with a drug-linker intermediate or linker reagent
followed by drug moiety reagent, then the resulting product is a
mixture of ADC compounds with a distribution of one or more drug
moieties attached to an antibody. The average number of drugs per
antibody may be calculated from the mixture by a dual ELISA
antibody assay, which is specific for antibody and specific for the
drug. Individual ADC molecules may be identified in the mixture by
mass spectroscopy and separated by HPLC, e.g. hydrophobic
interaction chromatography (see, e.g., Hamblett, K. J., et al.
"Effect of drug loading on the pharmacology, pharmacokinetics, and
toxicity of an anti-CD30 antibody-drug conjugate," Abstract No.
624, American Association for Cancer Research, 2004 Annual Meeting,
Mar. 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004;
Alley, S. C., et al. "Controlling the location of drug attachment
in antibody-drug conjugates," Abstract No. 627, American
Association for Cancer Research, 2004 Annual Meeting, Mar. 27-31,
2004, Proceedings of the AACR, Volume 45, March 2004). In certain
embodiments, a homogeneous ADC with a single loading value may be
isolated from the conjugation mixture by electrophoresis or
chromatography.
[0427] D. Metabolites of the Antibody-Drug Conjugates
[0428] Also falling within the scope of this invention are the in
vivo metabolic products of the ADC compounds described herein, to
the extent such products are novel and unobvious over the prior
art. Such products may result for example from the oxidation,
reduction, hydrolysis, amidation, esterification, enzymatic
cleavage, and the like, of the administered compound. Accordingly,
the invention includes novel and unobvious compounds produced by a
process comprising contacting a compound of this invention with a
mammal for a period of time sufficient to yield a metabolic product
thereof.
[0429] Metabolite products typically are identified by preparing a
radiolabeled (e.g. .sup.14C or .sup.3H) ADC, administering it
parenterally in a detectable dose (e.g. greater than about 0.5
mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to
man, allowing sufficient time for metabolism to occur (typically
about 30 seconds to 30 hours) and isolating its conversion products
from the urine, blood or other biological samples. These products
are easily isolated since they are labeled (others are isolated by
the use of antibodies capable of binding epitopes surviving in the
metabolite). The metabolite structures are determined in
conventional fashion, e.g. by MS, LC/MS or NMR analysis. In
general, analysis of metabolites is done in the same way as
conventional drug metabolism studies well-known to those skilled in
the art. The conversion products, so long as they are not otherwise
found in vivo, are useful in diagnostic assays for therapeutic
dosing of the ADC compounds of the invention.
VI. Methods of Preparing Immunconjugates
[0430] In the antibody drug conjugates (ADC) of the invention, an
antibody (Ab) is conjugated to one or more drug moieties (D), e.g.
about 1 to about 20 drug moieties per antibody, through a linker
(L). An ADC of Formula I, Ia, Ia', or II may be prepared by several
routes employing organic chemistry reactions, conditions, and
reagents known to those skilled in the art, including: (1) reaction
of a nucleophilic group of an antibody with a bivalent linker
reagent to form Ab-L via a covalent bond, followed by reaction with
a drug moiety D; and (2) reaction of a nucleophilic group of a drug
moiety with a bivalent linker reagent, to form D-L, via a covalent
bond, followed by reaction with a nucleophilic group of an
antibody. Exemplary methods for preparing an ADC of Formula I via
the latter route are described in U.S. Patent Publication No.
20050238649 A1, which is expressly incorporated herein by
reference.
[0431] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine,
and (iv) sugar hydroxyl or amino groups where the antibody is
glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic
and capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents including: (i) active
esters such as NHS esters, HOBt esters, haloformates, and acid
halides; (ii) alkyl and benzyl halides such as haloacetamides;
(iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain
antibodies have reducible interchain disulfides, i.e. cysteine
bridges. Antibodies may be made reactive for conjugation with
linker reagents by treatment with a reducing agent such as DTT
(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that the
antibody is fully or partially reduced. Each cysteine bridge will
thus form, theoretically, two reactive thiol nucleophiles.
Additional nucleophilic groups can be introduced into antibodies
through modification of lysine residues, e.g., by reacting lysine
residues with 2-iminothiolane (Traut's reagent), resulting in
conversion of an amine into a thiol. Reactive thiol groups may be
introduced into an antibody by introducing one, two, three, four,
or more cysteine residues (e.g., by preparing variant antibodies
comprising one or more non-native cysteine amino acid
residues).
[0432] Antibody-drug conjugates of the invention may also be
produced by reaction between an electrophilic group on an antibody,
such as an aldehyde or ketone carbonyl group, with a nucleophilic
group on a linker reagent or drug. Useful nucleophilic groups on a
linker reagent include, but are not limited to, hydrazide, oxime,
amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide. In one embodiment, an antibody is modified to
introduce electrophilic moieties that are capable of reacting with
nucleophilic substituents on the linker reagent or drug. In another
embodiment, the sugars of glycosylated antibodies may be oxidized,
e.g. with periodate oxidizing reagents, to form aldehyde or ketone
groups which may react with the amine group of linker reagents or
drug moieties. The resulting imine Schiff base groups may form a
stable linkage, or may be reduced, e.g. by borohydride reagents to
form stable amine linkages. In one embodiment, reaction of the
carbohydrate portion of a glycosylated antibody with either
galactose oxidase or sodium meta-periodate may yield carbonyl
(aldehyde and ketone) groups in the antibody that can react with
appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment, antibodies containing
N-terminal serine or threonine residues can react with sodium
meta-periodate, resulting in production of an aldehyde in place of
the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate
Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such an aldehyde can be
reacted with a drug moiety or linker nucleophile.
[0433] Nucleophilic groups on a drug moiety include, but are not
limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,
thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups
capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents including: (i) active
esters such as NHS esters, HOBt esters, haloformates, and acid
halides; (ii) alkyl and benzyl halides such as haloacetamides;
(iii) aldehydes, ketones, carboxyl, and maleimide groups.
[0434] The compounds of the invention expressly contemplate, but
are not limited to, ADC prepared with the following cross-linker
reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA,
SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS,
sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,
U.S.A; see pages 467-498, 2003-2004 Applications Handbook and
Catalog.
[0435] Immunoconjugates comprising an antibody and a cytotoxic
agent may also be made using a variety of bifunctional protein
coupling agents such as N-succinimidyl-3-(2-pyridyldithio)
propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0436] Alternatively, a fusion protein comprising an antibody and a
cytotoxic agent may be made, e.g., by recombinant techniques or
peptide synthesis. A recombinant DNA molecule may comprise regions
encoding the antibody and cytotoxic portions of the conjugate
either adjacent to one another or separated by a region encoding a
linker peptide which does not destroy the desired properties of the
conjugate.
[0437] The compounds of the invention include cysteine engineered
antibodies where one or more amino acids of a parent antibody are
replaced with a free cysteine amino acid. A cysteine engineered
antibody comprises one or more free cysteine amino acids having a
thiol reactivity value in the range of 0.6 to 1.0. A free cysteine
amino acid is a cysteine residue which has been engineered into the
parent antibody and is not part of a disulfide bridge.
[0438] In one aspect, the cysteine engineered antibody is prepared
by a process comprising:
(a) replacing one or more amino acid residues of a parent antibody
by cysteine; and (b) determining the thiol reactivity of the
cysteine engineered antibody by reacting the cysteine engineered
antibody with a thiol-reactive reagent. The cysteine engineered
antibody may be more reactive than the parent antibody with the
thiol-reactive reagent.
[0439] The free cysteine amino acid residues may be located in the
heavy or light chains, or in the constant or variable domains.
Antibody fragments, e.g. Fab, may also be engineered with one or
more cysteine amino acids replacing amino acids of the antibody
fragment, to form cysteine engineered antibody fragments.
[0440] Another aspect of the invention provides a method of
preparing (making) a cysteine engineered antibody, comprising:
[0441] (a) introducing one or more cysteine amino acids into a
parent antibody in order to generate the cysteine engineered
antibody; and
[0442] (b) determining the thiol reactivity of the cysteine
engineered antibody with a thiol-reactive reagent;
wherein the cysteine engineered antibody is more reactive than the
parent antibody with the thiol-reactive reagent.
[0443] Step (a) of the method of preparing a cysteine engineered
antibody may comprise:
[0444] (i) mutagenizing a nucleic acid sequence encoding the
cysteine engineered antibody;
[0445] (ii) expressing the cysteine engineered antibody; and
[0446] (iii) isolating and purifying the cysteine engineered
antibody.
[0447] Step (b) of the method of preparing a cysteine engineered
antibody may comprise expressing the cysteine engineered antibody
on a viral particle selected from a phage or a phagemid
particle.
[0448] Step (b) of the method of preparing a cysteine engineered
antibody may also comprise:
[0449] (i) reacting the cysteine engineered antibody with a
thiol-reactive affinity reagent to generate an affinity labeled,
cysteine engineered antibody; and
[0450] (ii) measuring the binding of the affinity labeled, cysteine
engineered antibody to a capture media.
[0451] Another aspect of the invention is a method of screening
cysteine engineered antibodies with highly reactive, unpaired
cysteine amino acids for thiol reactivity comprising:
[0452] (a) introducing one or more cysteine amino acids into a
parent antibody in order to generate a cysteine engineered
antibody;
[0453] (b) reacting the cysteine engineered antibody with a
thiol-reactive affinity reagent to generate an affinity labeled,
cysteine engineered antibody; and
[0454] (c) measuring the binding of the affinity labeled, cysteine
engineered antibody to a capture media; and
[0455] (d) determining the thiol reactivity of the cysteine
engineered antibody with the thiol-reactive reagent.
[0456] Step (a) of the method of screening cysteine engineered
antibodies may comprise:
[0457] (i) mutagenizing a nucleic acid sequence encoding the
cysteine engineered antibody;
[0458] (ii) expressing the cysteine engineered antibody; and
[0459] (iii) isolating and purifying the cysteine engineered
antibody.
[0460] Step (b) of the method of screening cysteine engineered
antibodies may comprise expressing the cysteine engineered antibody
on a viral particle selected from a phage or a phagemid
particle.
[0461] Step (b) of the method of screening cysteine engineered
antibodies may also comprise:
[0462] (i) reacting the cysteine engineered antibody with a
thiol-reactive affinity reagent to generate an affinity labeled,
cysteine engineered antibody; and
[0463] (ii) measuring the binding of the affinity labeled, cysteine
engineered antibody to a capture media.
[0464] Cysteine engineered antibodies may be useful in the
treatment of cancer and include antibodies specific for cell
surface and transmembrane receptors, and tumor-associated antigens
(TAA). Such antibodies may be used as naked antibodies
(unconjugated to a drug or label moiety) or as Formula I, Ia, Ia'
or II antibody-drug conjugates (ADC).
[0465] Embodiments of the methods for preparing and screening a
cysteine engineered antibody include where the parent antibody is
an antibody fragment, such as hu4D5Fabv8. The parent antibody may
also be a fusion protein comprising an albumin-binding peptide
sequence (ABP).
[0466] Cysteine engineered antibodies of the invention may be
site-specifically and efficiently coupled with a thiol-reactive
reagent. The thiol-reactive reagent may be a multifunctional linker
reagent, a capture label reagent, a fluorophore reagent, or a
drug-linker intermediate.
The cysteine engineered antibody may be labeled with a detectable
label, immobilized on a solid phase support and/or conjugated with
a drug moiety.
[0467] Another aspect of the invention is an antibody-drug
conjugate compound comprising a cysteine engineered antibody (Ab),
and a drug moiety (D) wherein the cysteine engineered antibody is
attached through one or more free cysteine amino acids by a linker
moiety (L) to D; the compound having Formula Ia:
Ab(LD).sub.p Ia
where p is 1, 2, 3, or 4; and wherein the cysteine engineered
antibody is prepared by a process comprising replacing one or more
amino acid residues of a parent antibody by one or more free
cysteine amino acids. Drug moieties include, but are not limited to
a maytansinoid, an auristatin, a dolastatin, a trichothecene,
CC1065, a calicheamicin and other enediyne antibiotics, a taxane,
an anthracycline, and stereoisomers, isosteres, analogs or
derivatives thereof. Exemplary drug moieties include DM1, MMAE, and
MMAF. Exemplary antibody-drug conjugates are set forth in U.S.
Patent Publication No. 20070092940 A1.
[0468] The antibody-drug conjugate of Formula Ia may further
comprise an albumin-binding peptide (ABP) sequence; the composition
having Formula Ia':
ABPAb(LD).sub.p Ia'
[0469] Antibodies of the invention comprising fusion proteins with
ABP sequences are taught by: (i) Dennis et al. (2002) J Biol. Chem.
277:35035-35043 at Tables III and IV, page 35038; (ii) US
20040001827 at [0076] SEQ ID NOS: 9-22; and (iii) WO 01/45746 at
pages 12-13, SEQ ID NOS: z1-z14, and all of which are incorporated
herein by reference.
VII. Cysteine Engineered Antibodies (ThioMAbs and ThioFabs)
[0470] The compounds of the invention include cysteine engineered
antibodies where one or more amino acids of a wild-type or parent
antibody are replaced with a cysteine amino acid. Any form of
antibody may be so engineered, i.e. mutated. For example, a parent
Fab antibody fragment may be engineered to form a cysteine
engineered Fab, referred to herein as "ThioFab." Similarly, a
parent monoclonal antibody may be engineered to form a "ThioMab."
It should be noted that a single site mutation yields a single
engineered cysteine residue in a ThioFab, while a single site
mutation yields two engineered cysteine residues in a ThioMab, due
to the dimeric nature of the IgG antibody. Mutants with replaced
("engineered") cysteine (Cys) residues are evaluated for the
reactivity of the newly introduced, engineered cysteine thiol
groups. The thiol reactivity value is a relative, numerical term in
the range of 0 to 1.0 and can be measured for any cysteine
engineered antibody. Thiol reactivity values of cysteine engineered
antibodies of the invention are in the ranges of 0.6 to 1.0; 0.7 to
1.0; or 0.8 to 1.0.
[0471] The design, selection, and preparation methods of the
invention enable cysteine engineered antibodies which are reactive
with electrophilic functionality. These methods further enable
antibody conjugate compounds such as antibody-drug conjugate (ADC)
compounds with drug molecules at designated, designed, selective
sites. Reactive cysteine residues on an antibody surface allow
specifically conjugating a drug moiety through a thiol reactive
group such as maleimide or haloacetyl. The nucleophilic reactivity
of the thiol functionality of a Cys residue to a maleimide group is
about 1000 times higher compared to any other amino acid
functionality in a protein, such as amino group of lysine residues
or the N-terminal amino group. Thiol specific functionality in
iodoacetyl and maleimide reagents may react with amine groups, but
higher pH (>9.0) and longer reaction times are required (Garman,
1997, Non-Radioactive Labelling: A Practical Approach, Academic
Press, London).
[0472] Cysteine engineered antibodies of the invention preferably
retain the antigen binding capability of their wild type, parent
antibody counterparts. Thus, cysteine engineered antibodies are
capable of binding, preferably specifically, to antigens. Such
antigens include, for example, tumor-associated antigens (TAA),
cell surface receptor proteins and other cell surface molecules,
transmembrane proteins, signalling proteins, cell survival
regulatory factors, cell proliferation regulatory factors,
molecules associated with (for e.g., known or suspected to
contribute functionally to) tissue development or differentiation,
lymphokines, cytokines, molecules involved in cell cycle
regulation, molecules involved in vasculogenesis and molecules
associated with (for e.g., known or suspected to contribute
functionally to) angiogenesis. The tumor-associated antigen may be
a cluster differentiation factor (i.e., a CD protein). An antigen
to which a cysteine engineered antibody is capable of binding may
be a member of a subset of one of the above-mentioned categories,
wherein the other subset(s) of said category comprise other
molecules/antigens that have a distinct characteristic (with
respect to the antigen of interest).
[0473] The parent antibody may also be a human or humanized
anti-Robo4 antibody or Fab having any one, two, three, four, five
or six of the HVR sequences disclosed herein. Cysteine engineered
antibodies of the invention may be site-specifically and
efficiently coupled with a thiol-reactive reagent. The
thiol-reactive reagent may be a multifunctional linker reagent, a
capture, i.e. affinity, label reagent (e.g. a biotin-linker
reagent), a detection label (e.g. a fluorophore reagent), a solid
phase immobilization reagent (e.g. SEPHAROSE.TM., polystyrene, or
glass), or a drug-linker intermediate. One example of a
thiol-reactive reagent is N-ethyl maleimide (NEM). In an exemplary
embodiment, reaction of a ThioFab with a biotin-linker reagent
provides a biotinylated ThioFab by which the presence and
reactivity of the engineered cysteine residue may be detected and
measured. Reaction of a ThioFab with a multifunctional linker
reagent provides a ThioFab with a functionalized linker which may
be further reacted with a drug moiety reagent or other label.
Reaction of a ThioFab with a drug-linker intermediate provides a
ThioFab drug conjugate.
[0474] Such an approach may be applied to the conjugation of other
thiol-reactive agents in which the reactive group is, for example,
a maleimide, an iodoacetamide, a pyridyl disulfide, or other
thiol-reactive conjugation partner (Haugland, 2003, Molecular
Probes Handbook of Fluorescent Probes and Research Chemicals,
Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2;
Garman, 1997, Non-Radioactive Labelling: A Practical Approach,
Academic Press, London; Means (1990) Bioconjugate Chem. 1:2;
Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San
Diego, pp. 40-55, 643-671). The partner may be a cytotoxic agent
(e.g. a toxin such as doxorubicin or pertussis toxin), a
fluorophore such as a fluorescent dye like fluorescein or
rhodamine, a chelating agent for an imaging or radiotherapeutic
metal, a peptidyl or non-peptidyl label or detection tag, or a
clearance-modifying agent such as various isomers of polyethylene
glycol, a peptide that binds to a third component, or another
carbohydrate or lipophilic agent.
[0475] The sites identified on the exemplary antibody fragment are
primarily in the constant domain of an antibody which is well
conserved across all species of antibodies. These sites should be
broadly applicable to other antibodies, without further need of
structural design or knowledge of specific antibody structures, and
without interference in the antigen binding properties inherent to
the variable domains of the antibody.
[0476] The PHESELECTOR assay (Phage ELISA for Selection of Reactive
Thiols, disclosed in WO2006/034488 (herein incorporated by
reference in its entirety)) allows for detection of reactive
cysteine groups in antibodies in an ELISA phage format. The process
of coating the protein (e.g. antibody) of interest on well
surfaces, followed incubation with phage particles and then HRP
labeled secondary antibody with absorbance detection is detailed in
WO2006/034488. Mutant proteins displayed on phage may be screened
in a rapid, robust, and high-throughput manner. Libraries of
cysteine engineered antibodies can be produced and subjected to
binding selection using the same approach to identify appropriately
reactive sites of free Cys incorporation from random protein-phage
libraries of antibodies or other proteins. This technique includes
reacting cysteine mutant proteins displayed on phage with an
affinity reagent or reporter group which is also
thiol-reactive.
[0477] Cysteine engineered antibodies which may be useful in the
treatment of cancer include, but are not limited to, antibodies
against cell surface receptors and tumor-associated antigens (TAA).
Such antibodies may be used as naked antibodies (unconjugated to a
drug or label moiety) or as Formula I, Ia, Ia', or II antibody-drug
conjugates (ADC). Tumor-associated antigens are known in the art,
and can prepared for use in generating antibodies using methods and
information which are well known in the art. In attempts to
discover effective cellular targets for cancer diagnosis and
therapy, researchers have sought to identify transmembrane or
otherwise tumor-associated polypeptides that are specifically
expressed on the surface of one or more particular type(s) of
cancer cell as compared to on one or more normal non-cancerous
cell(s) or expressed on the surface of angiogenic cells (such as
vascular endothelial cells) as compared to non-endothelial cells
unassociated with cancer. Often, such tumor-associated polypeptides
are more abundantly expressed on the surface of the cancer cells or
angiogenic cells as compared to on the surface of the non-cancerous
cells or non-angiogenic cells. The identification of such
tumor-associated cell surface antigen polypeptides has given rise
to the ability to specifically target cancer cells or angiogenic
cells for destruction via antibody-based therapies.
[0478] In yet another embodiment, an antibody may be conjugated to
a "receptor" (such as streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
VIII. Methods of Treatment Using Anti-Robo4 Antibodies
[0479] It is contemplated that the anti-Robo4 antibodies
(including, e.g., naked anti-Robo4 antibodies and ADC) of the
present invention may be used to treat various diseases or
disorders, e.g. characterized by the overexpression of a tumor or
angiogenesis associated antigen such as Robo4. Exemplary conditions
of hyperproliferative disorders include pathological conditions
associated with abnormal or unwanted endothelial cell
proliferation, such as abnormal or unwanted vascular cell
proliferation in disorders including, but not limited to, cancer,
angiogenesis and disorders associated with (e.g., augmented by
endothelial cell proliferation within the tissue experiencing the
disorder) solid tumors and metastasis, atherosclerosis, retrolental
fibroplasia, hemangiomas, chronic inflammation, intraocular
neovascular diseases such as proliferative retinopathies, e.g.,
diabetic retinopathy, age-related macular degeneration (AMD),
neovascular glaucoma, immune rejection of transplanted corneal
tissue and other tissues, rheumatoid arthritis, and psoriasis.
[0480] The anti-Robo4 antibodies and ADC compounds which are
identified in animal models and cell-based assays can be further
tested in tumor-bearing higher primates and human clinical trials.
Human clinical trials can be designed to test the efficacy of the
anti-Robo4 monoclonal antibody or immunoconjugate of the invention
in patients experiencing a cell proliferative disorder including
without limitation pathological conditions associated with abnormal
or unwanted endothelial cell proliferation, such as abnormal or
unwanted vascular cell proliferation in disorders including, but
not limited to, cancer, angiogenesis and disorders associated with
(e.g., augmented by endothelial cell proliferation within the
tissue experiencing the disorder) solid tumors and metastasis,
atherosclerosis, retrolental fibroplasia, hemangiomas, chronic
inflammation, intraocular neovascular diseases such as
proliferative retinopathies, e.g., diabetic retinopathy,
age-related macular degeneration (AMD), neovascular glaucoma,
immune rejection of transplanted corneal tissue and other tissues,
rheumatoid arthritis, and psoriasis. The clinical trial may be
designed to evaluate the efficacy of an anti-Robo4 antibody in
combinations with known therapeutic regimens, such as radiation
and/or chemotherapy involving known chemotherapeutic and/or
cytotoxic agents.
[0481] The cancer may comprise Robo4-expressing cells, such that an
anti-Robo4 antibody of the present invention is able to bind to the
Robo4-expressing endothelial cells within the cancer tissue. To
determine Robo4 expression in the cancer, various
diagnostic/prognostic assays are available. In one embodiment,
Robo4 overexpression may be analyzed by IHC. Paraffin-embedded
tissue sections from a tumor biopsy may be subjected to the IHC
assay and accorded a Robo4 protein staining intensity criteria with
respect to the degree of staining and in what proportion of tumor
cells examined. In one embodiment, Robo4 overexpression may be
analyzed by detection in vivo or in vitro of anti-Robo4 antibody
labeled with a detectable marker contacted with Robo4-expressing
cells in vivo. Detectable markers include without limitation
radioisotopes, fluorescent compounds, metallic and/or magnetic
nanoparticles, microbubbles, chelating ligands, and other
detectable markers as disclosed herein.
[0482] For the prevention or treatment of disease, the appropriate
dosage of an anti-Robo4 antibody will depend on the type of disease
to be treated, as defined above, the severity and course of the
disease, whether the molecule is administered for preventive or
therapeutic purposes, previous therapy, the patient's clinical
history and response to the antibody, and the discretion of the
attending physician. The molecule is suitably administered to the
patient at one time or over a series of treatments. Depending on
the type and severity of the disease, about 1 .mu.g/kg to 15 mg/kg
(e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. A typical
daily dosage might range from about 1 .mu.g/kg to 100 mg/kg or
more, depending on the factors mentioned above. An exemplary dosage
of ADC to be administered to a patient is in the range of about 0.1
to about 10 mg/kg of patient weight.
[0483] For repeated administrations over several days or longer,
depending on the condition, the treatment is sustained until a
desired suppression of disease symptoms occurs. An exemplary dosing
regimen comprises administering an initial loading dose of about 4
mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of an
anti-Robo4 antibody. Other dosage regimens may be useful. In yet
another embodiment, the dosage range is 275-375 mg/m2 The progress
of this therapy is easily monitored by conventional techniques and
assays.
[0484] A. Administration of Anti-Robo4 Antibodies
[0485] The anti-Robo4 antibodies (and adjunct therapeutic agent) of
the invention may be administered by any route appropriate to the
condition to be treated, including parenteral, subcutaneous,
intraperitoneal, intrapulmonary, and intranasal, and, if desired
for local treatment, intralesional administration. The anti-Robo4
antibodies will typically be administered parenterally, i.e.
infusion, subcutaneous, intramuscular, intravenous, intradermal,
intrathecal, epidural, intraarterial, and intraperitoneal. In
addition, the antibody is suitably administered by pulse infusion,
particularly with declining doses of the antibody. Dosing can be by
any suitable route, e.g. by injections, such as intravenous or
subcutaneous injections, depending in part on whether the
administration is brief or chronic.
[0486] For treating cancers or any disease in which angiogenisis
occurs, the anti-Robo4 antibody or antibody-drug conjugate is
administered via intravenous infusion. The dosage administered via
infusion is in the range of about 1 .mu.g/m.sup.2 to about 10,000
.mu.g/m.sup.2 per dose, generally one dose per week for a total of
one, two, three or four doses. Alternatively, the dosage range is
about 1 .mu.g/m.sup.2 to about 1000 .mu.g/m.sup.2, about 1
.mu.g/m.sup.2 to about 800 .mu.g/m.sup.2, about 1 .mu.g/m.sup.2 to
about 600 .mu.g/m.sup.2, about 1 .mu.g/m.sup.2 to about 400
.mu.g/m.sup.2, about 10 .mu.g/m.sup.2 to about 500 .mu.g/m.sup.2,
about 10 .mu.g/m.sup.2 to about 300 .mu.g/m.sup.2, about 10
.mu.g/m.sup.2 to about 200 .mu.g/m.sup.2, or about 1 .mu.g/m.sup.2
to about 200 .mu.g/m.sup.2. The dose may be administered once per
day, once per week, multiple times per week, but less than once per
day, multiple times per month but less than once per day, multiple
times per month but less than once per week, once per month or
intermittently to relieve or alleviate symptoms of the disease.
Administration may continue at any of the disclosed intervals until
the symptoms of the disease or disorder being treated are reduced,
alleviated, or eliminated (e.g., as indicated by shrinkage of a
tumor, reduction of elimination of symptoms of lymphoma, leukemia,
inhibition of metastatic spread without shrinkage of the primary
tumor (i.e., progression-free survival), inhibition of ocular
neovascularization). Administration may continue after remission or
relief of symptoms is achieved where such remission or relief is
prolonged by such continued administration.
[0487] The invention also provides a method of treating a cancer,
and/or a metastasis of a cancer and/or an ocular disorder
characterized by proliferation of vascular endothelial cells,
comprising administering to a patient suffering from such a
disorder, a therapeutically effective amount of an anti-Robo4
antibody of any one of the preceding embodiments, which antibody is
not conjugated to a cytotoxic molecule or a detectable molecule.
The antibody will typically be administered in a dosage range of
about 1 .mu.g/m.sup.2 to about 1000 mg/m.sup.2. Alternatively, the
dosage range is about 1 .mu.g/m.sup.2 to about 800 .mu.g/m.sup.2,
about 1 .mu.g/m.sup.2 to about 600 .mu.g/m.sup.2, about 1
.mu.g/m.sup.2 to about 400 .mu.g/m.sup.2, about 10 .mu.g/m.sup.2 to
about 500 .mu.g/m.sup.2, about 10 .mu.g/m.sup.2 to about 300
.mu.g/m.sup.2, about 10 .mu.g/m.sup.2 to about 200 .mu.g/m.sup.2,
or about 1 .mu.g/m.sup.2 to about 200 .mu.g/m.sup.2.
[0488] The invention also provides a method of treating a cancer,
and/or a metastasis of a cancer and/or an ocular disorder
characterized by proliferation of vascular endothelial cells,
comprising administering to a patient suffering from such a
disorder, a therapeutically effective amount of an anti-Robo4
antibody of any one of the preceding embodiments, which antibody is
conjugated to a cytotoxic molecule or a detectable molecule. The
antibody will typically be administered in a dosage range of about
1 .mu.g/m.sup.2 to about 1000 mg/m.sup.2. Alternatively, the dosage
range is about 1 .mu.g/m.sup.2 to about 800 .mu.g/m.sup.2, about 1
.mu.g/m.sup.2 to about 600 .mu.g/m.sup.2, about 1 .mu.g/m.sup.2 to
about 400 .mu.g/m.sup.2, about 10 .mu.g/m.sup.2 to about 500
.mu.g/m.sup.2, about 10 .mu.g/m.sup.2 to about 300 .mu.g/m.sup.2,
about 10 .mu.g/m.sup.2 to about 200 .mu.g/m.sup.2, or about 1
.mu.g/m.sup.2 to about 200 .mu.g/m.sup.2.
[0489] B. Pharmaceutical Formulations
[0490] In one aspect, the invention further provides pharmaceutical
formulations comprising at least one anti-Robo4 antibody of the
invention and/or at least one immunoconjugate thereof and/or at
least one anti-Robo4 antibody-drug conjugate of the invention. In
some embodiments, a pharmaceutical formulation comprises 1) an
anti-Robo4 antibody and/or an anti-Robo4 antibody-drug conjugate
and/or an immunoconjugate thereof, and 2) a pharmaceutically
acceptable carrier. In some embodiments, a pharmaceutical
formulation comprises 1) an anti-Robo4 antibody and/or an
immunoconjugate thereof, and optionally, 2) at least one additional
therapeutic agent.
[0491] Pharmaceutical formulations comprising an antibody or
immunoconjugate of the invention or the antibody-drug conjugate of
the invention are prepared for storage by mixing the antibody or
antibody-drug conjugate having the desired degree of purity with
optional physiologically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)) in the form of aqueous solutions or
lyophilized or other dried formulations. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, histidine and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride); phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
Pharmaceutical formulations to be used for in vivo administration
are generally sterile. This is readily accomplished by filtration
through sterile filtration membranes.
[0492] Active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0493] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0494] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody or
immunoconjugate of the invention, which matrices are in the form of
shaped articles, e.g., films, or microcapsule. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies or
immunoconjugates remain in the body for a long time, they may
denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for stabilization depending on the mechanism involved. For
example, if the aggregation mechanism is discovered to be
intermolecular S--S bond formation through thio-disulfide
interchange, stabilization may be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific
polymer matrix compositions.
[0495] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0496] C. Combination Therapy
[0497] An anti-Robo4 antibodies of the invention may be combined in
a pharmaceutical combination formulation, or dosing regimen as
combination therapy, with at least one additional compound having
anti-cancer properties. The at least one additional compound of the
pharmaceutical combination formulation or dosing regimen preferably
has complementary activities to the anti-Robo4 antibody composition
such that they do not adversely affect each other.
[0498] The at least one additional compound may be a
chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth
inhibitory agent, an anti-hormonal agent, and combinations thereof.
Such molecules are suitably present in combination in amounts that
are effective for the purpose intended. A pharmaceutical
composition containing an anti-Robo4 antibody of the invention may
also comprise a therapeutically effective amount of a
chemotherapeutic agent such as a tubulin-forming inhibitor, a
topoisomerase inhibitor, or a DNA binder.
[0499] In one aspect, the first compound is an anti-Robo4 ADC of
the invention and the at least one additional compound is a
therapeutic antibody other than an anti-Robo4 (naked antibody or an
ADC). In one embodiment, the at least one additional compound is an
antibody that binds a cancer cell surface marker. In one embodiment
the at least one additional compound is an anti-HER2 antibody,
trastuzumab (e.g., Herceptin.RTM., Genentech, Inc., South San
Francisco, Calif.). In one embodiment the at least one additional
compound is an anti-HER2 antibody, pertuzumab (Omnitarg.TM.,
Genentech, Inc., South San Francisco, Calif., see U.S. Pat. No.
6,949,245). In one embodiment, the at least one additional compound
is an anti-VEGF antibody (e.g., AVASTIN.RTM., Genentech, Inc.). In
an embodiment, the at least one additional compound is an antibody
(either a naked antibody or an ADC), and the additional antibody is
a second, third, fourth, fifth, sixth antibody or more, such that a
combination of such second, third, fourth, fifth, sixth, or more
antibodies (either naked or as an ADC) is efficacious in treating a
cell proliferative disease in a tissue expressing Robo4.
[0500] Other therapeutic regimens may be combined with the
administration of an anticancer agent identified in accordance with
this invention, including without limitation radiation therapy
and/or bone marrow and peripheral blood transplants, and/or a
cytotoxic agent, a chemotherapeutic agent, or a growth inhibitory
agent. In one of such embodiments, a chemotherapeutic agent is an
agent or a combination of agents such as, for example,
cyclophosphamide, hydroxydaunorubicin, adriamycin, doxorubincin,
vincristine (ONCOVIN.TM.), prednisolone, CHOP, CVP, or COP, or
immunotherapeutics such as anti-PSCA, anti-HER2 (e.g.,
HERCEPTIN.RTM., OMNITARG.TM.) or anti-VEGF (e.g., AVASTIN.RTM.).
The combination therapy may be administered as a simultaneous or
sequential regimen. When administered sequentially, the combination
may be administered in two or more administrations. The combined
administration includes coadministration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities.
[0501] In one embodiment, treatment with an anti-Robo4 antibody
involves the combined administration of an anticancer agent
identified herein, and one or more chemotherapeutic agents or
growth inhibitory agents, including coadministration of cocktails
of different chemotherapeutic agents. Chemotherapeutic agents
include taxanes (such as paclitaxel and docetaxel) and/or
anthracycline antibiotics. Preparation and dosing schedules for
such chemotherapeutic agents may be used according to
manufacturer's instructions or as determined empirically by the
skilled practitioner. Preparation and dosing schedules for such
chemotherapy are also described in "Chemotherapy Service", (1992)
Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md.
[0502] Suitable dosages for any of the above coadministered agents
are those presently used and may be lowered due to the combined
action (synergy) of the newly identified agent and other
chemotherapeutic agents or treatments.
[0503] The combination therapy may provide "synergy" and prove
"synergistic", i.e. the effect achieved when the active ingredients
used together is greater than the sum of the effects that results
from using the compounds separately. A synergistic effect may be
attained when the active ingredients are: (1) co-formulated and
administered or delivered simultaneously in a combined, unit dosage
formulation; (2) delivered by alternation or in parallel as
separate formulations; or (3) by some other regimen. When delivered
in alternation therapy, a synergistic effect may be attained when
the compounds are administered or delivered sequentially, e.g. by
different injections in separate syringes. In general, during
alternation therapy, an effective dosage of each active ingredient
is administered sequentially, i.e. serially, whereas in combination
therapy, effective dosages of two or more active ingredients are
administered together.
[0504] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with other agents such as chemotherapeutic agents) will
depend on the type of disease to be treated, the type of antibody,
the severity and course of the disease, whether the antibody is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the
antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg)
of antibody is an initial candidate dosage for administration to
the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment is sustained until a desired suppression
of disease symptoms occurs. One exemplary dosage of the antibody
would be in the range from about 0.05 mg/kg to about 10 mg/kg.
Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or
10 mg/kg (or any combination thereof) may be administered to the
patient. Such doses may be administered intermittently, e.g. every
week or every three weeks (e.g. such that the patient receives from
about two to about twenty, e.g. about six doses of the antibody).
An initial higher loading dose, followed by one or more lower doses
may be administered. An exemplary dosing regimen comprises
administering an initial loading dose of about 4 mg/kg, followed by
a weekly maintenance dose of about 2 mg/kg of the antibody.
However, other dosage regimens may be useful. The progress of this
therapy is easily monitored by conventional techniques and
assays.
IX. Labeled Antibodies and Uses Thereof
[0505] The invention also provides labeled anti-Robo4 antibodies.
Labeled antibodies may be useful in diagnostic assays, e.g., in
vitro, ex vivo, or in vivo assays for detecting expression of Robo4
in specific cells or tissues (e.g., to image angiogenesis,
neovascularization, and/or tumor vasculature). In some embodiments,
the labeled antibodies are used in in vivo imaging assays. The
anti-Robo4 antibodies of the invention, may be conjugated with any
label moiety which can be covalently attached to the antibody. In
some embodiments, the anti-Robo4 antibodies of the invention
(including, e.g., the cysteine engineered anti-Robo4 antibodies)
are covalently attached to the antibody through a reactive group on
the antibody, such as a reactive lysine or cysteine residue.
Covalent attachment through a reactive cysteine thiol group is
disclosed in Singh et al., Anal. Biochem. 304:147-15 (2002); Harlow
E. and Lane, D. (1999) Using Antibodies: A Laboratory Manual, Cold
Springs Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Lundblad
R. L. (1991) Chemical Reagents for Protein Modification, 2nd ed.
CRC Press, Boca Raton, Fla.
[0506] The attached label may function to: (i) provide a detectable
signal; (ii) interact with a second label to modify the detectable
signal provided by the first or second label, e.g. to give FRET
(fluorescence resonance energy transfer); (iii) stabilize
interactions or increase affinity of binding, with antigen or
ligand; (iv) affect mobility, e.g. electrophoretic mobility or
cell-permeability, by charge, hydrophobicity, shape, or other
physical parameters, or (v) provide a capture moiety, to modulate
ligand affinity, antibody/antigen binding, or ionic
complexation.
[0507] For diagnostic applications, the antibody will typically be
labeled with a detectable moiety. Numerous labels are available
which can be generally grouped into the following categories:
[0508] (a) In some embodiments, the anti-Robo4 antibodies are
labeled with radioisotopes (radionuclides), such as .sup.3H,
.sup.11C, .sup.14C, .sup.18F, .sup.19F, .sup.32P, .sup.35S,
.sup.64Cu, .sup.68Ga, .sup.86Y, .sup.99Tc, .sup.111In, .sup.123I,
.sup.124I, .sup.125I, .sup.131I, .sup.133Xe, .sup.177Lu,
.sup.211At, or .sup.213Bi. Radioisotope labeled anti-Robo4
antibodies are useful in receptor targeted imaging of cells
expressing Robo4, (e.g., for use in the diagnostic uses of the
invention such as in vivo imaging of tumor endothelial cells, tumor
vasculature, angiogenesis, and neovascularization).
[0509] (b) In some embodiments, the anti-Robo4 antibodies are
labeled with ligand reagents that bind, chelate or otherwise
complex a radioisotope metal where the reagent is reactive with the
engineered cysteine thiol of the antibody, using the techniques
described in Current Protocols in Immunology, Volumes 1 and 2,
Coligen et al, Ed. Wiley-Interscience, New York, N.Y., Pubs.
(1991). Chelating ligands which may complex a metal ion include
DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, Tex.).
Chelating linker reagents such as DOTA-maleimide
(4-maleimidobutyramidobenzyl-DOTA) can be prepared by the reaction
of aminobenzyl-DOTA with 4-maleimidobutyric acid (Fluka) activated
with isopropylchloroformate (Aldrich), following the procedure of
Axworthy et al. (2000) PNAS USA 97(4):1802-1807). DOTA-maleimide
reagents react with the free cysteine amino acids of the cysteine
engineered antibodies and provide a metal complexing ligand on the
antibody (Lewis et al., Bioconj. Chem. 9:72-86 (1998)). Chelating
linker labelling reagents such as DOTA-NHS
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono
(N-hydroxysuccinimide ester) are commercially available
(Macrocyclics, Dallas, Tex.). Radionuclides can be targeted via
complexation with the antibody-drug conjugates of the invention (Wu
et al. Nature Biotech. 23(9): 1137-1146 (2005)). Receptor target
imaging with radionuclide labeled antibodies can provide a marker
of increased target expression by detection and quantitation of
progressive accumulation of antibodies in tumor tissue (Albert et
al. Bioorg. Med. Chem. Lett. 8:1207-1210 (1998)). The conjugated
radio-metals may remain intracellular following lysosomal
degradation.
[0510] Metal-chelate complexes suitable as antibody labels for
imaging experiments are disclosed: U.S. Pat. No. 5,342,606; U.S.
Pat. No. 5,428,155; U.S. Pat. No. 5,316,757; U.S. Pat. No.
5,480,990; U.S. Pat. No. 5,462,725; U.S. Pat. No. 5,428,139; U.S.
Pat. No. 5,385,893; U.S. Pat. No. 5,739,294; U.S. Pat. No.
5,750,660; U.S. Pat. No. 5,834,456; Hnatowich et al. (1983) J.
Immunol. Methods 65:147-157; Meares et al., Anal. Biochem.
142:68-78 (1984); Mirzadeh et al., Bioconjugate Chem. 1:59-65
(1990); Meares et al., J. Cancer, Suppl. 10:21-26 (1990); Izard et
al., Bioconjugate Chem. 3:346-350 (1992); Nikula et al., Nucl. Med.
Biol. 22:387-90 (1995); Camera et al., Nucl. Med. Biol. 20:955-62
(1993); Kukis et al., J. Nucl. Med. 39:2105-2110 (1998); Verel et
al., J. Nucl. Med. 44:1663-1670 (2003); Camera et al., J. Nucl.
Med. 21:640-646 (1994); Ruegg et al., Cancer Res. 50:4221-4226
(1990); Verel et al., J. Nucl. Med. 44:1663-1670 (2003); Lee et
al., Cancer Res. 61:4474-4482 (2001); Mitchell, et al., J. Nucl.
Med. 44:1105-1112 (2003); Kobayashi et al., Bioconjugate Chem.
10:103-111 (1999); Miederer et al., J. Nucl. Med. 45:129-137
(2004); DeNardo et al., Clinical Cancer Research 4:2483-90 (1998);
Blend et al., Cancer Biotherapy & Radiopharmaceuticals
18:355-363 (2003); Nikula et al., J. Nucl. Med. 40:166-76 (1999);
Kobayashi et al., J. Nucl. Med. 39:829-36 (1998); Mardirossian et
al., Nucl. Med. Biol. 20:65-74 (1993); Roselli et al., Cancer
Biotherapy & Radiopharmaceuticals, 14:209-20 (1999).
[0511] (c) In some embodiments, the anti-Robo4 antibodies are
labeled with fluorescent labels such as rare earth chelates
(europium chelates), fluorescein types including FITC,
5-carboxyfluorescein, 6-carboxy fluorescein; rhodamine types
including TAMRA; dansyl; Lissamine; cyanines; phycoerythrins; Texas
Red; and analogs thereof. The fluorescent labels can be conjugated
to antibodies using the techniques disclosed in Current Protocols
in Immunology, supra, for example. Fluorescent dyes and fluorescent
label reagents include those which are commercially available from
Invitrogen/Molecular Probes (Eugene, Oreg.) and Pierce
Biotechnology, Inc. (Rockford, Ill.).
[0512] (d) In some embodiments, the anti-Robo4 antibodies are
labeled with various enzyme-substrate labels are available or
disclosed (U.S. Pat. No. 4,275,149). The enzyme generally catalyzes
a chemical alteration of a chromogenic substrate that can be
measured using various techniques. For example, the enzyme may
catalyze a color change in a substrate, which can be measured
spectrophotometrically. Alternatively, the enzyme may alter the
fluorescence or chemiluminescence of the substrate. Techniques for
quantifying a change in fluorescence are described above. The
chemiluminescent substrate becomes electronically excited by a
chemical reaction and may then emit light which can be measured
(using a chemiluminometer, for example) or donates energy to a
fluorescent acceptor. Examples of enzymatic labels include
luciferases (e.g., firefly luciferase and bacterial luciferase;
U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,
malate dehydrogenase, urease, peroxidase such as horseradish
peroxidase (HRP), alkaline phosphatase (AP), -galactosidase,
glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase,
galactose oxidase, and glucose-6-phosphate dehydrogenase),
heterocyclic oxidases (such as uricase and xanthine oxidase),
lactoperoxidase, microperoxidase, and the like. Techniques for
conjugating enzymes to antibodies are described in O'Sullivan et
al. (1981) "Methods for the Preparation of Enzyme-Antibody
Conjugates for use in Enzyme Immunoassay", in Methods in Enzym. (ed
J. Langone & H. Van Vunakis), Academic Press, New York,
73:147-166.
[0513] Examples of enzyme-substrate combinations include, for
example:
[0514] (i) Horseradish peroxidase (HRP) with hydrogen peroxidase as
a substrate, wherein the hydrogen peroxidase oxidizes a dye
precursor (e.g., orthophenylene diamine (OPD) or
3,3',5,5'-tetramethylbenzidine hydrochloride (TMB));
[0515] (ii) alkaline phosphatase (AP) with para-nitrophenyl
phosphate as chromogenic substrate; and
[0516] (iii) -D-galactosidase (-D-Gal) with a chromogenic substrate
(e.g., p-nitrophenyl-D-galactosidase) or fluorogenic substrate
4-methylumbelliferyl-D-galactosidase.
Numerous other enzyme-substrate combinations are available to those
skilled in the art. For a general review, see U.S. Pat. No.
4,275,149 and U.S. Pat. No. 4,318,980.
[0517] A label may be indirectly conjugated with an anti-Robo4
antibody (including, e.g., a cysteine engineered anti-Robo4
antibody). For example, the anti-Robo4 antibody can be conjugated
with biotin and any of the three broad categories of labels
mentioned above can be conjugated with avidin or streptavidin, or
vice versa. Biotin binds selectively to streptavidin and thus, the
label can be conjugated with the anti-Robo4 antibody in this
indirect manner. Alternatively, to achieve indirect conjugation of
the label with the anti-Robo4 antibody, the anti-Robo4 antibody is
conjugated with a small hapten (e.g., digoxin) and one of the
different types of labels mentioned above is conjugated with an
anti-hapten polypeptide variant (e.g., anti-digoxin antibody).
Thus, indirect conjugation of the label with the anti-Robo4
antibody can be achieved (Hermanson, G. (1996) in Bioconjugate
Techniques Academic Press, San Diego).
[0518] The anti-Robo4 antibody of the present invention may be
employed in any known assay method, such as ELISA, competitive
binding assays, direct and indirect sandwich assays, and
immunoprecipitation assays (Zola, (1987) Monoclonal Antibodies: A
Manual of Techniques, pp. 147-158, CRC Press, Inc.).
[0519] The labeled anti-Robo4 antibodies of the invention can
detect cell-surface receptors and are useful for localizing,
visualizing, and quantitating endothelial cells (e.g., tumor
endothelial cells), sites of angiogenesis, sites of
neovascularization, and/or tumor vasculature. Another use for
detectably labeled antibodies is a method of bead-based
immunocapture comprising conjugating a bead with a fluorescent
labeled antibody and detecting a fluorescence signal upon binding
of a ligand. Similar binding detection methodologies utilize the
surface plasmon resonance (SPR) effect to measure and detect
antibody-antigen interactions. The detectably labeled anti-Robo4
antibody of the present invention may be employed in any known
assay method, such as ELISA, competitive binding assays, direct and
indirect sandwich assays, and immunoprecipitation assays (Zola,
(1987) Monoclonal Antibodies: A Manual of Techniques, pp. 147-158,
CRC Press, Inc.).
[0520] Detection labels such as fluorescent dyes and
chemiluminescent dyes (Briggs et al, J. Chem. Soc., Perkin-Trans.
1:1051-1058 (1997)) provide a detectable signal and are generally
applicable for labelling antibodies, preferably with the following
properties: (i) the labeled antibody should produce a very high
signal with low background so that small quantities of antibodies
can be sensitively detected in both cell-free and cell-based
assays; and (ii) the labeled antibody should be photostable so that
the fluorescent signal may be observed, monitored and recorded
without significant photo bleaching. For applications involving
cell surface binding of labeled antibody to membranes or cell
surfaces, especially live cells, the labels preferably (iii) have
good water-solubility to achieve effective conjugate concentration
and detection sensitivity and (iv) are non-toxic to living cells so
as not to disrupt the normal metabolic processes of the cells or
cause premature cell death.
[0521] Direct quantification of cellular fluorescence intensity and
enumeration of fluorescently labeled events, e.g. cell surface
binding of peptide-dye conjugates may be conducted on an system
(FMAT.RTM. 8100 HTS System, Applied Biosystems, Foster City,
Calif.) that automates mix-and-read, non-radioactive assays with
live cells or beads (Miraglia, "Homogeneous cell- and bead-based
assays for high throughput screening using fluorometric microvolume
assay technology", (1999) J. of Biomolecular Screening 4:193-204).
Uses of labeled antibodies also include cell surface receptor
binding assays, immunocapture assays, fluorescence linked
immunosorbent assays (FLISA), caspase-cleavage (Zheng, "Caspase-3
controls both cytoplasmic and nuclear events associated with
Fas-mediated apoptosis in vivo", (1998) PNAS USA 95:618-23; U.S.
Pat. No. 6,372,907), apoptosis (Vermes, J. Immunol. Methods
184:39-51 (1995)) and cytotoxicity assays. Fluorometric microvolume
assay technology can be used to identify the up or down regulation
by a molecule that is targeted to the cell surface (Swartzman,
Anal. Biochem. 271:143-51 (1999)).
[0522] Labeled cysteine engineered antibodies of the invention are
useful as imaging biomarkers and probes by the various methods and
techniques of biomedical and molecular imaging such as: (i) MRI
(magnetic resonance imaging); (ii) X-ray computed tomography);
(iii) SPECT (single photon emission computed tomography); (iv) PET
(positron emission tomography) Chen et al. (2004) Bioconjugate
Chem. 15:41-49; (v) bioluminescence; (vi) fluorescence; and (vii)
ultrasound. Immunoscintigraphy is an imaging procedure in which
antibodies labeled with radioactive substances are administered to
an animal or human patient and a picture is taken of sites in the
body (e.g., tumors including tumor vasculature, or metastases
thereof) where the antibody localizes (U.S. Pat. No. 6,528,624).
Imaging biomarkers may be objectively measured and evaluated as an
indicator of normal biological processes, pathogenic processes, or
pharmacological responses to a therapeutic intervention. Biomarkers
may be of several types: Type 0 are natural history markers of a
disease and correlate longitudinally with known clinical indices,
e.g. MRI assessment of synovial inflammation in rheumatoid
arthritis; Type I markers capture the effect of an intervention in
accordance with a mechanism-of-action, even though the mechanism
may not be associated with clinical outcome; Type II markers
function as surrogate endpoints where the change in, or signal
from, the biomarker predicts a clinical benefit to "validate" the
targeted response, such as measured bone erosion in rheumatoid
arthritis by planar X-ray, MRI or CT. Imaging biomarkers thus can
provide pharmacodynamic (PD) therapeutic information about: (i)
expression of a target protein, (ii) binding of a therapeutic to
the target protein, i.e. selectivity, and (iii) clearance and
half-life pharmacokinetic data. Advantages of in vivo imaging
biomarkers relative to lab-based biomarkers include: non-invasive
treatment, quantifiable, whole body assessment, repetitive dosing
and assessment, i.e. multiple time points, and potentially
transferable effects from preclinical (small animal) to clinical
(human) results. For some applications, bioimaging supplants or
minimizes the number of animal experiments in preclinical
studies.
[0523] Peptide labelling methods are well known. See Haugland,
2003, Molecular Probes Handbook of Fluorescent Probes and Research
Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate
Chem. 3:2; Garman, (1997) Non-Radioactive Labelling: A Practical
Approach, Academic Press, London; Means (1990) Bioconjugate Chem.
1:2; Glazer et al. (1975) Chemical Modification of Proteins.
Laboratory Techniques in Biochemistry and Molecular Biology (T. S.
Work and E. Work, Eds.) American Elsevier Publishing Co., New York;
Lundblad, R. L. and Noyes, C. M. (1984) Chemical Reagents for
Protein Modification, Vols. I and II, CRC Press, New York;
Pfleiderer, G. (1985) "Chemical Modification of Proteins", Modern
Methods in Protein Chemistry, H. Tschesche, Ed., Walter DeGryter,
Berlin and New York; and Wong (1991) Chemistry of Protein
Conjugation and Cross-linking, CRC Press, Boca Raton, Fla.); De
Leon-Rodriguez et al. (2004) Chem. Eur. J. 10:1149-1155; Lewis et
al. (2001) Bioconjugate Chem. 12:320-324; Li et al. (2002)
Bioconjugate Chem. 13:110-115; Mier et al. (2005) Bioconjugate
Chem. 16:240-237.
[0524] Peptides and proteins labeled with two moieties, a
fluorescent reporter and quencher in sufficient proximity undergo
fluorescence resonance energy transfer (FRET). Reporter groups are
typically fluorescent dyes that are excited by light at a certain
wavelength and transfer energy to an acceptor, or quencher, group,
with the appropriate Stokes shift for emission at maximal
brightness. Fluorescent dyes include molecules with extended
aromaticity, such as fluorescein and rhodamine, and their
derivatives. The fluorescent reporter may be partially or
significantly quenched by the quencher moiety in an intact peptide.
Upon cleavage of the peptide by a peptidase or protease, a
detectable increase in fluorescence may be measured (Knight, C.
(1995) "Fluorimetric Assays of Proteolytic Enzymes", Methods in
Enzymology, Academic Press, 248:18-34).
[0525] The anti-Robo4 labeled antibodies of the invention may also
be used as an affinity purification agent. In this process, the
labeled antibody is immobilized on a solid phase such a Sephadex
resin or filter paper, using methods well known in the art. The
immobilized antibody is contacted with a sample containing the
antigen to be purified, and thereafter the support is washed with a
suitable solvent that will remove substantially all the material in
the sample except the antigen to be purified, which is bound to the
immobilized antibody. Finally, the support is washed with another
suitable solvent, such as glycine buffer, pH 5.0, that will release
the antigen from the antibody.
[0526] Labelling reagents typically bear reactive functionality
which may react (i) directly with a cysteine thiol of a cysteine
engineered antibody to form the labeled antibody, (ii) with a
linker reagent to form a linker-label intermediate, or (iii) with a
linker antibody to form the labeled antibody. Reactive
functionality of labelling reagents include: maleimide, haloacetyl,
iodoacetamide succinimidyl ester (e.g. NHS, N-hydroxysuccinimide),
isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl,
pentafluorophenyl ester, and phosphoramidite, although other
functional groups can also be used.
[0527] An exemplary reactive functional group is
N-hydroxysuccinimidyl ester (NHS) of a carboxyl group substituent
of a detectable label, e.g. biotin or a fluorescent dye. The NHS
ester of the label may be preformed, isolated, purified, and/or
characterized, or it may be formed in situ and reacted with a
nucleophilic group of an antibody. Typically, the carboxyl form of
the label is activated by reacting with some combination of a
carbodiimide reagent, e.g. dicyclohexylcarbodiimide,
diisopropylcarbodiimide, or a uronium reagent, e.g. TSTU
(O--(N-Succinimidyl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate, HBTU
(O-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate), or HATU
(O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate), an activator, such as 1-hydroxybenzotriazole
(HOBt), and N-hydroxysuccinimide to give the NHS ester of the
label. In some cases, the label and the antibody may be coupled by
in situ activation of the label and reaction with the antibody to
form the label-antibody conjugate in one step. Other activating and
coupling reagents include TBTU
(2-(1H-benzotriazo-1-yl)-1-1,3,3-tetramethyluronium
hexafluorophosphate), TFFH(N,N',N'',N'''-tetramethyluronium
2-fluoro-hexafluorophosphate), PyBOP
(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate, EEDQ
(2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline), DCC
(dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT
(1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole, and aryl
sulfonyl halides, e.g. triisopropylbenzenesulfonyl chloride.
[0528] A detectably labeled antibody of the invention may be
detectable by being linked to (including covalently conjugated to)
a magnetic particle or nanoparticle or metallic particle or
nanoparticle. Metallic particles are detectable by such methods as
magnetic resonance imaging (MRI), single-particle or
single-molecule tracking, immunocytochemistry, plasmon frequency
with dark-field illumination, differential interference contrast
and video enhancement, total internal reflection, and photothermal
interference contrast (PIC) technique (see, for example PNAS USA
100(20): 11350-11355 (2003); Sheetz et al. Nature 340:284-288
(1989); Baschong et al., Histochemistry 83:409-411 (1985); Slot and
Geuze, Eur. J. Cell Biol. 38:87-93 (1935); Frey and Frey J. Struct.
Biol. 127:94-100 (1999); Hainfeld and Powell, J. Histochem.
Cytochem. 48:471-480 (2000); Schultz et al. PNAS USA 97:996-1001
(2000); Gelles et al., Nature 331:450-453 (1988); Sonnichsen et
al., Appl Phys. Lett. 77:2949-2951 (2000); Boyer, D. et al.,
Science 297:1160-1163 (2002)). Anti-Robo4 antibodies of the
invention may comprise nanoparticulate agents including, but not
limited to, microbubbles (see Ellegala et al., Circulation
108:336-341 (2003))), also referred to as acoustically active
lipospheres (AALs) (see Tartis et al., Ultrasound Med. Biol.
32(11):1771-80 (2006)), superparamagnetic agents, liposomes,
perfluorocarbon nanoparticle emulsions (WO2005014051), and
dendrimers (see Caruthers et al., Methods in Molecular Medicine,
124:387-400 (2006) and references cited therein, all of which
references are hereby incorporated by reference in their
entirety).
[0529] When the labeled anti-Robo4 antibodies are used for
detection (e.g., in scintigraphic studies), the labels may comprise
a radioactive atom (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu) or a spin label for
nuclear magnetic resonance (NMR) imaging (also known as magnetic
resonance imaging, mri), such as Tc.sup.99m, .sup.123I, .sup.111In,
.sup.131I, .sup.19F, .sup.1H, .sup.14N, .sup.15N, .sup.17O,
.sup.23Na, .sup.31P, or .sup.13C, gadolinium, manganese, iron, or
iron oxides (e.g., super-paramagnetic iron oxide particles (SPIL)
or ultra-small paramagnetic iron oxide particles (USPIL)), or other
NMR observable agents.
X. Articles of Manufacture
[0530] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
etc. The containers may be formed from a variety of materials such
as glass or plastic. The container holds a composition which is by
itself or when combined with another composition effective for
treating, preventing and/or diagnosing the condition and may have a
sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). At least one active agent in the
composition is an antibody of the invention. The label or package
insert indicates that the composition is used for treating the
condition of choice, such as cancer. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antibody of
the invention; and (b) a second container with a composition
contained therein, wherein the composition comprises a further
cytotoxic agent. The article of manufacture in this embodiment of
the invention may further comprise a package insert indicating that
the first and second antibody compositions can be used to treat a
particular condition, e.g. cancer. Alternatively, or additionally,
the article of manufacture may further comprise a second (or third)
container comprising a pharmaceutically-acceptable buffer, such as
bacteriostatic water for injection (BWFI), phosphate-buffered
saline, Ringer's solution and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
and syringes.
[0531] The following are examples of the methods and compositions
of the invention. It is understood that various other embodiments
may be practiced, given the general description provided above.
EXAMPLES
[0532] Amino acid residues within antibody amino acid sequences are
numbered according to Kabat (Kabat et al., Sequences of proteins of
immunological interest, 5th Ed., Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). Single letter amino
acid abbreviations are used. DNA degeneracies are represented using
the IUB code (N=A/C/G/T, D=A/G/T, V=A/C/G, B=C/G/T, H=A/C/T, K=G/T,
M=A/C, R=A/G, S=G/C, W=A/T, Y=C/T).
Example 1
Antibodies Derived from Phage Encoding Hypervariable Regions of a
Starting Antibody
[0533] The nucleic acid sequences of the VL and VH domains of the
HERCEPTIN.RTM. anti-HER2 antibody rhuMAB 4D5-8 (Genentech, Inc.)
was used as the starting sequence for mutagenesis of the HVRs and
phage selection for binding to human Robo4 His-tagged antigen shown
in FIG. 5B or human Rob4-Fc fusion protein. Antibody 4D5 is a
humanized antibody specific for a cancer-associated antigen known
as Her-2 (erbB2). The antibody includes variable domains having
consensus framework regions; a few positions were reverted to mouse
sequence during the process of increasing affinity of the humanized
antibody. The sequence and crystal structure of humanized antibody
4D5 have been described in U.S. Pat. No. 6,054,297, Carter et al,
PNAS USA 89:4285 (1992), the crystal structure is shown in J. Mol.
Biol. 229:969 (1993) and online. The HERCEPTIN.RTM. VL and VH
domains comprise the consensus human kappa I VL domain and a
variant of the human subgroup III consensus VH domain,
respectively. The variant VH domain has 3 changes from the human
consensus: R71A, N73T and L78A.
[0534] The phagemid used for this work is a monovalent Fab-g3
display vector (pV0350-2B) having 2 open reading frames under
control of the phoA promoter, essentially as described in Lee et
al., J. Mol. Biol. (2004), 340(5): 1073-93. The first open reading
frame consists of the stII signal sequence fused to the VL and CH1
domains acceptor light chain and the second consists of the stII
signal sequence fused to the VH and CH1 domains of the acceptor
heavy chain followed by a truncated minor phage coat protein P3.
See Lee et al., J. Mol. Biol. (2004), 340(5):1073-93.
Example 2
Antibodies Generated by Mutagenesis of Heavy Chain HVRs
[0535] Fab clones YW71.6, YW71.1, YW71.22, and YW71.89 were
generated by mutagenesis of HVR-H1, H2, and H3 of huMAb 4D5-8
(HERCEPTIN.RTM. anti-BER2 antibody, Genentech, Inc.) heavy chain
and selection against human Robo4-His tagged antigen fusion
protein. In HVR-H1, Kabat positions 26 (G), 27 (F), 28 (T), 29 (I),
34 (I), and 35 (H) were held constant, and the amino acids at
positions 30-33 were varied. In HVR-H2, Kabat positions 51 (I), 52a
(P), 55 (G), 57 (T), 59 (Y), 60 (A), 61 (D), 62 (S), 63 (V), 64
(K), and 65 (G) were held constant, and positions 49, 50, 52, 53,
54, 56, and 58 were varied. In HVR-H3, Kabat positions 93 (A) and
102 (Y) were held constant, and positions 94-100, 100a-h, and 101
were varied. The light chain of YW71.22 was the modified huMAb
4D5-8 sequence (modified at positions 30, 66 and 91 resulting in
SEQ ID NO:168, comprising SEQ ID NOS:1, 2, and 3 as HVR-L1, L2, and
L3, respectively), the HVRs of which were not varied during phage
selection. Sequence diversity was introduced into each
hypervariable region by mutagenesis of selected amino acid
positions using standard mutagenesis techniques.
[0536] Generation of phage libraries--Randomized oligonucleotide
pools designed for each hypervariable region were phoshorylated
separately in six 20 .mu.l reactions containing 660 ng of
oligonucleotide, 50 mM Tris pH 7.5, 10 mM MgCl.sub.2, 1 mM ATP, 20
mM DTT, and 5 U polynucleotide kinase for 1 h at 37.degree. C. The
six phosphorylated oligonucleotide pools were then combined with 20
.mu.g of Kunkel template in 50 mM Tris pH 7.5, 10 mM MgCl.sub.2 in
a final volume of 500 .mu.l resulting in an oligonucleotide to
template ratio of 3. The mixture was annealed at 90.degree. C. for
4 min, 50.degree. C. for 5 min and then cooled on ice. Excess,
unannealed oligonucleotide was removed with a QIAQUICK.TM. PCR
purification kit (Qiagen kit 28106) using a modified protocol to
prevent excessive denaturation of the annealed DNA. To the 500
.mu.l of annealed mixture, 150 .mu.l of PB was added, and the
mixture was split between 2 silica columns. Following a wash of
each column with 750 .mu.l of PE and an extra spin to dry the
columns, each column was eluted with 110 .mu.l of 10 mM Tris, 1 mM
EDTA, pH 8. The annealed and cleaned-up template (220 .mu.l) was
then filled in by adding 1 .mu.l 100 mM ATP, 10 .mu.l 25 mM dNTPs
(25 mM each of dATP, dCTP, dGTP and dTTP), 15 .mu.l 100 mM DTT, 25
.mu.l 10.times.TM buffer (0.5 M Tris pH 7.5, 0.1 M MgCl.sub.2),
2400 U T4 ligase, and 30 U T7 polymerase for 3 h at room
temperature.
[0537] The filled in product was analyzed on
Tris-Acetate-EDTA/agarose gels (Sidhu et al., Methods in Enzymology
328:333-363 (2000)). Three bands were usually visible: the bottom
band is a correctly filled and ligated product, the middle band is
a filled but unligated product, and the top band is a strand
displaced product. The top band is produced by an intrinsic side
activity of T7 polymerase and is difficult to avoid (Lechner et
al., J. Biol. Chem. 258:11174-11184 (1983)); however, this band
transforms 30-fold less efficiently than the bottom band and
usually contributes little to the library. The middle band is due
to the absence of a 5' phosphate for the final ligation reaction;
this band transforms efficiently and gives mainly wild type
sequence.
[0538] The filled in product was then purified and electroporated
into SS320 cells and propagated in the presence of M13/KO7 helper
phage as described by Sidhu et al., Methods in Enzymology
328:333-363 (2000). Library sizes ranged from 1-2.times.10.sup.9
independent clones. Random clones from the initial libraries were
sequenced to assess library quality.
[0539] Phage Selection--The human Robo4-Fc and Robo4-His-tagged
proteins were used as the selection antigens. Human Robo4-Fc and
Robo4-His was coated on MaxiSorp microtiter plates (Nunc) at 10
.mu.g/mil in PBS and incubated overnight at 4 degrees. For the
first round of selection 12 wells of target were used. Wells were
blocked for 1 hour at room temperature using Phage Blocking Buffer
(1% BSA, 0.05% Tween.RTM. 20, PBS). Phage libraries were PEG
precipitates from frozen glycerol stocks, resuspended in Phage
Blocking Buffer and incubated for 1 hour at room temperature. Phage
libraries were then added to the blocked antigen plates incubated
overnight at room temperature. After overnight binding, unbound or
non-specifically bound phage were removed from the antigen plates
by washing with Wash Buffer (PBS, 05% Tween.RTM.20). Bound phage
were eluted by incubating the wells with 50 mM HCl, 0.5 M KCl for
30 min. Phage were amplified using XL-1 Blue cells and M13/KO7
helper phage and grown for 36 hours at 30.degree. C. in 2YT, 50
.mu.g/mil carbanecillin, 50 ug/ml kanamycin, 10 .mu.g/ml
tetracycline. Amplified phage were then recovered using a modified
PEG precipitation protocol (Clackson & Lowman 2004). The titers
of phage eluted from a target coated well were compared to titers
of phage recovered from a non-target coated well to assess
enrichment. Four rounds of phage selection were completed with the
number of target wells decreasing to 4 (round 2) and 2 (round 3 and
4). Casein Blocking Buffer (Pierce) was used as the blocking
reagent for antigen plates and phage for rounds 2 and 4. Selection
rounds 2-4 used a 3-4 hour phage-antigen binding period and
increased washing stringency. Four phage clones were selected:
YW71.6, YW7.1, YW71.22, and YW71.89. The sequences of the light and
heavy chain domains were determined and are shown in FIGS. 1A and
1B. The Robo4 binding characteristics were determined as disclosed
herein below.
Example 3
Antibodies Generated by Variation of HVRs H1, H2, H3 and L3
[0540] Clones YW79.1, YW79.8, and YW79.11 were generated by
mutagenesis of HVR-H1, H2, H3 and L3 of huMAb 4D5-8 (HERCEPTIN.RTM.
anti-HER2 antibody, Genentech, Inc.) heavy chain variable domain
and huMAb 4D5-8 modified light chain variable domain, SEQ ID
NO:168. In HVR-H1, Kabat positions 26 (G), 28 (T), 29 (F), 30 (S),
31 (S), and 35 (S) were held constant, and the amino acids at
positions 27, 32-34 were varied. In HVR-H2, Kabat positions 49 (S),
51 (I), 55 (G), 57 (T), 59 (Y), 60 (A), 61 (D), 62 (S), 63 (V), 64
(K), and 65 (G) were held constant, and positions 50, 52, 52a, 53,
54, 56, and 58 were varied. In HVR-H3, Kabat positions 93 (A), 94
(R), 100f-g (deletion) were held constant, and positions 95-100,
100a-e, 100h, and 102 were varied. In HVR-L3, Kabat positions 89
(Q), 90 (Q), 95 (P) and 97 (T) were held constant, and positions
91-94 and 96 were varied. The sequence of HVR-L1 was held constant
as RASQSISSYLA (SEQ ID NO:7) and the sequence of HVR-L2 was held
constant as GASSRAS (SEQ ID NO:8). Sequence diversity was
introduced into each hypervariable region by mutagenesis of
selected amino acid positions using standard mutagenesis
techniques. Anti-Robo4 antibody clones YW79.1, YW79.8, and YW79.11
were selected and sequenced. The sequences of the light chain and
heavy chain variable regions are shown in FIGS. 1A and 1B.
Example 4
Purification of Soluble Robo4 and Anti-Robo4 Antibodies
[0541] This example describes methods useful in the purification of
the Robo4 antigen and the anti-Robo4 antibodies of the invention.
Robo4 antigen was purified as a soluble Robo4 extracellular domain
fused to a histidine tag as shown in FIG. 5B.
[0542] Robo4 Antigen Purification
[0543] Human Robo4 constructs (amino acid M1 to amino acid L461 and
amino acid M1 to amino acid Y231) were cloned into the eukaryotic
expression vector pRK5 either as fusions to the Fc portion of human
IgG1 or to a C-terminal Histidine tag. Murine Robo4 (M1 to H232)
was cloned into pRK5 as fusion to a C-terminal Histidine tag only.
All proteins were produced by transient transfection of CHO cells.
Proteins were purified to >90% purity by affinity chromatography
using either protein-A Sepharose.TM. (GE Healthcare) for Fc fusion
proteins or NiNTA Superflow.TM. (Qiagen) for Histidine tag fusions.
If necessary an ion exchange chromatography step (Q- or
SP-Sepharose.TM., GE Healthcare) was added and/or a size exclusion
chromatography step (Superdex.TM. 75, GE Healthcare). Protein
identities were confirmed by N-terminal sequencing using the Edman
degradation method, concentrations were determined by the BCA assay
and by OD 280 absorption measurements, and purity was assessed by
size exclusion chromatography and SDS-PAGE.
[0544] Antibody Purification
[0545] Full length Robo4 antibodies were transiently expressed in
CHO cells and purified to >95% purity by affinity chromatography
using protein-A Sepharose.TM. (GE Healthcare), followed by ion
exchange chromatography using SP-Sepharose.TM. (GE Healthcare). If
necessary, an additional size exclusion chromatography step
(Superdex.TM. 200, GE Healthcare) was added. Antibody
concentrations were determined by the BCA assay according to
manufacturer's instructions (Pierce Chemical Co.) and by OD 280
absorption measurements, and purity was assessed by size exclusion
chromatography and SDS-PAGE. For all antibody purifications,
aggregate levels as determined by laser light scattering were below
5%, protein A levels as determined by protein A ELISA were below 50
ppm, and endotoxin levels as determined by the LAL (Limulus
Amoebocyte Lysate) chromogenic endotoxin assay were below 0.5
EU/mg.
Example 5
Affinity Maturation of YW71.22
[0546] To improve the affinity of anti-Robo4 antibody YW71.22,
three phage display libraries were generated in the background of
YW71.22, each targeting multiple HVRs for soft randomization
mutagenesis as described in Lee et al., J. Mol. Biol.
340(5):1073-93 (2004). To avoid re-selecting YW71.22 from a
potential high background of template, stop codons were introduced
into the HVR to be mutated prior to generating each library. A
solution sorting method was used to enhance the efficiency of the
affinity-based phage selection process. By manipulating the
biotinylated target concentration, reducing the phage capture time
to lower backgrounds and the addition of unbiotinylated target to
eliminate clones with faster off rates, high affinity clones can be
proficiently selected. Lee et al., J. Mol. Biol. (2004), 340(5):
1073-93. From the first round of selection, enrichment (target
dependent phage capture) was observed suggesting a large number of
clones were present in each library with reasonably high affinity
for human Robo4. Selection stringency was increased in subsequent
rounds. After 5 rounds of selection, clones from each library were
analyzed. New sequences were observed in libraries targeting each
of the six HVRs (FIGS. 2A and 2B). Selected clones were screened by
phage ELISA and then expressed as IgG protein and their affinity
characterized using Biacore.TM. binding analysis.
[0547] Phage libraries of affinity matured clones were sorted using
a solid/solution sorting method. Human Robo4-His was biotinylated
by mixing 500 .mu.l of 3.6 mg/ml human Robo4-His in PBS, and 10
.mu.l of 1 M potassium phosphate, pH 8 with 20 .mu.l 4 mM
Sulfo-NHS-LC-biotin (Pierce). For the first round of selection,
biotinylated Robo4-His was coated on MaxiSorp microtiter plates
(Nunc) at 10 .mu.g/ml in PBS and incubated overnight at 4.degree.
C. For the first round of selection 16 wells of target were used.
Wells were blocked for 1 hour at room temperature using SuperBlock
(Pierce). Maturation phage libries were diluted in SuperBlock
buffer and incubated 1 hour at room temperature. Phage libraries
were then added to the blocked antigen plates incubated 2 hours at
room temperature. After binding, unbound and non-specifically bound
phage were removed from the antigen plates by washing with Wash
Buffer (PBS, 05% Tween.RTM.20). Bound phage were eluted by
incubating the wells with 50 mM HCl, 0.5 M KCl for 30 min. Phage
were amplified using XL-1 Blue cells and M13/KO7 helper phage and
grown for 36 hours at 30.degree. C. in 2YT, 50 .mu.g/ml
carbanecillin, 50 ug/ml kanamycin, 10 .mu.g/ml tetracycline.
Amplified phage were then recovered using a modified PEG
precipitation protocol (Clackson & Lowman 2004). The titers of
phage eluted from a target coated well were compared to titers of
phage recovered from a non-target coated well to assess enrichment.
For selection rounds 2-5 a solution sorting protocol was
implemented. Microtiter wells were coated with 10 .mu.g/ml
neutravidin in PBS overnight at 4.degree. C. and then blocked for 1
hour using SuperBlock (Pierce). Recovered phage libraries were
suspended in SuperBlock and were mixed with 50 nM biotinylated
Robo4-His for 1 hour. Phage bound to biotinylated Robo4-His were
captured on neutravidin coated wells for 30 min and unbound phage
were washed away with Wash Buffer. Phage were eluted using 50 mM
HCl, 500 mM KCl for 30 min, neutralized, and propagated in XL1 blue
cells (Stratagene) in the presence of KO7 helper phage (New England
Biolabs). Subsequent rounds of sorting were performed similarly
with the following exceptions: in round 2 the final biotinylated
Robo4-His concentration was 50 nM, in round 3 the final
biotinylated Robo4-His concentration was 25 nM, in round 4 the
final biotinylated Robo4-His concentration was 5 nM and in round 5
the final biotinylated Robo4-His concentration was 0.5 nM with 50
nM unbiotinylated Robo4-His added to the mixture for 1 hour prior
to capture on neutravidin.
[0548] Several affinity matured clones were selected for binding to
human Robo4-His and sequenced. The variable region sequences of the
antibody clones derived by affinity maturation of clone YW71.22 are
shown in FIG. 2A (light chain variable regions) and FIG. 2B (heavy
chain variable region).
Example 6
Characterization of Selected Anti-Robo4 Antibody Clones
[0549] Phage ELISA--Phage competition binding assays were performed
to determine the approximate binding affinity (determined as phage
IC.sub.50) of phage-displayed Fabs for Robo4. The assays were
performed as follows. Briefly, purified phage supernatants from
each clone were produced using a modified PEG precipitation
protocol as described above. Purified phage supernatants were
serially diluted in Phage Blocking buffer, then incubated on plates
coated with Robo4-His (1 .mu.g/ml) for 15 minutes. The plates were
washed with Wash Buffer and were incubated for 30 minutes with
horseradish peroxidase/anti-M13 antibody conjugate (diluted 1:5000
in PBS buffer) (Amersham Pharmacia Biotech). The plates were
washed, developed with tetramethylbenzidine (TMB) substrate
(Kirkegaard and Perry Laboratories) and quenched with
0.1NH.sub.2SO.sub.4. Absorbance was measured spectrophotometrically
at 450 nm to determine the phage concentration giving about 50% of
the signal at saturation. A fixed, sub-saturating concentration of
phage was diluted in Phage Blocking buffer containing two-fold
serial dilutions of Robo4-His protein from 350 nM Robo4 to 5 nM
Robo4. The mixtures were incubated for one hour with gentle shaking
at room temperature, transferred to plates coated with Robo4-His (1
.mu.g/ml) and the plates were incubated for 20 minutes. The plates
were washed and treated as above. The binding affinities were
estimated as IC.sub.50 values (defined as the concentration of
antigen that blocked 50% of the phage binding to the immobilized
antigen). The IC.sub.50 results for the seven clones are shown in
Table 2.
TABLE-US-00012 TABLE 2 Anti-Robo4 Phage Competition Summary Clone
Phage IC.sub.50 versus human Robo4 YW71.6 15 nM YW71.22 5 nM YW71.1
15 nM YW71.89 20 nM YW79.1 15 nM YW79.11 <5 nM YW79.8 <5
nM
[0550] Fab Production and Affinity Determination--To express Fab
protein for affinity measurements, a stop codon was introduced
between the heavy chain and g3 in the phage display vector. Clones
were transformed into E. coli 34B8 cells and grown in AP5 media at
30 C (Presta et al. Cancer Res. 57: 4593-4599 (1997)). Cells were
harvested by centrifugation, suspended in 10 mM Tris, 1 mM EDTA pH
8 and broken open using a microfluidizer. Fab was purified with
Protein G affinity chromatography.
[0551] Affinity determinations were performed by surface plasmon
resonance using a BIAcore.TM. 2000 system (BIAcore, Piscataway,
N.J.). Robo4-His was immobilized (.about.1000 response units (RU))
on a CM5 chip and varied concentrations of Fab (4 to 500 nM) in
PBST were injected. After each injection the chip was regenerated
using 100 mM HCl.
[0552] The binding affinity of three phage-derived anti-Robo4
antibodies for soluble Robo4 extracellular domain (ECD) was
determined by surface plasmon resonance measurement using a
BIACORE.RTM. 3000 system (Biacore, Inc., Piscataway, N.J.). The
antibodies tested included YW71.6, YW71.22, and YW79.8. Briefly,
carboxymethylated dextran biosensor chips (CM5, Biacore Inc.) were
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. These activated chips were coated with
anti-Robo Fab by dilution to 5 ug/ml with 10 mM sodium acetate, pH
4.8, before injection at a flow rate of 5 .mu.l/minute to achieve
approximately 500 response units (RU) of coupled antibody. Next, 1M
ethanolamine was injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of human or mouse-ECD-His
tagged soluble antigen (approximately 500 nM to approximately 7.8
nM) were injected in PBS with 0.05% Tween.RTM. 20 at 25.degree. C.
at a flow rate of 25 .mu.l/min. Binding response was corrected by
subtracting the RU from a blank flow cell. Association rates
(k.sub.on) and dissociation rates (k.sub.off) were calculated using
a simple one-to-one Langmuir binding model (BIAevaluation Software
version 3.2). The equilibrium dissociation constant (K.sub.D) was
calculated as the ratio k.sub.dissociation/k.sub.association. The
results of this experiment are shown in Table 3 below. Antibody
YW71.22 cross-reacts with human and mouse Robo4.
TABLE-US-00013 TABLE 3 Anti-Robo4 BIAcore .TM. Binding Analysis
Summary Clone ka kd K.sub.D Human Robo4 YW71.22 2.90E+05 3.70E-03
1.30E-08 Murine Robo4 YW71.22 4.50E+05 2.80E-03 6.20E-09
[0553] An affinity matured anti-Robo4 antibody clone derived from
YW71.22 were tested for binding to human Robo4 expressed
endogenously in HUVEC cells and to murine Robo4 expressed
endogenously in MS1 cells. A FACS analysis revealed that clones
YW71.22.S1.16 and YW71.22.S1.21 bound to both human and murine
Robo4 and that binding was comparable to the YW71.22 antibody (FIG.
18).
Example 7
Binding Analysis of Affinity Matured Robo4 Antibodies
[0554] Affinity matured YW71.22.S1.16 was used to generate a
full-length IgG1 and a Fab fragment both with a Cys substitution at
position 118 of the heavy chain Fc region according to EU
numbering. The Cys is used for site specific conjugation of the
antibody to a label or drug of interest.
[0555] Binding affinities to human and mouse Robo4 were determined
by in vitro binding measurements and by radiolabeled cell binding
for full-length YW71.22.S1.16 (Mab), full-length YW71.22.S1.16 with
engineered Cys (thioMab), and a Fab fragment of YW71.22.S1.16 with
engineered Cys (ThioFab).
[0556] The in vitro binding experiments were performed by SPR
measurements on a ProteOn XPR36 instrument (Bio-Rad Laboratories,
Inc.) at 25.degree. C. Affinity matured Robo4 antibodies (Mab,
ThioMab and ThioFab) were immobilized at surface densities of
500-1000 RUs on an activated ProteOn GLC sensor chip (Bio-Rad
Laboratories, Inc.) using standard amine coupling procedures as
described by the manufacturer. Briefly, antibodies were injected at
a concentration of 10 .mu.g/ml in 20 mM sodium acetate, pH 4.5 and
at a flow rate of 30 .mu.l/min for 5 min. Unreacted groups were
blocked by injecting 1 M ethanolamine. To perform binding assays,
samples were injected at a flow rate of 30 .mu.l/min. For kinetics
measurements serial dilutions of either purified human or mouse
Robo4-His (approximately 25 nM to 0.8 nM) were injected in PBS with
0.01% Tween-20 and sensorgrams for association and dissociation
phases were recorded. Blank surfaces were used for background
corrections. There was no need to regenerate surfaces since the
ProteOn protein interaction array system allows to run up to six
binding experiments on an identical surface in parallel.
Association rates (k.sub.a) and dissociation rates (k.sub.d) were
calculated using a simple one-to-one Longmuir binding model
(ProteOn Manager Software V2.0, Bio-Rad Laboratories, Inc.) and the
equilibrium dissociation constant (K.sub.D) was calculated as the
ration k.sub.d/k.sub.a. The results are summarized in Table 4
below.
TABLE-US-00014 TABLE 4 Anti-Robo4 Antibody Affinities (K.sub.D)
Summary Antibody format In vitro (SPR) Radiolabeled Cell Binding
(YW71.22.S1.16) hRobo4 mRobo4 HUVEC MS1 thioMAb 1.2E-09 1.1E-09
1.2E-09 1.0E-09 MAb 1.1E-09 0.9E-09 ND ND thioFab 8.0E-09 1.5E-09
ND 3.2E-09
[0557] HUVECs and MS1 cells, which endogenously express Robo4
protein were used in radioligand cell binding experiments.
[0558] For the cell binding experiments anti-Robo4 thioMab and
thioFab antibodies were iodinated using the Iodogen method. Each
radiolabeled antibody was purified from free .sup.125I--Na by gel
filtration using a NAP-5 column; the purified thioMab antibody had
a specific activity of 6.96 .mu.Ci/.mu.g and the purified thioFab
antibody had a specific activity of 16.05 .mu.Ci/.mu.g. 50 .mu.L
competition mixtures containing a fixed concentration of iodinated
antibody and decreasing concentrations of the unlabeled antibody
were placed into 96-well plates. MS1 and HUVEC cells were cultured
in growth media at 37.degree. C. in 5% CO.sub.2 and then were
detached from tissue culture plates using a non-enzymatic cell
dissociating solution (Sigma #C5914) for subsequent binding
studies. Cells were washed with binding buffer (50:50 DMEM/F12
media containing 2% FBS, 2 mM sodium azide and 50 mM HEPES, pH 7.2)
and were placed into the 96-well plates containing the competition
mixtures at approximately 200,000 cells in 0.2 mL of binding
buffer. The final concentration of the iodinated Mab antibody in
each incubation with cells was .about.150 pM (.about.70,000 cpms
per 0.25 ml) and the final concentration of the unlabeled antibody
in the incubations with cells started at 400 nM and was serially
diluted by 2 fold for 10 concentrations. The final concentration of
the iodinated Fab antibody in each incubation with cells was
.about.400 pM (.about.150,000 cpms per 0.25 ml) and the final
concentration of the unlabeled antibody in the incubations with
cells started at 1.0 uM and was serially diluted by 2 fold for 10
concentrations. Competition reactions were assayed in triplicate.
The competition reactions were incubated for 2 hours at room
temperature. Then they were transferred to a Millipore Multiscreen
filter plate and washed three times with binding buffer to separate
the free from bound iodinated antibody. The filters were counted on
a Wallac Wizard 1470 gamma counter (PerkinElmer Life and Analytical
Sciences Inc. Wellesley, Mass.). The binding data was evaluated
using NewLigand software (Genentech), which uses the fitting
algorithm of Munson and Robard, Anal. Biochem. 107: 220-39 (1980),
to determine the binding affinity of the antibody and the
concentration of binding sites per well. The number of binding
sites per cell was determined by dividing the total binding sites
by the number of cells per well.
Example 8
Anti-Robo4 Antibody Inhibits HUVEC Tube Elongation
[0559] Anti-Robo4 antibody (YW71.22) significantly inhibits HUVEC
tube elongation in a bead outgrowth assay. Both total number and
length of the tubes are reduced as compared to the control antibody
E25 (FIG. 20). For example the number of tubes with a length of 300
.mu.m or more is reduced by 60%.
[0560] For the bead outgrowth assay, dextran-coated Cytodex 3
microcarrier beads (Amersham) were incubated with HUVECs (400 cells
per bead) in EGM-2, overnight at 37.degree. C. and 5% CO.sub.2. To
induce clotting, 0.5 ml cell-coated beads in PBS with 2.5 .mu.g/ml
fibrinogen (200 beads/ml) was added into one well of a 24-well
tissue culture plate containing 0.625 units thrombin and incubated
for 5 min at room temperature and then for 20 min at 37.degree. C.
The clot was equilibrated in EGM-2 for 30 min at 37.degree. C. The
medium was then replaced with EGM-2 containing skin fibroblast
cells (Detroit 551, 20,000 cells/ml). Antibodies (50 .mu.g/ml) were
added to each well, and the assay was monitored for 8 days with
change in medium every alternate day. Images of the beads were
captured by an inverted microscope, and concentric circles spaced
at 100, 200, and 300 .mu.m were drawn around the bead in each
image. The number of vessels crossing each circle was counted 10-12
beads were used per condition for quantification (Nakatsu et al.,
Microvascular Research 66: 102-112 (2003)).
Example 9
Anti-Robo4 Antibody and Anti-Robo4 Antibody Drug Conjugates as
Therapeutics
[0561] Anti-Robo4 ADCs were produced by conjugating anti-Robo4
antibodies to the drug-linker moieties MCC-DM1, SPDB-DM4, and
mPEO-DM1. Prior to conjugation, the antibodies were partially
reduced with TCEP using standard methods in accordance with the
methodology described in WO 2004/010957. The partially reduced
antibodies were conjugated to the above drug-linker moieties using
standard methods in accordance with the methodology described in
Doronina et al., Nat. Biotechnol. 21:778-784 (2003) and U.S. Patent
Application No. 2005/0238649. Briefly, the partially reduced
antibodies were combined with the drug linker moieties to allow
conjugation of the moieties to cysteine residues (including without
limitation, cysteine engineered residues of thioMAbs as disclosed
herein). The conjugation reactions were quenched, and the ADCs were
purified. The drug load (average number of drug moieties per
antibody) for each ADC was determined by HPLC. Additional
anti-Robo4 ADCs are made using e.g., drug linker moieties including
spp-DM1, smcc-DM1, MC-vc-PAB-MMAE; MC-vc-PAB-MMAF; MC-MMAE and
MC-MMAF (see, e.g., WO 2004/010957, and WO2006/034488 (each of
which is herein incorporated by reference in its entirety).
Example 10
In Vivo Tumor Volume Reduction Assay
[0562] Naked anti-Robo4 antibody: Tumor growth was not inhibited by
naked anti-Robo4 antibody YW71.22 (antibody not conjugated to a
cytotoxic agent or other agent) in mouse xenograft models of human
non-small cell lung carcinoma SK-MES-1. For this study, each HRLN
female nude mouse received a 1 mm.sup.3 tumor fragment s.c. implant
in the flank. Tumor growth was monitored twice weekly by caliper
measurements. When tumors reached an average size of 80-120
mm.sup.3, mice were sorted to give nearly identical group mean
tumor sizes, and treatment was started (Day 1). Animals were dosed
twice weekly i.p. with 10 mg/kg anti-Robo4 and/or 5 mg/kg anti-VEGF
or control antibody for 5 weeks. All treatments were body
weight-adjusted at 0.2 mg/20 g. The results are shown in FIG. 17.
In addition, no reduction of tumor growth in Fo5 mouse model with
MDA-MB-231 breast cancer xenograft was observed.
[0563] An anti-Robo4 antibody comprising an Fc region having
enhanced ADCC activity may be used to inhibit tumor growth. The
ADCC-enhanced anti-Robo4 antibody may be a naked antibody or an
antibody drug conjugate as disclosed herein. ADCC enhancement is
achieved, for example, by enhancing IgG1-FcgammaRIII interactions,
which are dependent on the carbohydrate moieties linked to the
antibody Fc region. Non-limiting exemplary methods for ADCC
enhancement include reducing antibody fucosylation by expressing
anti-Robo4 in a host cell overexpressing GnTIII, which encodes
N-acetylglucosaminyltransferase (Narishimhan, J. Biol. Chem.
257:10235-10242 (1982) and Umana et al., Nature Biotech. 17:176-180
(1999)); a host cell underexpressing or lacking expression of FUT8,
which encodes alpha-1,6-fucosyltransferase (Shinkawa et al., J.
Biol. Chem. 278:3466-3473 (2003)); or a host cell lacking or
underexpressing UDP-N-acetylglucosamine 2-epinerase (such as Lec3
CHO cells) (Hong and Stanley, J. Biol. Chem. 278:53045-53054
(2003)). In addition, ADCC is enhanced by amino acid changes in the
Fc region (see for example WO2000042072 and WO2004029207). Thus,
anti-Robo4 antibodies of the invention are useful as cytotoxic
agents include anti-Robo4 antibodies exhibiting enhanced ADCC
function relative to an anti-Robo4 antibody exhibiting wild type Fc
ADCC activity.
[0564] Anti-Robo4 antibody drug conjugate (Anti-Robo4 ADC): To test
the efficacy of toxin-conjugated anti-Robo4 ADCs for the ability to
reduce tumor volume in vivo, the following protocol is
employed.
[0565] SCID mice are each inoculated subcutaneously in the flank
with cells of a human cancer cell line. The human cancer cell lines
include, as non-limiting examples, SK-MES-1 human non-small lung
cancer cells and MDA-MB-231 breast cancer cells. When the tumors
reach a mean tumor volume of between approximately 80-200 mm.sup.3,
the mice are divided into groups, and treated by intravenous
injection with toxin-conjugated antibody or unconjugated
antibody.
[0566] Use of a Anti-Robo4 Maytansine Drug Conjugates to Reduce
Solid Tumor Volume
[0567] SCID mice are injected with human tumor cells subcutaneously
in a volume of 0.2 ml/20 g mouse body weight in the flank. Cells
are suspended in HBSS. When the mean tumor size reaches
approximately 80-200 mm.sup.3, mice are randomly grouped into
groups and given a single or multiple I.V. treatments (via the tail
vein) of either a anti-Robo4 ADC conjugated to maytansine or a
control antibody.
[0568] Mean tumor volume is monitored in each treatment group for
approximately 30 days post-antibody injection. Tumor may be
detected by, for example, fluorescence detection of remaining
luciferase where the tumor cells are engineered to express
luciferase. Efficacy of the toxin-conjugated anti-CD22 antibodies
is determined by comparison to control and unconjugated
antibodies.
[0569] The same experiment is performed using anti-Robo4 ADC
wherein the drug conjugated to the antibody is an auristatin.
[0570] The same experiment is repeated using an anti-Robo4 ADC
comprising an Fc region having enhanced ADCC activity.
Example 11
Anti-Robo4 Antibodies Conjugated to Detectable Markers
[0571] sDOTA-labeled anti-Robo4 antibodies are prepared as follows.
The DOTA-NHS-ester was dissolved in dimethylacetamide (DMA, Fluka
Chemika, Switzerland) and prepared to concentrations of 60-100
mg/mL. Typical procedures involved buffer exchanging the MAb into
PBS with 2 mM EDTA at pH 7.2. Reactions were performed at a ratio
of 1 molecule MAb to 4 DOTA molecules (1:4) and carried out at
25.degree. C. while gently stirring on a Thermomixer plate
(Eppendorf, Westbury, N.Y.). The DOTA-labeled anti-Robo4 antibody
is useful as a detectable marker in vitro or in vivo when
associated with, as non-limiting examples, yttrium .sup.90Y,
.sup.111In, .sup.177Lu and the like. The mAb conjugates are labeled
with, for example, .sup.111In by incubating the conjugate with
.sup.111InCl.sub.3 in 0.25 M ammonium acetate for 45 min at
43.degree. C. EDTA is added to a final concentration of 1 mM, and
the mixture is incubated at 37.degree. C. for 15 minutes. Labeling
with .sup.90Y, for example, is performed using a one-hour
incubation at 43.degree. C., after which DTPA is added to a final
concentration of 1 mM, and the mixture is incubated for an
additional 15 min at 37.degree. C. The radiometal-labeled mAb
conjugates are purified by size exclusion HPLC, using a TosoHaas
TSKgel G2000 SW column and a mobile phase of normal saline.
Example 12
Additional Characterization of Robo4 and Anti-Robo4 Antibodies
[0572] Robo4 does not block endothelial cell (EC) migration.
Robo4-Fc and Robo4-His were contacted with vascular ECs using a
HUVEC migration assay and in the presence or absence of VEGF. FIG.
8 shows the results of the assay. Robo4-Fc or Robo4-His alone did
not induce EC migration and VEGF-induced EC migration was not
enhanced by Robo4.
[0573] Robo4 does not bind to Slit2. Robo4-Fc contacted with Slit2
did not show binding by Biocore.TM. analysis, whereas Robo1 and
Slit 2 did show binding in the same assay (FIG. 9). Slit2-His
(histidine-tagged full-length Slit2) was immobilized on a CM5
Biacore.TM. chip at high density. A 5 .mu.l aliquot of each analyte
(Robo4-Fc or Robo1-Fc fusion proteins) was injected and allowed to
interact with the immobilized ligand in buffer (HEPES/EDTA/NaCl,
pH7.5).
[0574] Robo4 interacts in vitro with UNC5B, a cell surface receptor
involved in vascular guidance. It has been shown that netrin
binding to members of the uncoordinated-5 (UNC5) family of
receptors (UNC5A, B, C, and D) results in neural axon repression
(Klagsbrun, M. and Eichmann, A., Cytokine & Growth Factor
Reviews 16(4-5):535-548 (2005)). Netrin-UNC5B interaction has been
implicated in angiogenesis (Klagsbrun, M. and Eichmann, A., supra
(2005) and Lu, X. et al., Nature 432:179-186 (2004)). The
interaction of Robo4 with UNC5B in vitro was determined by SPR
binding measurements in which UNC5B-Fc fusion protein
(extracellular domain of UNC5B fused to an Fc region) was
immobilized on a chip and soluble Robo4-Fc or Robo4-His was allowed
to interact with it at different concentrations. SPR measurements
were performed as described in Example 7 and the interaction
results are shown in FIG. 10.
[0575] Robo4 is expressed in fetal mouse endothelium as shown by an
ISH assay using Robo4 specific RNA probes (FIG. 11). Robo4 is also
expressed in mouse tumor models of colon tumor (human HM-7 colon
tumor xenograft, FIGS. 12A and 12B) and in mammary tumor (human
MDA-MB-175 breast cancer tumor xenograft, FIGS. 12C and 12D). Robo4
shows increased expression in human tumor tissue relative to normal
human tissue as evidenced in FIGS. 13A and 13C (increased
expression in malignant melanoma) relative to normal tissue (FIGS.
13B and 13D). Robo4 expression is also seen in human small cell
lung cancer (FIGS. 14A-D), human colon cancer (FIGS. 14G-H), human
prostate tumor, and mouse angiosarcoma. Robo4 is expressed in tumor
endothelial cells in colon, non-small cell lung cancer, renal cell
carcinoma (RCC), transitional cell carcinoma (TCC, a common form of
bladder cancer), breast cancer, glioma, and sarcoma.
[0576] Anti-Robo4 antibody YW71.22 was internalized by
Robo4-expressing mouse microvascular endothelial MS1 cells over a
period of 2 hours. The YW71.22 antibody was allowed to bind Robo4
on mouse microvasculature endothelial MS1 cells at 0.degree. C. for
30 minutes, incubated at 37.degree. C. for 0-2 hours. At intervals,
cells were fixed and permeabilized or not permeabilized. Anti-Robo4
antibodies on the cell surface or internalized by the cell were
interacted with a fluorescently labeled (Alexa-labeled) anti-human
IgG secondary antibody. The results are shown in FIGS. 15A and 15B.
The YW71.22.S1.16 anti-Robo4 antibody was internalized by the same
protocol except that anti-Robo4 was incubated on MS1 cells at
37.degree. C. was for 0-4 hours. Cells were permeabilized and
stained as above. The results are shown in FIG. 15C.
[0577] Endothelial cell migration was not blocked by anti-Robo4
antibody in an in vitro assay using human umbilical vascular
endothelial cells (HUVEC) cellular migration assay. HUVECs
endogenously express human Robo4. The assay was performed as
follows. HUVECs were pre-incubated for 2 hours with VEGF. Cellular
migration was allowed to continue for 16 hours in the presence of
anti-Robo4 antibody YW71.22, anti-VEGF antibody, or Robo4-Fc or
Robo4-His. Neither anti-Robo4 nor the soluble Robo4 extracellular
domain fusion proteins (Robo4-Fc and Robo4-His) blocked EC
migration under these conditions (FIG. 16).
[0578] Anti-Robo4 antibody associates with vasculature in vivo.
Fifty micrograms of anti-Robo4 antibody 71.22 (FIG. 19A) or control
antibody (anti-HER2, FIG. 19B) were injected via tail vein and
allowed to circulate for 10 minutes. One hundred micrograms of
FITC-Lycopersicum esculentum was injected and allowed to circulate
for 5 minutes. Mice were perfused with 1% PFA in PBS, tissues were
collected into 30% sucrose and frozen in OCT. Antibodies were
detected with Cy3 goat anti-human IgG. FIG. 19A shows that the
location of FITC-Lycopersicum esculentum staining in vasculature
overlaps with the location of anti-Robo4 Cy3 staining, unlike the
control antibody which migrated into surrounding tissue.
[0579] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patents, patent applications, scientific references, and
Genbank Accession Nos. cited herein are expressly incorporated by
reference in their entirety for all purposes as if each patent,
patent application, scientific reference, and Genbank Accession No.
were specifically and individually incorporated by reference.
Sequence CWU 1
1
187111PRTArtificial sequenceantibody hypervariable region 1Arg Ala
Ser Gln Asp Val Ser Thr Ala Val Ala 5 1027PRTArtificial
sequenceantibody hypervariable region 2Ser Ala Ser Phe Leu Tyr Ser
539PRTArtificial sequenceantibody hypervariable region 3Gln Gln Ser
Tyr Thr Thr Pro Pro Thr 5410PRTArtificial sequenceantibody
hypervariable region 4Gly Phe Thr Ile Ser Gly Ser Trp Ile His 5
10518PRTArtificial sequenceantibody hypervariable region 5Ala Val
Ile Thr Pro Ala Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser1 5 10 15Val Lys
Gly618PRTArtificial sequenceantibody hypervariable region 6Ala Arg
Ser Asn Arg Tyr Ser Gly Gln Phe Val Pro Ala Tyr Ala1 5 10 15Met Asp
Tyr711PRTArtificial sequenceantibody hypervariable region 7Arg Ala
Ser Gln Ser Ile Ser Ser Tyr Leu Ala 5 1087PRTArtificial
sequenceantibody hypervariable region 8Gly Ala Ser Ser Arg Ala Ser
5923PRTArtificial sequenceantibody light chain framework region
9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys 201015PRTArtificial
sequenceantibody light chain framework region 10Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr1 5 10 151132PRTArtificial
sequenceantibody light chain framework region 11Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe1 5 10 15Thr Leu Thr Ile Ser
Ser Leu Gln Pro Glu Asp Phe Ala Thr Thr 20 25 30Tyr
Cys1210PRTArtificial sequenceantibody light chain framework region
12Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 5 101325PRTArtificial
sequenceantibody light chain framework region 13Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser 20 251413PRTArtificial sequenceantibody light
chain framework region 14Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 5 101530PRTArtificial sequenceantibody light chain
framework region 15Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr Leu1 5 10 15Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 20 25 301611PRTArtificial sequenceantibody heavy chain
framework region 16Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 5
101710PRTArtificial sequenceantibody hypervariable region 17Gly Phe
Thr Ile Asn Gly Tyr Tyr Ile His 5 101818PRTArtificial
sequenceantibody hypervariable region 18Gly Phe Ile Tyr Pro Ala Gly
Gly Asp Thr Asp Tyr Ala Asp Ser1 5 10 15Val Lys
Gly1917PRTArtificial sequenceantibody hypervariable region 19Ala
Arg Leu Ile Gly Asn Lys Phe Gly Trp Ser Ser Tyr Gly Met1 5 10 15Asp
Tyr209PRTArtificial sequenceantibody hypervariable region 20Gln Gln
Ser Arg Ser Asp His Pro Thr 5219PRTArtificial sequenceantibody
hypervariable region 21Gln Gln Ser Trp Ser Tyr Pro Leu Thr
5229PRTArtificial sequenceantibody hypervariable region 22Gln Gln
Ser Tyr Asn Thr Pro Phe Thr 5239PRTArtificial sequenceantibody
hypervariable region 23Gln Gln Tyr Tyr Ser Ser Pro Leu Thr
52410PRTArtificial sequenceantibody hypervariable region 24Gly Phe
Thr Ile Thr Asn Tyr Trp Ile His 5 102510PRTArtificial
sequenceantibody hypervariable region 25Gly Phe Thr Ile Asn Asn Asn
Tyr Ile His 5 102610PRTArtificial sequenceantibody hypervariable
region 26Gly Tyr Thr Phe Ser Ser Tyr Trp Ile Ser 5
102710PRTArtificial sequenceantibody hypervariable region 27Gly Phe
Thr Phe Ser Ser Arg Tyr Met Ser 5 102810PRTArtificial
sequenceantibody hypervariable region 28Gly Phe Thr Phe Ser Ser Tyr
Ala Met Ser 5 102918PRTArtificial sequenceantibody hypervariable
region 29Gly Gly Ile Tyr Pro Ala Asp Gly Tyr Thr Tyr Tyr Ala Asp
Ser1 5 10 15Val Lys Gly3018PRTArtificial sequenceantibody
hypervariable region 30Ala Ile Ile Ser Pro Thr Gly Gly Tyr Thr Asp
Tyr Ala Asp Ser1 5 10 15Val Lys Gly3118PRTArtificial
sequenceantibody hypervariable region 31Ser Thr Ile Tyr Gly Tyr Asp
Gly Ser Thr Asn Tyr Ala Asp Ser1 5 10 15Val Lys
Gly3218PRTArtificial sequenceantibody hypervariable region 32Ser
Gly Ile Tyr Pro Met Gly Gly Thr Thr Tyr Tyr Ala Asp Ser1 5 10 15Val
Lys Gly3318PRTArtificial sequenceantibody hypervariable region
33Ser Gly Ile Ser Pro Tyr Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser1 5 10
15Val Lys Gly3410PRTArtificial sequenceantibody hypervariable
region 34Ala Ser Gly Gly Tyr Ser Ser Leu Ala Tyr 5
103518PRTArtificial sequenceantibody hypervariable region 35Ala Arg
Asp Val Asn Val Tyr Ser Ala Arg Trp Trp Asp Tyr Val1 5 10 15Met Asp
Tyr3616PRTArtificial sequenceantibody hypervariable region 36Ala
Arg Met Ser Tyr Asn Trp Ser Ser Pro Gly His Gly Met Asp1 5 10
15Val3713PRTArtificial sequenceantibody hypervariable region 37Ala
Arg Gly Asn Tyr Tyr Ser Gly Ser Glu Phe Asp Tyr 5
103816PRTArtificial sequenceantibody hypervariable region 38Ala Arg
Trp Gly Tyr Tyr Met Pro Tyr Gly His Pro Val Met Asp1 5 10
15Val3911PRTArtificial sequenceantibody hypervariable region 39Arg
Ala Ser Gln Asp Gly Ala Arg Ser Leu Ala 5 104011PRTArtificial
sequenceantibody hypervariable region 40Arg Ala Ser Gln Asp Gly Ala
Ile Tyr Leu Ala 5 10417PRTArtificial sequenceantibody hypervariable
region 41Ser Ala Ser Phe Leu Ala Ser 5426PRTArtificial
sequenceantibody hypervariable region 42Ser Ala Ser Leu Glu Ser
5436PRTArtificial sequenceantibody hypervariable region 43Ser Ala
Thr Leu Ala Ser 5447PRTArtificial sequenceantibody hypervariable
region 44Ser Ala Ser Phe Leu Tyr Tyr 5457PRTArtificial
sequenceantibody hypervariable region 45Ser Ala Ser Asn Leu Ala Ser
5467PRTArtificial sequenceantibody hypervariable region 46Ser Ala
Ser Thr Leu Ala Ser 5479PRTArtificial sequenceantibody
hypervariable region 47Gln Gln Ser Phe Ala Thr Pro Ala Thr
5489PRTArtificial sequenceantibody hypervariable region 48Gln Gln
Ser Arg Ala Ala Leu Pro Thr 5499PRTArtificial sequenceantibody
hypervariable region 49Gln Gln Ser Arg Ala Asn Thr Pro Thr
5509PRTArtificial sequenceantibody hypervariable region 50Gln Gln
Ser Arg Thr Thr Pro Pro Thr 5519PRTArtificial sequenceantibody
hypervariable region 51Gln Gln Pro Phe Asp Leu Pro Met Thr
5529PRTArtificial sequenceantibody hypervariable region 52Gln Gln
Pro Asn Ser Thr Pro Phe Thr 5539PRTArtificial sequenceantibody
hypervariable region 53Gln Gln Ser Arg Phe Asp His Pro Thr
5549PRTArtificial sequenceantibody hypervariable region 54Gln Gln
Ser Tyr His Thr His Ser Thr 5559PRTArtificial sequenceantibody
hypervariable region 55Gln Gln Ser Arg Asp Ile Pro Pro Thr
5569PRTArtificial sequenceantibody hypervariable region 56Gln Gln
Ser Arg Asn Met Pro Ala Thr 5579PRTArtificial sequenceantibody
hypervariable region 57Gln Gln Thr Arg Val Met Pro Ala Thr
5589PRTArtificial sequenceantibody hypervariable region 58Gln Gln
Thr Tyr Thr Ile Pro Pro Thr 5599PRTArtificial sequenceantibody
hypervariable region 59Gln Gln Ser Tyr Ala Tyr Pro Phe Thr
5609PRTArtificial sequenceantibody hypervariable region 60Gln Gln
Ser Tyr Asp Leu Pro Phe Thr 5619PRTArtificial sequenceantibody
hypervariable region 61Gln Gln Ser Tyr Gly Gly Pro Phe Thr
5629PRTArtificial sequenceantibody hypervariable region 62Gln Gln
Ser Tyr Asn Tyr Pro Phe Thr 5639PRTArtificial sequenceantibody
hypervariable region 63Gln Gln Phe Tyr Ser Asp Pro Phe Thr
5649PRTArtificial sequenceantibody hypervariable region 64Gln Gln
Gly Tyr Asn Tyr Pro Phe Thr 56510PRTArtificial sequenceantibody
hypervariable region 65Gly Phe Ser Ile Tyr Ser Tyr Tyr Phe Glu 5
106610PRTArtificial sequenceantibody hypervariable region 66Gly Phe
Thr Leu Asp Gly Tyr Tyr Leu Gln 5 106710PRTArtificial
sequenceantibody hypervariable region 67Gly Phe Ser Ile Asn Gly Tyr
Tyr Asn Gln 5 106818PRTArtificial sequenceantibody hypervariable
region 68Gly Arg Ile Tyr Ser Ala Gly Gly His Thr Ala Tyr Ala Asp
Ser1 5 10 15Val Lys Gly6918PRTArtificial sequenceantibody
hypervariable region 69Gly Phe Ile Tyr Pro Ala Gly Gly Lys Thr Glu
Tyr Ala Asp Ser1 5 10 15Val Lys Gly7018PRTArtificial
sequenceantibody hypervariable region 70Gly Phe Ile Tyr Pro Ala Gly
Gly Ala Thr Ile Tyr Ala Asp Ser1 5 10 15Val Lys
Gly7118PRTArtificial sequenceantibody hypervariable region 71Gly
Phe Ile Tyr Pro Ala Leu Ser Val Ile Glu Tyr Ala Asp Ser1 5 10 15Val
Lys Gly72108PRTArtificial sequenceantibody light chain 72Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Ser Tyr
Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg73108PRTArtificial sequenceantibody light chain 73Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Ser Tyr
Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg74108PRTArtificial sequenceantibody light chain 74Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Ser Tyr
Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg75108PRTArtificial sequenceantibody light chain 75Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Ser Tyr
Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg76108PRTArtificial sequenceantibody light chain 76Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30Ser Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Gly Ala Ser Ser Arg Ala Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Ser Trp
Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg77108PRTArtificial sequenceantibody light chain 77Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30Ser Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Gly Ala Ser Ser Arg Ala Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Ser Tyr
Asn Thr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg78108PRTArtificial sequenceantibody light chain 78Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30Ser Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Gly Ala Ser Ser Arg Ala Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Tyr
Ser Ser Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg79108PRTArtificial sequenceantibody light chain 79Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Ser Phe
Ala Thr Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg80108PRTArtificial sequenceantibody light chain 80Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80
85 90Ser Arg Ala Ala Leu Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg81108PRTArtificial sequenceantibody light chain
81Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Ser Arg Ser Asp His Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg82108PRTArtificial sequenceantibody light chain
82Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Ser Arg Ala Asn Thr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg83108PRTArtificial sequenceantibody light chain
83Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Ser Arg Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg84108PRTArtificial sequenceantibody light chain
84Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Pro Phe Asp Leu Pro Met Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg85108PRTArtificial sequenceantibody light chain
85Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Pro Asn Ser Thr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg86108PRTArtificial sequenceantibody light chain
86Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Ser Arg Phe Asp His Pro Thr Phe Gly Gln Gly Thr Lys Met Glu 95
100 105Ile Lys Arg87108PRTArtificial sequenceantibody light chain
87Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Tyr Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Ser Tyr His Thr His Ser Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg88108PRTArtificial sequenceantibody light chain
88Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Ser Arg Asp Ile Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg89108PRTArtificial sequenceantibody light chain
89Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Ser Arg Asn Met Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg90108PRTArtificial sequenceantibody light chain
90Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Thr Arg Val Met Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg91108PRTArtificial sequenceantibody light chain
91Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Tyr Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Thr Tyr Thr Ile Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg92108PRTArtificial sequenceantibody light chain
92Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Phe Leu Ala Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Ser Tyr Ala Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg93107PRTArtificial sequenceantibody light chain
93Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25
30Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Ser Ala Ser Leu Glu Ser Gly Val Pro Ser Arg 50 55
60Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70
75Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser 80 85
90Tyr Asp Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 95
100 105Lys Arg94107PRTArtificial sequenceantibody light chain 94Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr
Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu
Leu Ile Tyr Ser Ala Thr Leu Ala Ser Gly Val Pro Ser Arg 50 55 60Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser 80 85 90Tyr
Gly Gly Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 95 100
105Lys Arg95108PRTArtificial sequenceantibody light chain 95Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ser Ala Ser Phe Leu Ala Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Ser Tyr
Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg96108PRTArtificial sequenceantibody light chain 96Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Gly Ala 20 25 30Arg Ser
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ser Ala Ser Asn Leu Ala Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Phe Tyr
Ser Asp Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg97108PRTArtificial sequenceantibody light chain 97Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Gly Ala 20 25 30Ile Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Gly Tyr
Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg98107PRTArtificial sequenceantibody light chain 98Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn 20 25 30Thr Ala
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90His Tyr
Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys9932PRTArtificial sequenceantibody light chain framework region
99Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe1 5 10
15Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 20 25
30Tyr Cys 10010PRTArtificial sequenceantibody light chain framework
region 100Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 5
1010114PRTArtificial sequenceantibody light chain framework region
101Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 5
1010223PRTArtificial sequenceantibody light chain framework region
102Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro1 5
10 15Gly Glu Pro Ala Ser Ile Ser Cys 2010315PRTArtificial
sequenceantibody light chain framework region 103Trp Tyr Leu Gln
Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr1 5 10
1510432PRTArtificial sequenceantibody light chain framework region
104Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe1 5
10 15Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 20
25 30Tyr Cys10523PRTArtificial sequenceantibody light chain
framework region 105Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser
Leu Ser Pro1 5 10 15Gly Glu Arg Ala Thr Leu Ser Cys
2010615PRTArtificial sequenceantibody light chain framework region
106Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr1 5
10 1510732PRTArtificial sequenceantibody light chain framework
region 107Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe1 5 10 15Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val
Tyr 20 25 30Tyr Cys10823PRTArtificial sequenceantibody light chain
framework region 108Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala
Val
Ser Leu1 5 10 15Gly Glu Arg Ala Thr Ile Asn Cys
2010915PRTArtificial sequenceantibody light chain framework region
109Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr1 5
10 1511032PRTArtificial sequenceantibody light chain framework
region 110Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe1 5 10 15Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr 20 25 30Tyr Cys11130PRTArtificial sequenceantibody heavy chain
framework region 111Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly1 5 10 15Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr 20 25 3011214PRTArtificial sequenceantibody heavy chain
framework region 112Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met Gly 5 1011332PRTArtificial sequenceantibody heavy chain
framework region 113Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr
Ala Tyr Met1 5 10 15Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 20 25 30Ala Arg11425PRTArtificial sequenceantibody
heavy chain framework region 114Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly1 5 10 15Ala Ser Val Lys Val Ser Cys Lys Ala
Ser 20 2511513PRTArtificial sequenceantibody heavy chain framework
region 115Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 5
1011631PRTArtificial sequenceantibody heavy chain framework region
116Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met1 5
10 15Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 20
25 30Ala11730PRTArtificial sequenceantibody heavy chain framework
region 117Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
Met1 5 10 15Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 20 25 3011830PRTArtificial sequenceantibody heavy chain
framework region 118Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser1 5 10 15Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly
Ser Val Ser 20 25 3011914PRTArtificial sequenceantibody heavy chain
framework region 119Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile Gly 5 1012032PRTArtificial sequenceantibody heavy chain
framework region 120Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu1 5 10 15Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys 20 25 30Ala Arg12125PRTArtificial sequenceantibody
heavy chain framework region 121Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser1 5 10 15Gln Thr Leu Ser Leu Thr Cys Thr Val
Ser 20 2512213PRTArtificial sequenceantibody heavy chain framework
region 122Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 5
1012331PRTArtificial sequenceantibody heavy chain framework region
123Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu1 5
10 15Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 20
25 30Ala12430PRTArtificial sequenceantibody heavy chain framework
region 124Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
Leu1 5 10 15Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys 20 25 3012530PRTArtificial sequenceantibody heavy chain
framework region 125Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser 20 25 3012614PRTArtificial sequenceantibody heavy chain
framework region 126Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val Ser 5 1012732PRTArtificial sequenceantibody heavy chain
framework region 127Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr Leu1 5 10 15Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 20 25 30Ala Arg12825PRTArtificial sequenceantibody
heavy chain framework region 128Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser 20 2512913PRTArtificial sequenceantibody heavy chain framework
region 129Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 5
1013031PRTArtificial sequenceantibody heavy chain framework region
130Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu1 5
10 15Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20
25 30Ala13130PRTArtificial sequenceantibody heavy chain framework
region 131Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu1 5 10 15Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 20 25 3013230PRTArtificial sequenceantibody heavy chain
framework region 132Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Asn Ile Lys 20 25 3013332PRTArtificial sequenceantibody heavy chain
framework region 133Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr Leu1 5 10 15Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 20 25 30Ser Arg13431PRTArtificial sequenceantibody
heavy chain framework region 134Arg Phe Thr Ile Ser Ala Asp Thr Ser
Lys Asn Thr Ala Tyr Leu1 5 10 15Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 20 25 30Ser13532PRTArtificial
sequenceantibody heavy chain framework region 135Arg Phe Thr Ile
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu1 5 10 15Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30Ala
Arg13631PRTArtificial sequenceantibody heavy chain framework region
136Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu1 5
10 15Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20
25 30Ala13730PRTArtificial sequenceantibody heavy chain framework
region 137Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
Leu1 5 10 15Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 20 25 301381007PRTHomo sapiens 138Met Gly Ser Gly Gly Asp Ser
Leu Leu Gly Gly Arg Gly Ser Leu1 5 10 15Pro Leu Leu Leu Leu Leu Ile
Met Gly Gly Met Ala Gln Asp Ser 20 25 30Pro Pro Gln Ile Leu Val His
Pro Gln Asp Gln Leu Phe Gln Gly 35 40 45Pro Gly Pro Ala Arg Met Ser
Cys Arg Ala Ser Gly Gln Pro Pro 50 55 60Pro Thr Ile Arg Trp Leu Leu
Asn Gly Gln Pro Leu Ser Met Val 65 70 75Pro Pro Asp Pro His His Leu
Leu Pro Asp Gly Thr Leu Leu Leu 80 85 90Leu Gln Pro Pro Ala Arg Gly
His Ala His Asp Gly Gln Ala Leu 95 100 105Ser Thr Asp Leu Gly Val
Tyr Thr Cys Glu Ala Ser Asn Arg Leu 110 115 120Gly Thr Ala Val Ser
Arg Gly Ala Arg Leu Ser Val Ala Val Leu 125 130 135Arg Glu Asp Phe
Gln Ile Gln Pro Arg Asp Met Val Ala Val Val 140 145 150Gly Glu Gln
Phe Thr Leu Glu Cys Gly Pro Pro Trp Gly His Pro 155 160 165Glu Pro
Thr Val Ser Trp Trp Lys Asp Gly Lys Pro Leu Ala Leu 170 175 180Gln
Pro Gly Arg His Thr Val Ser Gly Gly Ser Leu Leu Met Ala 185 190
195Arg Ala Glu Lys Ser Asp Glu Gly Thr Tyr Met Cys Val Ala Thr 200
205 210Asn Ser Ala Gly His Arg Glu Ser Arg Ala Ala Arg Val Ser Ile
215 220 225Gln Glu Pro Gln Asp Tyr Thr Glu Pro Val Glu Leu Leu Ala
Val 230 235 240Arg Ile Gln Leu Glu Asn Val Thr Leu Leu Asn Pro Asp
Pro Ala 245 250 255Glu Gly Pro Lys Pro Arg Pro Ala Val Trp Leu Ser
Trp Lys Val 260 265 270Ser Gly Pro Ala Ala Pro Ala Gln Ser Tyr Thr
Ala Leu Phe Arg 275 280 285Thr Gln Thr Ala Pro Gly Gly Gln Gly Ala
Pro Trp Ala Glu Glu 290 295 300Leu Leu Ala Gly Trp Gln Ser Ala Glu
Leu Gly Gly Leu His Trp 305 310 315Gly Gln Asp Tyr Glu Phe Lys Val
Arg Pro Ser Ser Gly Arg Ala 320 325 330Arg Gly Pro Asp Ser Asn Val
Leu Leu Leu Arg Leu Pro Glu Lys 335 340 345Val Pro Ser Ala Pro Pro
Gln Glu Val Thr Leu Lys Pro Gly Asn 350 355 360Gly Thr Val Phe Val
Ser Trp Val Pro Pro Pro Ala Glu Asn His 365 370 375Asn Gly Ile Ile
Arg Gly Tyr Gln Val Trp Ser Leu Gly Asn Thr 380 385 390Ser Leu Pro
Pro Ala Asn Trp Thr Val Val Gly Glu Gln Thr Gln 395 400 405Leu Glu
Ile Ala Thr His Met Pro Gly Ser Tyr Cys Val Gln Val 410 415 420Ala
Ala Val Thr Gly Ala Gly Ala Gly Glu Pro Ser Arg Pro Val 425 430
435Cys Leu Leu Leu Glu Gln Ala Met Glu Arg Ala Thr Gln Glu Pro 440
445 450Ser Glu His Gly Pro Trp Thr Leu Glu Gln Leu Arg Ala Thr Leu
455 460 465Lys Arg Pro Glu Val Ile Ala Thr Cys Gly Val Ala Leu Trp
Leu 470 475 480Leu Leu Leu Gly Thr Ala Val Cys Ile His Arg Arg Arg
Arg Ala 485 490 495Arg Val His Leu Gly Pro Gly Leu Tyr Arg Tyr Thr
Ser Glu Asp 500 505 510Ala Ile Leu Lys His Arg Met Asp His Ser Asp
Ser Gln Trp Leu 515 520 525Ala Asp Thr Trp Arg Ser Thr Ser Gly Ser
Arg Asp Leu Ser Ser 530 535 540Ser Ser Ser Leu Ser Ser Arg Leu Gly
Ala Asp Ala Arg Asp Pro 545 550 555Leu Asp Cys Arg Arg Ser Leu Leu
Ser Trp Asp Ser Arg Ser Pro 560 565 570Gly Val Pro Leu Leu Pro Asp
Thr Ser Thr Phe Tyr Gly Ser Leu 575 580 585Ile Ala Glu Leu Pro Ser
Ser Thr Pro Ala Arg Pro Ser Pro Gln 590 595 600Val Pro Ala Val Arg
Arg Leu Pro Pro Gln Leu Ala Gln Leu Ser 605 610 615Ser Pro Cys Ser
Ser Ser Asp Ser Leu Cys Ser Arg Arg Gly Leu 620 625 630Ser Ser Pro
Arg Leu Ser Leu Ala Pro Ala Glu Ala Trp Lys Ala 635 640 645Lys Lys
Lys Gln Glu Leu Gln His Ala Asn Ser Ser Pro Leu Leu 650 655 660Arg
Gly Ser His Ser Leu Glu Leu Arg Ala Cys Glu Leu Gly Asn 665 670
675Arg Gly Ser Lys Asn Leu Ser Gln Ser Pro Gly Ala Val Pro Gln 680
685 690Ala Leu Val Ala Trp Arg Ala Leu Gly Pro Lys Leu Leu Ser Ser
695 700 705Ser Asn Glu Leu Val Thr Arg His Leu Pro Pro Ala Pro Leu
Phe 710 715 720Pro His Glu Thr Pro Pro Thr Gln Ser Gln Gln Thr Gln
Pro Pro 725 730 735Val Ala Pro Gln Ala Pro Ser Ser Ile Leu Leu Pro
Ala Ala Pro 740 745 750Ile Pro Ile Leu Ser Pro Cys Ser Pro Pro Ser
Pro Gln Ala Ser 755 760 765Ser Leu Ser Gly Pro Ser Pro Ala Ser Ser
Arg Leu Ser Ser Ser 770 775 780Ser Leu Ser Ser Leu Gly Glu Asp Gln
Asp Ser Val Leu Thr Pro 785 790 795Glu Glu Val Ala Leu Cys Leu Glu
Leu Ser Glu Gly Glu Glu Thr 800 805 810Pro Arg Asn Ser Val Ser Pro
Met Pro Arg Ala Pro Ser Pro Pro 815 820 825Thr Thr Tyr Gly Tyr Ile
Ser Val Pro Thr Ala Ser Glu Phe Thr 830 835 840Asp Met Gly Arg Thr
Gly Gly Gly Val Gly Pro Lys Gly Gly Val 845 850 855Leu Leu Cys Pro
Pro Arg Pro Cys Leu Thr Pro Thr Pro Ser Glu 860 865 870Gly Ser Leu
Ala Asn Gly Trp Gly Ser Ala Ser Glu Asp Asn Ala 875 880 885Ala Ser
Ala Arg Ala Ser Leu Val Ser Ser Ser Asp Gly Ser Phe 890 895 900Leu
Ala Asp Ala His Phe Ala Arg Ala Leu Ala Val Ala Val Asp 905 910
915Ser Phe Gly Phe Gly Leu Glu Pro Arg Glu Ala Asp Cys Val Phe 920
925 930Ile Asp Ala Ser Ser Pro Pro Ser Pro Arg Asp Glu Ile Phe Leu
935 940 945Thr Pro Asn Leu Ser Leu Pro Leu Trp Glu Trp Arg Pro Asp
Trp 950 955 960Leu Glu Asp Met Glu Val Ser His Thr Gln Arg Leu Gly
Arg Gly 965 970 975Met Pro Pro Trp Pro Pro Asp Ser Gln Ile Ser Ser
Gln Arg Ser 980 985 990Gln Leu His Cys Arg Met Pro Lys Ala Gly Ala
Ser Pro Val Asp 995 1000 1005Tyr Ser13911PRTArtificial
sequenceantibody light chain framework region 139Phe Gly Gln Gly
Thr Lys Met Glu Ile Lys Arg 5 10140125PRTArtificial
sequenceantibody light chain 140Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Ile Ser 20 25 30Gly Ser Trp Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Ala Val Ile Thr Pro Ala
Gly Gly Tyr Thr Tyr Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Ser
Asn Arg Tyr Ser Gly Gln 95 100 105Phe Val Pro Ala Tyr Ala Met Asp
Tyr Trp Gly Gln Gly Thr Leu 110 115 120Val Thr Val Ser Ser
125141117PRTArtificial sequenceantibody heavy chain variable region
141Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Thr 20
25 30Asn Tyr Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45Glu Trp Val Gly Gly Ile Tyr Pro Ala Asp Gly Tyr Thr Tyr Tyr 50
55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65
70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Ser Gly Gly Tyr Ser Ser Leu Ala 95
100 105Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110
115142124PRTArtificial sequenceantibody heavy chain variable region
142Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Ile Asn 20 25 30Gly Tyr Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Phe Ile Tyr
Pro Ala Gly Gly Asp Thr Asp Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala
Arg Leu Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser Ser Tyr Gly Met
Asp Tyr Trp Gly Gln Gly Thr Leu Val 110 115 120Thr Val Ser
Ser143125PRTArtificial sequenceantibody heavy chain variable region
143Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn 20
25 30Asn Asn Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45Glu Trp Val Ala Ile Ile Ser Pro Thr Gly Gly Tyr Thr Asp Tyr 50
55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65
70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Asp Val Asn Val Tyr Ser Ala 95
100 105Arg Trp Trp Asp Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu
110 115 120Val Thr Val Ser Ser 125144123PRTArtificial
sequenceantibody heavy chain variable region 144Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser 20 25 30Ser Tyr Trp Ile Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Ser Thr
Ile Tyr Gly Tyr Asp Gly Ser Thr Asn Tyr 50 55 60Ala Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr
Cys Ala Arg Met Ser Tyr Asn Trp Ser Ser 95 100 105Pro Gly His Gly
Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr 110 115 120Val Ser
Ser145120PRTArtificial sequenceantibody heavy chain variable region
145Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20
25 30Ser Arg Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45Glu Trp Val Ser Gly Ile Tyr Pro Met Gly Gly Thr Thr Tyr Tyr 50
55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65
70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Gly Asn Tyr Tyr Ser Gly Ser 95
100 105Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
110 115 120146123PRTArtificial sequenceantibody heavy chain
variable region 146Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser 20 25 30Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 35 40 45Glu Trp Val Ser Gly Ile Ser Pro Tyr Gly Gly Tyr
Thr Tyr Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Trp Gly Tyr Tyr
Met Pro Tyr 95 100 105Gly His Pro Val Met Asp Val Trp Gly Gln Gly
Thr Leu Val Thr 110 115 120Val Ser Ser147124PRTArtificial
sequenceantibody heavy chain variable region 147Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Ser Ile Tyr 20 25 30Ser Tyr Tyr Phe Glu
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Arg
Ile Tyr Ser Ala Gly Gly His Thr Ala Tyr 50 55 60Ala Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr
Cys Ala Arg Leu Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser Ser Tyr
Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 110 115 120Thr Val Ser
Ser148124PRTArtificial sequenceantibody heavy chain variable region
148Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn 20
25 30Gly Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala Gly Gly Asp Thr Asp Tyr 50
55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65
70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ile Gly Asn Lys Phe Gly 95
100 105Trp Ser Ser Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
110 115 120Thr Val Ser Ser149124PRTArtificial sequenceantibody
heavy chain variable region 149Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Ile Asn 20 25 30Gly Tyr Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala
Gly Gly Asp Thr Asp Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu
Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser Ser Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val 110 115 120Thr Val Ser
Ser150124PRTArtificial sequenceantibody heavy chain variable region
150Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn 20
25 30Gly Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala Gly Gly Asp Thr Asp Tyr 50
55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65
70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ile Gly Asn Lys Phe Gly 95
100 105Trp Ser Ser Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
110 115 120Thr Val Ser Ser151124PRTArtificial sequenceantibody
heavy chain variable region 151Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Ile Asn 20 25 30Gly Tyr Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala
Gly Gly Asp Thr Asp Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu
Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser Ser Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val 110 115 120Thr Val Ser
Ser152124PRTArtificial sequenceantibody heavy chain variable region
152Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp 20
25 30Gly Tyr Tyr Leu Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala Gly Gly Lys Thr Glu Tyr 50
55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65
70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ile Gly Asn Lys Phe Gly 95
100 105Trp Ser Ser Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
110 115 120Thr Val Ser Ser153124PRTArtificial sequenceantibody
heavy chain variable region 153Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Ser Ile Asn 20 25 30Gly Tyr Tyr Asn Gln Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala
Gly Gly Ala Thr Ile Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu
Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser Ser Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val 110 115 120Thr Val Ser
Ser154124PRTArtificial sequenceantibody heavy chain variable region
154Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn 20
25 30Gly Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala Gly Gly Asp Thr Asp Tyr 50
55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65
70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ile Gly Asn Lys Phe Gly 95
100 105Trp Ser Ser Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
110 115 120Thr Val Ser Ser155124PRTArtificial sequenceantibody
heavy chain variable region 155Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Ile Asn 20 25 30Gly Tyr Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala
Leu Ser Val Ile Glu Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu
Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser Ser Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val 110 115 120Thr Val Ser
Ser156124PRTArtificial sequenceantibody heavy chain variable region
156Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn 20
25 30Gly Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala Gly Gly Asp Thr Asp Tyr 50
55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65
70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ile Gly Asn Lys Phe Gly 95
100 105Trp Ser Ser Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
110 115 120Thr Val Ser Ser157124PRTArtificial sequenceantibody
heavy chain variable region 157Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Ile Asn 20 25 30Gly Tyr Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala
Gly Gly Asp Thr Asp Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu
Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser Ser Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val 110 115 120Thr Val Ser
Ser158124PRTArtificial sequenceantibody heavy chain variable region
158Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn 20
25 30Gly Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala Gly Gly Asp Thr Asp Tyr 50
55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65
70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ile Gly Asn Lys Phe Gly 95
100 105Trp Ser Ser Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
110 115 120Thr Val Ser Ser159124PRTArtificial sequenceantibody
heavy chain variable region 159Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Ile Asn 20 25 30Gly Tyr Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala
Gly Gly Asp Thr Asp Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu
Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser Ser Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val 110 115 120Thr Val Ser
Ser160124PRTArtificial sequenceantibody heavy chain variable region
160Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1
5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn
20 25 30Gly Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala Gly Gly Asp Thr Asp Tyr
50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser
65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ile Gly Asn Lys Phe Gly
95 100 105Trp Ser Ser Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu
Val 110 115 120Thr Val Ser Ser161124PRTArtificial sequenceantibody
heavy chain variable region 161Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Ile Asn 20 25 30Gly Tyr Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala
Gly Gly Asp Thr Asp Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu
Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser Ser Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val 110 115 120Thr Val Ser
Ser162124PRTArtificial sequenceantibody heavy chain variable region
162Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn 20
25 30Gly Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala Gly Gly Asp Thr Asp Tyr 50
55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65
70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ile Gly Asn Lys Phe Gly 95
100 105Trp Ser Ser Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
110 115 120Thr Val Ser Ser163124PRTArtificial sequenceantibody
heavy chain variable region 163Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Ile Asn 20 25 30Gly Tyr Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala
Gly Gly Asp Thr Asp Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu
Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser Ser Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val 110 115 120Thr Val Ser
Ser164124PRTArtificial sequenceantibody heavy chain variable region
164Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn 20
25 30Gly Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala Gly Gly Asp Thr Asp Tyr 50
55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65
70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ile Gly Asn Lys Phe Gly 95
100 105Trp Ser Ser Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
110 115 120Thr Val Ser Ser165124PRTArtificial sequenceantibody
heavy chain variable region 165Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Ile Asn 20 25 30Gly Tyr Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Phe Ile Tyr Pro Ala
Gly Gly Asp Thr Asp Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Leu
Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser Ser Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val 110 115 120Thr Val Ser
Ser16610PRTArtificial sequenceantibody light chain framework region
166Phe Gly Gln Gly Thr Lys Met Glu Ile Lys 5 10167107PRTArtificial
sequenceantibody light chain 167Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Val Ser 20 25 30Thr Ala Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile Tyr Ser Ala Ser Phe Leu
Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln 80 85 90Ser Tyr Thr Thr Pro Pro Thr Phe Gly
Gln Gly Thr Lys Val Glu 95 100 105Ile Lys16832PRTArtificial
sequenceantibody light chain framework region 168Gly Val Pro Ser
Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe1 5 10 15Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 20 25 30Tyr
Cys169454PRTArtificial sequenceantibody heavy chain 169Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn 20 25 30Gly Tyr Tyr
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val
Gly Phe Ile Tyr Pro Ala Gly Gly Asp Thr Asp Tyr 50 55 60Ala Asp Ser
Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr
Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val
Tyr Tyr Cys Ala Arg Leu Ile Gly Asn Lys Phe Gly 95 100 105Trp Ser
Ser Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 110 115 120Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 125 130
135Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 140
145 150Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
155 160 165Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val 170 175 180Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 185 190 195Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn 200 205 210His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys 215 220 225Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu 230 235 240Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 245 250 255Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val 260 265 270Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr 275 280 285Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 290 295 300Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val 305 310 315Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 320 325 330Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 335 340 345Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 350 355 360Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 365 370
375Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 380
385 390Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
395 400 405Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp 410 415 420Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 425 430 435His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu 440 445 450Ser Pro Gly Lys170214PRTArtificial
sequenceantibody light chain 170Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Val Ser 20 25 30Thr Ala Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile Tyr Ser Ala Ser Phe Leu
Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr Thr Tyr Cys Gln Gln 80 85 90Ser Tyr Thr Thr Pro Pro Thr Phe Gly
Gln Gly Thr Lys Val Glu 95 100 105Ile Lys Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro 110 115 120Ser Asp Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu 125 130 135Leu Asn Asn Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val 140 145 150Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 155 160 165Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 170 175 180Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 185 190 195Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 200 205 210Arg
Gly Glu Cys171214PRTArtificial sequenceHis-tagged Robo4 171Gln Asp
Ser Pro Pro Gln Ile Leu Val His Pro Gln Asp Gln Leu1 5 10 15Phe Gln
Gly Pro Gly Pro Ala Arg Met Ser Cys Gln Ala Ser Gly 20 25 30Gln Pro
Pro Pro Thr Ile Arg Trp Leu Leu Asn Gly Gln Pro Leu 35 40 45Ser Met
Val Pro Pro Asp Pro His His Leu Leu Pro Asp Gly Thr 50 55 60Leu Leu
Leu Leu Gln Pro Pro Ala Arg Gly His Ala His Asp Gly 65 70 75Gln Ala
Leu Ser Thr Asp Leu Gly Val Tyr Thr Cys Glu Ala Ser 80 85 90Asn Arg
Leu Gly Thr Ala Val Ser Arg Gly Ala Arg Leu Ser Val 95 100 105Ala
Val Leu Arg Glu Asp Phe Gln Ile Gln Pro Arg Asp Met Val 110 115
120Ala Val Val Gly Glu Gln Phe Thr Leu Glu Cys Gly Pro Pro Trp 125
130 135Gly His Pro Glu Pro Thr Val Ser Trp Trp Lys Asp Gly Lys Pro
140 145 150Leu Ala Leu Gln Pro Gly Arg His Thr Val Ser Gly Gly Ser
Leu 155 160 165Leu Met Ala Arg Ala Glu Lys Ser Asp Glu Gly Thr Tyr
Met Cys 170 175 180Val Ala Thr Asn Ser Ala Gly His Arg Glu Ser Arg
Ala Ala Arg 185 190 195Val Ser Ile Gln Glu Pro Gln Asp Tyr Arg Ala
His His His His 200 205 210His His His His172243PRTArtificial
sequenceHis-tagged Robo4 172Met Gly Ser Gly Gly Thr Gly Leu Leu Gly
Thr Glu Trp Pro Leu1 5 10 15Pro Leu Leu Leu Leu Phe Ile Met Gly Gly
Glu Ala Leu Asp Ser 20 25 30Pro Pro Gln Ile Leu Val His Pro Gln Asp
Gln Leu Leu Gln Gly 35 40 45Ser Gly Pro Ala Lys Met Arg Cys Arg Ser
Ser Gly Gln Pro Pro 50 55 60Pro Thr Ile Arg Trp Leu Leu Asn Gly Gln
Pro Leu Ser Met Ala 65 70 75Thr Pro Asp Leu His Tyr Leu Leu Pro Asp
Gly Thr Leu Leu Leu 80 85 90His Arg Pro Ser Val Gln Gly Arg Pro Gln
Asp Asp Gln Asn Ile 95 100 105Leu Ser Ala Ile Leu Gly Val Tyr Thr
Cys Glu Ala Ser Asn Arg 110 115 120Leu Gly Thr Ala Val Ser Arg Gly
Ala Arg Leu Ser Val Ala Val 125 130 135Leu Gln Glu Asp Phe Gln Ile
Gln Pro Arg Asp Thr Val Ala Val 140 145 150Val Gly Glu Ser Leu Val
Leu Glu Cys Gly Pro Pro Trp Gly Tyr 155 160 165Pro Lys Pro Ser Val
Ser Trp Trp Lys Asp Gly Lys Pro Leu Val 170 175 180Leu Gln Pro Gly
Arg Arg Thr Val Ser Gly Asp Ser Leu Met Val 185 190 195Ser Arg Ala
Glu Lys Asn Asp Ser Gly Thr Tyr Met Cys Met Ala 200 205 210Thr Asn
Asn Ala Gly Gln Arg Glu Ser Arg Ala Ala Arg Val Ser 215 220 225Ile
Gln Glu Ser Gln Asp His Arg Arg Ala His His His His His 230 235
240His His His17311PRTArtificial sequenceantibody light chain
framework region 173Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 5
1017411PRTArtificial sequenceantibody hypervariable region 174Arg
Ala Ser Gln Xaa Xaa Xaa Xaa Xaa Xaa Ala 5 101757PRTArtificial
sequenceantibody hypervariable region 175Xaa Ala Xaa Xaa Xaa Xaa
Xaa 51769PRTArtificial sequenceantibody hypervariable region 176Gln
Gln Xaa Xaa Xaa Xaa Xaa Xaa Thr 517710PRTArtificial
sequenceantibody hypervariable region 177Gly Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 5 1017818PRTArtificial sequenceantibody
hypervariable region 178Xaa Xaa Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Tyr Ala Asp Ser1 5 10 15Val Lys Gly17918PRTArtificial
sequenceantibody hypervariable region 179Ala Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa
Xaa18010PRTArtificial sequenceantibody hypervariable region 180Gly
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 5 1018118PRTArtificial
sequenceantibody hypervariable region 181Xaa Xaa Ile Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Tyr Ala Asp Ser1 5 10 15Val Lys
Gly18216PRTArtificial sequenceantibody hypervariable region 182Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp1 5 10
15Xaa18311PRTArtificial sequenceantibody hypervariable region
183Arg Ala Ser Gln Asp Xaa Xaa Xaa Xaa Xaa Ala 5
101847PRTArtificial sequenceantibody hypervariable region 184Ser
Ala Xaa Xaa Xaa Xaa Xaa 51859PRTArtificial sequenceantibody
hypervariable region 185Gln Gln Xaa Xaa Xaa Xaa Xaa Xaa Thr
518610PRTArtificial
sequenceantibody hypervariable region 186Gly Phe Xaa Xaa Xaa Gly
Tyr Tyr Xaa Xaa 5 1018718PRTArtificial sequenceantibody
hypervariable region 187Gly Xaa Ile Tyr Xaa Ala Xaa Gly Xaa Thr Xaa
Tyr Ala Asp Ser1 5 10 15Val Lys Gly
* * * * *