U.S. patent application number 17/369542 was filed with the patent office on 2022-06-02 for monoclonal antibodies against her2.
The applicant listed for this patent is GENMAB A/S. Invention is credited to Ole BAADSGAARD, Bart DE GOEIJ, Simone DE HAIJ, Rene HOET, Aran Frank LABRIJN, Joyce MEESTERS, Paul PARREN, Thilo RIEDL, David SATIJN, Janine SCHUURMAN, Jan VAN DE WINKEL, Edward N. VAN DEN BRINK.
Application Number | 20220169738 17/369542 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-02 |
United States Patent
Application |
20220169738 |
Kind Code |
A1 |
DE GOEIJ; Bart ; et
al. |
June 2, 2022 |
MONOCLONAL ANTIBODIES AGAINST HER2
Abstract
Isolated monoclonal antibodies which bind to human epidermal
growth factor receptor 2 (HER2), and related antibody-based
compositions and molecules, are disclosed. Pharmaceutical
compositions comprising the antibodies and therapeutic and
diagnostic methods for using the antibodies are also disclosed.
Inventors: |
DE GOEIJ; Bart; (Utrecht,
NL) ; DE HAIJ; Simone; (Weesp, NL) ; RIEDL;
Thilo; (Arnhem, NL) ; HOET; Rene; (Boxmeer,
NL) ; BAADSGAARD; Ole; (Hellerup, DK) ; VAN DE
WINKEL; Jan; (Zeist, NL) ; SATIJN; David;
(Utrecht, NL) ; PARREN; Paul; (Odijk, NL) ;
LABRIJN; Aran Frank; (Nigtevecht, NL) ; MEESTERS;
Joyce; (Utrecht, NL) ; SCHUURMAN; Janine;
(Diemen, NL) ; VAN DEN BRINK; Edward N.; (Halfweg,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENMAB A/S |
Copenhagen V |
|
DK |
|
|
Appl. No.: |
17/369542 |
Filed: |
July 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15832337 |
Dec 5, 2017 |
11091553 |
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17369542 |
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13700341 |
Mar 14, 2013 |
9862769 |
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PCT/EP2011/058779 |
May 27, 2011 |
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15832337 |
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61349180 |
May 27, 2010 |
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International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/32 20060101 C07K016/32 |
Foreign Application Data
Date |
Code |
Application Number |
May 27, 2010 |
DK |
PA201000467 |
Apr 20, 2011 |
DK |
PA201100312 |
Apr 20, 2011 |
EP |
PCT/EP2011/056388 |
Claims
1. A monoclonal antibody which binds human epidermal growth factor
receptor 2 (HER2) and which comprises a VH region comprising a CDR3
sequence selected from the group consisting of: a) SEQ ID NO:133,
such as the sequence of SEQ ID NO:4 (169); b) SEQ ID NO:11 (050,
049, 051, 055); c) SEQ ID NO:130, such as the sequence of SEQ ID
NO:18 (084); d) SEQ ID NO:136, such as the sequence of SEQ ID NO:25
(025); e) SEQ ID NO:139, such as the sequence of SEQ ID NO: 31
(091); f) SEQ ID NO:142, such as the sequence of SEQ ID NO:38
(129); g) SEQ ID NO:148, such as the sequence of SEQ ID NO:48
(127); h) SEQ ID NO:52 (159); i) SEQ ID NO:145, such as the
sequence of SEQ ID NO:59 (098); j) SEQ ID NO:154, such as the
sequence of SEQ ID NO: 66 (153); and k) SEQ ID NO:151, such as the
sequence of SEQ ID NO: 73 (132).
2-3. (canceled)
4. The antibody of claim 1, comprising a VH region and a VL region
selected from the groups consisting of a) a VH region comprising
the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:2, 3 and 4,
respectively; and a VL region comprising the CDR1, CDR2 and CDR3
sequences of SEQ ID NOs:6, DAS, and SEQ ID NO:7, respectively
(169); b) a VH region comprising the CDR1, CDR2 and CDR3 sequences
of SEQ ID NOs:9, 10 and 11, respectively; and a VL region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:13, AAS,
and SEQ ID NO:14, respectively (050); c) a VH region comprising the
CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:16, 17 and 18,
respectively; and a VL region comprising the CDR1, CDR2 and CDR3
sequences of SEQ ID NOs:20, VAS, and SEQ ID NO:21, respectively
(084); d) a VH region comprising the CDR1, CDR2 and CDR3 sequences
of SEQ ID NOs:23, 24 and 25, respectively; and a VL region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:27, AAS,
and SEQ ID NO:28, respectively (025); e) a VH region comprising the
CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:30, 163 and 31,
respectively; and a VL region comprising the CDR1, CDR2 and CDR3
sequences of SEQ ID NOs:33, AAS, and SEQ ID NO:34, respectively
(091); f) a VH region comprising the CDR1, CDR2 and CDR3 sequences
of SEQ ID NOs:36, 37 and 38, respectively; and a VL region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:40, DAS,
and SEQ ID NO:41, respectively (129); g) a VH region comprising the
CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:43, 44 and 45,
respectively; and a VL region comprising the CDR1, CDR2 and CDR3
sequences of SEQ ID NOs:47, AAS, and SEQ ID NO:48, respectively
(127); h) a VH region comprising the CDR1, CDR2 and CDR3 sequences
of SEQ ID NOs:50, 51 and 52, respectively; and a VL region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:54, AAS,
and SEQ ID NO:55, respectively (159); i) a VH region comprising the
CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:57, 58 and 59,
respectively; and a VL region comprising the CDR1, CDR2 and CDR3
sequences of SEQ ID NOs:60, AAS, and SEQ ID NO:61, respectively
(098); j) a VH region comprising the CDR1, CDR2 and CDR3 sequences
of SEQ ID NOs:64, 65 and 66, respectively; and a VL region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:68, DAS,
and SEQ ID NO:69, respectively (153); and k) a VH region comprising
the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:71, 72 and 73,
respectively; and a VL region comprising the CDR1, CDR2 and CDR3
sequences of SEQ ID NOs:75, DAS, and SEQ ID NO:76, respectively
(132).
5-8. (canceled)
9. A monoclonal antibody comprising a VH region and a VL region
selected from the group consisting of: a) a VH region comprising
the sequence of SEQ ID NO:1 and, preferably, a VL region comprising
the sequence of SEQ ID NO:5 (169); b) a VH region comprising the
sequence of SEQ ID NO:8 and, preferably, a VL region comprising the
sequence of SEQ ID NO:12 (050); c) a VH region comprising the
sequence of SEQ ID NO:15 and, preferably, a VL region comprising
the sequence of SEQ ID NO:19 (084); d) a VH region comprising the
sequence of SEQ ID NO:77 and, preferably, a VL region comprising
the sequence of SEQ ID NO:78 (049); e) a VH region comprising the
sequence of SEQ ID NO:79 and, preferably, a VL region comprising
the sequence of SEQ ID NO:80 (051); f) a VH region comprising the
sequence of SEQ ID NO:81 and, preferably, a VL region comprising
the sequence of SEQ ID NO:82 (055); g) a VH region comprising the
sequence of SEQ ID NO:83 and, preferably, a VL region comprising
the sequence of SEQ ID NO:84 (123); h) a VH region comprising the
sequence of SEQ ID NO:85 and, preferably, a VL region comprising
the sequence of SEQ ID NO:86 (161); i) a VH region comprising the
sequence of SEQ ID NO:87 and, preferably, a VL region comprising
the sequence of SEQ ID NO:88 (124); j) a VH region comprising the
sequence of SEQ ID NO:22 and, preferably, a VL region comprising
the sequence of SEQ ID NO:26 (025); k) a VH region comprising the
sequence of SEQ ID NO:29 and, preferably, a VL region comprising
the sequence of SEQ ID NO:32 (091); l) a VH region comprising the
sequence of SEQ ID NO:35 and, preferably, a VL region comprising
the sequence of SEQ ID NO:39 (129); m) a VH region comprising the
sequence of SEQ ID NO:89 and, preferably, a VL region comprising
the sequence of SEQ ID NO:90 (001); n) a VH region comprising the
sequence of SEQ ID NO:91 and, preferably, a VL region comprising
the sequence of SEQ ID NO:92 (143); o) a VH region comprising the
sequence of SEQ ID NO:93 and, preferably, a VL region comprising
the sequence of SEQ ID NO:94 (019); p) a VH region comprising the
sequence of SEQ ID NO:95 and, preferably, a VL region comprising
the sequence of SEQ ID NO:96 (021); q) a VH region comprising the
sequence of SEQ ID NO:97 and, preferably, a VL region comprising
the sequence of SEQ ID NO:98 (027); r) a VH region comprising the
sequence of SEQ ID NO:99 and, preferably, a VL region comprising
the sequence of SEQ ID NO:100 (032); s) a VH region comprising the
sequence of SEQ ID NO:101 and, preferably, a VL region comprising
the sequence of SEQ ID NO:102 (035); t) a VH region comprising the
sequence of SEQ ID NO:103 and, preferably, a VL region comprising
the sequence of SEQ ID NO:104 (036); u) a VH region comprising the
sequence of SEQ ID NO:105 and, preferably, a VL region comprising
the sequence of SEQ ID NO:106 (054); v) a VH region comprising the
sequence of SEQ ID NO:106 and, preferably, a VL region comprising
the sequence of SEQ ID NO:108 (094); w) a VH region comprising the
sequence of SEQ ID NO:46 and, preferably, a VL region comprising
the sequence of SEQ ID NO:49 (127); x) a VH region comprising the
sequence of SEQ ID NO:49 and, preferably, a VL region comprising
the sequence of SEQ ID NO:53 (159); y) a VH region comprising the
sequence of SEQ ID NO:56 and, preferably, a VL region comprising
the sequence of SEQ ID NO:60 (098); z) a VH region comprising the
sequence of SEQ ID NO:63 an, preferably, a VL region comprising the
sequence of SEQ ID NO:67 (153); aa) a VH region comprising the
sequence of SEQ ID NO:70 and, preferably, a VL region comprising
the sequence of SEQ ID NO:74 (132); bb) a VH region comprising the
sequence of SEQ ID NO:109 and, preferably, a VL region comprising
the sequence of SEQ ID NO:110 (105); cc) a VH region comprising the
sequence of SEQ ID NO:111 and, preferably, a VL region comprising
the sequence of SEQ ID NO:112 (100); dd) a VH region comprising the
sequence of SEQ ID NO:113 and, preferably, a VL region comprising
the sequence of SEQ ID NO:114 (125); ee) a VH region comprising the
sequence of SEQ ID NO:115 and, preferably, a VL region comprising
the sequence of SEQ ID NO:116 (162); ff) a VH region comprising the
sequence of SEQ ID NO:117 and, preferably, a VL region comprising
the sequence of SEQ ID NO:118 (033); gg) a VH region comprising the
sequence of SEQ ID NO:119 and, preferably, a VL region comprising
the sequence of SEQ ID NO:120 (160) hh) a VH region comprising the
sequence of SEQ ID NO:121 and, preferably, a VL region comprising
the sequence of SEQ ID NO:122 (166); ii) a VH region comprising the
sequence of SEQ ID NO:123 and, preferably, a VL region comprising
the sequence of SEQ ID NO:124 (152); jj) a VH region comprising the
sequence of SEQ ID NO:125 and, preferably, a VL region comprising
the sequence of SEQ ID NO:126 (167); and kk) a variant of any of
said antibodies, wherein said variant preferably has at most 1, 2
or 3 amino-acid modifications, more preferably amino-acid
substitutions, such as conservative amino acid substitutions and
substitutions where the new amino acid is one at the same position
in an aligned sequence in FIG. 1 or 2, particularly at positions
indicated by "X" in the corresponding consensus sequence.
10-20. (canceled)
21. A bispecific antibody comprising the antigen-binding region of
an antibody as defined in claim 1, and a second antigen-binding
site having a different binding specificity, such as a binding
specificity for a human effector cell, a human Fc receptor, a T
cell receptor or a non-blocking epitope of HER2.
22-27. (canceled)
28. The antibody of claim 1, wherein the antibody is a full-length
antibody, preferably an IgG1 antibody, in particular an
IgG1,.kappa. antibody.
29. The antibody of claim 1, wherein the antibody is conjugated to
another moiety, such as a cytotoxic moiety, a radioisotope, a drug,
or a cytokine.
30. The antibody of claim 29, wherein the cytotoxic moiety is
selected from the group consisting of taxol; cytochalasin B;
gramicidin D; ethidium bromide; emetine; mitomycin; etoposide;
tenoposide; vincristine; vinblastine; colchicin; doxorubicin;
daunorubicin; dihydroxy anthracin dione; a tubulin-inhibitor such
as maytansine or an analog or derivative thereof; mitoxantrone;
mithramycin; actinomycin D; 1-dehydrotestosterone; a
glucocorticoid; procaine; tetracaine; lidocaine; propranolol;
puromycin; calicheamicin or an analog or derivative thereof an
antimetabolite such as methotrexate, 6 mercaptopurine, 6
thioguanine, cytarabine, fludarabin, 5 fluorouracil, decarbazine,
hydroxyurea, asparaginase, gemcitabine, or cladribine; an
alkylating agent such as mechlorethamine, thioepa, chlorambucil,
melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide,
busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC),
procarbazine, mitomycin C, cisplatin, carboplatin, duocarmycin A,
duocarmycin SA, rachelmycin (CC-1065), or an analog or derivative
thereof; an antibiotic such as dactinomycin, bleomycin,
daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin,
mitoxantrone, plicamycin, anthramycin (AMC)); an antimitotic agent
such as monomethyl auristatin E or F or an analog or derivative
thereof; diphtheria toxin and related molecules such as diphtheria
A chain and active fragments thereof and hybrid molecules, ricin
toxin such as ricin A or a deglycosylated ricin A chain toxin,
cholera toxin, a Shiga-like toxin such as SLT I, SLT II, SLT IIV,
LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin,
soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin,
alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A
chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phytolacca americana proteins such as PAPI, PAPII, and PAP S,
Momordica charantia inhibitor, curcin, crotin, Sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, and enomycin toxins; ribonuclease (RNase); DNase I,
Staphylococcal enterotoxin A; pokeweed antiviral protein;
diphtherin toxin; and Pseudomonas endotoxin.
31. The antibody of claim 29, which is conjugated to a cytotoxic
moiety selected from the group consisting of maytansine,
calicheamicin, duocarmycin, rachelmycin (CC-1065), monomethyl
auristatin E, monomethyl auristatin F or an analog, derivative, or
prodrug of any thereof.
32. The antibody of claim 29, which is conjugated to a cytokine
selected from the group consisting of IL-2, IL-4, IL-6, IL-7,
IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a,
IL-28b, IL-29, KGF, IFN.alpha., IFN.beta., IFN.gamma., GM-CSF,
CD40L, Flt3 ligand, stem cell factor, ancestim, and TNF.alpha..
33. (canceled)
34. A nucleotide sequence encoding one or more of the amino acid
sequences selected from the group consisting of SEQ ID NO: 1, 5, 8,
12, 15, 19, 22, 26, 29, 32, 35, 39, 42, 46, 49, 53, 56, 60, 63, 67,
70, 74, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,
120, 121, 122, 123, 124, 125 and 126.
35. An expression vector comprising a nucleotide sequence according
to claim 34, wherein the vector further encodes an
operatively-linked constant region of a light chain, constant
region of a heavy chain or both light and heavy chains of an
antibody.
36. A recombinant eukaryotic or prokaryotic host cell which
produces an antibody as defined in claim 1.
37. A pharmaceutical composition comprising the antibody of claim 1
and a pharmaceutically acceptable carrier.
38-45. (canceled)
46. A method for inhibiting growth and/or proliferation of one or
more tumor cells expressing HER2, comprising administration, to an
individual in need thereof, of an antibody according to claim
1.
47. The method of claim 46, wherein the one or more tumor cells
coexpress HER2 and EGFR and/or HER3.
48. A method for treating cancer, comprising a) selecting a subject
suffering from a cancer comprising tumor cells co-expressing HER2
and EGFR and/or HER3, and b) administering to the subject the
antibody according to claim 1.
49. The method of claim 48, wherein the cancer is selected from the
group consisting of breast cancer, colorectal cancer,
endometrial/cervical cancer, lung cancer, malignant melanoma,
ovarian cancer, pancreatic cancer, prostate cancer, testis cancer,
a soft-tissue tumor such as synovial sarcoma, and bladder
cancer.
50. A method for producing an antibody, said method comprising the
steps of a) culturing a host cell of claim 36, and b) purifying the
antibody from the culture media.
51. A method for detecting the presence of HER2 in a sample,
comprising: contacting the sample with an antibody of claim 1 under
conditions that allow for formation of a complex between the
antibody and HER2; and analyzing whether a complex has been
formed.
52. A kit for detecting the presence of HER2 in a sample comprising
the antibody of claim 1; and instructions for use of the kit.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/832,337, filed Dec. 5, 2017 (now U.S. Pat. No.
11,091,553), which is a divisional of U.S. patent application Ser.
No. 13/700,341, filed Mar. 14, 2013 (now U.S. Pat. No. 9,862,769),
which is a 35 U.S.C. 371 national stage filing of
PCT/EP2011/058779, filed May 27, 2011, which claims priority to
U.S. Provisional Application No. 61/349,180, filed May 27, 2010,
International Patent Application No. PCT/EP2011/056388, filed Apr.
20, 2011, and Danish Patent Application Nos. PA201100312, filed
Apr. 20, 2011, and PA201000467, filed May 27, 2010. The contents of
the aforementioned applications are hereby incorporated by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jul. 7, 2021, is named GMI_131USDV4_Sequence_Listing.txt and is
149,745 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to monoclonal antibodies
directed to human epidermal growth factor receptor 2 (HER2) and to
uses of such antibodies, in particular their use in the treatment
of cancer.
BACKGROUND OF THE INVENTION
[0004] HER2 is a 185-kDa cell surface receptor tyrosine kinase and
member of the epidermal growth factor receptor (EGFR) family that
comprises four distinct receptors: EGFR/ErbB-1, HER2/ErbB-2,
HER3/ErbB-3, and HER4/ErbB-4. Both homo- and heterodimers are
formed by the four members of the EGFR family, with HER2 being the
preferred and most potent dimerization partner for other ErbB
receptors (Graus-Porta et al., Embo J 1997; 16:1647-1655; Tao et
al., J Cell Sci 2008; 121:3207-3217). HER2 can be activated by
overexpression or by heterodimerization with other ErbBs that can
be activated by ligand binding (Riese and Stern, Bioessays 1998;
20:41-48). For HER2, no ligand has been identified. HER2 activation
leads to receptor phosphorylation, which triggers a cascade of
downstream signals through multiple signaling pathways, such as
MAPK, phosphoinositol 3-kinase/AKT, JAK/STAT and PKC, which
ultimately results in the regulation of multiple cellular
functions, such as growth, survival and differentiation (Huang et
al., Expert Opin Biol Ther 2009; 9:97-110).
[0005] Much of the attention on HER2 in tumors has been focused on
its role in breast cancer, in which HER2 overexpression is reported
in approximately 20% of the cases and is correlated with poor
prognosis (Reese et al., Stem Cells 1997; 15:1-8; Andrechek et al.,
Proc Natl Acad Sci USA 2000; 97:3444-3449; and Slamon et al.,
Science 1987; 235:177-182). Besides breast cancer, HER2 expression
has also been associated with other human carcinoma types,
including prostate cancer, non-small cell lung cancer, bladder
cancer, ovarian cancer, gastric cancer, colon cancer, esophageal
cancer and squamous cell carcinoma of the head & neck (Garcia
de Palazzo et al., Int J Biol Markers 1993; 8:233-239; Ross et al.,
Oncologist 2003; 8:307-325; Osman et al., J Urol 2005;
174:2174-2177; Kapitanovic et al., Gastroenterology 1997;
112:1103-1113; Turken et al., Neoplasma 2003; 50:257-261; and
Oshima et al., Int J Biol Markers 2001; 16:250-254).
[0006] Trastuzumab (Herceptin.RTM.) is a recombinant, humanized
monoclonal antibody directed against domain IV of the HER2 protein,
thereby blocking ligand-independent HER2 homodimerization, and to a
lesser extend heterodimerization of HER2 with other family members
in cells with high HER2 overexpression (Cho et al., Nature 2003;
421:756-760 and Wehrman et al., Proc Natl Acad Sci USA 2006;
103:19063-19068). In cells with modest HER2 expressing levels,
trastuzumab was found to inhibit the formation of HER2/EGFR
heterodimers (Wehrman et al., (2006), supra; Schmitz et al., Exp
Cell Res 2009; 315:659-670). Trastuzumab mediates
antibody-dependent cellular cytotoxicity (ADCC) and prevents
ectodomain shedding, which would otherwise result in the formation
of a truncated constitutively active protein in HER2 overexpressing
cells. Also inhibition of both in vitro and in vivo proliferation
of tumor cells expressing high levels of HER2 has been reported for
trastuzumab (reviewed in Nahta and Esteva, Oncogene 2007;
26:3637-3643). Herceptin.RTM. has been approved both for first-line
and adjuvant treatment of HER2 overexpressing metastatic breast
cancer, either in combination with chemotherapy, or as a single
agent following one or more chemotherapy regimens. Trastuzumab has
been found to be effective only in 20-50% of HER2 overexpressing
breast tumor patients and many of the initial responders show
relapse after a few months (Dinh et al., Clin Adv Hematol Oncol
2007; 5:707-717).
[0007] Pertuzumab (Omnitarg.TM.) is another humanized monoclonal
antibody. It is directed against domain II of the HER2 protein,
resulting in inhibition of ligand-induced heterodimerization (i.e.,
HER2 dimerizing with another member of the ErbB family to which a
ligand has bound); a mechanism reported to not strictly require
high HER2 expression levels (Franklin et al., Cancer Cell 2004;
5:317-328.). Although pertuzumab also mediates ADCC, the main
mechanism of action of pertuzumab relies on its dimerization
blockade (Hughes et al., Mol Cancer Ther 2009; 8:1885-1892).
Moreover, pertuzumab was found to enhance EGFR internalization and
downregulation by inhibiting the formation of EGFR/HER2
heterodimers, which otherwise tethers EGFR at the plasma membrane
(Hughes et al., 2009, supra). This correlates with the observation
that EGFR homodimers internalize more efficient than EGFR/HER2
dimers (Pedersen et al., Mol Cancer Res 2009; 7:275-284. The
complementary mechanisms of action of pertuzumab and trastuzumab
reportedly results in enhanced anti-tumor effects and efficacy when
combined in patients who progressed during prior trastuzumab
therapy (Baselga et al., J Clin Oncol 2010; 28:1138-1144), and a
phase III trial to evaluate this antibody combination together with
Docetaxel in previously untreated HER2-positive metastatic breast
cancer is underway.
[0008] An alternative approach to improve targeted antibody therapy
is by delivering cytotoxic cells or drugs specifically to the
antigen-expressing cancer cells. For example, the so-called
trifunctional antibodies are bispecific antibodies, targeting with
one arm the antigen on the tumor cell and with the other arm for
instance CD3 on T cells. Upon binding, a complex of T cells, tumor
cells and effector cells that bind Fc is formed, leading to killing
of the tumor cells (Muller and Kontermann, BioDrugs 2010;
24:89-98.). Ertumaxomab is one such trifunctional antibody against
HER2, which induces cytotoxicity in cell lines with low HER2
expression and which is in Phase II clinical development in
metastatic breast cancer (Jones et al., Lancet Oncol 2009;
10:1179-1187 and Kiewe et al., Clin Cancer Res 2006;
12:3085-3091).
[0009] A HER2 antibody drug conjugate (ADC) is currently in
clinical development. T-DM1 consists of trastuzumab conjugated to
the fungal toxin maytansine. In Phase II trials, responses in a
heavily pretreated patient cohort including prior trastuzumab
and/or lapatinib therapy were reported Burris et al, 2011, J Clin
Oncol 29: 398-405 and Lewis Phillips et al., Cancer Res 2008;
68:9280-9290). Preliminary data from a Phase II trial determining
efficacy and safety of T-DM1 versus trastuzumab plus docetaxel in
her2-positive metastatic breast cancer patients with no rior
chemotherapy for metastatic disease were reported (Perez et al,
Abstract BA3, European Society for Medical Oncology meeting 2010).
A Phase III trial to evaluate T-DM1 efficacy and safety versus
capecitabine+lapatinib in patients with HER2-positive locally
advanced or metastatic breast cancer who received prior trastuzumab
therapy is ongoing.
[0010] While many factors are involved in selecting a suitable
antibody for HER2 targeted therapy, it is typically an advantage
for an ADC approach if the HER2-antibody complex efficiently
internalizes upon antibody binding. Studies on murine HER2
antibodies have shown that certain combinations of antibodies
instigate HER2 endocytosis (Ben-Kasus et al., PNAS 2009;
106:3294-9). Human HER2 antibodies F5 and C1 have been reported to
internalize relatively rapidly on their own and to bind the same
epitope (WO 99/55367 and WO 2006/116107). As compared to EGFR,
however, internalization of HER2 is impaired. Indeed, EGFR
homodimers internalize much more efficiently than HER2 homodimers
(Dinh et al., Clin Adv Hematol Oncol 2007; 5:707-717). EGFR, and
also HER3, can increase endocytosis of HER2 by the formation of
EGFR/HER2 and HER3/HER2 heterodimers, respectively (Baulida et al.,
J Biol Chem 1996; 271:5251-5257; Pedersen N M, et al., Mol Cancer
Res 2009; 7:275-84).
[0011] The complex mechanisms regulating the function of HER2
warrant further research on new and optimized therapeutic
strategies against this proto-oncogene. Accordingly, there remains
a need for effective and safe products for treating HER2-related
diseases, such as cancer.
SUMMARY OF THE INVENTION
[0012] It is an object of the present invention to provide novel
highly specific and effective monoclonal HER2 antibodies for
medical use. The antibodies of the invention exhibit HER2 binding
characteristics that differ from antibodies described in the art.
Particularly, although most of the antibodies apparently bind to
HER2 segments overlapping with those bound by trastuzumab,
pertuzumab or F5/C1 as shown in a cross-blocking HER2 binding
assay, the novel antibodies are characterized by a higher
efficiency in killing HER2-expressing tumor cells in an ADC assay,
improved internalization and/or other advantages over the known
antibodies.
[0013] In preferred embodiments, the antibodies of the invention
are fully human, bind to novel epitopes, and/or have favorable
properties for therapeutic use in human patients. Exemplary
properties include, but are not limited to, favorable binding
characteristics to cancer cells expressing human HER2 at high or
low levels, specific binding rhesus epithelial cells expressing a
HER2 ortholog, efficient internalization upon binding to HER2, high
capacity for killing cancer cells expressing high- or low-levels of
HER2 when administered as an ADC, promoting proliferation of
HER2-expressing cells less than F5, a neutral or inhibitory effect
on the proliferation of HER2-expressing cancer cells, and provide
for effective ADCC-mediated killing of HER2-expressing cells, as
well as any combination of the foregoing properties.
[0014] These and other aspects of the invention are described in
further detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A-1K: Alignment of HuMab heavy chain variable region
(VH) sequences with germline (reference) sequences (FIGS. 1A-1K).
In each VH sequence, the amino acids that differ from those of the
germline (reference) at specific positions are highlighted.
Consensus VH sequences are shown, where "X" indicates positions at
which alternative amino acids (selected from those aligned at each
position) are possible. The CDR1, CDR2, and CDR3 sequences are
underlined in each VH sequence. The consensus CDR sequences are
further defined in Table 4.
[0016] FIGS. 2A-2F: Alignment of HuMab light chain variable region
(VL) sequences with germline (reference) sequences (FIGS. 2A-2B).
In each VL sequence, the amino acids that differ from those of the
germline (reference) at specific positions are highlighted. In FIG.
2A, all VL sequences derived from the same V-segment (IgKV1-12-01),
but the closest J-segment differed between antibodies. Consensus VL
sequences are shown, where "X" indicates positions at which
alternative amino acids (selected from those aligned at the
indicated position) are possible. The CDR1, CDR2, and CDR3
sequences are underlined in each VL sequence. The consensus CDR
sequences are further defined in Table 4.
[0017] FIGS. 3A-3D: Binding curves of HER2 antibodies to (FIG. 3A,
FIG. 3B) high (AU565) and (FIG. 3C, FIG. 3D) low (A431) HER2
expressing cell lines, determined as described in Example 12. Data
shown are mean fluorescence intensities (MFI) of one representative
experiment for each cell line. The EC.sub.50 values indicate the
apparent affinities.
[0018] FIG. 4: Binding of HER2 antibodies to HER2 expressed on
monkey Rhesus epithelial cells. Data shown are mean fluorescence
intensities (MFI) of one experiment, described in Example 13.
[0019] FIG. 5: Chromium-release (ADCC) assay of HER2 antibodies,
showing PBMC-mediated lysis of .sup.51Cr-labeled SK-BR-3 cells
after incubation with HER2 antibody. Values depicted are the mean
maximum percentages .sup.51Cr-release t the standard deviation from
one representative in vitro ADCC experiment with SK-BR-3 cells. See
Example 15 for details.
[0020] FIG. 6: Effect of HER2 antibodies on the proliferation of
AU565 cells, as compared to untreated cells (set to 100%). Data
shown are percentages proliferation of AU565 cells compared to
untreated cells measured in three independent experiments t the
standard deviation. * Significant (P<0.05). See Example 16 for
details.
[0021] FIG. 7: Percentage of viable MCF7 cells stimulated with
Heregulin-01 and treated with the indicated HER2 antibodies,
relative to cells stimulated with Heregulin-01 only. As a control,
the percentage proliferation of unstimulated cells is shown (none).
Data was obtained from three independent experiments t the stdev. *
Significant inhibition of Heregulin-01-induced proliferation
(P<0.05). See Example 17 for details.
[0022] FIGS. 8A-8D: ADC assay, showing killing of AU565 cells (FIG.
8A, FIG. 8B) or A431 cells (FIG. 8C, FIG. 8D) via
anti-kappa-ETA'-conjugated HER2 antibodies. (FIG. 8A, FIG. 8B) Data
shown are fluorescence intensities (FI) of one representative
experiment with AU565 cells treated with non-conjugated and
anti-kappa-ETA'-conjugated HER2 antibodies. (FIG. 8C, FIG. 8D) Data
shown are mean fluorescence intensities (MFI) of one representative
experiment with A431 cells treated with non-conjugated and
anti-kappa-ETA'-conjugated HER2 antibodies. See Example 18 for
details.
[0023] FIG. 9: Killing of A431 cells induced by anti-kappa-ETA'
pre-incubated HER2.times.HER2 bispecific antibodies. The viability
of A431 cells after 3 days incubation with HER2 antibodies,
pre-incubated with anti-kappa-ETA'. Cell viability was quantified
using Alamarblue. Data shown are fluorescence intensities (FI) of
one experiment with A431 cells treated with
anti-kappa-ETA'-conjugated HER2 antibodies and HER2.times.HER2
bispecific antibodies. Staurosporin was used as positive control,
whereas an isotype control antibody was used as negative
control.
[0024] FIG. 10: HER2.times.HER2 bispecific molecules induced
downmodulation of HER2 receptor. Relative percentage of HER2
expression levels in AU565 cell lysates after 3 days incubation
with 10 .mu.g/mL mAb. The amount of HER2 was quantified using a
HER2-specific capture ELISA and depicted as percentage inhibition
compared to untreated cells. Data shown is the mean of two
experiments plus standard deviation, except for combinations of
monospecific IgG1 antibodies which were tested once.
[0025] FIGS. 11A and 11B: Colocalization analysis of
HER2.times.HER2 bispecific antibodies (FITC) with lysosomal marker
LAMP1 (Cy5). FITC pixel intensity overlapping with Cy5 for various
monospecific HER2 antibodies and HER2.times.HER2 bispecific
antibodies (FIG. 11A) FITC pixel intensity in LAMP1/Cy5 positive
pixels of three different images is plotted for each antibody
tested. Monospecifics show lower FITC pixel intensities in the
LAMP1/Cy5 positive pixels compared to bispecifics. FIG. 11B
represents the mean value of FITC pixel intensity per LAMP1/Cy5
positive pixel calculated from the three different images. Together
these results indicate that after internalization higher levels of
bispecific antibodies, compared to monospecifics antibodies,
localize to Lamp1/Cy5 positive vesicles.
[0026] FIG. 12: Inhibition of proliferation by HER-2 mono and
bispecific antibodies. AU565 cells were seeded in the presence of
10 .mu.g/mL HER2 antibody or HER2.times.HER2 bispecific antibody in
serum-free cell culture medium. After three days, the amount of
viable cells was quantified with Alamarblue and cell viability was
presented as a percentage relative to untreated cells. An isotype
control antibody was used as negative control. Data shown are
percentage viable AU565 cells compared to untreated cells measured
in five-fold t the standard deviation. * indicates only one data
point was depicted.
[0027] FIG. 13: T cell mediated cytotoxicity of AU565 cells by
HER2.times.CD3 bispecific antibodies as well as by N297Q mutants of
HER2.times.CD3 bispecific antibodies (bispecific indicated as Duo
in the figure).
[0028] FIG. 14: Antibody induced downmodulation of HER2. Relative
percentage of HER2 expressed in AU565 cell lysate after 3 days
incubation with 10 .mu.g/mL antibody. The amount of HER2 was
quantified using a HER2-specific capture ELISA and plotted as a
percentage relative to untreated cells. Data shown are mean of
three experiments.+-.standard deviation.
[0029] FIG. 15: Colocalization analysis of HER2 antibodies (FITC)
with lysosomal marker LAMP1 (Cy5). FITC pixel intensity overlapping
with Cy5 for various monospecific HER2 antibodies. FITC pixel
intensity in LAMP1/Cy5 positive pixels of three different images is
plotted for each antibody. Group 3 antibodies 098 and 153 show
higher FITC pixel intensities in the LAMP1/Cy5 positive
compartments compared to antibodies 025 and pertuzumab from Group 2
and 169 and Herceptin from Group 1.
[0030] FIG. 16: HER2 antibody binding to CHO--S cells transfected
with different HER2 ECD construct analyzed by means of flow
cytometry. Hu-HER2=fully human HER2, Hu-HER2-ch(I) CR1=hu-HER2 with
chicken domain I, Hu-HER2-ch(II)=hu-HER2 with chicken domain II,
hu-HER2-ch(III)=hu-HER2 with chicken domain III and
Hu-HER2-ch(IV)=hu-HER2 with chicken domain IV. Data shown are mean
fluorescence intensities (MFI) of one representative antibody,
TH1014-153. See example 25 for details.
[0031] FIGS. 17A and 17B: In vivo effect of HER2-HuMabs in the
NCI-N87 human gastric carcinoma xenograft model in female CB.17
severe combined immunodeficiency (SCID) mice. Data shown are mean
tumorsize S.E.M. per group (n=10 mice per group) (FIG. 17A) and
survival (FIG. 17B). See example 29 for details.
[0032] FIGS. 18A and 18B: In vivo effect of HER2 HuMabs in BT-474
breast tumor xenografts in Balb/C nude mice. Data shown are mean
tumorsize t S.E.M. per group (n=8 mice per group) (FIG. 18A) and
survival (FIG. 18B). See example 30 for details.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0033] The term "HER2" (also known as ErbB-2, NEU, HER-2, and
CD340), when used herein, refers to human epidermal growth factor
receptor 2 (SwissProt P04626) and includes any variants, isoforms
and species homologs of HER2 which are naturally expressed by
cells, including tumor cells, or are expressed on cells transfected
with the HER2 gene. Species homologs include rhesus monkey HER2
(Macaca mulatta; Genbank accession No. GI:109114897).
[0034] The term "immunoglobulin" refers to a class of structurally
related glycoproteins consisting of two pairs of polypeptide
chains, one pair of light (L) low molecular weight chains and one
pair of heavy (H) chains, all four inter-connected by disulfide
bonds. The structure of immunoglobulins has been well
characterized. See for instance Fundamental Immunology Ch. 7 (Paul,
W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy
chain typically is comprised of a heavy chain variable region
(abbreviated herein as V.sub.H or VH) and a heavy chain constant
region. The heavy chain constant region typically is comprised of
three domains, C.sub.H1, C.sub.H2, and C.sub.H3. Each light chain
typically is comprised of a light chain variable region
(abbreviated herein as V.sub.L or VL) and a light chain constant
region. The light chain constant region typically is comprised of
one domain, C.sub.L. The V.sub.H and V.sub.L regions may be further
subdivided into regions of hypervariability (or hypervariable
regions which may be hypervariable in sequence and/or form of
structurally defined loops), also termed complementarity
determining regions (CDRs), interspersed with regions that are more
conserved, termed framework regions (FRs). Each V.sub.H and V.sub.L
is typically composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol.
Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted
by context, CDR sequences herein are identified according to IMGT
rules (Brochet X., Nucl Acids Res. 2008; 36:W503-508 and Lefranc M
P., Nucleic Acids Research 1999; 27:209-212; see also internet http
address
imgt.cines.fr/IMGT_vquest/vquest?livret=0&Option=humanIg.
However, the numbering of amino acid residues in an antibody
sequence can also be performed by the method described in Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991) (phrases such as "variable domain residue numbering as in
Kabat", "Kabat position" or "according to Kabat" herein refer to
this numbering system). Particularly, for numbering of amino acids
in the constant region, the EU index numbering system according to
Kabat et al, supra, can be used. 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.
[0035] The term "antibody" (Ab) in the context of the present
invention refers to an immunoglobulin molecule, a fragment of an
immunoglobulin molecule, or a derivative of either thereof, which
has the ability to specifically bind to an antigen under typical
physiological conditions with a half life of significant periods of
time, such as at least about 30 minutes, at least about 45 minutes,
at least about one hour, at least about two hours, at least about
four hours, at least about 8 hours, at least about 12 hours, about
24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or
more days, etc., or any other relevant functionally-defined period
(such as a time sufficient to induce, promote, enhance, and/or
modulate a physiological response associated with antibody binding
to the antigen and/or time sufficient for the antibody to recruit
an effector activity). The variable regions of the heavy and light
chains of the immunoglobulin molecule contain a binding domain that
interacts with an antigen. The constant regions of the antibodies
(Abs) may mediate the binding of the immunoglobulin to host tissues
or factors, including various cells of the immune system (such as
effector cells) and components of the complement system such as
C1q, the first component in the classical pathway of complement
activation. A HER2 antibody may also be a bispecific antibody,
diabody, or similar molecule (see for instance PNAS USA 90(14),
6444-8 (1993) for a description of diabodies). Indeed, bispecific
antibodies, diabodies, and the like, provided by the present
invention may bind any suitable target in addition to a portion of
HER2. As indicated above, the term antibody herein, unless
otherwise stated or clearly contradicted by context, includes
fragments of an antibody that are antigen-binding fragments, i.e.,
retain the ability to specifically bind to the antigen. It has been
shown that the antigen-binding function of an antibody may be
performed by fragments of a full-length antibody. Examples of
antigen-binding fragments encompassed within the term "antibody"
include (i) a Fab' or Fab fragment, a monovalent fragment
consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains,
or a monovalent antibody as described in WO2007059782 (Genmab);
(ii) F(ab').sub.2 fragments, bivalent fragments comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting essentially of the V.sub.H and C.sub.H1
domains; (iv) a Fv fragment consisting essentially of the V.sub.L
and V.sub.H domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., Nature 341, 544-546 (1989)), which consists
essentially of a V.sub.H domain and also called domain antibodies
(Holt et al; Trends Biotechnol. 2003 November; 21(11):484-90); (vi)
camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005
January; 5(1):111-24) and (vii) an isolated complementarity
determining region (CDR). Furthermore, although the two domains of
the Fv fragment, V.sub.L and V.sub.H, are coded for by separate
genes, they may be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the V.sub.L and V.sub.H regions pair to form
monovalent molecules (known as single chain antibodies or single
chain Fv (scFv), see for instance Bird et al., Science 242, 423-426
(1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such
single chain antibodies are encompassed within the term antibody
unless otherwise noted or clearly indicated by context. Although
such fragments are generally included within the meaning of
antibody, they collectively and each independently are unique
features of the present invention, exhibiting different biological
properties and utility. These and other useful antibody fragments
in the context of the present invention, as well as bispecific
formats of such fragments, are discussed further herein. It also
should be understood that the term antibody, unless specified
otherwise, also includes polyclonal antibodies, monoclonal
antibodies (mAbs), antibody-like polypeptides, such as chimeric
antibodies and humanized antibodies, and antibody fragments
retaining the ability to specifically bind to the antigen
(antigen-binding fragments) provided by any known technique, such
as enzymatic cleavage, peptide synthesis, and recombinant
techniques. An antibody as generated can possess any isotype.
[0036] As used herein, "isotype" refers to the immunoglobulin class
(for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that
is encoded by heavy chain constant region genes.
[0037] The term "monovalent antibody" means in the context of the
present invention that an antibody molecule is capable of binding a
single molecule of the antigen, and thus is not able of antigen
crosslinking.
[0038] An "antibody deficient in effector function" or an
"effector-function-deficient antibody" refers to an antibody which
has a significantly reduced or no ability to activate one or more
effector mechanisms, such as complement activation or Fc receptor
binding. Thus, effector-function deficient antibodies have
significantly reduced or no ability to mediate antibody-dependent
cell-mediated cytotoxicity (ADCC) and/or complement-dependent
cytotoxicity (CDC). An example of such an antibody is IgG4.
[0039] A "HER2 antibody" or "anti-HER2 antibody" is an antibody as
described above, which binds specifically to the antigen HER2.
[0040] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo). However, the term "human antibody", as used
herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework
sequences.
[0041] As used herein, a human antibody is "derived from" a
particular germline sequence if the antibody is obtained from a
system using human immunoglobulin sequences, for instance by
immunizing a transgenic mouse carrying human immunoglobulin genes
or by screening a human immunoglobulin gene library, and wherein
the selected human antibody is at least 90%, such as at least 95%,
for instance at least 96%, such as at least 97%, for instance at
least 98%, or such as at least 99% identical in amino acid sequence
to the amino acid sequence encoded by the germline immunoglobulin
gene. Typically, outside the heavy chain CDR3, a human antibody
derived from a particular human germline sequence will display no
more than 20 amino acid differences, e.g. no more than 10 amino
acid differences, such as no more than 9, 8, 7, 6 or 5, for
instance no more than 4, 3, 2, or 1 amino acid difference from the
amino acid sequence encoded by the germline immunoglobulin
gene.
[0042] In a preferred embodiment, the antibody of the invention is
isolated. An "isolated antibody," as used herein, is intended to
refer to an antibody which is substantially free of other
antibodies having different antigenic specificities (for instance
an isolated antibody that specifically binds to HER2 is
substantially free of antibodies that specifically bind antigens
other than HER2). An isolated antibody that specifically binds to
an epitope, isoform or variant of HER2 may, however, have
cross-reactivity to other related antigens, for instance from other
species (such as HER2 species homologs). Moreover, an isolated
antibody may be substantially free of other cellular material
and/or chemicals. In one embodiment of the present invention, two
or more "isolated" monoclonal antibodies having different
antigen-binding specificities are combined in a well-defined
composition.
[0043] When used herein in the context of two or more antibodies,
the term "competes with" or "cross-competes with" indicates that
the two or more antibodies compete for binding to HER2, e.g.
compete for HER2 binding in the assay described in Example 14. An
antibody "blocks" or "cross-blocks" one or more other antibodies
from binding to HER2 if the antibody competes with the one or more
other antibodies 25% or more, with 25%-74% representing "partial
block" and 75%-100% representing "full block", preferably as
determined using the assay of Example 14. For some pairs of
antibodies, competition or blocking in the assay of the Examples is
only observed when one antibody is coated on the plate and the
other is used to compete, and not vice versa. Unless otherwise
defined or negated by context, the terms "competes with",
"cross-competes with", "blocks" or "cross-blocks" when used herein
is also intended to cover such pairs of antibodies.
[0044] The term "epitope" means a protein determinant capable of
specific binding to an antibody. Epitopes usually consist of
surface groupings of molecules such as amino acids or sugar side
chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics.
Conformational and nonconformational epitopes are distinguished in
that the binding to the former but not the latter is lost in the
presence of denaturing solvents. The epitope may comprise amino
acid residues directly involved in the binding (also called
immunodominant component of the epitope) and other amino acid
residues, which are not directly involved in the binding, such as
amino acid residues which are effectively blocked or covered by the
specifically antigen binding peptide (in other words, the amino
acid residue is within the footprint of the specifically antigen
binding peptide).
[0045] The term "monoclonal antibody" as used herein refers to a
preparation of antibody molecules of single molecular composition.
A monoclonal antibody composition displays a single binding
specificity and affinity for a particular epitope. Accordingly, the
term "human monoclonal antibody" refers to antibodies displaying a
single binding specificity which have variable and constant regions
derived from human germline immunoglobulin sequences. The human
monoclonal antibodies may be generated by a hybridoma which
includes a B cell obtained from a transgenic or transchromosomal
nonhuman animal, such as a transgenic mouse, having a genome
comprising a human heavy chain transgene and a light chain
transgene, fused to an immortalized cell.
[0046] As used herein, the term "binding" in the context of the
binding of an antibody to a predetermined antigen or epitope
typically is a binding with an affinity corresponding to a K.sub.D
of about 10.sup.-7 M or less, such as about 10.sup.-8 M or less,
such as about 10.sup.-9 M or less, about 10.sup.-10 M or less, or
about 10.sup.-11 M or even less when determined by for instance
surface plasmon resonance (SPR) technology in a BIAcore 3000
instrument using the antigen as the ligand and the antibody as the
analyte, and binds to the predetermined antigen with an affinity
corresponding to a K.sub.D that is at least ten-fold lower, such as
at least 100 fold lower, for instance at least 1,000 fold lower,
such as at least 10,000 fold lower, for instance at least 100,000
fold lower than its affinity for binding to a non-specific antigen
(e.g., BSA, casein) other than the predetermined antigen or a
closely-related antigen. The amount with which the affinity is
lower is dependent on the K.sub.D of the antibody, so that when the
K.sub.D of the antibody is very low (that is, the antibody is
highly specific), then the amount with which the affinity for the
antigen is lower than the affinity for a non-specific antigen may
be at least 10,000 fold.
[0047] The term "k.sub.d" (sec.sup.-1), as used herein, refers to
the dissociation rate constant of a particular antibody-antigen
interaction. Said value is also referred to as the k.sub.off
value.
[0048] The term "k.sub.a" (M.sup.-1.times.sec.sup.-1), as used
herein, refers to the association rate constant of a particular
antibody-antigen interaction.
[0049] The term "K.sub.D" (M), as used herein, refers to the
dissociation equilibrium constant of a particular antibody-antigen
interaction.
[0050] The term "K.sub.A" (M.sup.-1), as used herein, refers to the
association equilibrium constant of a particular antibody-antigen
interaction and is obtained by dividing the k.sub.a by the
k.sub.d.
[0051] As used herein, the term "inhibits proliferation" (e.g.
referring to cells, such as tumor cells) is intended to include any
substantial decrease in the cell proliferation when contacted with
a HER2 antibody as compared to the proliferation of the same cells
not in contact with a HER2 antibody, e.g., the inhibition of
proliferation of a cell culture by at least about 10%, at least
about 20% or at least about 30%, or at least as much as a reference
antibody such as trastuzumab, e.g., as determined by an assay in
the Examples, e.g. example 16.
[0052] As used herein, the term "promotes proliferation" (e.g.
referring to cells, such as tumor cells) is intended to include any
substantial increase in the cell proliferation when contacted with
a HER2 antibody as compared to the proliferation of the same cells
not in contact with a HER2 antibody, e.g., the promotion of
proliferation of a cell culture by at least about 10%, at least
about 20% or at least about 30%, or at least as much as a reference
antibody as F5, e.g., as determined by an assay in the
Examples.
[0053] As used herein, the term "internalization", when used in the
context of a HER2 antibody includes any mechanism by which the
antibody is internalized into a HER2-expressing cell from the
cell-surface and/or from surrounding medium, e.g., via endocytosis.
The internalization of an antibody can be evaluated using a direct
assay measuring the amount of internalized antibody (such as, e.g.,
the fab-CypHer5E assay described in Example 18), or an indirect
assay where the effect of an internalized antibody-toxin conjugate
is measured (such as, e.g., the anti-kappa-ETA' assay of Example
17).
[0054] The present invention also provides antibodies comprising
functional variants of the V.sub.L region, V.sub.H region, or one
or more CDRs of the antibodies of the examples. A functional
variant of a V.sub.L, V.sub.H, or CDR used in the context of a HER2
antibody still allows the antibody to retain at least a substantial
proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of
the affinity/avidity and/or the specificity/selectivity of the
parent antibody and in some cases such a HER2 antibody may be
associated with greater affinity, selectivity and/or specificity
than the parent antibody.
[0055] Such functional variants typically retain significant
sequence identity to the parent antibody. The percent identity
between two sequences is a function of the number of identical
positions shared by the sequences (i.e., % homology=# of identical
positions/total # of positions.times.100), taking into account the
number of gaps, and the length of each gap, which need to be
introduced for optimal alignment of the two sequences. The percent
identity between two nucleotide or amino acid sequences may e.g. be
determined using the algorithm of E. Meyers and W. Miller, Comput.
Appl. Biosci 4, 11-17 (1988) which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4. In addition, the
percent identity between two amino acid sequences may be determined
using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970)
algorithm.
[0056] Exemplary variants include those which differ from a parent
antibody VH and/or VL sequence shown in FIGS. 1 and 2 at one or
more "variant" amino acid positions, denoted "X" in the
corresponding consensus sequence. Preferred variants are those in
which the new amino acid is selected from those at the
corresponding position in one of the aligned sequences in FIG. 1 or
2 (for details on CDR sequence variants, see Table 4).
Alternatively or additionally, the sequence of VH, VL or CDR
variants may differ from the sequence of the VH, VL or CDR of the
parent antibody sequences mainly by conservative substitutions; for
instance at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1
of the substitutions in the variant are conservative amino acid
residue replacements.
[0057] In the context of the present invention, conservative
substitutions may be defined by substitutions within the classes of
amino acids reflected in the following table:
TABLE-US-00001 Amino acid residue classes for conservative
substitutions Acidic Residues Asp (D) and Glu (E) Basic Residues
Lys (K), Arg (R), and His (H) Hydrophilic Uncharged Ser (S), Thr
(T), Asn (N), and Residues Gln (Q) Aliphatic Uncharged Gly (G), Ala
(A), Val (V), Leu (L), Residues and Ile (I) Non-polar Uncharged Cys
(C), Met (M), and Pro (P) Residues Aromatic Residues Phe (F), Tyr
(Y), and Trp (W)
[0058] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which an
expression vector has been introduced, e.g. an expression vector
encoding an antibody of the invention. Recombinant host cells
include, for example, transfectomas, such as CHO cells, HEK293
cells, NS/0 cells, and lymphocytic cells.
[0059] The term "transgenic non-human animal" refers to a non-human
animal having a genome comprising one or more human heavy and/or
light chain transgenes or transchromosomes (either integrated or
non-integrated into the animal's natural genomic DNA) and which is
capable of expressing fully human antibodies. For example, a
transgenic mouse can have a human light chain transgene and either
a human heavy chain transgene or human heavy chain transchromosome,
such that the mouse produces human HER2 antibodies when immunized
with HER2 antigen and/or cells expressing HER2. The human heavy
chain transgene may be integrated into the chromosomal DNA of the
mouse, as is the case for transgenic mice, for instance HuMAb mice,
such as HCo7, HCo12, or HCo17 mice, or the human heavy chain
transgene may be maintained extrachromosomally, as is the case for
transchromosomal KM mice as described in WO02/43478. Similar mice,
having a larger human Ab gene repertoire, include HCo7 and HCo20
(see e.g. WO2009097006). Such transgenic and transchromosomal mice
(collectively referred to herein as "transgenic mice") are capable
of producing multiple isotypes of human monoclonal antibodies to a
given antigen (such as IgG, IgA, IgM, IgD and/or IgE) by undergoing
V-D-J recombination and isotype switching. Transgenic, nonhuman
animal can also be used for production of antibodies against a
specific antigen by introducing genes encoding such specific
antibody, for example by operatively linking the genes to a gene
which is expressed in the milk of the animal.
[0060] "Treatment" refers to the administration of an effective
amount of a therapeutically active compound of the present
invention with the purpose of easing, ameliorating, arresting or
eradicating (curing) symptoms or disease states.
[0061] An "effective amount" or "therapeutically effective amount"
refers to an amount effective, at dosages and for periods of time
necessary, to achieve a desired therapeutic result. A
therapeutically effective amount of a HER2 antibody may vary
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the HER2 antibody to
elicit a desired response in the individual. A therapeutically
effective amount is also one in which any toxic or detrimental
effects of the antibody or antibody portion are outweighed by the
therapeutically beneficial effects.
[0062] An "anti-idiotypic" antibody is an antibody which recognizes
unique determinants generally associated with the antigen-binding
site of an antibody.
Further Aspects and Embodiments of the Invention
[0063] As described above, in a first aspect, the invention relates
to a monoclonal antibody which binds HER2.
[0064] Monoclonal antibodies of the present invention may be
produced, e.g., by the hybridoma method first described by Kohler
et al., Nature 256, 495 (1975), or may be produced by recombinant
DNA methods. Monoclonal antibodies may also be isolated from phage
antibody libraries using the techniques described in, for example,
Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J.
Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may be
obtained from any suitable source. Thus, for example, monoclonal
antibodies may be obtained from hybridomas prepared from murine
splenic B cells obtained from mice immunized with an antigen of
interest, for instance in form of cells expressing the antigen on
the surface, or a nucleic acid encoding an antigen of interest.
Monoclonal antibodies may also be obtained from hybridomas derived
from antibody-expressing cells of immunized humans or non-human
mammals such as rats, dogs, primates, etc.
[0065] In one embodiment, the antibody of the invention is a human
antibody. Human monoclonal antibodies directed against HER2 may be
generated using transgenic or transchromosomal mice carrying parts
of the human immune system rather than the mouse system. Such
transgenic and transchromosomic mice include mice referred to
herein as HuMAb mice and KM mice, respectively, and are
collectively referred to herein as "transgenic mice".
[0066] The HuMAb mouse contains a human immunoglobulin gene
miniloci that encodes unrearranged human heavy (.mu. and .gamma.)
and .kappa. light chain immunoglobulin sequences, together with
targeted mutations that inactivate the endogenous .mu. and .kappa.
chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)).
Accordingly, the mice exhibit reduced expression of mouse IgM or
.kappa. and in response to immunization, the introduced human heavy
and light chain transgenes, undergo class switching and somatic
mutation to generate high affinity human IgG,.kappa. monoclonal
antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg,
N. Handbook of Experimental Pharmacology 113, 49-101 (1994),
Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93
(1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci 764
536-546 (1995)). The preparation of HuMAb mice is described in
detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295
(1992), Chen, J. et al., International Immunology 5, 647-656
(1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor,
L. et al., International Immunology 6, 579-591 (1994), Fishwild, D.
et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat.
Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650,
5,877,397, 5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807,
WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and
WO 01/09187.
[0067] The HCo7, HCo12, HCo17 and HCo20 mice have a JKD disruption
in their endogenous light chain (kappa) genes (as described in Chen
et al., EMBO J. 12, 821-830 (1993)), a CMD disruption in their
endogenous heavy chain genes (as described in Example 1 of WO
01/14424), and a KCo5 human kappa light chain transgene (as
described in Fishwild et al., Nature Biotechnology 14, 845-851
(1996)). Additionally, the Hco7 mice have a HCo7 human heavy chain
transgene (as described in U.S. Pat. No. 5,770,429), the HCo12 mice
have a HCo12 human heavy chain transgene (as described in Example 2
of WO 01/14424), the HCo17 mice have a HCo17 human heavy chain
transgene (as described in Example 2 of WO 01/09187) and the HCo20
mice have a HCo20 human heavy chain transgene. The resulting mice
express human immunoglobulin heavy and kappa light chain transgenes
in a background homozygous for disruption of the endogenous mouse
heavy and kappa light chain loci.
[0068] In the KM mouse strain, the endogenous mouse kappa light
chain gene has been homozygously disrupted as described in Chen et
al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy
chain gene has been homozygously disrupted as described in Example
1 of WO 01/09187. This mouse strain carries a human kappa light
chain transgene, KCo5, as described in Fishwild et al., Nature
Biotechnology 14, 845-851 (1996). This mouse strain also carries a
human heavy chain transchromosome composed of chromosome 14
fragment hCF (SC20) as described in WO 02/43478. HCo12-Balb/C mice
can be generated by crossing HCo12 to KCo5[J/K](Balb) as described
in WO/2009/097006.
[0069] Splenocytes from these transgenic mice may be used to
generate hybridomas that secrete human monoclonal antibodies
according to well known techniques.
[0070] Further, human antibodies of the present invention or
antibodies of the present invention from other species may be
identified through display-type technologies, including, without
limitation, phage display, retroviral display, ribosomal display,
and other techniques, using techniques well known in the art and
the resulting molecules may be subjected to additional maturation,
such as affinity maturation, as such techniques are well known in
the art (see for instance Hoogenboom et al., J. Mol. Biol. 227, 381
(1991) (phage display), Vaughan et al., Nature Biotech 14, 309
(1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942
(1997) (ribosomal display), Parmley and Smith, Gene 73, 305-318
(1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et
al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. Acids
Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews
130, 43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84
(1992), and U.S. Pat. No. 5,733,743). If display technologies are
utilized to produce antibodies that are not human, such antibodies
may be humanized.
Antibodies of Cross-Block Group 1
[0071] In one aspect of the antibody of the invention, the antibody
binds the same epitope on HER2 as one or more of the novel human
antibodies of cross-block group 1 described herein.
[0072] In one embodiment, the antibody cross-blocks the binding to
soluble HER2 of trastuzumab, when determined as described in
Example 14.
[0073] In one embodiment, the antibody binds the same epitope as a
reference antibody comprising a VH region comprising the sequence
of SEQ ID NO:1 and a VL region comprising the sequence of SEQ ID
NO:5 (169).
[0074] In one embodiment, the antibody binds the same epitope as a
reference antibody comprising a VH region comprising the sequence
of SEQ ID NO:8 and a VL region comprising the sequence of SEQ ID
NO: 12 (050).
[0075] In one embodiment, the antibody binds the same epitope as a
reference antibody comprising a VH region comprising the sequence
of SEQ ID NO:15 and a VL region comprising the sequence of SEQ ID
NO:84 (084).
[0076] In one embodiment, the antibody binds to the same epitope as
a reference antibody comprising VH and VL regions selected from the
group consisting of: [0077] a) a VH region comprising the sequence
of SEQ ID NO:77 and a VL region comprising the sequence of SEQ ID
NO:78 (049); [0078] b) a VH region comprising the sequence of SEQ
ID NO:79 and a VL region comprising the sequence of SEQ ID NO:80
(051); [0079] c) a VH region comprising the sequence of SEQ ID
NO:81 and a VL region comprising the sequence of SEQ ID NO:82
(055); [0080] d) a VH region comprising the sequence of SEQ ID
NO:83 and a VL region comprising the sequence of SEQ ID NO:84
(123); [0081] e) a VH region comprising the sequence of SEQ ID
NO:85 and a VL region comprising the sequence of SEQ ID NO:86
(161); and [0082] f) a VH region comprising the sequence of SEQ ID
NO:87 and a VL region comprising the sequence of SEQ ID NO:88
(124).
[0083] In another additional or alternative aspect of the antibody
of the invention, the antibody binds to HER2 and comprises a VH
CDR3, VH region and/or VL region sequence similar or identical to a
sequence of the novel antibodies described herein.
[0084] In one embodiment, the antibody comprises a VH CDR3 region
having a sequence selected from the group consisting of
[0085] SEQ ID NO:11 (050, 049, 051, 055), optionally wherein the VH
region is derived from the IgHV3-21-1 germline sequence;
[0086] SEQ ID No: 130, such as the sequence of SEQ ID NO: 18 (084),
optionally wherein the VH region is derived from the IgHV1-69-04
germline sequence;
[0087] SEQ ID NO: 133 (169, 123, 161, 124), such as the sequence of
SEQ ID NO:4 (169), optionally wherein the VH region is derived from
the IgHV1-18-1 germline sequence; or
[0088] In one embodiment, the antibody comprises a VH CDR3 region
of one of antibodies 123, 161, or 124, as shown in FIG. 1,
optionally wherein the VH region is derived from an IgHV1-18-1
germline.
[0089] In one embodiment, the antibody comprises a VH region
selected from the group consisting of [0090] a) a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:9, 127
and 11, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 9,
10 and 11 (050); optionally where the VH region is derived from an
IgHV3-23-1 germline; [0091] b) a VH region comprising the CDR1,
CDR2 and CDR3 sequences of SEQ ID NOs:128, 129 and 130, such the
CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:16, 17 and 18,
respectively (084), optionally where the VH region is derived from
an IgHV1-69-04 germline; and [0092] c) a VH region comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:131, 132, and 133,
such as the CRD1, CDR2, and CDR3 sequences of SEQ ID NOs: 2, 3 and
4 (169), respectively, optionally where the VH region is derived
from an IgHV1-18-1 germline.
[0093] In one embodiment, the antibody comprises a VH region
selected from the preceding embodiments (a) or (b) and a VL region
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 13, XAS
(wherein X is A or V), and SEQ ID No: 155, respectively, such as a
CDR1 sequence selected from SEQ ID Nos: 13 or 20, a CDR2 which is
AAS or VAS, and a CDR3 sequence selected from SEQ ID NOs:14 and 21
(050, 084); respectively, optionally where the VL region is derived
from an IgKV1-12-01 germline.
[0094] In one embodiment, the antibody comprises a VH region which
is the preceding embodiment (c) and a VL region comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6, DXS (wherein X=A or
T), and SEQ ID NO:156 (169), respectively, optionally wherein the
VL region is derived from IgKV3-11-01.
[0095] In one embodiment, the antibody comprises a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:2, 3 and
4, respectively; and a VL region comprising the CDR1, CDR2 and CDR3
sequences of SEQ ID NOs:6, DAS, and SEQ ID NO:7, respectively
(169).
[0096] In one embodiment, the antibody comprises a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:9, 10
and 11, respectively; and a VL region comprising the CDR1, CDR2 and
CDR3 sequences of SEQ ID NOs:13, AAS, and SEQ ID NO: 14,
respectively (050).
[0097] In one embodiment, the antibody comprises a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:16, 17
and 18, respectively; and a VL region comprising the CDR1, CDR2 and
CDR3 sequences of SEQ ID NOs:20, VAS, and SEQ ID NO:21,
respectively (084).
[0098] In separate embodiments, the antibody comprises: [0099] a) a
VH region comprising the sequence of SEQ ID NO:1 and, preferably, a
VL region comprising the sequence of SEQ ID NO:5 (169); [0100] b) a
VH region comprising the sequence of SEQ ID NO:8 and, preferably, a
VL region comprising the sequence of SEQ ID NO: 12 (050); [0101] c)
a VH region comprising the sequence of SEQ ID NO: 15 and,
preferably, a VL region comprising the sequence of SEQ ID NO: 19
(084); [0102] d) a VH region comprising the sequence of SEQ ID
NO:77 and, preferably, a VL region comprising the sequence of SEQ
ID NO:78 (049); [0103] e) a VH region comprising the sequence of
SEQ ID NO:79 and, preferably, a VL region comprising the sequence
of SEQ ID NO:80 (051); [0104] f) a VH region comprising the
sequence of SEQ ID NO:81 and, preferably, a VL region comprising
the sequence of SEQ ID NO:82 (055); [0105] g) a VH region
comprising the sequence of SEQ ID NO:83 and, preferably, a VL
region comprising the sequence of SEQ ID NO:84 (123); [0106] h) a
VH region comprising the sequence of SEQ ID NO:85 and, preferably,
a VL region comprising the sequence of SEQ ID NO:86 (161); [0107]
i) a VH region comprising the sequence of SEQ ID NO:87 and,
preferably, a VL region comprising the sequence of SEQ ID NO:88
(124); and/or [0108] j) a variant of any of said antibodies,
wherein said variant preferably has at most 1, 2 or 3 amino-acid
modifications, more preferably amino-acid substitutions, such as
conservative amino acid substitutions and substitutions where the
new amino acid is one at the same position in an aligned sequence
in FIG. 1 or 2, particularly at positions indicated by "X" in the
corresponding consensus sequence.
Antibodies of Cross-Block Group 2
[0109] In one aspect of the antibody of the invention, the antibody
binds the same epitope on HER2 as one or more of the novel human
antibodies of cross-block group 2 described herein.
[0110] In one embodiment, the antibody cross-blocks the binding to
soluble HER2 of pertuzumab, when determined as described in Example
14.
[0111] In one embodiment, the antibody binds the same epitope as a
reference antibody comprising a VH region comprising the sequence
of SEQ ID NO:22 and a VL region comprising the sequence of SEQ ID
NO:26 (025).
[0112] In one embodiment, the antibody binds the same epitope as a
reference antibody comprising a VH region comprising the sequence
of SEQ ID NO:29 and a VL region comprising the sequence of SEQ ID
NO:32 (091).
[0113] In one embodiment, the antibody binds the same epitope as a
reference antibody comprising a VH region comprising the sequence
of SEQ ID NO:35 and a VL region comprising the sequence of SEQ ID
NO:39 (129).
[0114] In one embodiment, the antibody binds to the same epitope as
a reference antibody comprising VH and VL regions selected from the
group consisting of: [0115] a) a VH region comprising the sequence
of SEQ ID NO:89 and a VL region comprising the sequence of SEQ ID
NO:90 (001); [0116] b) a VH region comprising the sequence of SEQ
ID NO:91 and a VL region comprising the sequence of SEQ ID NO:92
(143); [0117] c) a VH region comprising the sequence of SEQ ID
NO:93 and a VL region comprising the sequence of SEQ ID NO:94
(019); [0118] d) a VH region comprising the sequence of SEQ ID
NO:95 and a VL region comprising the sequence of SEQ ID NO:96
(021); [0119] e) a VH region comprising the sequence of SEQ ID
NO:97 and a VL region comprising the sequence of SEQ ID NO:98
(027); [0120] f) a VH region comprising the sequence of SEQ ID
NO:99 and a VL region comprising the sequence of SEQ ID NO: 100
(032) [0121] g) a VH region comprising the sequence of SEQ ID NO:
101 and a VL region comprising the sequence of SEQ ID NO: 102
(035); [0122] h) a VH region comprising the sequence of SEQ ID NO:
103 and a VL region comprising the sequence of SEQ ID NO: 104
(036); [0123] i) a VH region comprising the sequence of SEQ ID NO:
105 and a VL region comprising the sequence of SEQ ID NO: 106
(054); and [0124] j) a VH region comprising the sequence of SEQ ID
NO: 107 and a VL region comprising the sequence of SEQ ID NO: 108
(094).
[0125] In another additional or alternative aspect of the antibody
of the invention, the antibody binds to HER2 and comprises a VH
CDR3, VH region and/or VL region sequence similar or identical to a
sequence of the novel antibodies described herein.
[0126] In one embodiment, the antibody comprises a VH CDR3 region
having a sequence selected from the group consisting of
[0127] SEQ ID NO: 136, such as the sequence of SEQ ID NO:25 (025),
optionally wherein the VH region is derived from the IgHV4-34-1
germline sequence;
[0128] SEQ ID NO: 139, such as the sequence of SEQ ID NO:31 (091),
optionally wherein the VH region is derived from the IgHV4-34-01
germline sequence; and
[0129] SEQ ID NO: 142, such as the sequence of SEQ ID NO:38 (129),
optionally wherein the VH region is derived from the IgHV3-30-01
germline sequence.
[0130] In one embodiment, the antibody comprises a VH CDR3 region
of one of antibodies 001, 143, 019, 021, 027, 032, 035, 036, 054 or
094 as shown in FIG. 1, optionally wherein the VH region is derived
from an IgHV4-34-1 germline.
[0131] In one embodiment, the antibody comprises a VH region
selected from the group consisting of [0132] a) a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:134, 135
and 136, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS:
23, 24 and 25 (025); optionally where the VH region is derived from
an IgHV4-34-1 germline; [0133] b) a VH region comprising the CDR1,
CDR2 and CDR3 sequences of SEQ ID NOs:137, 138 and 139, such the
CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:30, 163, and 31,
respectively (091), optionally where the VH region is derived from
an IgHV4-34-01 germline; and [0134] c) a VH region comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:140, 141 and 142, such
as the CRD1, CDR2, and CDR3 sequences of SEQ ID NOs: 36, 37 and 38
(129), respectively, optionally where the VH region is derived from
an IgHV3-30-01 germline.
[0135] In one embodiment, the antibody comprises a VH region
selected from the preceding embodiment (a) and a VL region
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 157,
AAS, and SEQ ID No: 164, respectively, such as the CDR1, CDR2, and
CDR3 sequences of SEQ ID Nos:27, AAS, and SEQ ID NO:28 (025);
respectively, optionally where the VL region is derived from an
IgKV1D-16-01 germline.
[0136] In one embodiment, the antibody comprises a VH region
selected from the preceding embodiment (b) and a VL region
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:33,
AX.sub.1X.sub.2 (wherein X.sub.1 is A or T, preferably A; and
X.sub.2 is S or F, preferably S), and SEQ ID No: 158, respectively,
such as the CDR1, CDR2 and CDR3 sequences of SEQ ID Nos:33, AAS,
and SEQ ID NO:34 (091); respectively, optionally where the VL
region is derived from an IgKV1D-16-01 germline.
[0137] In one embodiment, the antibody comprises a VH region which
is the preceding embodiment (c) and a VL region comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NO:40, DAS and SEQ ID
NO:41 (129), respectively, optionally wherein the VL region is
derived from IgKV3-11-01.
[0138] In one embodiment, the antibody comprises a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:23, 24
and 25, respectively; and a VL region comprising the CDR1, CDR2 and
CDR3 sequences of SEQ ID NOs:27, AAS, and SEQ ID NO:28,
respectively (025).
[0139] In one embodiment, the antibody comprises a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:30, 163
and 31, respectively; and a VL region comprising the CDR1, CDR2 and
CDR3 sequences of SEQ ID NOs:33, AAS, and SEQ ID NO:34,
respectively (091).
[0140] In one embodiment, the antibody comprises a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:36, 37
and 38, respectively; and a VL region comprising the CDR1, CDR2 and
CDR3 sequences of SEQ ID NOs:40, DAS, and SEQ ID NO:41,
respectively (129).
[0141] In separate embodiments, the antibody comprises: [0142] a) a
VH region comprising the sequence of SEQ ID NO:22 and, preferably,
a VL region comprising the sequence of SEQ ID NO:26 (025); [0143]
b) a VH region comprising the sequence of SEQ ID NO:29 and,
preferably, a VL region comprising the sequence of SEQ ID NO:32
(091); [0144] c) a VH region comprising the sequence of SEQ ID
NO:35 and, preferably, a VL region comprising the sequence of SEQ
ID NO:39 (129); [0145] d) a VH region comprising the sequence of
SEQ ID NO:89 and, preferably, a VL region comprising the sequence
of SEQ ID NO:90 (001); [0146] e) a VH region comprising the
sequence of SEQ ID NO:91 and, preferably, a VL region comprising
the sequence of SEQ ID NO:92 (143); [0147] f) a VH region
comprising the sequence of SEQ ID NO:93 and, preferably, a VL
region comprising the sequence of SEQ ID NO:94 (019); [0148] g) a
VH region comprising the sequence of SEQ ID NO:95 and, preferably,
a VL region comprising the sequence of SEQ ID NO:96 (021); [0149]
h) a VH region comprising the sequence of SEQ ID NO:97 and,
preferably, a VL region comprising the sequence of SEQ ID NO:98
(027); [0150] i) a VH region comprising the sequence of SEQ ID
NO:99 and, preferably, a VL region comprising the sequence of SEQ
ID NO: 100 (032); [0151] j) a VH region comprising the sequence of
SEQ ID NO: 101 and, preferably, a VL region comprising the sequence
of SEQ ID NO: 102 (035); [0152] k) a VH region comprising the
sequence of SEQ ID NO: 103 and, preferably, a VL region comprising
the sequence of SEQ ID NO: 104 (036); [0153] l) a VH region
comprising the sequence of SEQ ID NO: 105 and, preferably, a VL
region comprising the sequence of SEQ ID NO: 106 (054); [0154] m) a
VH region comprising the sequence of SEQ ID NO: 106 and,
preferably, a VL region comprising the sequence of SEQ ID NO: 108
(094); and/or [0155] n) a variant of any of said antibodies,
wherein said variant preferably has at most 1, 2 or 3 amino-acid
modifications, more preferably amino-acid substitutions, such as
conservative amino acid substitutions and substitutions where the
new amino acid is one at the same position in an aligned sequence
in FIG. 1 or 2, particularly at positions indicated by "X" in the
corresponding consensus sequence.
Antibodies of Cross-Block Group 3
[0156] In one aspect of the antibody of the invention, the antibody
binds the same epitope on HER2 as one or more of the novel human
antibodies of cross-block group 3 described herein.
[0157] In one embodiment, the antibody cross-blocks the binding to
soluble HER2 of F5 and/or C5, when determined as described in
Example 14.
[0158] In one embodiment, the antibody binds the same epitope as a
reference antibody comprising a VH region comprising the sequence
of SEQ ID NO:46 and a VL region comprising the sequence of SEQ ID
NO:49 (127).
[0159] In one embodiment, the antibody binds the same epitope as a
reference antibody comprising a VH region comprising the sequence
of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID
NO:53 (159).
[0160] In one embodiment, the antibody binds the same epitope as a
reference antibody comprising a VH region comprising the sequence
of SEQ ID NO:56 and a VL region comprising the sequence of SEQ ID
NO:60 (098).
[0161] In one embodiment, the antibody binds the same epitope as a
reference antibody comprising a VH region comprising the sequence
of SEQ ID NO:63 and a VL region comprising the sequence of SEQ ID
NO:67 (153).
[0162] In one embodiment, the antibody binds the same epitope as a
reference antibody comprising a VH region comprising the sequence
of SEQ ID NO:70 and a VL region comprising the sequence of SEQ ID
NO:74 (132).
[0163] In one embodiment, the antibody binds to the same epitope as
a reference antibody comprising VH and VL regions selected from the
group consisting of: [0164] k) a VH region comprising the sequence
of SEQ ID NO: 109 and a VL region comprising the sequence of SEQ ID
NO:110 (105); [0165] l) a VH region comprising the sequence of SEQ
ID NO: 111 and a VL region comprising the sequence of SEQ ID NO:112
(100); [0166] m) a VH region comprising the sequence of SEQ ID
NO:113 and a VL region comprising the sequence of SEQ ID NO:114
(125); [0167] n) a VH region comprising the sequence of SEQ ID NO:
115 and a VL region comprising the sequence of SEQ ID NO:116 (162);
[0168] o) a VH region comprising the sequence of SEQ ID NO:117 and
a VL region comprising the sequence of SEQ ID NO:118 (033); [0169]
p) a VH region comprising the sequence of SEQ ID NO:119 and a VL
region comprising the sequence of SEQ ID NO: 120 (160) [0170] q) a
VH region comprising the sequence of SEQ ID NO: 121 and a VL region
comprising the sequence of SEQ ID NO: 122 (166); [0171] r) a VH
region comprising the sequence of SEQ ID NO: 123 and a VL region
comprising the sequence of SEQ ID NO: 124 (152); and [0172] s) a VH
region comprising the sequence of SEQ ID NO: 125 and a VL region
comprising the sequence of SEQ ID NO: 126 (167).
[0173] In another additional or alternative aspect of the antibody
of the invention, the antibody binds to HER2 and comprises a VH
CDR3, VH region and/or VL region sequence similar or identical to a
sequence of the novel antibodies described herein.
[0174] In one embodiment, the antibody comprises a VH CDR3 region
having a sequence selected from the group consisting of
[0175] SEQ ID NO: 148, such as the sequence of SEQ ID NO:48 (127),
optionally wherein the VH region is derived from the IgHV5-51-01
germline sequence;
[0176] SEQ ID NO:52 (159), optionally wherein the VH region is
derived from the IgHV5-51-01 germline sequence;
[0177] SEQ ID NO:145, such as the sequence of SEQ ID NO:59 (098),
optionally wherein the VH region is derived from the IgHV3-23-01
germline sequence;
[0178] SEQ ID NO: 154, such as the sequence of SEQ ID NO:66 (153),
optionally wherein the VH region is derived from the IgHV3-30-03-01
germline sequence; and
[0179] SEQ ID NO: 151, such as the sequence of SEQ ID NO:73 (132),
optionally wherein the VH region is derived from the IgHV1-18-01
germline sequence.
[0180] In one embodiment, the antibody comprises a VH CDR3 region
of one of antibodies 105, 100, 125 or 162 as shown in FIG. 1,
optionally wherein the VH region is derived from an IgHV3-23-1
germline.
[0181] In one embodiment, the antibody comprises a VH CDR3 region
of one of antibodies 033, 160, 166, 152 or 167 as shown in FIG. 1,
optionally wherein the VH region is derived from an IgHV3-30-3-01
germline.
[0182] In one embodiment, the antibody comprises a VH region
selected from the group consisting of [0183] a) a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:146, 147
and 148, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS:
43, 44 and 45 (127); optionally where the VH region is derived from
an IgHV5-51-01 germline; [0184] b) a VH region comprising the CDR1,
CDR2 and CDR3 sequences of SEQ ID NOs:149, 51 and 52, such as the
CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:50, 51 and 52,
respectively (159), optionally where the VH region is derived from
an IgHV5-51-01 germline; [0185] c) a VH region comprising the CDR1,
CDR2, and CDR3 sequences of SEQ ID NOs:143, 144 and 145, such as
the CRD1, CDR2, and CDR3 sequences of SEQ ID NOs: 57, 58 and 59
(098), respectively, optionally where the VH region is derived from
an IgHV3-23-01 germline; [0186] d) a VH region comprising the CDR1,
CDR2 and CDR3 sequences of SEQ ID NOs:152, 153 and 154, such as the
CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:64, 65 and 66,
respectively (153), optionally where the VH region is derived from
an IgHV3-30-03-01 germline; and [0187] e) a VH region comprising
the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:71, 150 and 151,
such as the CRD1, CDR2, and CDR3 sequences of SEQ ID NOs: 71, 72
and 73 (132), respectively, optionally where the VH region is
derived from an IgHV1-18-01 germline.
[0188] In one embodiment, the antibody comprises a VH region
selected from the preceding embodiment (a) and a VL region
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:47, AAS
and SEQ ID NO:48, respectively (127); respectively, optionally
where the VL region is derived from an IgKV1D-8-01 germline.
[0189] In one embodiment, the antibody comprises a VH region
selected from the preceding embodiment (b) and a VL region
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:54, AAS,
and SEQ ID No:55 (159); respectively, optionally where the VL
region is derived from an IgKV1D-16-01 germline.
[0190] In one embodiment, the antibody comprises a VH region which
is the preceding embodiment (c) and a VL region comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 159, AAS and SEQ ID
NO: 160, respectively, such as the VL CDR1, CDR2 and CDR3 sequences
of SEQ ID NOS: 61, AAS and SEQ ID NO:62 (098), optionally wherein
the VL region is derived from IgKV1D-16-01.
[0191] In one embodiment, the antibody comprises a VH region which
is the preceding embodiment (d) and a VL region comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 161, XAS (wherein X=D
or A, preferably D), and SEQ ID NO: 162 (153), respectively, such
as the VL CDR sequences of SEQ ID NO:68, DAS, and 69, optionally
wherein the VL region is derived from IgKV1D-16-01.
[0192] In one embodiment, the antibody comprises a VH region which
is the preceding embodiment (e) and a VL region comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NO:75, DAS and SEQ ID
NO:76 (132), respectively, optionally wherein the VL region is
derived from IgKV3-11-01.
[0193] In one embodiment, the antibody comprises a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:43, 44
and 45, respectively; and a VL region comprising the CDR1, CDR2 and
CDR3 sequences of SEQ ID NOs:47, AAS, and SEQ ID NO:48,
respectively (127).
[0194] In one embodiment, the antibody comprises a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:50, 51
and 52, respectively; and a VL region comprising the CDR1, CDR2 and
CDR3 sequences of SEQ ID NOs:54, AAS, and SEQ ID NO:55,
respectively (159).
[0195] In one embodiment, the antibody comprises a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:57, 58
and 59, respectively; and a VL region comprising the CDR1, CDR2 and
CDR3 sequences of SEQ ID NOs:60, AAS, and SEQ ID NO:61,
respectively (098).
[0196] In one embodiment, the antibody comprises a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:64, 65
and 66, respectively; and a VL region comprising the CDR1, CDR2 and
CDR3 sequences of SEQ ID NOs:68, DAS, and SEQ ID NO:69,
respectively (153).
[0197] In one embodiment, the antibody comprises a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:71, 72
and 73, respectively; and a VL region comprising the CDR1, CDR2 and
CDR3 sequences of SEQ ID NOs:75, DAS, and SEQ ID NO:76,
respectively (132).
[0198] In separate embodiments, the antibody comprises: [0199] a) a
VH region comprising the sequence of SEQ ID NO:46 and, preferably,
a VL region comprising the sequence of SEQ ID NO:49 (127); [0200]
b) a VH region comprising the sequence of SEQ ID NO:49 and,
preferably, a VL region comprising the sequence of SEQ ID NO:53
(159); [0201] c) a VH region comprising the sequence of SEQ ID
NO:56 and, preferably, a VL region comprising the sequence of SEQ
ID NO:60 (098); [0202] d) a VH region comprising the sequence of
SEQ ID NO:63 an, preferably, a VL region comprising the sequence of
SEQ ID NO:67 (153); [0203] e) a VH region comprising the sequence
of SEQ ID NO:70 and, preferably, a VL region comprising the
sequence of SEQ ID NO:74 (132); [0204] f) a VH region comprising
the sequence of SEQ ID NO: 109 and, preferably, a VL region
comprising the sequence of SEQ ID NO:110 (105); [0205] g) a VH
region comprising the sequence of SEQ ID NO: 111 and, preferably, a
VL region comprising the sequence of SEQ ID NO:112 (100); [0206] h)
a VH region comprising the sequence of SEQ ID NO: 113 and,
preferably, a VL region comprising the sequence of SEQ ID NO:114
(125); [0207] i) a VH region comprising the sequence of SEQ ID NO:
115 and, preferably, a VL region comprising the sequence of SEQ ID
NO:116 (162); [0208] j) a VH region comprising the sequence of SEQ
ID NO: 117 and, preferably, a VL region comprising the sequence of
SEQ ID NO:118 (033); [0209] k) a VH region comprising the sequence
of SEQ ID NO:119 and, preferably, a VL region comprising the
sequence of SEQ ID NO: 120 (160) [0210] l) a VH region comprising
the sequence of SEQ ID NO: 121 and, preferably, a VL region
comprising the sequence of SEQ ID NO: 122 (166); [0211] m) a VH
region comprising the sequence of SEQ ID NO: 123 and, preferably, a
VL region comprising the sequence of SEQ ID NO: 124 (152); [0212]
o) a VH region comprising the sequence of SEQ ID NO: 125 and,
preferably, a VL region comprising the sequence of SEQ ID NO: 126
(167); and/or [0213] p) a variant of any of said antibodies,
wherein said variant preferably has at most 1, 2 or 3 amino-acid
modifications, more preferably amino-acid substitutions, such as
conservative amino acid substitutions and substitutions where the
new amino acid is one at the same position in an aligned sequence
in FIG. 1 or 2, particularly at positions indicated by "X" in the
corresponding consensus sequence.
Bispecific Antibodies
[0214] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody comprising antigen-binding region
of an antibody as defined herein, e.g. an antibody of cross-block
1, 2 or 3, or the VH and VL region comprising the sequences of
(005), and (ii) a second antibody comprising an antigen-binding
region of an antibody which binds to CD3.
[0215] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody comprising an antigen-binding
region of an antibody as defined herein or the VH and VL region
comprising the sequences of (005), and (ii) a second antibody
comprising antigen-binding region of an antibody as defined herein
or the VH and VL region comprising sequences of (005), wherein the
first antigen-binding region binds to a different epitope than the
second antigen-binding region.
[0216] In one embodiment the first antibody comprises a VH region
comprising a CDR3 sequence of an antibody of cross-block 1, 2 or 3
as defined herein, such as SEQ ID NO: 4, 25 or 66 (169, 025 or
153), or a CDR3 sequence of SEQ ID NO: 168 (005).
[0217] In one embodiment the first antibody comprises a VH region
comprising CDR1, CDR2 and CDR3 sequences of an antibody of
cross-block 1, 2 or 3 as defined herein, such as CDR1, CDR2, and
CDR3 sequences SEQ ID NOs: 2, 3 and 4 (169), or CDR1, CDR2 and CDR3
sequences of SEQ ID NOs:23, 24 and 25 (025), or CDR1, CDR2 and CDR3
sequences of SEQ ID NOs: 64, 65 and 66 (153), or CDR1, CDR2 CDR3
sequence of SEQ ID NOs: 166, 167 and 168 (005).
[0218] In a further or alternative embodiment the first antibody
comprises a VH region comprising a CDR3 sequence of an antibody of
cross-block 1, 2 or 3 as defined herein, such as CDR3 sequence an
antibody of cross-block 1 of SEQ ID NO: 11 (050), or SEQ ID NO: 18
(084); or a CDR3 sequence of an antibody of cross-block 2 of SEQ ID
NO: 31 (091), or SEQ ID NO: 38 (129), or a CDR3 sequence of an
antibody of cross-block 3 of SEQ ID NO: 45 (127), or SEQ ID NO:52
(159), or SEQ ID NO:59 (098), or SEQ ID NO:73 (132).
[0219] In one embodiment the first antibody comprises a VH region
comprising CDR1, CDR2 and CDR3 sequences of an antibody of
cross-block 1, 2 or 3 as defined herein, such as CDR1, CDR2, and
CDR3 sequences SEQ ID NOs: 2, 3 and 4 (169), or CDR1, CDR2 and CDR3
sequences of SEQ ID NOs:23, 24 and 25 (025), or CDR1, CDR2 and CDR3
sequences of SEQ ID NOs: 64, 65 and 66 (153), or CDR1, CDR2 CDR3
sequence of SEQ ID NOs: 166, 167 and 168 (005).
[0220] In one embodiment the first antibody comprises a VH region
comprising CDR1, CDR2 and CDR3 sequences of an antibody of
cross-block 1, 2 or 3 as defined herein a VL region comprising
CDR1, CDR2 and CDR3 sequences of an antibody of cross-block 1, 2 or
3 as defined herein.
[0221] In a further or alternative embodiment the first antibody
comprises a VH region comprising CDR1, CDR2 and CDR3 sequences of
an antibody of cross-block 1, 2 or 3 as defined herein, such as
CDR1, CDR2, and CDR3 sequences of an antibody of cross-block 1 of
SEQ ID NOs: 9, 10 and 11 (050), or SEQ ID NOs: 16, 17 and 18 (084);
or CDR1, CDR2, and CDR3 sequences of an antibody of cross-block 2
of SEQ ID NOs: 30, 163 and 31 (091), or SEQ ID NOs: 36, 37 and 38
(129), or CDR1, CDR2, and CDR3 sequences of an antibody of
cross-block 3 SEQ ID NOs: 43, 44 and 45 (127), or SEQ ID NOs:50, 51
and 52 (159), or SEQ ID NOs:57, 58 and 59 (098), or SEQ ID NOs:71,
72 and 73 (132).
[0222] In one embodiment the first antibody comprises a VH region
and a VL region selected from the group consisting of: [0223] a) a
VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID
NOs: 2, 3 and 4; and a VL region comprising the CDR1, CDR2 and CDR3
sequences of SEQ ID: 6, GAS and SEQ ID NO:7, respectively (169);
[0224] b) a VH region comprising the CDR1, CDR2, and CDR3 sequences
of SEQ ID NOs: 23, 24 and 25; and a VL region comprising the CDR1,
CDR2 and CDR3 sequences of SEQ ID NO: 27, AAS and SEQ ID NO:28,
respectively (025); [0225] c) a VH region comprising the CDR1, CDR2
and CDR3 sequences of SEQ ID NOs:64, 65 and 66; and a VL region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 68, DAS
and SEQ ID NO:69 (153); and [0226] d) a VH region comprising the
CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:166, 167 and 168; and a
VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID
NO: 169, GAS and SEQ ID NO: 170 (005).
[0227] In a further or alternative embodiment the first antibody
comprises a VH region and a VL region selected from the group
consisting of: [0228] a) a VH region comprising the CDR1, CDR2 and
CDR3 sequences of SEQ ID NOs:9, 127 and 11, such as the CDR1, CDR2
and CDR3 sequences of SEQ ID NOS: 9, 10 and 11 (050); optionally
where the VH region is derived from an IgHV3-23-1 germline; [0229]
b) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ
ID NOs:128, 129 and 130, such the CDR1, CDR2 and CDR3 sequences of
SEQ ID NOs:16, 17 and 18, respectively (084), optionally where the
VH region is derived from an IgHV1-69-04 germline; and [0230] c) a
VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID
NOs:137, 138 and 139, such the CDR1, CDR2 and CDR3 sequences of SEQ
ID NOs:30, 163, and 31, respectively (091), optionally where the VH
region is derived from an IgHV4-34-01 germline; and [0231] d) a VH
region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID
NOs:140, 141 and 142, such as the CRD1, CDR2, and CDR3 sequences of
SEQ ID NOs: 36, 37 and 38 (129), respectively, optionally where the
VH region is derived from an IgHV3-30-01 germline. [0232] e) a VH
region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID
NOs:146, 147 and 148, such as the CDR1, CDR2 and CDR3 sequences of
SEQ ID NOS: 43, 44 and 45 (127); optionally where the VH region is
derived from an IgHV5-51-01 germline; [0233] f) a VH region
comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:149, 51
and 52, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:50,
51 and 52, respectively (159), optionally where the VH region is
derived from an IgHV5-51-01 germline; [0234] g) a VH region
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:143,
144 and 145, such as the CRD1, CDR2, and CDR3 sequences of SEQ ID
NOs: 57, 58 and 59 (098), respectively, optionally where the VH
region is derived from an IgHV3-23-01 germline; [0235] h) a VH
region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID
NOs:71, 150 and 151, such as the CRD1, CDR2, and CDR3 sequences of
SEQ ID NOs: 71, 72 and 73 (132), respectively, optionally where the
VH region is derived from an IgHV1-18-01 germline.
[0236] In one embodiment the second antibody is one of the previous
embodiment described for the first antibody, but wherein the second
antibody binds to a different epitope than the first antibody.
[0237] In one embodiment the second antibody is a CD3 antibody. In
one embodiment a CD3 antibody may be an antibody comprising a VH
region comprising the sequence of SEQ ID NO: 171 (YTH12.5) and VL
region comprising the sequence of SEQ ID NO:172 (YTH12.5). Another
example of a CD3 antibody is an antibody comprising a VH region
comprising the sequence of SEQ ID NO: 173 (huCLB-T3/4) and VL
region comprising the sequence of SEQ ID NO: 174 (huCLB-T3/4).
[0238] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody having an Fc region and VH and VL
sequences, which antibody comprises the VH and VL region sequences
of (005), (025), (153) or (169), and which antibody comprises an
IgG1 wildtype Fc region, wherein the CH3 region contains Ile at
position 350, a Thr at position 370, and a Leu at position 405 and
(ii) a second antibody having an Fc region and VH and VL sequences,
which antibody comprising the VH and VL region sequences of (005),
(025), (153) or (169), and which antibody comprises a IgG1 wildtype
Fc region, wherein the CH3 region contains an Arg at position 409.
Specific embodiments are disclosed in examples.
[0239] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO: 164, and the VL region comprises the amino acid
sequence of SEQ ID NO: 165 (005), optionally wherein the first
antibody comprises an IgG1,.kappa. Fc region, wherein the CH3
region contains Ile at position 350, a Thr at position 370, and a
Leu at position 405; and (ii) a second antibody having an Fc region
and VH and VL sequences, wherein the VH region comprises the amino
acid sequence of SEQ ID NO:1 and the VL region comprises the amino
acid sequence of SEQ ID NO:5 (169), optionally wherein the second
antibody comprises an IgG1,.kappa. Fc region having an Arg at
position 409.
[0240] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO:22, and the VL region comprises the amino acid
sequence of SEQ ID NO:26 (025), optionally wherein the first
antibody comprises an IgG1,.kappa. Fc region, wherein the CH3
region contains Ile at position 350, a Thr at position 370, and a
Leu at position 405; and (ii) a second antibody having an Fc region
and VH and VL sequences, wherein the VH region comprises the amino
acid sequence of SEQ ID NO: 164 and the VL region comprises the
amino acid sequence of SEQ ID NO:165 (005), optionally wherein the
second antibody comprises an IgG1,.kappa. Fc region having an Arg
at position 409.
[0241] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO:22, and the VL region comprises the amino acid
sequence of SEQ ID NO:26 (025), optionally wherein the first
antibody comprises an IgG1,.kappa. Fc region, wherein the CH3
region contains Ile at position 350, a Thr at position 370, and a
Leu at position 405; and (ii) a second antibody having an Fc region
and VH and VL sequences, wherein the VH region comprises the amino
acid sequence of SEQ ID NO:63 and the VL region comprises the amino
acid sequence of SEQ ID NO:37 (153), optionally wherein the second
antibody comprises an IgG1,.kappa. Fc region having an Arg at
position 409.
[0242] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO:22, and the VL region comprises the amino acid
sequence of SEQ ID NO:26 (025), optionally wherein the first
antibody comprises an IgG1,.kappa. Fc region, wherein the CH3
region contains Ile at position 350, a Thr at position 370, and a
Leu at position 405; and (ii) a second antibody having an Fc region
and VH and VL sequences, wherein the VH region comprises the amino
acid sequence of SEQ ID NO:1 and the VL region comprises the amino
acid sequence of SEQ ID NO:5 (169), optionally wherein the second
antibody comprises an IgG1,.kappa. Fc region having an Arg at
position 409.
[0243] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO:63, and the VL region comprises the amino acid
sequence of SEQ ID NO:67 (153), optionally wherein the first
antibody comprises an IgG1,.kappa. Fc region, wherein the CH3
region contains Ile at position 350, a Thr at position 370, and a
Leu at position 405; and (ii) a second antibody having an Fc region
and VH and VL sequences, wherein the VH region comprises the amino
acid sequence of SEQ ID NO: 164 and the VL region comprises the
amino acid sequence of SEQ ID NO:165 (005), optionally wherein the
second antibody comprises an IgG1,.kappa. Fc region having an Arg
at position 409.
[0244] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO:63, and the VL region comprises the amino acid
sequence of SEQ ID NO:67 (153), optionally wherein the first
antibody comprises an IgG1,.kappa. Fc region, wherein the CH3
region contains Ile at position 350, a Thr at position 370, and a
Leu at position 405; and (ii) a second antibody having an Fc region
and VH and VL sequences, wherein the VH region comprises the amino
acid sequence of SEQ ID NO:1 and the VL region comprises the amino
acid sequence of SEQ ID NO:5 (169), optionally wherein the second
antibody comprises an IgG1,.kappa. Fc region having an Arg at
position 409.
[0245] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO:63, and the VL region comprises the amino acid
sequence of SEQ ID NO:67 (153), optionally wherein the first
antibody comprises an IgG1,.kappa. Fc region having Arg at position
409, or Gln at position 297, or Arg at position 409 and Gln at
position 297; and (ii) a second antibody having an Fc region and VH
and VL sequences, wherein the VH region comprises the amino acid
sequence of SEQ ID NO: 171 and the VL region comprises the amino
acid sequence of SEQ ID NO: 172 (YTH12.5), optionally wherein the
second antibody comprises an IgG1,.kappa. Fc region having an Gln
at position 297, or Leu at position 405, or Gln at position 297 and
Leu at position 405.
[0246] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO: 1, and the VL region comprises the amino acid
sequence of SEQ ID NO:5 (169), optionally wherein the first
antibody comprises an IgG1,.kappa. Fc region having Arg at position
409; and (ii) a second antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO: 171 and the VL region comprises the amino acid
sequence of SEQ ID NO: 172 (YTH12.5), optionally wherein the second
antibody comprises an IgG1,.kappa. Fc region having an Gln at
position 297, or Leu at position 405, or Gln at position 297 and
Leu at position 405.
[0247] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO:63, and the VL region comprises the amino acid
sequence of SEQ ID NO:67 (153), optionally wherein the first
antibody comprises an IgG1,.kappa. Fc region having Arg at position
409, or Gln at position 297, or Arg at position 409 and Gln at
position 297; and (ii) a second antibody having an Fc region and VH
and VL sequences, wherein the VH region comprises the amino acid
sequence of SEQ ID NO: 173 and the VL region comprises the amino
acid sequence of SEQ ID NO: 174 (huCLB-T3/4), optionally wherein
the second antibody comprises an IgG1,.kappa. Fc region having an
Gln at position 297, or Leu at position 405, or Gln at position 297
and Leu at position 405.
[0248] In one embodiment, the antibody is a bispecific antibody,
comprising (i) a first antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO: 1, and the VL region comprises the amino acid
sequence of SEQ ID NO:5 (169), optionally wherein the first
antibody comprises an IgG1,.kappa. Fc region having Arg at position
409; and (ii) a second antibody having an Fc region and VH and VL
sequences, wherein the VH region comprises the amino acid sequence
of SEQ ID NO: 173 and the VL region comprises the amino acid
sequence of SEQ ID NO: 174 (huCLB-T3/4), optionally wherein the
second antibody comprises an IgG1,.kappa. Fc region having an Gln
at position 297, or Leu at position 405, or Gln at position 297 and
Leu at position 405.
[0249] A CD3 antibody is an antibody with a VH region comprising
the sequence of SEQ ID NO: 171 (VH YTH12.5) and VL region
comprising the sequence of SEQ ID NO: 172 (VL YTH12.5). Another
example is a CD3 antibody with a VH region comprising the sequence
of SEQ ID NO: 173 (VH huCLB-T3/4) and VL region comprising the
sequence of SEQ ID NO:174 (VL huCLB-T3/4).
[0250] In one embodiment a bispecific antibody of the present
invention may be selected from the group consisting of:
IgG1-005-ITL.times.IgG1-169-K409R,
IgG1-025-ITL.times.IgG1-005-K409R,
IgG1-025-ITL.times.IgG1-153-K409R,
IgG1-025-ITL.times.IgG1-169-K409R,
IgG1-153-ITL.times.IgG1-005-K409R; and
IgG1-153-ITL.times.IgG1-169-K409R, wherein IgG1-005-ITL means 005
IgG1,.kappa. having Ile at position 350, Thr at position 370, and
Leu at position 405, IgG1-005-K409R means 005 IgG1,.kappa. having
an Arg at position 409, IgG1-025-ITL means 025 IgG1,.kappa. having
Ile at position 350, Thr at position 370, and Leu at position 405,
IgG1-153-ITL means 153 IgG1,.kappa. having contains Ile at position
350, Thr at position 370, and Leu at position 405, IgG1-153-K409R
means 153 IgG1,.kappa. having an Arg at position 409,
IgG1-169-K409R means 169 IgG1,.kappa. having an Arg at position
409, and wherein the bold numbers refer to antibodies described
herein with the VH and VL regions comprising the sequences
described in table 1 and for 005 in example 21; i.e. SEQ ID NOs:164
and 165.
[0251] In one embodiment the bispecific antibody may be selected
from the group consisting of:
IgG1-HER2-153-K409R.times.IgG1-YTH12.5-F405L,
IgG1-HER2-153-K409R.times.IgG1-YTH12.5-N297Q-F405L,
IgG1-HER2-153-K409R.times.IgG1-hu-CLB-T3/4-F405L,
IgG1-HER2-153-K409R.times.IgG1-hu-CLB-T3/4-N297Q-F405L,
IgG1-HER2-153-N297Q-K409R.times.IgG1-YTH12.5-F405L,
IgG1-HER2-153-N297Q-K409R.times.IgG1-YTH12.5-N297Q-F405L,
IgG1-HER2-153-N297Q-K409R.times.IgG1-hu-CLB-T3/4-F405L,
IgG1-HER2-153-N297Q-K409R.times.IgG1-hu-CLB-T3/4-N297Q-F405L,
IgG1-HER2-169-K409R.times.IgG1-hu-CLB-T3/4-F405L,
IgG1-HER2-169-K409R.times.IgG1-hu-CLB-T3/4-N297Q-F405L,
IgG1-HER2-169-K409R.times.IgG1-YTH12.5-F405L and
IgG1-HER2-169-K409R.times.IgG1-YTH12.5-N297Q-F405L.
Functional Properties of Group 1, 2 and 3 Antibodies and Bispecific
Antibodies
[0252] In another aspect of the antibody of the invention, the
antibody binds to the same HER2 epitope as one or more of the novel
Group 1, 2 or 3 antibodies described herein, preferably when
determined as described in Example 14; and is further characterized
by one or more properties determined as described in Examples 12,
13, 15, 16, 17, 18 and 19.
[0253] In one embodiment, the HER2 antibody has a lower EC.sub.50
value (half maximal effective concentration) than trastuzumab in
binding to A431 cells, preferably an EC.sub.50 value lower than
0.80 .mu.g/ml, 0.50 .mu.g/ml, or 0.30 .mu.g/ml, when determined as
described in Example 12, and preferably binds the same epitope as
at least one reference antibody comprising the VH and VL regions
selected from the group consisting of [0254] a) a VH region
comprising the sequence of SEQ ID NO:1 and a VL region comprising
the sequence of SEQ ID NO:5 (169); [0255] b) a VH region comprising
the sequence of SEQ ID NO: 15 and a VL region comprising the
sequence of SEQ ID NO: 19 (084); [0256] c) a VH region comprising
the sequence of SEQ ID NO:22 and a VL region comprising the
sequence of SEQ ID NO:26 (025); [0257] d) a VH region comprising
the sequence of SEQ ID NO:29 and a VL region comprising the
sequence of SEQ ID NO:32 (091); [0258] e) a VH region comprising
the sequence of SEQ ID NO:46 and a VL region comprising the
sequence of SEQ ID NO:49 (127); [0259] f) a VH region comprising
the sequence of SEQ ID NO:49 and a VL region comprising the
sequence of SEQ ID NO:53 (159); [0260] g) a VH region comprising
the sequence of SEQ ID NO:56 and a VL region comprising the
sequence of SEQ ID NO:60 (098); [0261] h) a VH region comprising
the sequence of SEQ ID NO:63 and a VL region comprising the
sequence of SEQ ID NO:67 (153); and [0262] i) a VH region
comprising the sequence of SEQ ID NO:70 and a VL region comprising
the sequence of SEQ ID NO:74 (132).
[0263] In an additional or alternative embodiment, the anti-HER2
antibody specifically binds HER2-positive Rhesus epithelial cells,
when determined as described in Example 13, and preferably binds
the same epitope as at least one reference antibody comprising the
VH and VL regions selected from the group consisting of the VH and
VL regions of any of antibodies 169, 050, 084, 025, 091, 129, 127,
159, 098, 153 and 132.
[0264] In an additional or alternative embodiment, the anti-HER2
antibody efficiently induces ADCC (antibody-dependent cell-mediated
cytotoxicity), preferably achieving a specific .sup.51Cr-release of
at least 30%, more preferably at least 40%, when determined as
described in Example 15, and preferably binds the same epitope as
at least one reference antibody comprising the VH and VL regions
selected from the group consisting of: [0265] a) a VH region
comprising the sequence of SEQ ID NO:1 and a VL region comprising
the sequence of SEQ ID NO:5 (169); [0266] b) a VH region comprising
the sequence of SEQ ID NO:8 and a VL region comprising the sequence
of SEQ ID NO: 12 (050); [0267] c) a VH region comprising the
sequence of SEQ ID NO: 15 and a VL region comprising the sequence
of SEQ ID NO: 19 (084); [0268] d) a VH region comprising the
sequence of SEQ ID NO:22 and a VL region comprising the sequence of
SEQ ID NO:26 (025); [0269] e) a VH region comprising the sequence
of SEQ ID NO:29 and a VL region comprising the sequence of SEQ ID
NO:32 (091); [0270] f) a VH region comprising the sequence of SEQ
ID NO:35 and a VL region comprising the sequence of SEQ ID NO:39
(129); and [0271] g) a VH region comprising the sequence of SEQ ID
NO:63 an, preferably, a VL region comprising the sequence of SEQ ID
NO:67 (153).
[0272] In an additional or alternative embodiment, the HER2
antibody specifically binds HER2-expressing AU565 cells but
promotes ligand-independent proliferation of the cells less than
any of F5 and C1 when determined as described in Example 16, and
preferably binds the same epitope as at least one reference
antibody comprising the VH and VL regions selected from the group
consisting of [0273] a) a VH region comprising the sequence of SEQ
ID NO:1 and a VL region comprising the sequence of SEQ ID NO:5
(169); [0274] b) a VH region comprising the sequence of SEQ ID NO:8
and a VL region comprising the sequence of SEQ ID NO: 12 (050);
[0275] c) a VH region comprising the sequence of SEQ ID NO: 15 and
a VL region comprising the sequence of SEQ ID NO: 19 (084); [0276]
d) a VH region comprising the sequence of SEQ ID NO:22 and a VL
region comprising the sequence of SEQ ID NO:26 (025); [0277] e) a
VH region comprising the sequence of SEQ ID NO:29 and a VL region
comprising the sequence of SEQ ID NO:32 (091); [0278] f) a VH
region comprising the sequence of SEQ ID NO:35 and a VL region
comprising the sequence of SEQ ID NO:39 (129); [0279] g) a VH
region comprising the sequence of SEQ ID NO:46 and a VL region
comprising the sequence of SEQ ID NO:49 (127); [0280] h) a VH
region comprising the sequence of SEQ ID NO:49 and a VL region
comprising the sequence of SEQ ID NO:53 (159); [0281] i) a VH
region comprising the sequence of SEQ ID NO:56 and a VL region
comprising the sequence of SEQ ID NO:60 (098); [0282] j) a VH
region comprising the sequence of SEQ ID NO:63 and a VL region
comprising the sequence of SEQ ID NO:67 (153); and [0283] k) a VH
region comprising the sequence of SEQ ID NO:70 and a VL region
comprising the sequence of SEQ ID NO:74 (132).
[0284] In an additional or alternative embodiment, the HER2
antibody specifically binds HER2-expressing AU565 cells and
inhibits ligand-independent proliferation of the cells, preferably
inhibiting proliferation by at least 20%, more preferably at least
25%, when determined as described in Example 16, and preferably
binds the same epitope as at least one reference antibody
comprising the VH and VL regions selected from the group consisting
of: [0285] a) a VH region comprising the sequence of SEQ ID NO:1
and a VL region comprising the sequence of SEQ ID NO:5 (169); and
[0286] b) a VH region comprising the sequence of SEQ ID NO:8 and a
VL region comprising the sequence of SEQ ID NO: 12 (050).
[0287] In an additional or alternative embodiment, the HER2
antibody specifically binds HER2-expressing AU565 cells but has no
significant effect on, or does not promote, ligand-induced
proliferation of the cells, preferably inhibiting proliferation by
no more than 25%, more preferably by no more than 15%, when
determined as described in Example 17, and binds the same epitope
as at least one reference antibody comprising the VH and VL regions
selected from the group consisting of: [0288] a) a VH region
comprising the sequence of SEQ ID NO:1 and a VL region comprising
the sequence of SEQ ID NO:5 (169); [0289] b) a VH region comprising
the sequence of SEQ ID NO:8 and a VL region comprising the sequence
of SEQ ID NO: 12 (050); [0290] c) a VH region comprising the
sequence of SEQ ID NO: 15 and a VL region comprising the sequence
of SEQ ID NO: 19 (084); and [0291] d) a VH region comprising the
sequence of SEQ ID NO:56 and a VL region comprising the sequence of
SEQ ID NO:60 (098).
[0292] In an additional or alternative embodiment, the HER2
antibody specifically binds HER2-expressing MCF-7 cells and
inhibits ligand-induced proliferation, e.g. it may completely
inhibit the ligand-induced effect or inhibit the total
proliferation by 50%, e.g. 60% or 70% or 80%, of the cells when
determined as described in Example 17, and binds the same epitope
as at least one reference antibody comprising the VH and VL regions
selected from the group consisting of: [0293] a) a VH region
comprising the sequence of SEQ ID NO:22 and a VL region comprising
the sequence of SEQ ID NO:26 (025); [0294] b) a VH region
comprising the sequence of SEQ ID NO:29 and a VL region comprising
the sequence of SEQ ID NO:32 (091); [0295] c) a VH region
comprising the sequence of SEQ ID NO:35 and a VL region comprising
the sequence of SEQ ID NO:39 (129); and [0296] d) a VH region
comprising the sequence of SEQ ID NO:63 an, preferably, a VL region
comprising the sequence of SEQ ID NO:67 (153).
[0297] In an additional or alternative embodiment, the antibody,
when conjugated directly or indirectly to a therapeutic moiety such
as a truncated form of the pseudomonas-exotoxin A, is more
effective than trastuzumab in killing AU565 cells, A431 cells, or
both AU565 and A431 cells, when determined as described in Example
18.
[0298] In one embodiment, the conjugated antibody has an EC.sub.50
value of less than 70 ng/ml, less than 50 ng/ml, or less than 30
ng/ml in killing AU565 cells and/or A431 cells, when determined as
described in Example 18, and binds the same epitope as at least one
reference antibody comprising the VH and VL regions of an antibody
selected from the group consisting of 169, 091, 050, 084, 098, 05,
153, 129, 132, 127 and 159; preferably selected from antibodies
153, 129, 098, 091 and 025.
[0299] In one embodiment, the conjugated antibody has or results in
a higher percentage of killed AU565 cells than trastuzumab and
pertuzumab when determined as described in Example 18, preferably
killing at least 49%, more preferably at least 60% of the AU565
cells, and binds the same epitope as at least one reference
antibody comprising the VH and VL regions of an antibody selected
from the group consisting of 169, 091, 050, 084, 098, 025, 153,
129, 132, 127 and 159; preferably selected from antibodies 153,
132, 127, 129, 159 and 025.
[0300] In a preferred embodiment, the conjugated antibody binds to
the same epitope as a reference antibody comprising a VH region
comprising the sequence of SEQ ID NO:49 and a VL region comprising
the sequence of SEQ ID NO:53 (159).
[0301] In one embodiment, the conjugated antibody has a higher
percentage of killed AU431 cells than trastuzumab and pertuzumab
when determined as described in Example 18, preferably killing at
least 50%, more preferably at least 70%, and binds the same epitope
as at least one reference antibody comprising the VH and VL regions
of an antibody selected from the group consisting of 025, 084, 091,
098, 129 and 153; preferably selected from antibodies 025, 091,
098, 129 and 153.
[0302] In a preferred embodiment, the conjugated antibody binds to
the same epitope as a reference antibody comprising a VH region
comprising the sequence of SEQ ID NO:56 and a VL region comprising
the sequence of SEQ ID NO:60 (098).
[0303] In an additional or alternative embodiment, the antibody is
internalized by tumor cells expressing HER2, such as AU565 cells,
to a higher degree than trastuzumab and pertuzumab, preferably more
than twice or three times the amount of internalized trastuzumab,
preferably when determined according to Example 18, and binds to
the same epitope as an antibody comprising VH and VL regions
selected from the group consisting of: [0304] a) a VH region
comprising the sequence of SEQ ID NO:46 and a VL region comprising
the sequence of SEQ ID NO:49 (127); [0305] b) a VH region
comprising the sequence of SEQ ID NO:49 and a VL region comprising
the sequence of SEQ ID NO:53 (159); [0306] c) a VH region
comprising the sequence of SEQ ID NO:56 and a VL region comprising
the sequence of SEQ ID NO:60 (098); [0307] d) a VH region
comprising the sequence of SEQ ID NO:63 and a VL region comprising
the sequence of SEQ ID NO:67 (153); and [0308] e) a VH region
comprising the sequence of SEQ ID NO:70 and a VL region comprising
the sequence of SEQ ID NO:74 (132).
[0309] Preferably, the antibody binds to the same epitope as an
antibody comprising VH and VL regions selected from [0310] a) a VH
region comprising the sequence of SEQ ID NO:46 and a VL region
comprising the sequence of SEQ ID NO:49 (127) and [0311] b) a VH
region comprising the sequence of SEQ ID NO:56 and a VL region
comprising the sequence of SEQ ID NO:60 (098).
[0312] In a further embodiment, the antibody binds to Domain II or
IV of HER2, preferably wherein the antibody does not significantly
promote proliferation of HER2 expressing cells, and is more
efficiently internalized, or is internalized to a higher degree,
than trastuzumab or pertuzumab into HER2-expressing tumor cells,
preferably when determined as described in the Examples, e.g.
examples 16 and 19, respectively.
[0313] In a further embodiment the antibody enhanced HER2
downmodulation more than trastuzumab, e.g. the antibody enhanced
HER2 downmodulation by more 30%, such as more than 40% or more than
50% when determined as described in example 22, preferably wherein
the antibody binds to the same epitope as an antibody of
cross-block group 3 of the present invention, e.g. an antibody
binding to the same epitope as an antibody comprising VH and VL
regions selected from the group consisting of: [0314] a) a VH
region comprising the sequence of SEQ ID NO:56 and a VL region
comprising the sequence of SEQ ID NO:60 (098); [0315] b) a VH
region comprising the sequence of SEQ ID NO:63 and a VL region
comprising the sequence of SEQ ID NO:67 (153).
[0316] In another or alternative embodiment the antibody decreased
tumour growth and improved survival in vivo more than trastuzumab,
when determined as described in example 29, preferably wherein the
antibody binds to the same epitope as an antibody of cross-block 1
or cross-block 2 of the present invention, e.g. an antibody binding
to the same epitope as an antibody comprising VH and VL regions
selected from the group consisting of: [0317] a) a VH region
comprising the sequence of SEQ ID NO:1 and a VL region comprising
the sequence of SEQ ID NO:5 (169); [0318] b) a VH region comprising
the sequence of SEQ ID NO: 15 and a VL region comprising the
sequence of SEQ ID NO: 19 (084); and [0319] c) a VH region
comprising the sequence of SEQ ID NO:29 and a VL region comprising
the sequence of SEQ ID NO:32 (091).
[0320] In another or alternative embodiment the antibody decreased
tumour growth and improved survival in vivo more than trastuzumab,
when determined as described in example 30, preferably wherein the
antibody binds to the same epitope as an antibody of cross-block 2
or cross-block 3 of the present invention, e.g. an antibody binding
to the same epitope as an antibody comprising VH and VL regions
selected from the group consisting of: [0321] a) a VH region
comprising the sequence of SEQ ID NO:22 and a VL region comprising
the sequence of SEQ ID NO:26 (025); [0322] b) a VH region
comprising the sequence of SEQ ID NO:29 and a VL region comprising
the sequence of SEQ ID NO:32 (091); [0323] c) a VH region
comprising the sequence of SEQ ID NO:35 and a VL region comprising
the sequence of SEQ ID NO:39 (129); and [0324] d) a VH region
comprising the sequence of SEQ ID NO:63 and a VL region comprising
the sequence of SEQ ID NO:67 (153).
[0325] More particularly, wherein the antibody binds to the same
epitope as an antibody comprising VH and VL regions selected from
the group consisting of: [0326] a) a VH region comprising the
sequence of SEQ ID NO:22 and a VL region comprising the sequence of
SEQ ID NO:26 (025); and [0327] b) a VH region comprising the
sequence of SEQ ID NO:29 and a VL region comprising the sequence of
SEQ ID NO:32 (091).
[0328] In one embodiment the antibody is a bispecific antibody.
[0329] In a further embodiment the antibody is a bispecific
antibody which enhanced HER2 downmodulation, in particular more
than their monospecific counterparts, e.g. the antibody enhanced
HER2 downmodulation by more 20%, such as more than 30% or more than
40% when determined as described in example 22, preferably wherein
the antibody binds to the same epitopes as bispecific antibody
selected from the group consisting of
IgG1-005-ITL.times.IgG1-169-K409R,
IgG1-025-ITL.times.IgG1-005-K409R,
IgG1-025-ITL.times.IgG1-153-K409R,
IgG1-025-ITL.times.IgG1-169-K409R,
IgG1-153-ITL.times.IgG1-005-K409R; and
IgG1-153-ITL.times.IgG1-169-K409R.
[0330] In an additional or alternative embodiment, the bispecific
antibody specifically binds HER2-expressing AU565 cells and
inhibits ligand-induced proliferation of the cells when determined
as described in Example 24, and binds the same epitopes as at least
one bispecific antibody selected from the group consisting of:
IgG1-005-ITL.times.IgG1-169-K409R,
IgG1-025-ITL.times.IgG1-005-K409R,
IgG1-025-ITL.times.IgG1-153-K409R,
IgG1-025-ITL.times.IgG1-169-K409R,
IgG1-153-ITL.times.IgG1-005-K409R; and
IgG1-153-ITL.times.IgG1-169-K409R. In particular the bispecific
antibody inhibits proliferation of the AU565 cells more than their
monospecific counterparts and is selected from the group consisting
of IgG1-005-ITL.times.IgG1-169-K409R and
IgG1-025-ITL.times.IgG1-005-K409R.
[0331] In an additional or alternative embodiment the bispecific
antibody is a HER2.times.CD3 bispecific antibody induce T cell
mediated cytotoxicity of AU565 as described in example 25, and
binds the same epitopes as at least one of the bispecific
antibodies selected from the group consisting of: Duo
huCLB-Q/153-Q, Duo huCLB-Q/B12-Q, Duo YTH12.5/153-Q and Duo
YTH12.5/B12-Q (Duo indicating bispecific antibody).
Antibody Formats
[0332] The present invention provides HER2 antibodies which
efficiently bind to and internalize into HER2-expressing tumor
cells, typically without significantly promoting ligand-independent
proliferation of the cells. Depending on the desired functional
properties for a particular use, particular antibodies can be
selected from the set of antibodies provided in the present
invention and/or their format can be adapted to change these
properties, as described below.
[0333] The antibody of the invention can be of any isotype. The
choice of isotype typically will be guided by the desired effector
functions, such as ADCC induction. Exemplary isotypes are IgG1,
IgG2, IgG3, and IgG4. Either of the human light chain constant
regions, kappa or lambda, may be used. If desired, the class of a
HER2 antibody of the present invention may be switched by known
methods. For example, an antibody of the present invention that was
originally IgM may be class switched to an IgG antibody of the
present invention. Further, class switching techniques may be used
to convert one IgG subclass to another, for instance from IgG1 to
IgG2. Thus, the effector function of the antibodies of the present
invention may be changed by isotype switching to, e.g., an IgG1,
IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various
therapeutic uses. In one embodiment an antibody of the present
invention is an IgG1 antibody, for instance an IgG1,.kappa..
[0334] In a further embodiment, the antibody of the invention is
glyco-engineered to reduce fucose and thus enhance ADCC, e.g. by
addition of compounds to the culture media during antibody
production as described in US2009317869 or as described in van
Berkel et al. (2010) Biotechnol. Bioeng. 105:350 or by using FUT8
knockout cells, e.g. as described in Yamane-Ohnuki et al (2004)
Biotechnol. Bioeng 87:614. ADCC may alternatively be optimized
using the method described by Umaha et al. (1999) Nature Biotech
17:176.
[0335] In a further embodiment, the antibody of the invention has
been engineered to enhance complement activation, e.g. as described
in Natsume et al. (2009) Cancer Sci. 100:2411.
[0336] In one embodiment, the antibody of the invention is a
full-length antibody, preferably an IgG1 antibody, in particular an
IgG1,.kappa. antibody. In another embodiment, the antibody of the
invention is an antibody fragment or a single-chain antibody.
[0337] Antibody fragments may e.g. be obtained by fragmentation
using conventional techniques, and the fragments screened for
utility in the same manner as described herein for whole
antibodies. For example, F(ab').sub.2 fragments may be generated by
treating an antibody with pepsin. The resulting F(ab').sub.2
fragment may be treated to reduce disulfide bridges with a reducing
agent, such as dithiothreitol, to produce Fab' fragments. Fab
fragments may be obtained by treating an antibody with papain. A
F(ab').sub.2 fragment may also be produced by binding Fab'
fragments via a thioether bond or a disulfide bond. Antibody
fragments may also be generated by expression of nucleic acids
encoding such fragments in recombinant cells (see for instance
Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). For example, a
chimeric gene encoding a portion of an F(ab').sub.2 fragment could
include DNA sequences encoding the C.sub.H1 domain and hinge region
of the H chain, followed by a translational stop codon to yield
such a truncated antibody fragment molecule.
[0338] As explained above, in one embodiment, the HER2 antibody of
the invention is a bivalent antibody, i.e. an antibody capable of
binding two antigens or epitopes on the same antigen.
[0339] In another embodiment, the HER2 antibody of the invention is
a monovalent antibody.
[0340] In one embodiment, the antibody of the invention is a Fab
fragment or a one-armed antibody, such as described in
US20080063641 (Genentech) or other monovalent antibody, e.g. such
as described in WO2007048037 (Amgen).
[0341] In a preferred embodiment, a monovalent antibody has a
structure as described in WO2007059782 (Genmab) (incorporated
herein by reference) having a deletion of the hinge region.
Accordingly, in one embodiment, the antibody is a monovalent
antibody, wherein said HER2 antibody is constructed by a method
comprising:
[0342] i) providing a nucleic acid construct encoding the light
chain of said monovalent antibody, said construct comprising a
nucleotide sequence encoding the VL region of a selected antigen
specific HER2 antibody and a nucleotide sequence encoding the
constant CL region of an Ig, wherein said nucleotide sequence
encoding the VL region of a selected antigen specific antibody and
said nucleotide sequence encoding the CL region of an Ig are
operably linked together, and wherein, in case of an IgG1 subtype,
the nucleotide sequence encoding the CL region has been modified
such that the CL region does not contain any amino acids capable of
forming disulfide bonds or covalent bonds with other peptides
comprising an identical amino acid sequence of the CL region in the
presence of polyclonal human IgG or when administered to an animal
or human being;
[0343] ii) providing a nucleic acid construct encoding the heavy
chain of said monovalent antibody, said construct comprising a
nucleotide sequence encoding the VH region of a selected antigen
specific antibody and a nucleotide sequence encoding a constant CH
region of a human Ig, wherein the nucleotide sequence encoding the
CH region has been modified such that the region corresponding to
the hinge region and, as required by the Ig subtype, other regions
of the CH region, such as the CH3 region, does not comprise any
amino acid residues which participate in the formation of
disulphide bonds or covalent or stable non-covalent inter-heavy
chain bonds with other peptides comprising an identical amino acid
sequence of the CH region of the human Ig in the presence of
polyclonal human IgG or when administered to an animal human being,
wherein said nucleotide sequence encoding the VH region of a
selected antigen specific antibody and said nucleotide sequence
encoding the CH region of said Ig are operably linked together;
[0344] iii) providing a cell expression system for producing said
monovalent antibody;
[0345] iv) producing said monovalent antibody by co-expressing the
nucleic acid constructs of (i) and (ii) in cells of the cell
expression system of (iii).
[0346] Similarly, in one embodiment, the HER2 antibody is a
monovalent antibody, which comprises
[0347] (i) a variable region of an antibody of the invention as
described herein or an antigen binding part of the said region,
and
[0348] (ii) a C.sub.H region of an immunoglobulin or a fragment
thereof comprising the C.sub.H2 and C.sub.H3 regions, wherein the
C.sub.H region or fragment thereof has been modified such that the
region corresponding to the hinge region and, if the immunoglobulin
is not an IgG4 subtype, other regions of the C.sub.H region, such
as the C.sub.H3 region, do not comprise any amino acid residues,
which are capable of forming disulfide bonds with an identical
C.sub.H region or other covalent or stable non-covalent inter-heavy
chain bonds with an identical C.sub.H region in the presence of
polyclonal human IgG.
[0349] In a further embodiment hereof, the heavy chain of the
monovalent HER2 antibody has been modified such that the entire
hinge has been deleted.
[0350] In another further embodiment, the immunoglobulin referred
to in step ii) above is of the IgG4 subtype.
[0351] In another further embodiment, said monovalent antibody is
of the IgG4 subtype, but the C.sub.H3 region has been modified so
that one or more of the following amino acid substitutions have
been made:
TABLE-US-00002 Numbering of CH3 mutations EU index KABAT* G4*
Mutations E378 E357 E357A or E357T or E357V or E357I S387 S364
S364R or S364K T389 T366 T366A or T366R or T366K or T366N L391 L368
L368A or L368V or L368E or L368G or L368S or L368T D427 D399 D399A
or D399T or D399S F436 F405 F405A or F405L or F405T or F405D or
F405R or F405Q or F405K or F405Y Y438 Y407 Y407A or Y407E or Y407Q
or Y407K or Y407F F436 and F405 and (F405T and Y407E) or (F405D and
Y407E) Y438 Y407 D427 and D399 and (D399S and Y407Q) or (D399S and
Y407K) Y438 Y407 or (D399S and Y407E) *KABAT indicates amino acid
numbering according to Kabat (Kabat et al., Sequences of Proteins
of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, MD. (1991). EU index indicates
amino acid numbering according to EU index as outlined in Kabat et
al., (supra).
[0352] In another further embodiment, the sequence of said
monovalent antibody has been modified so that it does not comprise
any acceptor sites for N-linked glycosylation. HER2 antibodies of
the invention also include single chain antibodies. Single chain
antibodies are peptides in which the heavy and light chain Fv
regions are connected. In one embodiment, the present invention
provides a single-chain Fv (scFv) wherein the heavy and light
chains in the Fv of a HER2 antibody of the present invention are
joined with a flexible peptide linker (typically of about 10, 12,
15 or more amino acid residues) in a single peptide chain. Methods
of producing such antibodies are described in for instance U.S.
Pat. No. 4,946,778, Pluckthun in The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New
York, pp. 269-315 (1994), Bird et al., Science 242, 423-426 (1988),
Huston et al., PNAS USA 85, 5879-5883 (1988) and McCafferty et al.,
Nature 348, 552-554 (1990). The single chain antibody may be
monovalent, if only a single VH and VL are used, bivalent, if two
VH and VL are used, or polyvalent, if more than two VH and VL are
used.
[0353] In one embodiment, the HER2 antibody of the invention is an
effector-function-deficient antibody. In one embodiment, the
effector-function-deficient HER2 antibody is a human stabilized
IgG4 antibody, which has been modified to prevent Fab-arm exchange
(van der Neut Kolfschoten et al. (2007) Science 317(5844):1554-7).
Examples of suitable human stabilized IgG4 antibodies are
antibodies, wherein arginine at position 409 in a heavy chain
constant region of human IgG4, which is indicated in the EU index
as in Kabat et al., is substituted with lysine, threonine,
methionine, or leucine, preferably lysine (described in
WO2006033386 (Kirin)) and/or wherein the hinge region has been
modified to comprise a Cys-Pro-Pro-Cys sequence.
[0354] In one embodiment, the stabilized IgG4 HER2 antibody is an
IgG4 antibody comprising a heavy chain and a light chain, wherein
said heavy chain comprises a human IgG4 constant region having a
residue selected from the group consisting of: Lys, Ala, Thr, Met
and Leu at the position corresponding to 409 and/or a residue
selected from the group consisting of: Ala, Val, Gly, Ile and Leu
at the position corresponding to 405, and wherein said antibody
optionally comprises one or more further substitutions, deletions
and/or insertions, but does not comprise a Cys-Pro-Pro-Cys sequence
in the hinge region. Preferably, said antibody comprises a Lys or
Ala residue at the position corresponding to 409 or the CH3 region
of the antibody has been replaced by the CH3 region of human IgG1,
of human IgG2 or of human IgG3. See also WO2008145142 (Genmab).
[0355] In an even further embodiment, the stabilized IgG4 HER2
antibody is an IgG4 antibody comprising a heavy chain and a light
chain, wherein said heavy chain comprises a human IgG4 constant
region having a residue selected from the group consisting of: Lys,
Ala, Thr, Met and Leu at the position corresponding to 409 and/or a
residue selected from the group consisting of: Ala, Val, Gly, Ile
and Leu at the position corresponding to 405, and wherein said
antibody optionally comprises one or more further substitutions,
deletions and/or insertions and wherein said antibody comprises a
Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said
antibody comprises a Lys or Ala residue at the position
corresponding to 409 or the CH3 region of the antibody has been
replaced by the CH3 region of human IgG1, of human IgG2 or of human
IgG3.
[0356] In a further embodiment, the effector-function-deficient
HER2 antibody is an antibody of a non-IgG4 type, e.g. IgG1, IgG2 or
IgG3 which has been mutated such that the ability to mediate
effector functions, such as ADCC, has been reduced or even
eliminated. Such mutations have e.g. been described in Dall'Acqua W
F et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J
Virol.; 75(24):12161-12168 (2001).
Conjugates
[0357] In a further embodiment, the present invention provides a
HER2 antibody or an HER2 bispecific antibody linked or conjugated
to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic
drug, a cytokine, an immunosuppressant, or a radioisotope. Such
conjugates are referred to herein as "immunoconjugates".
Immunoconjugates which include one or more cytotoxins are referred
to as "immunotoxins".
[0358] A cytotoxin or cytotoxic agent includes any agent that is
detrimental to (e.g., kills) cells. Suitable therapeutic agents for
forming immunoconjugates of the present invention include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, maytansine or
an analog or derivative thereof, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin; calicheamicin or
analogs or derivatives thereof; antimetabolites (such as
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase,
gemcitabine, cladribine), alkylating agents (such as
mechlorethamine, thioepa, chlorambucil, melphalan, carmustine
(BSNU), lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine,
mitomycin C, cisplatin and other platinum derivatives, such as
carboplatin; as well as duocarmycin A, duocarmycin SA, CC-1065
(a.k.a. rachelmycin), or analogs or derivatives of CC-1065),
antibiotics (such as dactinomycin (formerly actinomycin),
bleomycin, daunorubicin (formerly daunomycin), doxorubicin,
idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin,
anthramycin (AMC)), anti-mitotic agents (e.g., tubulin-inhibitors)
such as monomethyl auristatin E, monomethyl auristatin F, or other
analogs or derivatives of dolastatin 10; diphtheria toxin and
related molecules (such as diphtheria A chain and active fragments
thereof and hybrid molecules); ricin toxin (such as ricin A or a
deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like
toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin,
pertussis toxin, tetanus toxin, soybean Bowman-Birk protease
inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin,
gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolacca americana proteins
(PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin,
crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, and enomycin toxins. Other suitable
conjugated molecules include antimicrobial/lytic peptides such as
CLIP, Magainin 2, mellitin, Cecropin, and P18; ribonuclease
(RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral
protein, diphtherin toxin, and Pseudomonas endotoxin. See, for
example, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif.
A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents
that may be administered in combination with a HER2 antibody of the
present invention as described elsewhere herein, such as, e.g.,
anti-cancer cytokines or chemokines, are also candidates for
therapeutic moieties useful for conjugation to an antibody of the
present invention.
[0359] In one embodiment, a HER2 antibody of the invention
comprises a conjugated nucleic acid or nucleic acid-associated
molecule. In one such embodiment, the conjugated nucleic acid is a
cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory
RNA molecule (e.g., a siRNA molecule) or an immunostimulatory
nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA
molecule). In another embodiment, a HER2 antibody of the invention
is conjugated to an aptamer or a ribozyme.
[0360] In one embodiment, HER2 antibodies comprising one or more
radiolabeled amino acids are provided. A radiolabeled HER2 antibody
may be used for both diagnostic and therapeutic purposes
(conjugation to radiolabeled molecules is another possible
feature). Non-limiting examples of labels for polypeptides include
3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re.
[0361] In one embodiment, the antibody is conjugated to a
radioisotope or to a radioisotope-containing chelate. For example,
the antibody can be conjugated to a chelator linker, e.g. DOTA,
DTPA or tiuxetan, which allows for the antibody to be complexed
with a radioisotope. The antibody may also or alternatively
comprise or be conjugated to one or more radiolabeled amino acids
or other radiolabeled molecule. A radiolabeled CD74 Ab may be used
for both diagnostic and therapeutic purposes. Non-limiting examples
of radioisotopes include .sup.3H, .sup.14C, .sup.15N, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.125I, .sup.111In, .sup.131In, .sup.186Re,
.sup.213Bs, .sup.225Ac and .sup.227Th.
[0362] HER2 antibodies may also be chemically modified by covalent
conjugation to a polymer to for instance increase their circulating
half-life. Exemplary polymers, and methods to attach them to
peptides, are illustrated in for instance U.S. Pat. Nos. 4,766,106,
4,179,337, 4,495,285 and 4,609,546. Additional polymers include
polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., a PEG
with a molecular weight of between about 1,000 and about 40,000,
such as between about 2,000 and about 20,000).
[0363] Any method known in the art for conjugating the HER2
antibody to the conjugated molecule(s), such as those described
above, may be employed, including the methods described by Hunter
et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014
(1974), Pain et al., J. Immunol. Meth. 40, 219 (1981) and Nygren,
J. Histochem. and Cytochem. 3, 407 (1982). Such antibodies may be
produced by chemically conjugating the other moiety to the
N-terminal side or C-terminal side of the HER2 antibody or fragment
thereof (e.g., a HER2 antibody H or L chain) (see, e.g., Antibody
Engineering Handbook, edited by Osamu Kanemitsu, published by
Chijin Shokan (1994)). Such conjugated antibody derivatives may
also be generated by conjugation at internal residues or sugars,
where appropriate.
[0364] The agents may be coupled either directly or indirectly to a
HER2 antibody of the present invention. One example of indirect
coupling of a second agent is coupling via a spacer moiety to
cysteine or lysine residues in the antibody. In one embodiment, a
HER2 antibody is conjugated to a prodrug molecule that can be
activated in vivo to a therapeutic drug via a spacer or linker.
After administration, the spacers or linkers are cleaved by
tumor-cell associated enzymes or other tumor-specific conditions,
by which the active drug is formed. Examples of such prodrug
technologies and linkers are described in WO02083180, WO2004043493,
WO2007018431, WO2007089149, and WO2009017394 by Syntarga B V, et
al. Suitable antibody-prodrug technology and duocarmycin analogs
can also be found in U.S. Pat. No. 6,989,452 (Medarex).
[0365] In one embodiment, the HER2 antibody of the present
invention is attached to a chelator linker, e.g. tiuxetan, which
allows for the antibody to be conjugated to a radioisotope.
Bispecific Antibodies
[0366] In a further aspect, the invention relates to a bispecific
molecule comprising a first antigen binding site from a HER2
antibody of the invention as described herein above and a second
antigen binding site with a different binding specificity, such as
a binding specificity for a human effector cell, a human Fc
receptor, a T cell receptor, a B cell receptor or a binding
specificity for a non-overlapping epitope of HER2, i.e. a
bispecific antibody wherein the first and second antigen binding
sites do not cross-block each other for binding to HER2, e.g. when
tested as described in Example 14.
[0367] Exemplary bispecific antibody molecules of the invention
comprise (i) two antibodies, one with a specificity to HER2 and
another to a second target that are conjugated together, (ii) a
single antibody that has one chain or arm specific to HER2 and a
second chain or arm specific to a second molecule, (iii) a single
chain antibody that has specificity to HER2 and a second molecule,
e.g., via two scFvs linked in tandem by an extra peptide linker;
(iv) a dual-variable-domain antibody (DVD-Ig), where each light
chain and heavy chain contains two variable domains in tandem
through a short peptide linkage (Wu et al., Generation and
Characterization of a Dual Variable Domain Immunoglobulin
(DVD-Ig.TM.) Molecule, In: Antibody Engineering, Springer Berlin
Heidelberg (2010)); (v) a chemically-linked bispecific (Fab').sub.2
fragment; (vi) a Tandab, which is a fusion of two single chain
diabodies resulting in a tetravalent bispecific antibody that has
two binding sites for each of the target antigens; (vii) a
flexibody, which is a combination of scFvs with a diabody resulting
in a multivalent molecule; (viii) a so called "dock and lock"
molecule, based on the "dimerization and docking domain" in Protein
Kinase A, which, when applied to Fabs, can yield a trivalent
bispecific binding protein consisting of two identical Fab
fragments linked to a different Fab fragment; (ix) a so-called
Scorpion molecule, comprising, e.g., two scFvs fused to both
termini of a human Fc-region; and (x) a diabody. In one embodiment,
the bispecific antibody of the present invention is a diabody, a
cross-body, or a bispecific obtained via a controlled Fab arm
exchange as those described in the present invention.
[0368] Examples of platforms useful for preparing bispecific
antibodies include but are not limited to BiTE (Micromet), DART
(MacroGenics), Fcab and Mab.sup.2 (F-star), Fc-engineered IgG1
(Xencor) or DuoBody (based on Fab arm exchange, Genmab, this
application).
[0369] Examples of different classes of bispecific antibodies
include but are not limited to [0370] asymmetric IgG-like
molecules, wherein the one side of the molecule contains the Fab
region or part of the Fab region of at least one antibody, and the
other side of the molecule contains the Fab region or parts of the
Fab region of at least one other antibody; in this class, asymmetry
in the Fc region could also be present, and be used for specific
linkage of the two parts of the molecule; [0371] symmetric IgG-like
molecules, wherein the two sides of the molecule each contain the
Fab region or part of the Fab region of at least two different
antibodies; [0372] IgG fusion molecules, wherein full length IgG
antibodies are fused to extra Fab regions or parts of Fab regions;
[0373] Fc fusion molecules, wherein single chain Fv molecules or
stabilized diabodies are fused to Fc.gamma. regions or parts
thereof; [0374] Fab fusion molecules, wherein different
Fab-fragments are fused together; [0375] ScFv- and diabody-based
molecules wherein different single chain Fv molecules or different
diabodies are fused to each other or to another protein or carrier
molecule.
[0376] Examples of asymmetric IgG-like molecules include but are
not limited to the Triomab/Quadroma (Trion Pharma/Fresenius
Biotech), the Knobs-into-Holes (Genentech), CrossMAbs (Roche) and
the electrostatically-matched (Amgen), the LUZ-Y (Genentech), the
Strand Exchange Engineered Domain body (EMD Serono), the Biclonic
(Merus) and the DuoBody (Genmab A/S).
[0377] Example of symmetric IgG-like molecules include but are not
limited to Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one
Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center),
mAb.sup.2 (F-Star) and CovX-body (CovX/Pfizer).
[0378] Examples of IgG fusion molecules include but are not limited
to Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific
(ImClone/Eli Lilly), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics),
HERCULES (Biogen Idec) and TvAb (Roche).
[0379] Examples of Fc fusion molecules include but are not limited
to ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent
BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting
Technology (Fc-DART) (MacroGenics) and Dual(ScFv).sub.2-Fab
(National Research Center for Antibody Medicine--China).
[0380] Examples of class V bispecific antibodies include but are
not limited to F(ab).sub.2 (Medarex/AMGEN), Dual-Action or Bis-Fab
(Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent
Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).
[0381] Examples of ScFv- and diabody-based molecules include but
are not limited to Bispecific T Cell Engager (BiTE) (Micromet9,
Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting
Technology (DART) (MacroGenics), Single-chain Diabody (Academic),
TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv
Fusion (Merrimack) and COMBODY (Epigen Biotech).
[0382] In one embodiment, the second molecule is a cancer
antigen/tumor-associated antigen such as carcinoembryonic antigen
(CEA), prostate specific antigen (PSA), RAGE (renal antigen),
.alpha.-fetoprotein, CAMEL (CTL-recognized antigen on melanoma), CT
antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10;
NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g.,
MUC1, mucin-CA125, etc.), ganglioside antigens, tyrosinase, gp75,
c-Met, C-myc, Marti, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM or
a cancer-associated integrin, such as a503 integrin. In another
embodiment, the second molecule is a T cell and/or NK cell antigen,
such as CD3 or CD16. In another embodiment, the second molecule is
an angiogenic factor or other cancer-associated growth factor, such
as a vascular endothelial growth factor, a fibroblast growth
factor, epidermal growth factor, angiogenin or a receptor of any of
these, particularly receptors associated with cancer progression
(for instance another one of the HER receptors; HER1, HER3, or
HER4). In one embodiment, the second antigen-binding site binds a
different, preferably non-blocking, site on HER2 than the one bound
by the antibody of the invention. For example, the second molecule
may be derived from, or cross-block HER2-binding of, trastuzumab,
pertuzumab, F5, or C1.
[0383] Methods of preparing bispecific antibodies include those
described in WO 2008119353 (Genmab) and reported van der
Neut-Kolfschoten et al. (Science. 2007 Sep. 14; 317(5844):1554-7)
and it may for example be performed as described in example 20 of
the present invention.
Nucleic Acid Sequences, Vectors and Host Cells
[0384] In a further aspect, the invention relates to nucleic acid
sequences, such as DNA sequences, encoding heavy and light chains
of an antibody of the invention.
[0385] In one embodiment, the nucleic acid sequence encodes an
amino acid sequence selected from the group consisting of: SEQ ID
NO: 1, 5, 8, 12, 15, 19, 22, 26, 29, 32, 35, 39, 42, 46, 49, 53,
56, 60, 63, 67, 70, 74, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125 and 126.
[0386] In another particular embodiment, the nucleic acid sequence
encodes a VH amino acid sequence selected from the group consisting
of: SEQ ID NO: 1, 8, 15, 22, 29, 35, 42, 49, 56, 63, 70, 77, 79,
81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,
111, 113, 115, 117, 119, 121, 123, and 125.
[0387] In another particular embodiment, the nucleic acid sequence
encodes a VL amino acid sequence selected from the group consisting
of: SEQ ID NO: 5, 12, 19, 26, 32, 39, 46, 53, 60, 67, 74, 78, 80,
82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,
112, 114, 116, 118, 120, 122, 124, and 126.
[0388] In an even further aspect, the invention relates to an
expression vector, or a set of expression vectors, encoding an
antibody of the invention. The heavy and light chain of the
antibody may be encoded by the same vector or by different
vector.
[0389] Such expression vectors may be used for recombinant
production of antibodies of the invention.
[0390] In one embodiment, the expression vector of the invention
comprises a nucleotide sequence encoding one or more of the amino
acid sequences selected from the group consisting of: SEQ ID NO: 1,
5, 8, 12, 15, 19, 22, 26, 29, 32, 35, 39, 42, 46, 49, 53, 56, 60,
63, 67, 70, 74, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 121, 122, 123, 124, 125 and 126.
[0391] In another particular embodiment, the expression vector of
the invention comprises a nucleotide sequence encoding one or more
of the VH amino acid sequences selected from the group consisting
of: SEQ ID NO: 1, 8, 15, 22, 29, 35, 42, 49, 56, 63, 70, 77, 79,
81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,
111, 113, 115, 117, 119, 121, 123, and 125.
[0392] In another particular embodiment, the expression vector of
the invention comprises a nucleotide sequence encoding one or more
of the VL amino acid sequences selected from the group consisting
of: SEQ ID NO: 5, 12, 19, 26, 32, 39, 46, 53, 60, 67, 74, 78, 80,
82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,
112, 114, 116, 118, 120, 122, 124, and 126.
[0393] In a further embodiment, the expression vector further
comprises a nucleotide sequence encoding the constant region of a
light chain, a heavy chain or both light and heavy chains of an
antibody, e.g. a human antibody.
[0394] An expression vector in the context of the present invention
may be any suitable vector, including chromosomal, non-chromosomal,
and synthetic nucleic acid vectors (a nucleic acid sequence
comprising a suitable set of expression control elements). Examples
of such vectors include derivatives of SV40, bacterial plasmids,
phage DNA, baculovirus, yeast plasmids, vectors derived from
combinations of plasmids and phage DNA, and viral nucleic acid (RNA
or DNA) vectors. In one embodiment, a HER2 antibody-encoding
nucleic acid is comprised in a naked DNA or RNA vector, including,
for example, a linear expression element (as described in for
instance Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a
compacted nucleic acid vector (as described in for instance U.S.
Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such as
pBR322, pUC 19/18, or pUC 118/119, a "midge" minimally-sized
nucleic acid vector (as described in for instance Schakowski et
al., Mol Ther 3, 793-800 (2001)), or as a precipitated nucleic acid
vector construct, such as a CaPO4-precipitated construct (as
described in for instance WO 00/46147, Benvenisty and Reshef, PNAS
USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and
Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such
nucleic acid vectors and the usage thereof are well known in the
art (see for instance U.S. Pat. Nos. 5,589,466 and 5,973,972).
[0395] Exemplary expression vectors for the antibodies of the
invention are also described in Examples 2 and 3.
[0396] In one embodiment, the vector is suitable for expression of
the HER2 antibody in a bacterial cell. Examples of such vectors
include expression vectors such as BlueScript (Stratagene), pIN
vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509
(1989), pET vectors (Novagen, Madison Wis.) and the like).
[0397] An expression vector may also or alternatively be a vector
suitable for expression in a yeast system. Any vector suitable for
expression in a yeast system may be employed. Suitable vectors
include, for example, vectors comprising constitutive or inducible
promoters such as alpha factor, alcohol oxidase and PGH (reviewed
in: F. Ausubel et al., ed. Current Protocols in Molecular Biology,
Greene Publishing and Wiley InterScience New York (1987), and Grant
et al., Methods in Enzymol 153, 516-544 (1987)).
[0398] An expression vector may also or alternatively be a vector
suitable for expression in mammalian cells, e.g. a vector
comprising glutamine synthetase as a selectable marker, such as the
vectors described in Bebbington (1992) Biotechnology (NY)
10:169-175.
[0399] A nucleic acid and/or vector may also comprises a nucleic
acid sequence encoding a secretion/localization sequence, which can
target a polypeptide, such as a nascent polypeptide chain, to the
periplasmic space or into cell culture media. Such sequences are
known in the art, and include secretion leader or signal
peptides.
[0400] In an expression vector of the invention, HER2
antibody-encoding nucleic acids may comprise or be associated with
any suitable promoter, enhancer, and other expression-facilitating
elements. Examples of such elements include strong expression
promoters (e. g., human CMV IE promoter/enhancer as well as RSV,
SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A)
termination sequences, an origin of replication for plasmid product
in E. coli, an antibiotic resistance gene as selectable marker,
and/or a convenient cloning site (e.g., a polylinker). Nucleic
acids may also comprise an inducible promoter as opposed to a
constitutive promoter such as CMV IE.
[0401] In one embodiment, the HER2 antibody-encoding expression
vector may be positioned in and/or delivered to the host cell or
host animal via a viral vector.
[0402] In an even further aspect, the invention relates to a
recombinant eukaryotic or prokaryotic host cell, such as a
transfectoma, which produces an antibody of the invention as
defined herein. Examples of host cells include yeast, bacterial,
and mammalian cells, such as CHO or HEK cells. For example, in one
embodiment, the present invention provides a cell comprising a
nucleic acid stably integrated into the cellular genome that
comprises a sequence coding for expression of a HER2 antibody of
the present invention. In another embodiment, the present invention
provides a cell comprising a non-integrated nucleic acid, such as a
plasmid, cosmid, phagemid, or linear expression element, which
comprises a sequence coding for expression of a HER2 antibody of
the invention.
[0403] In a further aspect, the invention relates to a hybridoma
which produces an antibody of the invention as defined herein. In
an even further aspect, the invention relates to a transgenic
non-human animal or plant comprising nucleic acids encoding a human
heavy chain and a human light chain, wherein the animal or plant
produces an antibody of the invention of the invention.
[0404] In a further aspect, the invention relates to a method for
producing a HER2 antibody of the invention, said method comprising
the steps of [0405] a) culturing a hybridoma or a host cell of the
invention as described herein above, and [0406] b) purifying the
antibody of the invention from the culture media.
Compositions
[0407] In a further main aspect, the invention relates to a
pharmaceutical composition comprising: [0408] a HER2 antibody as
defined herein, and [0409] a pharmaceutically-acceptable
carrier.
[0410] The pharmaceutical composition of the present invention may
contain one antibody of the present invention or a combination of
different antibodies of the present invention.
[0411] The pharmaceutical compositions may be formulated in
accordance with conventional techniques such as those disclosed in
Remington: The Science and Practice of Pharmacy, 19th Edition,
Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995. A
pharmaceutical composition of the present invention may e.g.
include diluents, fillers, salts, buffers, detergents (e. g., a
nonionic detergent, such as Tween-20 or Tween-80), stabilizers (e.
g., sugars or protein-free amino acids), preservatives, tissue
fixatives, solubilizers, and/or other materials suitable for
inclusion in a pharmaceutical composition.
[0412] Pharmaceutically acceptable carriers include any and all
suitable solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonicity agents, antioxidants and absorption
delaying agents, and the like that are physiologically compatible
with a compound of the present invention. Examples of suitable
aqueous and nonaqueous carriers which may be employed in the
pharmaceutical compositions of the present invention include water,
saline, phosphate buffered saline, ethanol, dextrose, polyols (such
as glycerol, propylene glycol, polyethylene glycol, and the like),
and suitable mixtures thereof, vegetable oils, carboxymethyl
cellulose colloidal solutions, tragacanth gum and injectable
organic esters, such as ethyl oleate, and/or various buffers.
Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. Proper
fluidity may be maintained, for example, by the use of coating
materials, such as lecithin, by the maintenance of the required
particle size in the case of dispersions, and by the use of
surfactants.
[0413] Pharmaceutical compositions of the present invention may
also comprise pharmaceutically acceptable antioxidants for instance
(1) water soluble antioxidants, such as ascorbic acid, cysteine
hydrochloride, sodium bisulfate, sodium metabisulfite, sodium
sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol, and the
like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0414] Pharmaceutical compositions of the present invention may
also comprise isotonicity agents, such as sugars, polyalcohols,
such as mannitol, sorbitol, glycerol or sodium chloride in the
compositions.
[0415] The pharmaceutical compositions of the present invention may
also contain one or more adjuvants appropriate for the chosen route
of administration such as preservatives, wetting agents,
emulsifying agents, dispersing agents, preservatives or buffers,
which may enhance the shelf life or effectiveness of the
pharmaceutical composition. The compounds of the present invention
may be prepared with carriers that will protect the compound
against rapid release, such as a controlled release formulation,
including implants, transdermal patches, and microencapsulated
delivery systems. Such carriers may include gelatin, glyceryl
monostearate, glyceryl distearate, biodegradable, biocompatible
polymers such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid
alone or with a wax, or other materials well known in the art.
Methods for the preparation of such formulations are generally
known to those skilled in the art.
[0416] Sterile injectable solutions may be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients e.g.
as enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients e.g. from those enumerated above. In the case of
sterile powders for the preparation of sterile injectable
solutions, examples of methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0417] The actual dosage levels of the active ingredients in the
pharmaceutical compositions may be varied so as to obtain an amount
of the active ingredient which is effective to achieve the desired
therapeutic response for a particular patient, composition, and
mode of administration, without being toxic to the patient. The
selected dosage level will depend upon a variety of pharmacokinetic
factors including the activity of the particular compositions of
the present invention employed, or the amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0418] The pharmaceutical composition may be administered by any
suitable route and mode. In one embodiment, a pharmaceutical
composition of the present invention is administered parenterally.
"Administered parenterally" as used herein means modes of
administration other than enteral and topical administration,
usually by injection, and include epidermal, intravenous,
intramuscular, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac, intradermal, intraperitoneal,
intratendinous, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal,
intracranial, intrathoracic, epidural and intrasternal injection
and infusion.
[0419] In one embodiment that pharmaceutical composition is
administered by intravenous or subcutaneous injection or
infusion.
Uses
[0420] In a further main aspect, the invention relates to a HER2
antibody of the invention for use as a medicament.
[0421] The HER2 antibodies of the invention may be used for a
number of purposes. In particular, the antibodies of the invention
may be used for the treatment of various forms of cancer, including
metastatic cancer and refractory cancer.
[0422] In one embodiment, the HER2 antibodies of the invention are
used for the treatment of breast cancer, including primary,
metastatic, and refractory breast cancer.
[0423] In one embodiment, the HER2 antibodies of the invention are
used for the treatment of a form of cancer selected from the group
consisting of prostate cancer, non-small cell lung cancer, bladder
cancer, ovarian cancer, gastric cancer, colorectal cancer,
esophageal cancer, squamous cell carcinoma of the head & neck,
cervical cancer, pancreatic cancer, testis cancer, malignant
melanoma and a soft-tissue cancer (e.g. synovial sarcoma).
[0424] Similarly, the invention relates to a method for killing a
tumor cell expressing HER2, comprising administration, to an
individual in need thereof, of an effective amount of an antibody
of the invention, such as an antibody drug-conjugate (ADC).
[0425] In one embodiment, said tumor cell is involved in a form of
cancer selected from the group consisting of: breast cancer,
prostate cancer, non-small cell lung cancer, bladder cancer,
ovarian cancer, gastric cancer, colorectal cancer, esophageal
cancer and squamous cell carcinoma of the head & neck, cervical
cancer, pancreatic cancer, testis cancer, malignant melanoma, and a
soft-tissue cancer (e.g., synovial sarcoma).
[0426] In one embodiment, the tumor cell is one that co-expresses
HER2 and at least one other member of the EGFR family, preferably
EGFR, HER3, or both of EGFR and HER3, and is a tumor cell involved
in breast cancer, colorectal cancer, endometrial/cervical cancer,
lung cancer, malignant melanoma, ovarian cancer, pancreatic cancer,
prostate cancer, testis cancer, a soft-tissue tumor (e.g., synovial
sarcoma), or bladder cancer.
[0427] In one aspect, the invention relates to a method for
treating cancer in a subject, comprising selecting a subject
suffering from a cancer comprising tumor cells co-expressing HER2
and EGFR and/or HER3, and administering to the subject an antibody
of the invention, optionally in the form of an antibody conjugated
to a cytotoxic agent or drug. In one embodiment, the subject
suffers from a cancer selected from the group consisting of breast
cancer, colorectal cancer, endometrial/cervical cancer, lung
cancer, malignant melanoma, ovarian cancer, pancreatic cancer,
prostate cancer, testis cancer, a soft-tissue tumor (e.g., synovial
sarcoma), or bladder cancer.
[0428] Also, the invention relates to the use of a monoclonal
antibody that binds to human HER2 for the preparation of a
medicament for the treatment of cancer, such as one of the specific
cancer indications mentioned above.
[0429] The invention further relates to a monoclonal antibody for
use in the treatment of cancer, such as one of the cancer
indications mentioned above.
[0430] In a further embodiment of the methods of treatment of the
present invention, the efficacy of the treatment is being monitored
during the therapy, e.g. at predefined points in time, by
determining tumor burden or HER2 expression levels on the relevant
tumor cells.
[0431] Dosage regimens in the above methods of treatment and uses
are adjusted to provide the optimum desired response (e.g., a
therapeutic response). For example, a single bolus may be
administered, several divided doses may be administered over time
or the dose may be proportionally reduced or increased as indicated
by the exigencies of the therapeutic situation. Parenteral
compositions may be formulated in dosage unit form for ease of
administration and uniformity of dosage.
[0432] The efficient dosages and the dosage regimens for the HER2
antibodies depend on the disease or condition to be treated and may
be determined by the persons skilled in the art. An exemplary,
non-limiting range for a therapeutically effective amount of a
compound of the present invention is about 0.1-100 mg/kg, such as
about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about
0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1,
about 3, about 5, or about 8 mg/kg.
[0433] A physician or veterinarian having ordinary skill in the art
may readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the HER2 antibody employed in the
pharmaceutical composition at levels lower than that required in
order to achieve the desired therapeutic effect and gradually
increase the dosage until the desired effect is achieved. In
general, a suitable daily dose of a composition of the present
invention will be that amount of the compound which is the lowest
dose effective to produce a therapeutic effect. Administration may
e.g. be parenteral, such as intravenous, intramuscular or
subcutaneous. In one embodiment, the HER2 antibodies may be
administered by infusion in a weekly dosage of from 10 to 500
mg/m.sup.2, such as of from 200 to 400 mg/m.sup.2. Such
administration may be repeated, e.g., 1 to 8 times, such as 3 to 5
times. The administration may be performed by continuous infusion
over a period of from 2 to 24 hours, such as of from 2 to 12 hours.
In one embodiment, the HER2 antibodies may be administered by slow
continuous infusion over a long period, such as more than 24 hours,
in order to reduce toxic side effects.
[0434] In one embodiment the HER2 antibodies may be administered in
a weekly dosage of from 250 mg to 2000 mg, such as for example 300
mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times,
such as from 4 to 6 times when given once a week. Such regimen may
be repeated one or more times as necessary, for example, after 6
months or 12 months. The dosage may be determined or adjusted by
measuring the amount of compound of the present invention in the
blood upon administration by for instance taking out a biological
sample and using anti-idiotypic antibodies which target the antigen
binding region of the HER2 antibodies of the present invention.
[0435] The efficient dosages and the dosage regimens for the
bispecific antibodies depend on the disease or condition to be
treated and may be determined by the persons skilled in the art. An
exemplary, non-limiting range for a therapeutically effective
amount of a bispecific antibody of the present invention is about
0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20
mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about
such as 0.3, about 1, about 3, about 5, or about 8 mg/kg.
[0436] In one embodiment, the HER2 antibodies may be administered
by maintenance therapy, such as, e.g., once a week for a period of
6 months or more.
[0437] A HER2 antibody may also be administered prophylactically in
order to reduce the risk of developing cancer, delay the onset of
the occurrence of an event in cancer progression, and/or reduce the
risk of recurrence when a cancer is in remission.
[0438] HER2 antibodies may also be administered in combination
therapy, i.e., combined with other therapeutic agents relevant for
the disease or condition to be treated. Accordingly, in one
embodiment, the antibody-containing medicament is for combination
with one or more further therapeutic agent, such as a cytotoxic,
chemotherapeutic or anti-angiogenic agent.
[0439] Such combined administration may be simultaneous, separate
or sequential. For simultaneous administration the agents may be
administered as one composition or as separate compositions, as
appropriate. The present invention thus also provides methods for
treating a disorder involving cells expressing HER2 as described
above, which methods comprise administration of a HER2 antibody of
the present invention combined with one or more additional
therapeutic agents as described below.
[0440] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing HER2 in a
subject, which method comprises administration of a therapeutically
effective amount of a HER2 antibody of the present invention, and
optionally at least one additional therapeutic agent, or an
antibody binding to a different epitope than said HER2 antibody, to
a subject in need thereof.
[0441] In one embodiment, the present invention provides a method
for treating or preventing cancer, which method comprises
administration of a therapeutically effective amount of a HER2
antibody of the present invention and at least one additional
therapeutic agent to a subject in need thereof.
[0442] In one embodiment, such an additional therapeutic agent may
be selected from an antimetabolite, such as methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine,
5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine
or cladribine.
[0443] In another embodiment, such an additional therapeutic agent
may be selected from an alkylating agent, such as mechlorethamine,
thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine
(CCNU), cyclophosphamide, busulfan, dibromomannitol,
streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C,
cisplatin and other platinum derivatives, such as carboplatin.
[0444] In another embodiment, such an additional therapeutic agent
may be selected from an anti-mitotic agent, such as taxanes, for
instance docetaxel, and paclitaxel, and vinca alkaloids, for
instance vindesine, vincristine, vinblastine, and vinorelbine.
[0445] In another embodiment, such an additional therapeutic agent
may be selected from a topoisomerase inhibitor, such as topotecan
or irinotecan, or a cytostatic drug, such as etoposide and
teniposide.
[0446] In another embodiment, such an additional therapeutic agent
may be selected from a growth factor inhibitor, such as an
inhibitor of ErbB1 (EGFR) (such as an EGFR antibody, e.g.
zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR
inhibitors, such as gefitinib or erlotinib), another inhibitor of
ErbB2 (HER2/neu) (such as a HER2 antibody, e.g. trastuzumab,
trastuzumab-DM1 or pertuzumab) or an inhibitor of both EGFR and
HER2, such as lapatinib).
[0447] In another embodiment, such an additional therapeutic agent
may be selected from a tyrosine kinase inhibitor, such as imatinib
(Glivec, Gleevec STI571) or lapatinib, PTK787/ZK222584.
[0448] In another embodiment, the present invention provides a
method for treating a disorder involving cells expressing HER2 in a
subject, which method comprises administration of a therapeutically
effective amount of an HER2 antibody of the present invention and
at least one inhibitor of angiogenesis, neovascularization, and/or
other vascularization to a subject in need thereof
[0449] Examples of such angiogenesis inhibitors are urokinase
inhibitors, matrix metalloprotease inhibitors (such as marimastat,
neovastat, BAY 12-9566, AG 3340, BMS-275291 and similar agents),
inhibitors of endothelial cell migration and proliferation (such as
TNP-470, squalamine, 2-methoxyestradiol, combretastatins,
endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plough
Corp, Madison, N.J.), R115777 (Janssen Pharmaceutica, Inc,
Titusville, N.J.) and similar agents), antagonists of angiogenic
growth factors (such as such as ZD6474, SU6668, antibodies against
angiogenic agents and/or their receptors (such as VEGF (e.g.
bevacizumab), bFGF, and angiopoietin-1), thalidomide, thalidomide
analogs (such as CC-5013), Sugen 5416, SU5402, antiangiogenic
ribozyme (such as angiozyme), interferon .alpha. (such as
interferon .alpha.2a), suramin and similar agents), VEGF-R kinase
inhibitors and other anti-angiogenic tyrosine kinase inhibitors
(such as SU011248), inhibitors of endothelial-specific
integrin/survival signaling (such as vitaxin and similar agents),
copper antagonists/chelators (such as tetrathiomolybdate, captopril
and similar agents), carboxyamido-triazole (CAI), ABT-627, CM101,
interleukin-12 (IL-12), IM862, PNU145156E as well as nucleotide
molecules inhibiting angiogenesis (such as antisense-VEGF-cDNA,
cDNA coding for angiostatin, cDNA coding for p53 and cDNA coding
for deficient VEGF receptor-2).
[0450] Other examples of such inhibitors of angiogenesis,
neovascularization, and/or other vascularization are
anti-angiogenic heparin derivatives (e.g., heperinase III),
temozolomide, NK4, macrophage migration inhibitory factor,
cyclooxygenase-2 inhibitors, inhibitors of hypoxia-inducible factor
1, anti-angiogenic soy isoflavones, oltipraz, fumagillin and
analogs thereof, somatostatin analogues, pentosan polysulfate,
tecogalan sodium, dalteparin, tumstatin, thrombospondin, NM-3,
combrestatin, canstatin, avastatin, antibodies against other
targets, such as anti-alpha-v/beta-3 integrin and anti-kininostatin
antibodies.
[0451] In one embodiment, a therapeutic agent for use in
combination with a HER2 antibody for treating the disorders as
described above may be an anti-cancer immunogen, such as a cancer
antigen/tumor-associated antigen (e.g., epithelial cell adhesion
molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen
(CEA), tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A,
MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines (e.g.,
human papillomavirus vaccines) or tumor-derived heat shock
proteins,
[0452] In one embodiment, a therapeutic agent for use in
combination with a HER2 antibody for treating the disorders as
described above may be an anti-cancer cytokine, chemokine, or
combination thereof. Examples of suitable cytokines and growth
factors include IFN.gamma., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12,
IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29,
KGF, IFN.alpha. (e.g., INF.alpha.2b), IFN.beta., GM-CSF, CD40L,
Flt3 ligand, stem cell factor, ancestim, and TNF.alpha.. Suitable
chemokines may include Glu-Leu-Arg (ELR)-negative chemokines such
as IP-10, MCP-3, MIG, and SDF-1.alpha. from the human CXC and C--C
chemokine families. Suitable cytokines include cytokine
derivatives, cytokine variants, cytokine fragments, and cytokine
fusion proteins.
[0453] In one embodiment, a therapeutic agent for use in
combination with a HER2 antibody for treating the disorders as
described above may be a cell cycle control/apoptosis regulator (or
"regulating agent"). A cell cycle control/apoptosis regulator may
include molecules that target and modulate cell cycle
control/apoptosis regulators such as (i) cdc-25 (such as NSC
663284), (ii) cyclin-dependent kinases that overstimulate the cell
cycle (such as flavopiridol (L868275, HMR1275),
7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine
(R-roscovitine, CYC202)), and (iii) telomerase modulators (such as
BIBR1532, SOT-095, GRN163 and compositions described in for
instance U.S. Pat. Nos. 6,440,735 and 6,713,055). Non-limiting
examples of molecules that interfere with apoptotic pathways
include TNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2
ligand (Apo-2L), antibodies that activate TRAIL receptors,
IFNs,.quadrature. and anti-sense Bcl-2.
[0454] In one embodiment, a therapeutic agent for use in
combination with a HER2 antibody for treating the disorders as
described above may be a hormonal regulating agent, such as agents
useful for anti-androgen and anti-estrogen therapy. Examples of
such hormonal regulating agents are tamoxifen, idoxifene,
fulvestrant, droloxifene, toremifene, raloxifene,
diethylstilbestrol, ethinyl estradiol/estinyl, an antiandrogene
(such as flutaminde/eulexin), a progestin (such as such as
hydroxyprogesterone caproate, medroxy-progesterone/provera,
megestrol acepate/megace), an adrenocorticosteroid (such as
hydrocortisone, prednisone), luteinizing hormone-releasing hormone
(and analogs thereof and other LHRH agonists such as buserelin and
goserelin), an aromatase inhibitor (such as anastrazole/arimidex,
aminoglutethimide/cytraden, exemestane) or a hormone inhibitor
(such as octreotide/sandostatin).
[0455] In one embodiment, a therapeutic agent for use in
combination with a HER2 antibody for treating the disorders as
described above may be an anti-anergic agent, such as compounds are
molecules that block the activity of CTLA-4, e.g. ipilimumab.
[0456] In one embodiment, a therapeutic agent for use in
combination with a HER2 antibody for treating the disorders as
described above may be an anti-cancer nucleic acid or an
anti-cancer inhibitory RNA molecule.
[0457] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with a HER2 antibody
for treating the disorders as described above are differentiation
inducing agents, retinoic acid analogues (such as all trans
retinoic acid, 13-cis retinoic acid and similar agents), vitamin D
analogues (such as seocalcitol and similar agents), inhibitors of
ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa, PDGFRbeta, Flk2,
Flt4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA, TRKC, RON (such as an
anti-RON antibody), Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7
and similar agents.
[0458] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with a HER2 antibody
for treating the disorders as described above are estramustine and
epirubicin.
[0459] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with a HER2 antibody
for treating the disorders as described above are a HSP90 inhibitor
like 17-allyl amino geld-anamycin, antibodies directed against a
tumor antigen such as PSA, CA125, KSA, integrins, e.g. integrin 01,
or inhibitors of VCAM.
[0460] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with a HER2 antibody
for treating the disorders as described above are
calcineurin-inhibitors (such as valspodar, PSC 833 and other MDR-1
or p-glycoprotein inhibitors), TOR-inhibitors (such as sirolimus,
everolimus and rapamcyin). and inhibitors of "lymphocyte homing"
mechanisms (such as FTY720), and agents with effects on cell
signaling such as adhesion molecule inhibitors (for instance
anti-LFA).
[0461] In one embodiment, the HER2 antibody of the invention is for
use in combination with one or more other therapeutic antibodies,
such as ofatumumab, zanolimumab, daratumumab, ranibizumab,
nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab
(Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab
(Tysabri), omalizumab (Xolair), efalizumab (Raptiva) and/or
rituximab.
[0462] In another embodiment, two or more different antibodies of
the invention as described herein are used in combination for the
treatment of disease. Particularly interesting combinations include
two or more non-blocking antibodies. Such combination therapy may
lead to binding of an increased number of antibody molecules per
cell, which may give increase efficacy, e.g. via activation of
complement-mediated lysis.
[0463] In addition to the above, other embodiments of combination
therapies of the invention include the following:
[0464] For the treatment of breast cancer, a HER2 antibody or a
therapeutic conjugate thereof, in combination with methotrexate,
paclitaxel, doxorubicin, carboplatin, cyclophosphamide,
daunorubicin, epirubicin, 5-fluorouracil, gemcitabine, ixabepilone,
mutamycin, mitoxantrone, vinorelbine, docetaxel, thiotepa,
vincristine, capecitabine, an EGFR antibody (e.g. zalutumumab,
cetuximab, panitumumab or nimotuzumab) or other EGFR inhibitor
(such as gefitinib or erlotinib), another HER2 antibody or
-conjugate (such as, e.g., trastuzumab, trastuzumab-DM1 or
pertuzumab), an inhibitor of both EGFR and HER2 (such as
lapatinib), and/or in combination with a HER3 inhibitor.
[0465] For the treatment of non-small-cell lung cancer, a HER2
antibody in combination with EGFR inhibitors, such as an EGFR
antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab
or other EGFR inhibitors (such as gefitinib or erlotinib), or in
combination with an another HER2 agent (such as a HER2 antibody,
e.g. trastuzumab, trastuzumab-DM1 or pertuzumab) or in combination
with an inhibitor of both EGFR and HER2, such as lapatinib, or in
combination with a HER3 inhibitor.
[0466] For the treatment of colorectal cancer a HER2 antibody in
combination with one or more compounds selected from: gemcitabine,
bevacizumab, FOLFOX, FOLFIRI, XELOX, IFL, oxaliplatin, irinotecan,
5-FU/LV, Capecitabine, UFT, EGFR targeting agents, such as
cetuximab, panitumumab, zalutumumab; VEGF inhibitors, or tyrosine
kinase inhibitors such as sunitinib.
[0467] For the treatment of prostate cancer a HER2 antibody in
combination with one or more compounds selected from:
hormonal/antihormonal therapies; such as antiandrogens, Luteinizing
hormone releasing hormone (LHRH) agonists, and chemotherapeutics
such as taxanes, mitoxantrone, estramustine, 5FU, vinblastine, and
ixabepilone.
Radiotherapy--Surgery
[0468] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing HER2 in a
subject, which method comprises administration of a therapeutically
effective amount of a HER2 antibody, such as a HER2 antibody of the
present invention, and radiotherapy to a subject in need
thereof.
[0469] In one embodiment, the present invention provides a method
for treating or preventing cancer, which method comprises
administration of a therapeutically effective amount of a HER2
antibody, such as a HER2 antibody of the present invention, and
radiotherapy to a subject in need thereof.
[0470] In one embodiment, the present invention provides the use of
a HER2 antibody, such as a HER2 antibody of the present invention,
for the preparation of a pharmaceutical composition for treating
cancer to be administered in combination with radiotherapy.
[0471] Radiotherapy may comprise radiation or associated
administration of radiopharmaceuticals to a patient is provided.
The source of radiation may be either external or internal to the
patient being treated (radiation treatment may, for example, be in
the form of external beam radiation therapy (EBRT) or brachytherapy
(BT)). Radioactive elements that may be used in practicing such
methods include, e.g., radium, cesium-137, iridium-192,
americium-241, gold-198, cobalt-57, copper-67, technetium-99,
iodide-123, iodide-131, and indium-111.
[0472] In a further embodiment, the present invention provides a
method for treating or preventing cancer, which method comprises
administration to a subject in need thereof of a therapeutically
effective amount of a HER2 antibody, such as a HER2 antibody of the
present invention, in combination with surgery.
Diagnostic Uses
[0473] The HER2 antibodies of the invention may also be used for
diagnostic purposes. Thus, in a further aspect, the invention
relates to a diagnostic composition comprising a HER2 antibody as
defined herein.
[0474] In one embodiment, the HER2 antibodies of the present
invention may be used in vivo or in vitro for diagnosing diseases
wherein activated cells expressing HER2 play an active role in the
pathogenesis, by detecting levels of HER2, or levels of cells which
contain HER2 on their membrane surface. This may be achieved, for
example, by contacting a sample to be tested, optionally along with
a control sample, with the HER2 antibody under conditions that
allow for formation of a complex between the antibody and HER2.
[0475] Thus, in a further aspect, the invention relates to a method
for detecting the presence of HER2 antigen, or a cell expressing
HER2, in a sample comprising: [0476] contacting the sample with a
HER2 antibody of the invention under conditions that allow for
formation of a complex between the antibody and HER2; and [0477]
analyzing whether a complex has been formed.
[0478] In one embodiment, the method is performed in vitro.
[0479] More specifically, the present invention provides methods
for the identification of, and diagnosis of invasive cells and
tissues, and other cells targeted by HER2 antibodies of the present
invention, and for the monitoring of the progress of therapeutic
treatments, status after treatment, risk of developing cancer,
cancer progression, and the like.
[0480] Suitable labels for the HER2 antibody and/or secondary
antibodies used in such techniques are well-known in the art.
[0481] In a further aspect, the invention relates to a kit for
detecting the presence of HER2 antigen, or a cell expressing HER2,
in a sample comprising [0482] a HER2 antibody of the invention or a
bispecific molecule of the invention; and [0483] instructions for
use of the kit.
[0484] In one embodiment, the present invention provides a kit for
diagnosis of cancer comprising a container comprising a HER2
antibody, and one or more reagents for detecting binding of the
HER2 antibody to HER2. Reagents may include, for example,
fluorescent tags, enzymatic tags, or other detectable tags. The
reagents may also include secondary or tertiary antibodies or
reagents for enzymatic reactions, wherein the enzymatic reactions
produce a product that may be visualized.
Anti-Idiotypic Antibodies
[0485] In a further aspect, the invention relates to an
anti-idiotypic antibody which binds to a HER2 antibody of the
invention as described herein.
[0486] An anti-idiotypic (Id) antibody is an antibody which
recognizes unique determinants generally associated with the
antigen-binding site of an antibody. An Id antibody may be prepared
by immunizing an animal of the same species and genetic type as the
source of a HER2 mAb with the mAb to which an anti-Id is being
prepared. The immunized animal typically can recognize and respond
to the idiotypic determinants of the immunizing antibody by
producing an antibody to these idiotypic determinants (the anti-Id
antibody).
[0487] An anti-Id antibody may also be used as an "immunogen" to
induce an immune response in yet another animal, producing a
so-called anti-anti-Id antibody. An anti-anti-Id may be
epitopically identical to the original mAb, which induced the
anti-Id. Thus, by using antibodies to the idiotypic determinants of
a mAb, it is possible to identify other clones expressing
antibodies of identical specificity.
[0488] The present invention is further illustrated by the
following examples, which should not be construed as limiting the
scope of the invention.
[0489] The present invention is further illustrated by the
following examples, which should not be construed as limiting the
scope of the invention.
EXAMPLES
Example 1--Expression Constructs for HER2 and HER2 Variants
[0490] Fully codon-optimized constructs for expression of full
length HER2 (1255 aa, Swissprot P04626), the extracellular domain
(ECD) of HER2 (Her2-ECDHis, aa 1-653 with a C-terminal His6 tag),
the naturally occurring HER2 splice variant (Her2-delex16,
resulting from exon 16 deletion and lacking aa 633-648) and a
truncated form of the HER2 receptor (Her2-stumpy, aa 648-1256),
were generated. The construct contained suitable restriction sites
for cloning and an optimal Kozak sequence (Kozak, M., Gene 1999;
234(2):187-208.). The constructs were cloned in the mammalian
expression vector pEE13.4 (Lonza Biologics; Bebbington, C. R., et
al., Biotechnology (N Y) 1992; 10(2):169-75) and fully sequenced to
confirm the correctness of the construct.
Example 2--Expression Constructs for Pertuzumab, C1 and F5
[0491] Fully codon-optimized constructs for expression of the heavy
chain (HC) and the light chain (LC) of the IgG1 antibodies
pertuzumab, C1 and F5 in HEK cells, were generated. The variable
regions encoded by these constructs are identical to those
described in U.S. Pat. No. 6,949,245 for pertuzumab heavy chain and
light chain and U.S. Pat. No. 7,244,826 for C1 and F5 heavy and
light chain. For C1 and F5, the mammalian expression vectors p33G1f
and p33K or p33L (pcDNA3.3 (Invitrogen)) containing the fully codon
optimized constant region for the human IgG1 heavy chain (allotype
f), the human kappa light chain or the human lambda light chain,
respectively, were used. For pertuzumab, the mammalian expression
vectors pG1f (pEE12.4 (Lonza Biologics) and pKappa (pEE6.4 (Lonza
Biologics), containing the fully codon-optimized constant region
for the human IgG1 heavy chain (allotype f) and the human kappa
light chain, respectively, were used.
[0492] Trastuzumab (Herceptin.RTM.) can be produced in the same
manner, using the heavy and light chain sequences described in,
e.g., U.S. Pat. No. 7,632,924.
Example 3--Transient Expression in HEK-293 or CHO Cells
[0493] Freestyle.TM. 293-F (a HEK-293 subclone adapted to
suspension growth and chemically defined Freestyle medium,
(HEK-293F)) cells were obtained from Invitrogen and transfected
with the appropriate plasmid DNA, using 293fectin (Invitrogen)
according to the manufacturer's instructions. In the case of
antibody expression, the appropriate heavy chain and light chain
expression vectors were co-expressed.
[0494] pEE13.4Her2, pEE13.4Her2-delex16 and pEE13.4Her2-stumpy were
transiently transfected in the Freestyle.TM. CHO--S(Invitrogen)
cell line using Freestyle MAX transfection reagent (Invitrogen).
Expression of HER2 and Her2-delex16 was tested by means of FACS
analysis as described below.
Example 4--Stable Polyclonal Pool Expression in NS0
[0495] pEE13.4Her2, pEE13.4Her2-delex16 and pEE13.4Her2-stumpy were
stably transfected in NS0 cells by nucleofection (Amaxa). A pool of
stably transfected cells was established after selection on
glutamine dependent growth, based on the integrated glutamine
synthetase selection marker (Barnes, L. M., et al., Cytotechnology
2000; 32(2):109-123).
Example 5--Purification of His-Tagged HER2
[0496] Her2ECDHis was expressed in HEK-293F cells. The His-tag in
Her2ECDHis enabled purification with immobilized metal affinity
chromatography, since the His-tagged protein binds strongly to the
resin beads, while other proteins present in the culture
supernatant do not bind strongly.
[0497] In this process, a chelator fixed onto the chromatographic
resin was charged with Co.sup.2+ cations. Her2ECDHis containing
supernatant was incubated with the resin in batch mode (i.e.
solution). After incubation, the beads were retrieved from the
supernatant and packed into a column. The column was washed in
order to remove weakly bound proteins. The strongly bound
Her2ECDHis proteins were then eluted with a buffer containing
imidazole, which competes with the binding of His to Co.sup.2+. The
eluent was removed from the protein by buffer exchange on a
desalting column.
Example 6--Immunization Procedure of Transgenic Mice
[0498] Antibodies 001, 019, 021, 025, 027, 032, 033, 035, 036, 049,
050, 051, 054, 055, 084, 091, 094, 098, 100, 105, 123 and 124 were
derived from the following immunization: three female HCo12 mice,
one male and two female HCo12-Balb/C mice, one male HCo17 mouse and
one male HCo20 mouse (Medarex, San Jose, Calif., USA) were
immunized alternating with 5.times.10.sup.6 NS0 cells transiently
transfected with Her2ECD intraperitoneal (IP) and 20 .mu.g
Her2ECDHis protein coupled to the hapten Keyhole Limpet Hemocyanin
(KLH) subcutaneous (SC) at the tail base, with an interval of
fourteen days. A maximum of eight immunizations was performed per
mouse (four IP and four SC immunizations). The first immunization
with cells was done in complete Freunds' adjuvant (CFA; Difco
Laboratories, Detroit, Mich., USA). For all other immunizations,
cells were injected IP in PBS and KLH coupled Her2ECD was injected
SC using incomplete Freunds' adjuvant (IFA; Difco Laboratories,
Detroit, Mich., USA).
[0499] Antibodies 125, 127, 129, 132, 152, 153 and 159 were derived
from the following immunization: one male and two female
HCo12-Balb/C mice, one female HCo20 mouse, and one female HCo12
mouse (Medarex) were immunized alternating with 5.times.10.sup.6
NS0 cells transiently transfected with Her2delex16 IP and 20 .mu.g
Her2ECDHis protein coupled to the hapten Keyhole Limpet Hemocyanin
(KLH) SC at the tail base, with an interval of fourteen days. A
maximum of eight immunizations was performed per mouse (four IP and
four SC immunizations). The first immunization with cells was done
in complete Freunds' adjuvant (CFA; Difco Laboratories, Detroit,
Mich., USA). For all other immunizations, cells were injected IP in
PBS and KLH coupled Her2ECD was injected SC using incomplete
Freunds' adjuvant (IFA; Difco Laboratories, Detroit, Mich.,
USA).
[0500] Antibody 143, 160, 161, 162, 166 and 169 were derived from
the following immunization: one female and one male Hco12 mouse,
one female Hco12-Balb/C mouse, one male HCo17 mouse and one male
HCo20 mouse (Medarex) were immunized alternating with 20 .mu.g
Her2ECDHis protein coupled to the hapten Keyhole Limpet Hemocyanin
(KLH), alternating IP and SC at the tail base with an interval of
fourteen days. A maximum of eight immunizations was performed per
mouse (four IP and four SC immunizations). The first immunization
was done IP in complete Freunds' adjuvant (CFA; Difco Laboratories,
Detroit, Mich., USA). The other immunizations were injected using
incomplete Freunds' adjuvant (IFA; Difco Laboratories, Detroit,
Mich., USA).
[0501] Mice with at least two sequential titers against
TC1014-Her2, TC1014-Her2delex16 or TC1014-Her2stumpy in the antigen
specific FMAT screening assay (as described in example 7), were
considered positive and fused.
Example 7--Homogeneous Antigen Specific Screening Assay
[0502] The presence of HER2 antibodies in sera of immunized mice or
HuMab (human monoclonal antibody) hybridoma or transfectoma culture
supernatant was determined by homogeneous antigen specific
screening assays (four quadrant) using Fluorometric Micro volume
Assay Technology (FMAT; Applied Biosystems, Foster City, Calif.,
USA). For this, a combination of 4 cell based assays was used.
Binding to TC1014-Her2 (HEK-293F cells transiently expressing the
HER2 receptor; produced as described above), TC1014-Her2delex16
(HEK-293F cells transiently expressing the extracellular domain of
Her2-delex (a 16 amino acid deletion mutant of the HER2 receptor;
produced as described above) and TC1014-Her2stumpy (HEK-293F cells
transiently expressing the extracellular stumpy domain of the HER2
receptor; produced as described above) as well as HEK293 wild type
cells (negative control cells which do not express HER2) was
determined. Samples were added to the cells to allow binding to
HER2. Subsequently, binding of HuMab was detected using a
fluorescent conjugate (Goat anti-Human IgG-Cy5; Jackson
ImmunoResearch). TH1014-Pertuzumab (produced in HEK-293F cells) was
used as a positive control and HuMab-mouse pooled serum and
HuMab-KLH were used as negative controls. The samples were scanned
using an Applied Biosystems 8200 Cellular Detection System (8200
CDS) and `counts.times.fluorescence` was used as read-out. Samples
were stated positive when counts were higher than 50 and
counts.times.fluorescence were at least three times higher than the
negative control.
Example 8--HuMab Hybridoma Generation
[0503] HuMab mice with sufficient antigen-specific titer
development (defined as above) were sacrificed and the spleen and
lymph nodes flanking the abdominal aorta and vena cava were
collected. Fusion of splenocytes and lymph node cells to a mouse
myeloma cell line was done by electrofusion using a CEEF 50
Electrofusion System (Cyto Pulse Sciences, Glen Burnie, Md., USA),
essentially according to the manufacturer's instructions. Next, the
primary wells were sub cloned using the ClonePix system (Genetix,
Hampshire, UK). To this end specific primary well hybridoma's were
seeded in semisolid medium made from 40% CloneMedia (Genetix,
Hampshire, UK) and 60% HyQ 2.times. complete media (Hyclone,
Waltham, USA). The sub clones were retested in the antigen-specific
binding assay as described in Example 7 and IgG levels were
measured using an Octet (Fortebio, Menlo Park, USA) in order to
select the most specific and best producing clone per primary well
for further expansion. Further expansion and culturing of the
resulting HuMab hybridomas were done based upon standard protocols
(e.g. as described in Coligan J. E., Bierer, B. E., Margulies, D.
H., Shevach, E. M. and Strober, W., eds. Current Protocols in
Immunology, John Wiley & Sons, Inc., 2006). Clones derived by
this process were designated PC1014.
Example 9--Mass Spectrometry of Purified Antibodies
[0504] Small aliquots of 0.8 mL antibody containing supernatant
from 6-well or Hyperflask stage were purified using PhyTip columns
containing Protein G resin (PhyNexus Inc., San Jose, USA) on a
Sciclone ALH 3000 workstation (Caliper Lifesciences, Hopkinton,
USA). The PhyTip columns were used according to manufacturer's
instructions, although buffers were replaced by: Binding Buffer PBS
(B.Braun, Medical B.V., Oss, Netherlands) and Elution Buffer 0.1M
Glycine-HCl pH 2.7 (Fluka Riedel-de Haen, Buchs, Germany). After
purification, samples were neutralized with 2M Tris-HCl, pH 9.0
(Sigma-Aldrich, Zwijndrecht, Netherlands). Alternatively, in some
cases larger volumes of culture supernatant were purified using
MabSelect SuRe.
[0505] After purification, the samples were placed in a 384-well
plate (Waters, 100 .mu.l square well plate, part #186002631).
Samples were deglycosylated overnight at 37.degree. C. with
N-glycosidase F (Roche cat no 11365177001. DTT (15 mg/mL) was added
(1 .mu.L/well) and incubated for 1 h at 37.degree. C. Samples (5 or
6 .mu.L) were desalted on an Acquity UPLC.TM. (Waters, Milford,
USA) with a BEH300 C18, 1.7 .mu.m, 2.1.times.50 mm column at
60.degree. C. MQ water and LC-MS grade acetonitrile (Biosolve, cat
no 01204101, Valkenswaard, The Netherlands) with both 0.1% formic
acid (Fluka, cat no 56302, Buchs, Germany), were used as Eluens A
and B, respectively. Time-of-flight electrospray ionization mass
spectra were recorded on-line on a micrOTOF.TM. mass spectrometer
(Bruker, Bremen, Germany) operating in the positive ion mode. Prior
to analysis, a 900-3000 m/z scale was calibrated with ES tuning mix
(Agilent Technologies, Santa Clara, USA). Mass spectra were
deconvoluted with DataAnalysis.TM. software v. 3.4 (Bruker) using
the Maximal Entropy algorithm searching for molecular weights
between 5 and 80 kDa.
[0506] After deconvolution, the resulting heavy and light chain
masses for all samples were compared in order to find duplicate
antibodies. This was sometimes due to the presence of an extra
light chain, but in the comparison of the heavy chains, the
possible presence of C-terminal lysine variants was also taken into
account. This resulted in a list of unique antibodies, i.e., a
unique combination of specific heavy and light chains. In case
duplicate antibodies were found, one unique antibody was selected
based on results from other tests.
Example 10--Sequence Analysis of the HER2 Antibody Variable Domains
and Cloning in Expression Vectors
[0507] Total RNA of the HER2 HuMabs was prepared from
5.times.10.sup.6 hybridoma cells and 5'-RACE-Complementary DNA
(cDNA) was prepared from 100 ng total RNA, using the SMART RACE
cDNA Amplification kit (Clontech), according to the manufacturer's
instructions. VH and VL coding regions were amplified by PCR and
cloned directly, in frame, in the pG1f and pKappa expression
vectors, by ligation independent cloning (Aslanidis, C. and P. J.
de Jong, Nucleic Acids Res 1990; 18(20): 6069-74). Clones derived
by this process were designated TH1014. For each antibody, 16 VL
clones and 8 VH clones were sequenced. Clones which predicted heavy
and light chain mass in agreement with the mass of the hybridoma
derived material of the same antibody (as determined by mass
spectrometry) were selected for further study and expression.
[0508] The resulting sequences are shown in FIGS. 1 and 2 and in
the Sequence Listing. Selected sequences are also described in more
detail below. CDR sequences were defined according to IMGT (Lefranc
M P. et al., Nucleic Acids Research, 27, 209-212, 1999 and Brochet
X. Nucl. Acids Res. 36, W503-508 (2008)). Table 1, Table 2 and
Table 3 give an overview of antibody sequence information or
germline sequences, and Table 4 shows consensus sequences.
TABLE-US-00003 TABLE 1 Heavy chain variable region (VH), light
chain variable region (VL) and CDR sequences of HuMabs 169, 050,
084, 025, 091, 129, 127, 159, 098, 153, and 132. SEQ ID No: 1 VH
169 QVQLVQSGAEVKKPG ASVKVSCKASGYTFT NYGISWVRQAPGQGL EWMGWLSAYSGNTIY
AQKLQGRVTMTTDTS TTTAYMELRSLRSDD TAVYYCARDRIVVRP DYFDYWGQGTLVTVS S
SEQ ID No: 2 VH 169, CDR1 GYTFTNYG SEQ ID No: 3 VH 169, CDR2
LSAYSGNT SEQ ID No: 4 VH 169, CDR3 ARDRIVVRPDYFDY SEQ ID No: 5 VL
169 EIVLTQSPATLSLSP GERATLSCRASQSVS SYLAWYQQKPGQAPR LLIYDASNRATGIPA
RFSGSGSGTDFTLTI SSLEPEDFAVYYCQQ RSNWPRTFGQGTKVE IK SEQ ID No: 6 VL
169, CDR1 QSVSSY VL 169, CDR2 DAS SEQ ID No: 7 VL 169, CDR3
QQRSNWPRT SEQ ID No: 8 VH 050 EVQLLESGGGLVQPG GSLRLSCAASGFTFS
SYAMNWVRQAPGKGL EWVSAISGRGGTTYY ADSVKGRFTISRDNS KNTLYLQMSSLRAED
TAVYYCAKARANWDY FDYWGQGTLVTVSS SEQ ID No: 9 VH 050, CDR1 GFTFSSYA
SEQ ID No: 10 VH 050, CDR2 ISGRGGTT SEQ ID No: 11 VH 050, CDR3
AKARANWDYFDY SEQ ID No: 12 VL 050 DIQMTQSPSSVSASV GDRVTITCRASQGIS
SWLAWYQHKPGKAPK LLIYAASILQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ
ANSFPITFGQGTRLE IK SEQ ID No: 13 VL 050, CDR1 QGISSW VL 050, CDR2
AAS SEQ ID No: 14 VL 050, CDR3 QQANSFPIT SEQ ID No: 15 VH 084
QVQLVQSGAEVKKPG SSVKVSCKASGGTFR TYAINWVRQAPGQGL EWMGRINTVLGIVNH
AQKFQGRVTITADKS TNTAYMELNSLRSED TAVYYCAREKGVDYY YGIEVWGQGTTVTVS S
SEQ ID No: 16 VH 084, CDR1 GGTFRTYA SEQ ID No: 17 VH 084, CDR2
INTVLGIV SEQ ID No: 18 VH 084, CDR3 AREKGVDYYYGIEV SEQ ID No: 19 VL
084 DIQMTQSPSSVSASV GDRVTITCRASQGIS SWLAWYQHKPGKAPK LLIYVASTLQSGVPS
RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ ANSFPLTFGGGTKVE IK SEQ ID No: 20 VL
084, CDR1 QGISSW VL 084, CDR2 VAS SEQ ID No: 21 VL 084, CDR3
QQANSFPLT SEQ ID No: 22 VH 025 QVQLQQWGAGLLKPS ETLSLTCAVYGGSFS
DYYWNWIRQPPGKGL EWIGEIHHSGSTNYN PSLKSRVTISVDTSK NQFSLKLSSVTAADT
AVYYCARGYYDSGVY YFDYWAQGTLVTVSS SEQ ID No: 23 VH 025, CDR1 GGSFSDYY
SEQ ID No: 24 VH 025, CDR2 IHHSGST SEQ ID No: 25 VH 025, CDR3
ARGYYDSGVYYFDY SEQ ID No :26 VL 025 DIQMTQSPSSLSASV GDRVTITCRASQGIS
RWLAWYQQKPEKAPK SLIYAASSLRSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ
YNSYPITFGQGTRLE IK SEQ ID No: 27 VL 025, CDR1 QGISRW VL 025, CDR2
AAS SEQ ID No: 28 VL 025, CDR3 QQYNSYPIT SEQ ID No: 29 VH 091
QVQLQQWGAGLLKPS ETLSLTCAVSGGSFS GYYWTWIRQPPGKGL EWIGEIYHSGDTNYN
PSLKSRVTISVDTSK NQFSLKLYSVTAADT AVYYCARLYFGSGIY YLDYWGQGTLVTVSS SEQ
ID No: 30 VH 091, CDR1 GGSFSGYY SEQ ID No: 163 VH 091, CDR2 IYHSGDT
SEQ ID No: 31 VH 091, CDR3 ARLYFGSGIYYLDY SEQ ID No: 32 VL 091
DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLVWYQQKPEKAPK SLIYAASSLQSGVPS
RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSFPPTFGQGTKVE IK SEQ ID No: 33 VL
091, CDR1 QGISSW VL 091, CDR2 AAS SEQ ID No: 34 VL 091, CDR3
QQYNSFPPT SEQ ID No: 35 VH 129 QVQLVESGGGVVQPG RSLRLSCAASGFTFS
TFAIHWVRQAPGKGL EWVAVISYDGGHKFY ADSVKGRFTISRDNS KNTLYLQMNSLRAED
TAMYYCARGLGVWGA FDYWGQGTLVTVSS SEQ ID No: 36 VH 129, CDR1 GFTFSTFA
SEQ ID No: 37 VH 129, CDR2 ISYDGGHK SEQ ID No: 38 VH 129, CDR3
ARGLGVWGAFDY SEQ ID No :39 VL 129 EIVLTQSPATLSLSP GERATLSCRASQSVS
SYLAWYQQKPGQAPR LLIYDASNRATGIPA RFSGSGSGTDFTLTI SSLEPEDFAVYYCQQ
RSNWWTFGQGTKVEI K SEQ ID No: 40 VL 129, CDR1 QSVSSY VL 129, CDR2
DAS SEQ ID No: 41 VL 129, CDR3 QQRSNWWT SEQ ID No: 42 VH 127
EVQLVQSGAEVKKPG ESLTISCKGSGYSFS IYWIGWVRQMPGKGL EWMGIIFPGDSDIRY
SPSFQGQVTISADKS ISTAYLQWSSLKASD TAMYYCARQPGDWSP RHWYFDLWGRGTLVT VSS
SEQ ID No: 43 VH 127, CDR1 GYSFSIYW SEQ ID No: 44 VH 127, CDR2
IFPGDSDI SEQ ID No: 45 VH 127, CDR3 ARQPGDWSPRHWYFDL SEQ ID No: 46
VL 127 VIWMTQSPSLLSAST GDRVTISCRMSQGIS SYLAWYQQKPGKAPE
LLIYAASTLQSGVPS RFSGSGSGTDFTLTI SYLQSEDFATYYCQQ YYSFPLTFGGGTKVE IK
SEQ ID No: 47 VL 127, CDR1 QGISSY VL 127, CDR2 AAS SEQ ID No: 48 VL
127, CDR3 QQYYSFPLT SEQ ID No: 49 VH 159 EVQLVQSGAEVKKPG
ESLKISCKGSGYNFT SYWIGWVRQMPGKGL EWMGIIYPGDSDTRY SPSFQGQVTISADKS
ISTAYLQWSSLKASD TAMYYCARWGTYYDI LTGYFNWFDPWGQGT LVTVSS SEQ ID No:
50 VH 159, CDR1 GYNFTSYW SEQ ID No: 51 VH 159, CDR2 IYPGDSDT SEQ ID
No: 52 VH 159, CDR3 ARWGTYYDILTGYFN SEQ ID No: 53 VL 159
DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLAWYQQKPEKAPK SLIYAASSLQSGVPS
RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YYIYPWTFGQGTKVE IK SEQ ID No: 54 VL
159, CDR1 QGISSW VL 159, CDR2 AAS SEQ ID No: 55 VL 159, CDR3
QQYYIYPWT SEQ ID No: 56 VH 098 EVQLLESGGGLVQPG GSLRLSCAASGFTFS
NYGMSWVRQAPGKGL EWVSAISGSAYSTYY ADSVKGRFTISRDNS KNTLWLQMNSLRAED
TAVYYCAKAHYHGSG SYYTLFDYWGQGTLV
TVSS SEQ ID No: 57 VH 098, CDR1 GFTFSNYG SEQ ID No: 58 VH 098, CDR2
ISGSAYST SEQ ID No: 59 VH 098, CDR3 AKAHYHGSGSYYTLFDY SEQ ID No: 60
VL 098 DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLAWYQQKPEKAPK
SLIYAASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSYPYTFGQGTKLE IK
SEQ ID No: 61 VL 098, CDR1 QGISSW VL 098, CDR2 AAS SEQ ID No: 62 VL
098, CDR3 QQYNSYPYT SEQ ID No: 63 VH 153 QVQLVESGGGVVQPG
RSLRLSCAASGFTFS DYVIHWVRQAPGKGL EWVTVISYDGSNKYY ADSVKGRFTISRDNS
KNTLYLQMNSLSAED TAMYYCARGGITGTT GVFDYWGQGTLVTVS S SEQ ID No: 64 VH
153, CDR1 GFTFSDYV SEQ ID No: 65 VH 153, CDR2 ISYDGSNK SEQ ID No:
66 VH 153, CDR3 ARGGITGTTGVFDY SEQ ID No: 67 VL 153 DIQMTQSPSSLSASV
GDRVTITCRASQGIS SWLAWYQQKPEKAPK SLIYDASSLQSGVPS RFSGSGYGTDFSLTI
SSLQPEDFAIYYCQQ YKSYPITFGQGTRLE IK SEQ ID No: 68 VL 153, CDR1
QGISSW VL 153, CDR2 DAS SEQ ID No: 69 VL 153, CDR3 QQYKSYPIT SEQ ID
No: 70 VH 132 QVQLVQSGAEVKKPG ASVKVSCKASGYTFT SYGISWVRQAPGQGL
EWMGWISAYNGNSNY VQKFQGRVTMTTDTT TSTAYMELRSLTSDD TAVYYCAREYSYDSG
TYFYYGMDVWGQGTT VTVSS SEQ ID No: 71 VH 132, CDR1 GYTFTSYG SEQ ID
No: 72 VH 132, CDR2 ISAYNGNS SEQ ID No: 73 VH 132, CDR3
AREYSYDSGTYFYYG MDV SEQ ID No: 74 VL 132 EIVLTQSPATLSLSP
GERATLSCRASQSVS SYLAWYQQKPGQAPR LLIYDASNRATGIPA RFSGSGSGTDFTLTI
SSLEPEDFAVYYCQQ RSNWPMYTFGQGTKL EIK SEQ ID No: 75 VL 132, CDR1
QSVSSY VL 132, CDR2 DAS SEQ ID No: 76 VL 132, CDR3 QQRSNWPMYT
TABLE-US-00004 TABLE 2 Mouse origin and heavy and light chain
sequence homologies of selected HuMabs. HuMab: Mouse: Strain:
Germline VH: Germline VL: 169 361494 HCo20 IgHV1-18-01 IgKV3-11-01
050 350633 HCo12 IgHV3-23-01 IgKV1-12-01 084 350615 HCo12-BalbC
IgHV1-69-04 IgKV1-12-01 025 350631 HCo12 IgHV4-34-01 IgKV1D-16-01
091 350630 HCo12 IgHV4-34-01 IgKV1D-16-01 129 359783 HCo12-BalbC
IgHV3-30-3-01 IgKV3-11-01 127 359783 HCo12-BalbC IgHV5-51-01
IgKV1D-8-01 159 363503 HCo12 IgHV5-51-01 IgKV1D-16-01 098 350659
HCo17 IgHV3-23-01 IgKV1D-16-01 153 359785 HCo12-BalbC IgHV3-30-3-01
IgKV1D-16-01 132 361487 HCo20 IgHV1-18-01 IgKV3-11-01
TABLE-US-00005 TABLE 3 Heavy chain variable region (VH), light
chain variable region (VL) sequences of HuMabs 049, 051, 055, 123,
161, 124, 001, 143, 019, 021, 027, 032, 035, 036, 054, 094. The
respective CDRs correspond to those underlined in FIGS. 1 and 2,
for VH and VL sequences, respectively. SEQ ID No: 77 VH 049
EVQLLESGGDLVQPG GSLRLSCAASGFTFS SYAMSWVRQAPGKGL EWVSAISGRGGTTYY
ADSVKGRFTISRDNS KSTLCLQMNSLRAED TAVYYCAKARANWDY FDYWGQGTLVTVSS SEQ
ID No: 78 VL 049 DIQMTQSPSSVSASV GDRVTITCRASQGIS SWLAWYQHKPGKAPK
LLIYAASILQSGVPS RFSGSGSGTDFTLTI SSLRPEDFATYYCQQ ANSFPITFGQGTRLE IK
SEQ ID No: 79 VH 051 EVQLLESGGDLVQPG GSLRLSCAASGFTFS
SYAMSWVRQAPGKGL EWVSAISGRGGTTYY ADSVKGRFTISRDNS KSTLCLQMNSLRAED
TAVYYCAKARANWDY FDYWGQGTLVTVSS SEQ ID No: 80 VL 051 DIQMTQSPSSVSASV
GDRVTITCRASQGIS SWLAWYQHKPGKAPK LLIYAASILQSGVPS RFSGSGSGTDFTLTI
SSLRPEDFATYYCQQ ANSFPITFGQGTRLE IK SEQ ID No: 81 VH 055
EVQLLESGGGLVQPG GSLRLSCAASGFTFS SYAMNWVRQAPGKGL EWVSAISGRGGTTYY
ADSVKGRFTISRDNS KSTLCLQMNSLRAED TAVYYCAKARANWDY FDYWGQGTLVTVSS SEQ
ID No: 82 VL 055 DIQMTQSPSSVSASV GDRVTITCRASQGIS SWLAWYQHKPGKAPK
LLIYAASILQSGVPS RFSGSGSGTDFTLTI SSLRPEDFATYYCQQ ANSFPITFGQGTRLE IK
SEQ ID No: 83 VH 123 QVQLVQSGAEVKKPG ASVKVSCKAAGYTFT
NYGISWVRQAPGQAL EWMGWITTYSSNTIY AQKLQGRVTMTTDTS TSTAYMELRSLRSDD
TAVYYCARDRVVVRP DYFDYWGQGTLVTVS S SEQ ID No: 84 VL 123
EIVLTQSPATLSLSP GERATLSCRASQSVS SYLAWYQQKPGQAPR LLIYDTSNRATGIPA
RFSGSGSGTDFTLTI SSLEPEDFAVYYCQQ RSHWPRTFGQGTKVE IK SEQ ID No: 85 VH
161 QVQLVQSGAEVKKPG ASVKVSCKASGYTFT NYGISWVRQAPGQGL EWMGWLSAYSGNTIY
AQKLQGRVTMTTDTS TTTAYMELRSLRSDD TAVYYCARDRIVVRP DYFDYWGQGTLVTVS S
SEQ ID No: 86 VL 161 EIVLTQSPATLSLSP GERATLSCRASQSVS
SYLAWYQQKPGQAPR LLIYDASNRATGIPA RFSGSGSGTDFTLTI SSLEPEDFAVYYCQQ
RSNWPRTFGQGTKVE IK SEQ ID No: 87 VH 124 QVQLVQSGAEVKKPG
ASVKVSCKAAGYTFT NYGISWVRQAPGQGL EWMGWIITYNGNTIY AQRFQDRVTMTTDTS
TSTAYMELRSLRSDD TAVYYCARDRIIVRP DYFDYWGQGTLVTVS S SEQ ID No: 88 VL
124 EIVLTQSPATLSLSP GERATLSCRASQSVS SYLAWYQQKPGQAPR LLIYDASNRATGIPA
RFSGSGSGTDFTLTI SSLEPEDFAVYYCQQ RSNWPRTFGQGTKVE IK SEQ ID No: 89 VH
001 QVQLQQWGAGLLKPS ETLSLTCAVYGGSFS GYYWNWIRQPPGKGL EWIGEINHSGSTNYN
PSLKSRVTISVDTSK NQFSLKLSSVTAADT AVYYCARGNYGSGYY YFDLWGRGTQVTVSS SEQ
ID No: 90 VL 001 DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLAWYQQKPEKAPK
SLIFAASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YISFPITFGQGTRLE IK
SEQ ID No: 91 VH 143 QVQLQQWGAGLLKPS ETLSLTCAVYGGSFS
GYYWNWIRQPPGKGL EWIGEIHHSGSANYN PSLMSRVTISVDTSK NQFSLQLSSVTAADT
AVYYCARGYYGSGYY YFDYWGQGTLVTVSS SEQ ID No: 92 VL 143
DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLAWYQQKPEKAPK SLIYAASRLQSGVPS
RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSYPITFGQGTRLE IK SEQ ID No: 93 VH
019 QVQLQQWGAGLLKPS ETLSLTCAVYGGSFS DYYWNWIRQPPGKGL EWIGEIHHVGSTNYN
PSLKSRVTISVDTSK SQFSLKLSSVTAADT AVYYCARGYYDSGVY YFDYWAQGTLVTVSS SEQ
ID No: 94 VL 019 DIQMTQSPSSLSASV GDRVTITCRASQGIS RWLAWYQQKPEKAPK
SLIYAASSLRSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSYPITFGQGTRLE IK
SEQ ID No: 95 VH 021 QVQLQQWGAGLLKPS ETLSLTCAVYGGSFS
DYYWNWIRQPPGKGL EWIGEIHHSGSTNYN PSLKSRVTISVDTSK NQFSLKLSSVTAADT
AVYYCARGYYASGVY YFDYWGQGTLVTVSS SEQ ID No: 96 VL 021
DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLAWYQQKPEKAPK SLIYAASSLQSGVPS
RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSYPITFGQGTRLE IK SEQ ID No: 97 VH
027 QVQLQQWGAGLLKPS ETLSLTCAVYGGSFS DYFWNWIRQPPGKGL EWIGEIHHSGSTNYN
PSLKSRVTISVDTSK NQFSLNLSSVTAADT AVYYCARGLIGSGYY YFDYWDQGTLVTVSS SEQ
ID No: 98 VL 027 DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLAWYQQKPEKAPK
SLIYAASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSYPITFGQGTRLE IK
SEQ ID No: 99 VH 032 QVQLQQWGAGLLKPS ETLSLTCAVYGGSFS
GYYWSWIRQPPGKGL EWIGEINHSGDTNYN PSLTSRVTISVDTSK NQFSLKLSSVTAADT
AVYYCARLFYGSGIY YFDYWGQGTLVTVSS SEQ ID No: 100 VL 032
DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLAWYQQKPEKAPK SLIYATFRLQSGVPS
RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSFPPTFGQGTKVE IK SEQ ID No: 101
VH 035 QVQLQQWGAGLLKPS ETLSLTCAIYGGSFS GYYWSWIRQPPGKGL
EWIGEINHSGDTNYN PSLTSRVTISVDTSK NQFSLKLSSVTAADT AVYYCARLFYGSGIY
YFDYWGQGTLVTVSS SEQ ID No: 102 VL 035 DIQMTQSPSSLSASV
GDRVTITCRASQGIS SWLAWYQQKPEKAPK SLIYATFRLQSGVPS RFSGSGSGTDFTLTI
SSLQPEDFATYYCQQ YNSFPPTFGQGTKVE IK SEQ ID No: 103 VH 036
QVQLQQWGAGLLKPS ETLSLTCAVYGGSFS
DYYWSWIRQPPGKGL EWIGEINHSGSTNYN PSLKSRVTISVDTSK NQFSLKLSSVTAADT
AVYYCARLYYGSGTY YFDYWGQGTLVTVSS SEQ ID No: 104 VL 036
DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLTWYQQKPEKAPK SLIYAASRLQSGVPS
RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSFPPTFGQGTKVE IK SEQ ID No: 105
VH 054 QVQLQQWGAGLLKPS ETLSLTCAVYGGSFS GYYWSWIRQPPGKGL
EWIGEIHHSGSTNYN PSLKSRVTISVDTSK NQFSLKLSSVTAADT AVYYCARLWYGSGSY
YFDYWGQGTLVTVSS SEQ ID No: 106 VL 054 DIQMTQSPSSLSASV
GDRVTITCRASQGIS SWLAWYQQKPEKAPK SLIYAASSLQSGVPS RFSGSGSGTDFTLTI
SSLQPEDFATYYCQQ YNSFPPTFGGGTKVE IK SEQ ID No: 107 VH 094
QVQLQQWGAGLLKPS ETLSLTCAVSGGSFS GYYWTWIRQPPGKGL EWIGEIYHSGDTNYN
PSLKSRVTISVDTSK NQFSLKLYSVTAADT AVYYCARLYFGSGIY YLDYWGQGTLVTVSS SEQ
ID No: 108 VL 094 DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLVWYQQKPEKAPK
SLIYAASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSFPPTFGQGTKVE IK
SEQ ID No: 109 VH 105 EVQLLESGGGLVQPG GSLRLSCAASGFTFS
NYGMSWVRQAPGKGL EWVSAISGSAYSTYY ADSVKGRFTISRDNS KNTLWLQMNSLRAED
TAVYYCAKAHYHGSG SYYTLFDYWGQGTLV TVSS SEQ ID No: 110 VL 105
DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLAWYQQKPEKAPK SLIYAASSLQSGVPS
RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSYPYTFGQGTKLE IK SEQ ID No: 111
VH 100 EVQLLESGGGLVQPG GSLRLSCAASGFTFN NYGMNWVRQAPGKGL
EWVSAISGTGYSTYY ADSVKGRFTISRDNS KNTLYLQMNSLRAED TAVYYCAKAHYFGSG
SYYTLFDYWGQGTLV TVSS SEQ ID No: 112 VL 100 DIQMTQSPSSLSASV
GDRVTITCRASQGIS SWLAWYQQKPEKAPK SLIYAASSLQSGVPS RFSGSGSGTDFTLTI
SSLQPEDFATYYCQQ YNSYPYTFGQGTKLE IK SEQ ID No: 113 VH 125
EVQLLESGGGLVQPG GSLRLSCAASGFTFT DYAMNWVRQAPGKGL EWVSTISGSGYATYY
ADSVKGRFTISRDNS KTTLYLQMNSLRAED TAVYYCAKGHTLGSG SYYTLFDYWGQGTLV
TVSS SEQ ID No: 114 VL 125 DIQMTQSPSSLSASV GDRVTITCRASQGIN
SWLAWYQQKPEKAPK SLIYAASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ
YNSYPYTFGQGTKLE IK SEQ ID No: 115 VH 162 EVQLWESGGGSVQPG
GSLRLSCAASGFTFS SYGMSWVRQAPGKGL EWVSGISGSGYSTYY ADSVKGRFTISRDNS
KNTLYLQMNSLRAED TAVYYCAKGYYHGSG SYYTSFDYWGQGTLV TVSS SEQ ID No: 116
VL 162 DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLAWYQQKPEKAPK
SLIYAASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSYPLTFGGGTKVE IK
SEQ ID No: 117 VH 033 QVQLVESGGGVVQTG RSLRLSCAASGFTFS
SHAMHWVRQAPGKGL EWVAAISYDGSNKYY ADSVKGRFTISRDNS KNTLYLQMNSLRAED
TAVYYCARGDYISSS GVFDYWGQGTLVTVS S SEQ ID No: 118 VL 033
DIQMTQSPSSLSASV GDRVTITCRASQGIS SWLAWYQQKPEKAPK SLIYAASSLQSGVPS
RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ YNSYPITFGQGTRLE IK SEQ ID No: 119
VH 160 QVQLVESGGGVVQPG RSLRLSCAASGFTFS SHAMHWVRQAPGKGL
EWVAAISYDGSNKYY ADSVKGRFTISRDNS KNTMYLQMNSLRAED TAMCYCARGSITGST
GVFDYWGQGTLVTVS S SEQ ID No: 120 VL 160 DIQMTQSPSSLSASV
GDRVTITCRASQDIS SWLAWYQQKPEKAPK SLIYAASSLQSGVPS RFSGSGSGTDFTLTI
SSLQPEDFATYYCQQ YNSYPITFGQGTRLE IK SEQ ID No: 121 VH 166
QVQLVESGGGVVQPG RSLRLSCAASGFTFS SYAMHWVRQAPGKGL EWVAVISYDGSNEYY
ADSVKGRFTISRDNS KNTLYLQMNSLRAED TAVYYCARGSIIGST GVFDYWGQGTLVTVS S
SEQ ID No: 122 VL 166 DIQMTQSPSSLSASV GDRVTITCRASQGIS
NWLAWYQQKPEKAPK SLIYDASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ
YNSYPITFGQGTRLE IK SEQ ID No: 123 VH 152 QVQVVESGGGVVQPG
RSLRLSCAASGFTFS SYAMHWVRQAPGKGL EWVAVISYDGSYKYY ADSVKGRFTISRDNS
KNTLYLQMNSLRAED TAVYYCARGSITGST GVFDYWGQGTLVTVS S SEQ ID No: 124 VL
152 DIQMTQSPSSLSASV GDRVTITCRASQGIN SWLAWYQQKPEKAPK SLIYDASSLQSGVPS
RFSGSGSGTDFTLTI SSLQPENFATYYCQQ YNSYPITFGQGTRLE IK SEQ ID No: 125
VH 167 QVQLVESGGGVVQPG RSLRLSCAASGFTFS SYAIHWVRQAPGKGL
EWVAVISYDGSNKYY ADSVKGRFTISRDNS KNTLYLQMNSLRAED TAVYYCARGSITGST
GVFDYWGQGTLVTVS S SEQ ID No: 126 VL 167 DIQMTQSPSSLSASV
GDRVTITCRASQGIS NWLAWYQQKPEKAPK SLIYDASSLQSGVPS RFSGSGSGTDFTLTI
SSLQPEDFATYYCQQ YNSYPITFGQGTRLE IK
TABLE-US-00006 TABLE 4 Consensus CDRs based on sequence alignments
shown in FIGS. 1 and 2. SEQ ID No: 9 IgHV3-23-1 VH GFTFSSYA
050-049-051- CDR1 055 SEQ ID No: 127 IgHV3-23-1 VH ISGX1GGX2T
Wherein X1 = R or S, 050-049-051- CDR2 and 055 X2 = T or S;
preferably, wherein X1 = R and X2 = T SEQ ID No: 11 IgHV3-23-1 VH
AKARANWDYFD 050-049-051- CDR3 Y 055 SEQ ID No: 128 IgHV1-69-04 VH
GGTFX1X2YA Wherein X1 = R or S, 084 CDR1 and X2 = T or S;
preferably, wherein X1 = R and X2 = T SEQ ID No: 129 IgHV1-69-04 VH
IX2X3X3LGIX4 Wherein X1 = N or I, 084 CDR2 X2 = T or P, X3 = V or
I, and X4 = V or A, preferably, wherein X1 = N, X2 = T, X3 = V, and
X4 = V SEQ ID No: 130 IgHV1-69-04 VH AREKGVDYYYG Wherein X1 = I or
M, 084 CDR3 X1X2 X2 = E or D; preferably, wherein X1 = I, X2 = E
SEQ ID No: 131 IgHV1-18-1 VH GYTFTXYG Wherein X = N or S,
169-123-161- CDR1 preferably 124 N SEQ ID No: 132 IgHV1-18-1 VH
IX1X2YX3GNT Wherein X1 = S, 169-123-161- CDR2 T, or I; 124 X2 = A
or T; X3 = S or N; preferably, wherein X1 = S, X2 = A, and X3 = S
SEQ ID No: 133 IgHV1-18-1 VH ARDRX1X2VRP Wherein X1 = I or
169-123-161- CDR3 DYFDY V, X2 = V or 124 I; preferably, wherein X1
= I and X2 = V SEQ ID No: 134 IgHV4-34-01 VH GGSFSX1YX2 Wherein X1
= D or G 025-001-143- CDR1 and 019-021-027 X2 = Y or F; preferably,
wherein X1 = D and X2 = Y SEQ ID No: 135 IgHV4-34-01 VH IX1HX2GSX3
Wherein X1 = H or N, 025-001-143- CDR2 X2 = S or V, 019-021-027 and
X3 = T or A; preferably, wherein X1 = H, X2 = S, and X3 = T SEQ ID
No: 136 IgHV4-34-01 VH ARGX1X2X3SG Wherein X1 = Y, N or L;
025-001-143- CDR3 X4YYFDX5 X2 = Y or I, 019-021-027 X3 = D, G or A;
X4 = V or Y; and X5 = Y or L; preferably, wherein X1 = Y, X2 = Y,
X3 = D, X4 = V, and X5 = Y SEQ ID No: 137 IgHV4-34-01 VH GGSFSX1YY
Wherein X1 = G or D, 091-032-035- CDR1 preferably G 036-054-094 SEQ
ID No: 138 IgHV4-34-01 VH IX1HSGX2T Wherein X1 = Y, 091-032-035-
CDR2 N or H; and 036-054-094 X2 = D or S; preferably, wherein X1 =
Y and X2 = D SEQ ID No: 139 IgHV4-34-01 VH ARLX1X2GSGX Wherein X1 =
Y, F or W; 091-032-035- CDR3 3YYX4DY X2 = F or Y; 036-054-094 X3 =
I, T or S; and X4 = L or F; preferably, wherein X1 = Y, X2 = F, X3
= I, and X4 = L SEQ ID No: 140 IgHV3-30-01 VH GFTFSX1X2A Wherein X1
= T or F, 129 CDR1 X2 = F or Y; preferably, wherein X1 = T and X2 =
F SEQ ID No: 141 IgHV3-30-01 VH ISYDGX1X2K Wherein X1 = G or S, 129
CDR2 X2 = H or N; preferably, wherein X1 = G and X2 = H SEQ ID No:
142 IgHV3-30-01 VH ARGLGVWGX1F Wherein X1 = A or Y, 129 CDR3 DY
preferably A SEQ ID No: 143 IgHV3-23-01 VH GFTFX1X2YX3 Wherein X1 =
S, N or T; 098-105-100- CDR1 X2 = N, D or S; 125-162 and X3 = G or
A; preferably, wherein X1 = S, X2 = N and X3 = G SEQ ID No: 144
IgHV3-23-01 VH ISGX1X2X3X4T Wherein X1 = S or T, 098-105-100- CDR2
X2 = A or G, 125-162 X3 = Y or G, X4 = S or A; preferably, wherein
X1 = S, X2 = A, X3 = Y, X4 = S SEQ ID No: 145 IgHV3-23-01 VH
AKX1X2X3X4G Wherein X1 = A or G; 098-105-100- CDR3 SGSYYTX5FDY X2 =
H or Y; X3 = Y or T; 125-162 X4 = H, F or L; X5 = L or S;
preferably, wherein X1 = A; X2 = H; X3 = Y; X4 = H;X5 = L SEQ ID
No: 146 IgHV5-51-01 VH GYSFX1X2YW Wherein X1 = S or T, 127 CDR1 X2
= I or S; preferably, wherein X1 = S, X2 = I SEQ ID No: 147
IgHV5-51-01 VH IX1PGDSDX2 Wherein X1 = F or Y, 127 CDR2 X2 = I or
T; preferably, wherein X1 = F, X2 = I SEQ ID No: 148 IgHV5-51-01 VH
ARQPGDWSPR 127 CDR3 HWYFDL SEQ ID No: 149 IgHV5-51-01 VH GYXFTSYW
Wherein X = N or S, 159 CDR1 Preferably N SEQ ID No: 51 IgHV5-51-01
VH IYPGDSDT 159 CDR2 SEQ ID No: 52 IgHV5-51-01 VH ARWGTYYDILT 159
CDR3 GYFN SEQ ID No: 71 IgHV1-18-01 VH GYTFTSYG 132 CDR1 SEQ ID No:
150 IgHV1-18-01 VH ISAYNGNX Wherein X = S or T, 132 CDR2 Preferably
S SEQ ID No: 151 IgHV1-18-01 VH AREYSYDSGTY 132 CDR3 FYYGMDV SEQ ID
No: 152 IgHV3-30- VH GFTFSX1X2X3 Wherein X1 = D or S, 153-033-160-
03-01 CDR1 X2 = Y or H, 166-152-167 X3 = V or A; preferably,
wherein X1 = D, X2 = Y, X3 = V SEQ ID No: 153 IgHV3-30- VH
ISYDGSX1X2 Wherein X1 = N or Y, 153-033-160- 03-01 CDR2 X2 = K or
E, 166-152-167 preferably wherein X1 = N and X2 = K SEQ ID No: 154
IgHV3-30- VH ARGX1X2X3X4 Wherein X1 = G, 153-033-160- 03-01 CDR3
X5X6GX7FDY D or S; X2 = I 166-152-167 or Y; X3 = T or I; X4 = G or
S; X5 = T or S; X6 = T or S; X7 = Y or V; preferably, wherein X1 =
G; X2 = I; X3 = T; X4 = G; X5 = T; X6 = T; and X7 = V SEQ ID No: 13
IgKV1-12-01 VL QGISSW 050-084-049- CDR1 051-055 050-084-049-
IgKV1-12-01 VL XAS Wherein X = A or V 051-055 CDR2 SEQ ID No: 155
IgKV1-12-01 VL QQANSFPXT Wherein X = I or L 050-084-049- CDR3
051-055 SEQ ID No: 6 IgKV3-11-01 VL QSVSSY 169-124-161- CDR1 123
169-124-161- IgKV3-11-01 VL DXS Wherein X = A or T, 123 CDR2
preferably A SEQ ID No: 156 IgKV3-11-01 VL QQRSXWPRT Wherein X = N
or H, 169-124-161- CDR3 preferably 123 N SEQ ID No: 157 IgKV1D-16-
VL QGISXW Wherein X = R or S, 025-001-019- 01 CDR1 preferably
143-021-027 R 025-001-019- IgKV1D-16- VL AAS 143-021-027 01 CDR2
SEQ ID No: 164 IgKV1D-16- VL QQYNSXPIT Wherein X = Y or F,
025-001-019- 01 CDR3 preferably 143-021-027 Y SEQ ID No: 33
IgKV1D-16- VL QGISSW 091-032-035- 01 CDR1 036-054-094 091-032-035-
IgKV1D-16- VL AX1X2 Wherein X1 = A or T, 036-054-094 01 CDR2 and X2
= S or F; preferably, wherein X1 = A and X2 = S SEQ ID No: 158
IgKV1D-16- VL QQYNSFPPT 091-032-035- 01 CDR3 036-054-094 SEQ ID No:
159 IgKV1D-16- VL QGIXSW Wherein X = S or N, 098-100-105- 01 CDR1
preferably 125-162 S
098-100-105- IgKV1D-16- VL AAS 125-162 01 CDR2 SEQ ID No: 160
IgKV1D-16- VL QQYNSYPXT Wherein X = Y or L, 098-100-105- 01 CDR3
preferably 125-162 Y SEQ ID No: 161 IgKV1D-16- VL QGIX1X2W Wherein
X1 = S or N; 153-152-166- 01 CDR1 X2 = S or 167-160-033 N;
preferably, wherein X1 = X2 = S 153-152-166- IgKV1D-16- VL XAS
Wherein X = D or A, 167-160-033 01 CDR2 preferably D SEQ ID No: 162
IgKV1D-16- VL QQYXSYPIT Wherein X = K or N, 153-152-166- 01 CDR3
preferably 167-160-033 K
Example 11--Purification of Antibodies
[0509] Culture supernatant was filtered over 0.2 .mu.m dead-end
filters, loaded on 5 ml MabSelect SuRe columns (GE Health Care) and
eluted with 0.1 M sodium citrate-NaOH, pH 3. The eluate was
immediately neutralized with 2M Tris-HCl, pH 9 and dialyzed
overnight to 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B.Braun).
Alternatively, subsequent to purification, the eluate was loaded on
a HiPrep Desalting column and the antibody was exchanged into 12.6
mM NaH2PO4, 140 mM NaCl, pH 7.4 (B.Braun) buffer. After dialysis or
exchange of buffer, samples were sterile filtered over 0.2 .mu.m
dead-end filters. Purity was determined by SDS-PAGE and
concentration was measured by nephelometry and absorbance at 280
nm. Purified antibodies were stored at 4.degree. C. Mass
spectrometry was performed to identify the molecular mass of the
antibody heavy and light chains expressed by the hybridomas as
described in Example 9.
Example 12--Binding of HER2 Clones to Tumor Cells Expressing
Membrane-Bound HER2 Measured by Means of FACS Analysis
[0510] The binding of HER2 antibodies to AU565 cells (purchased at
ATCC, CRL-2351) and A431 cells (purchased at ATCC, CRL-1555), was
tested using flow cytometry (FACS Canto II, BD Biosciences). Qifi
analysis (Dako, Glostrup, Denmark) revealed that AU565 cells
expressed on average 1,000,000 copies of HER2 protein per cell,
whereas A431 cells expressed on average 15,000 copies per cell.
Binding of HER2 antibodies was detected using a Phycoerythrin
(PE)-conjugated goat-anti-human IgG antibody (Jackson). Trastuzumab
(clinical-grade Herceptin.RTM.) was used as positive control
antibody, and an isotype control antibody was used as negative
control antibody. EC.sub.50 values were determined by means of
non-linear regression (sigmoidal dose-response with variable slope)
using GraphPad Prism V4.03 software (GraphPad Software, San Diego,
Calif., USA).
[0511] As shown in FIG. 3, all tested HER2 antibodies bound to HER2
expressed on both AU565 and A431 cells in a dose-dependent manner.
The EC.sub.50 values for binding varied between 0.336-2.290
.mu.g/mL for AU565 cells and 0.068-1.135 .mu.g/mL for A431 cells.
Especially on A431 cells, large differences in EC.sub.50 values
were observed between the tested antibodies. However, antibody 098
had the best (i.e., lowest) EC.sub.50 value on both types of cells.
Also some differences in maximum binding levels were observed
between different antibodies, on both AU565 and A431 cells. Of the
tested antibodies, antibody 098 also had the highest maximum
binding level on AU565 cells, whereas antibody 025 had the highest
maximum binding level on A431 cells.
Example 13--Binding of HER2 Antibodies to Membrane-Bound HER2
Expressed on Rhesus Epithelial Cells Measured by Means of FACS
Analysis
[0512] To determine cross-reactivity with Rhesus HER2, the binding
of HER2 antibodies to HER2-positive Rhesus epithelial cells (4MBr-5
purchased at ATCC) was tested using flow cytometry (FACS Canto II,
BD Biosciences). A Phycoerythrin-conjugated goat-anti-human IgG
antibody (Jackson) was used as a secondary conjugate. An isotype
control antibody was used as negative control antibody.
[0513] As shown in FIG. 4, all tested HER2 antibodies were
cross-reactive with Rhesus monkey HER2. At both tested
concentrations (1 .mu.g/mL and 10 .mu.g/mL), the HER2 antibodies
were able to bind specifically to Rhesus monkey HER2. Antibody 127
demonstrated poor binding at 1 .mu.g/mL concentration, but showed
good binding at 10 .mu.g/mL concentration. Antibody 098 had the
highest binding level at both antibody concentrations. No binding
was observed with the isotype control antibody.
Example 14--Competition of HER2 Antibodies for Binding to Soluble
Her2ECDHis Measured in Sandwich-ELISA
[0514] The optimal coating concentrations of the tested HER2
antibodies and optimal Her2ECDHis concentration were determined in
the following manner: ELISA wells were coated overnight at
4.degree. C. with HER2 HuMabs serially diluted in PBS (0.125-8
.mu.g/mL in 2-fold dilutions). Next, the ELISA wells were washed
with PBST (PBS supplemented with 0.05% Tween-20 [Sigma-Aldrich,
Zwijndrecht, The Netherlands]) and blocked for one hour at room
temperature (RT) with PBSTC (PBST supplemented 2% [v/v] chicken
serum [Gibco, Paisley, Scotland]). The ELISA wells were then washed
with PBST and incubated for one hour at RT with Her2ECDHis serially
diluted in PBSTC (0.25-2 .mu.g/mL in 2-fold dilutions). Unbound
Her2ECDHis was washed away with PBST, and bound Her2ECDHis was
incubated for one hour at RT with 0.25 .mu.g/mL biotinylated
rabbit-anti-6.times.his-biot (Abcam, Cambridge, UK). The plate was
thereafter washed with PBST and incubated for one hour with 0.1
.mu.g/mL Streptavidin-poly-HRP (Sanquin, Amsterdam, The
Netherlands) diluted in PBST. After washing, the reaction was
visualized through a 15 minutes incubation with 2,2'-azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid (ABTS: one ABTS tablet
diluted in 50 mL ABTS buffer (Roche Diagnostics, Almere, The
Netherlands)) at RT protected from light. The colorization was
stopped by adding an equal volume of oxalic acid (Sigma-Aldrich,
Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured
on a microtiter plate reader (Biotek Instruments, Winooski, USA).
The antibody concentrations that resulted in sub-optimal binding of
each antibody were determined and used for the following
cross-block experiments.
[0515] Each HER2 antibody was coated to the ELISA wells at the
sub-optimal dose that was determined as described above. After
blocking of the ELISA wells, the wells were incubated with the
predetermined concentration of 1 .mu.g/mL biotinylated Her2ECDHis
in the presence or absence of an excess of a second (competitor)
HER2 antibody. The ELISA was then performed as described above.
Residual binding of Her2ECDHis to the coated antibody was expressed
as a percentage relative to the binding observed in the absence of
competitor antibody. Percentage competition was then determined as
100 minus the percentage of inhibition. 75% competition was
considered as complete cross-block, whereas 25-74% competition was
considered as partial cross-block, and 0-24% competition was
considered non-blocking.
[0516] As shown in Table 5, all HER2 antibodies were found to be
able to block binding to Her2ECDHis, at least partially, for
themselves. After dividing the antibodies into 3 major cross-block
groups, all antibodies were tested for competition with at least
one representative antibody from each group.
[0517] The first group comprised trastuzumab and antibodies 169,
050 and 084, which blocked each other for binding to Her2ECDHis,
but did not cross-block antibodies from other groups.
[0518] The second group comprised pertuzumab and antibodies 025,
091 and 129, which blocked each other for binding to Her2ECDHis,
except for antibodies 129 and 091 which both cross-blocked
pertuzumab and 025, but not each other. None of the antibodies of
group 2 blocked antibodies from other groups.
[0519] A third group comprised antibodies C1, F5, 127, 098, 132,
153 and 159, which did not cross-block any antibody from the other
groups. Within this group 3, some variation was observed. Antibody
127 was the only antibody that was able to cross-block all other
antibodies in this group for binding to Her2ECDHis; antibody 159
cross-blocked all other antibodies within this group, except 132;
clone 098 cross-blocked all antibodies of group 3, except 132 and
153; antibody 153 cross-blocked 127, 132 and 159 for binding to
Her2ECDHis, but not 098, C1 or F5; clone 132 cross-blocked 127, 132
and 153. When added as competitor antibodies, F5 and C1 only
demonstrated cross-blocking of each other. However, the reverse
reaction also revealed competition with antibodies 127, 098 and
159, but not 153 and 132. Possibly, these differences may have
resulted from lower affinities of antibodies C1 and F5 for
Her2ECDHis.
[0520] Values higher than 100% can be explained by avidity effects
and the formation of antibody-Her2ECDHis complexes containing two
non-competing antibodies.
TABLE-US-00007 TABLE 5 Competition and cross-blocking of HER2
antibodies for binding to Her2ECDHis Immobilized Competing mAb
.fwdarw. mAb .dwnarw. tras 169 050 084 pert 025 091 129 C1 F5 127
159 098 153 132 Trastuzumab 6 15 6 51 100 107 100 85 103 99 115 90
101 101 101 TH1014-169 19 45 21 73 101 98 105 106 ND ND ND ND 105
102 ND TH1014-050 13 30 12 74 95 104 98 110 ND ND ND ND 102 104 ND
TH1014-084 74 73 76 20 101 106 104 104 ND ND ND ND 109 98 ND
TH1014-pert 104 100 94 95 9 20 19 39 106 125 116 81 103 100 109
TH1014-025 98 98 100 104 8 18 21 15 ND ND ND ND 102 99 ND
TH1014-091 99 99 95 100 5 13 15 78 ND ND ND ND 98 98 ND TH1014-129
93 99 97 92 22 55 76 12 ND ND ND ND 106 98 ND TH1014-C1 89 ND ND ND
ND ND ND ND 65 58 73 53 58 77 90 TH1014-F5 197 ND ND ND ND ND ND ND
70 21 62 15 16 80 125 TH1014-127 102 ND ND ND ND ND ND ND 112 88 11
8 58 21 44 TH1014-159 111 ND ND ND 112 ND ND ND 96 86 15 6 11 40 79
TH1014-098 107 102 100 103 104 108 104 107 125 96 21 9 17 110 142
TH1014-153 134 111 103 107 121 97 102 106 257 96 27 23 115 28 33
TH1014-132 353 ND ND ND 288 ND ND ND 422 379 30 131 309 41 32
Cross-block 1 1 1 1 2 2 2 2b 3a 3a 3a 3a 3a 3b 3b group
[0521] Depicted values are mean percentages of binding relative to
the binding observed in the absence of competitor antibody, of two
independent experiments. Competition experiments with HEK produced
TH1014-C1 and TH1014-F5 were performed once. Trastuzumab (clinical
grade Herceptin.COPYRGT.) and HEK-produced pertuzumab (TH1014-pert)
were also tested.
Example 15--Antibody-Dependent Cell-Mediated Cytotoxicity
(ADCC)
[0522] SK-BR-3 cells (purchased at ATCC, HTB-30) were harvested
(5.times.10.sup.6 cells), washed (twice in PBS, 1500 rpm, 5 min)
and collected in 1 mL RPMI 1640 medium supplemented with 10% cosmic
calf serum (CCS) (HyClone, Logan, Utah, USA), to which 200 .mu.Ci
.sup.51Cr (Chromium-51; Amersham Biosciences Europe GmbH,
Roosendaal, The Netherlands) was added. The mixture was incubated
in a shaking water bath for 1.5 hours at 37.degree. C. After
washing of the cells (twice in PBS, 1500 rpm, 5 min), the cells
were resuspended in RPMI 1640 medium supplemented with 10% CCS,
counted by trypan blue exclusion and diluted to a concentration of
1.times.10.sup.5 cells/mL.
[0523] Meanwhile, peripheral blood mononuclear cells (PBMCs) were
isolated from fresh buffy coats (Sanquin, Amsterdam, The
Netherlands) using standard Ficoll density centrifugation according
to the manufacturer's instructions (lymphocyte separation medium;
Lonza, Verviers, France). After resuspension of cells in RPMI 1640
medium supplemented with 10% CCS, cells were counted by trypan blue
exclusion and concentrated to 1.times.10.sup.7 cells/mL.
[0524] Trastuzumab was produced in CHO cells resulting in an
(increased) non-core fucosylation grade of 12.4%, whereas the other
HER2 antibodies were produced in HEK cells, resulting on average in
4% non-core fucosylation.
[0525] For the ADCC experiment, 50 .mu.L .sup.51Cr-labeled SK-BR-3
cells (5.000 cells) were pre-incubated with 15 .mu.g/mL HER2
antibody (IgG1,.kappa.) in a total volume of 100 .mu.L RPMI medium
supplemented with 10% CCS in a 96-well microtiter plate. After 15
min at RT, 50 .mu.L PBMCs (500,000 cells) were added, resulting in
an effector to target ratio of 100:1. The maximum amount of cell
lysis was determined by incubating 50 .mu.L .sup.51Cr-labeled
SK-BR-3 cells (5000 cells) with 100 .mu.L 5% Triton-X100. The
amount of spontaneous lysis was determined by incubating 5000
.sup.51Cr-labeled SK-BR-3 cells in 150 .mu.L medium, without any
antibody or effector cells. The level of antibody-independent cell
lysis was determined by incubating 5000 SK-BR-3 cells with 500,000
PBMCs without antibody. Subsequently, the cells were incubated 4 hr
at 37.degree. C., 5% CO.sub.2. To determine the amount of cell
lysis, the cells were centrifuged (1200 rpm, 3 min) and 75 .mu.L of
supernatant was transferred to micronic tubes, after which the
released .sup.51Cr was counted using a gamma counter. The measured
counts per minute (cpm) were used to calculate the percentage of
antibody-mediated lysis as follows:
(cpm sample-cpm Ab-independent lysis)/(cpm max. lysis-cpm
spontaneous lysis).times.100%
[0526] As shown in FIG. 5, HER2 antibodies from cross-block groups
1 and 2 induced efficient lysis of SK-BR-3 cells through ADCC. From
group 3, antibody 153 was the only antibody that induced efficient
ADCC, antibody 132 induced about 10% ADCC, and clones 098, 159 and
127 did not induce ADCC.
Example 16--Inhibition of Ligand-Independent Proliferation of AU565
Cells
[0527] HER2 antibodies were tested for their ability to inhibit
proliferation of AU565 cells in vitro. Due to the high HER2
expression levels on AU565 cells (.about.1,000,000 copies per cell
as described in Example 12), HER2 is constitutively active in these
cells and thus not dependent on ligand-induced
heterodimerization.
[0528] In a 96-well tissue culture plate (Greiner bio-one,
Frickenhausen, Germany), 9000 AU565 cells were seeded per well in
the presence of 10 .mu.g/mL HER2 antibody in serum-free cell
culture medium. As a control, cells were seeded in serum-free
medium without antibody. After 3 days, the amount of viable cells
was quantified with Alamarblue (BioSource International, San
Francisco, US) according to the manufacturer's instructions.
Fluorescence was monitored using the EnVision 2101 Multilabel
reader (PerkinElmer, Turku, Finland) with standard Alamarblue
settings. The Alamarblue signal of antibody-treated cells was
plotted as a percentage relative to untreated cells. Dunnett's test
was applied for statistical analysis.
[0529] The results are shown in FIG. 6, depicting the percentage
proliferation of AU565 cells after HER2 antibody treatment compared
to untreated cells, which was set to 100%. Of the tested Group 1
antibodies, trastuzumab, 050 and 169 demonstrated significant
inhibition of AU565 cell proliferation (P<0.05), whereas 084 had
no effect. None of the tested antibodies from group 2 (Pertuzumab,
025, 092 and 129) was able to inhibit AU565 cell proliferation. The
tested antibodies from group 3 (098 and 153) did not inhibit AU565
proliferation. In contrast, both antibodies induced enhanced
proliferation of AU565 cells compared to untreated cells (098 more
than 153). Enhancing proliferation can be an advantage in some
therapeutic applications of ADC-conjugates, e.g., where the
cytotoxic action of the drug relies on, or is enhanced by, cell
proliferation. For trastuzumab and pertuzumab, this was in
accordance with the results described by Juntilla et al. (Cancer
Cell 2009; 15(5):353-355).
Example 17--Inhibition of Ligand-Induced Proliferation of MCF-7
Cells
[0530] Since HER2 is an orphan receptor, its signaling is mainly
dependent on activation of other ErbB-family members such as EGFR
and Her3. Upon ligand binding, these two receptors can bind to and
activate the HER2 receptor, resulting in e.g. proliferation.
Various publications describe that pertuzumab efficiently inhibits
Heregulin-01-induced proliferation (Franklin M C. Cancer Cell
2004/Landgraf R. BCR 2007). For trastuzumab, it has been described
that it has little effect on Heregulin-.beta.1-induced HER2/HER3
heterodimerization and proliferation (Larsen S S., et al., Breast
Cancer Res Treat 2000; 58:41-56; Agus D B., et al., Cancer Cell
2002; 2:127-137; Wehrman et al. (2006), supra).
[0531] To investigate the ability of the present human HER2
antibodies to interfere with Heregulin-01-induced HER2/HER3
heterodimers, a Heregulin-01-induced proliferation assay was
performed. Therefore, MCF7 cells (purchased at ATCC, HTB-22)
expressing .about.20.000 HER2 molecules per cell, were seeded in a
96-wells tissue culture plate (Greiner bio-one) (2.500 cells/well)
in complete cell culture medium. After 4 hours, the cell culture
medium was replaced with starvation medium containing 1% Cosmic
Calf Serum (CCS) and 10 .mu.g/mL HER2 antibody. Next, Heregulin-01
(PeproTech, Princeton Business Park, US) diluted in 1% CCS
containing starvation medium was added to the wells to a final
concentration of 1.5 ng/ml. After 4 days incubation, the amount of
viable cells was quantified with Alamarblue (BioSource
International) according to the manufacturer's instructions.
[0532] Fluorescence was monitored using the EnVision 2101
Multilabel reader (PerkinElmer) with standard Alamarblue settings.
The Alamarblue signal of HER2 antibody-treated ligand-induced cells
was plotted as a percentage signal compared to ligand-induced cells
incubated without HER2 antibody. Dunnett's test was applied for
statistical analysis.
[0533] FIG. 7 shows the percentage of viable MCF7 cells stimulated
with Heregulin-01 and treated with the indicated HER2 antibody,
relative to the viable cells after stimulation with Heregulin-01 in
the absence of HER2 antibody, which was set to 100%. MCF-7
proliferation in absence of both Heregulin-01 and antibody was also
depicted (none). Antibodies 025, 091, 129, 153 and pertuzumab
(TH1014-pert) demonstrated significant inhibition of
Heregulin-01-induced MCF-7 proliferation (P<0.05). Also
trastuzumab showed some inhibition of Heregulin-01-induced
proliferation of MCF-7 cells, although not as efficient as the
other tested HER2 antibodies. It has been reported that domain IV
of HER2 is involved in the stabilization of EGFR/HER2 heterodimers,
but without details on its contribution to HER2/HER3 heterodimers
(Wehrman et al., supra). Antibodies 050, 084, 169 and 098 had no
statistically significant effect on Heregulin-01-induced
proliferation of MCF-7 cells. Without being limited to theory, this
suggests that these antibodies do not inhibit ligand-induced
HER2/HER3 heterodimerization.
Example 18--Anti-Kappa-ETA' Assay
[0534] To investigate the suitability of HER2 antibodies for an
antibody-drug conjugate approach, a generic in vitro cell-based
killing assay using kappa-directed pseudomonas-exotoxin A
(anti-kappa-ETA') was developed. The assay makes use of a high
affinity anti-kappa domain antibody conjugated to a truncated form
of the pseudomonas-exotoxin A. Upon internalization, the
anti-kappa-ETA' domain antibody undergoes proteolysis and
disulfide-bond reduction, separating the catalytic from the binding
domain. The catalytic domain is transported from the Golgi to the
endoplasmic reticulum via the KDEL retention motif, and
subsequently translocated to the cytosol where it inhibits protein
synthesis and induces apoptosis (ref. Kreitman R J. BioDrugs 2009;
23(1):1-13). In this assay, to identify HER2 antibodies that enable
internalization and killing through the toxin, HER2 antibodies are
preconjugated with the anti-kappa-ETA' before incubation with
HER2-positive cells.
[0535] First, the optimal concentration of anti-kappa-ETA' was
determined for each cell line, i.e. the maximally tolerated dose
that does not lead to induction of non-specific cell death. AU565
cells (7500 cells/well) and A431 cells (2500 cells/well) were
seeded in normal cell culture medium in 96-wells tissue culture
plate (Greiner bio-one) and allowed to adhere for at least 4 hours.
Next, cells were incubated with 100, 10, 1, 0.1, 0.01, 0.001 and 0
.mu.g/mL anti-kappa-ETA' dilutions in normal cell culture medium.
After 3 days, the amount of viable cells was quantified with
Alamarblue (BioSource International, San Francisco, US) according
to the manufacturer's instruction. Fluorescence was monitored using
the EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland)
with standard Alamarblue settings. The highest concentration
anti-kappa-ETA' that did not kill the cells by itself was used for
following experiments (0.5 .mu.g/mL for AU565 and 1 .mu.g/mL for
A431).
[0536] Next, antibody-mediated internalization and killing by the
toxin was tested for different HER2 antibodies. Cells were seeded
as described above. Dilution-series of HER2 antibodies were
pre-incubated for 30 minutes with the predetermined concentration
anti-kappa-ETA' before adding them to the cells. After 3 days of
incubation, the amount of viable cells was quantified as described
above. The Alamarblue signal of cells treated with anti-kappa-ETA'
conjugated antibodies was plotted compared to cells treated with
antibody alone. 23.4 .mu.g/mL Staurosporin was used as positive
control for cell killing. An isotype control antibody was used as
negative control.
[0537] As shown in FIG. 8A,B and Table 6, all
anti-kappa-ETA'-conjugated HER2 antibodies were able to kill AU565
cells in a dose-dependent manner. All tested
anti-kappa-ETA'-conjugated HER2 antibodies demonstrated better
killing of AU565 cells compared to both anti-kappa-ETA'-conjugated
trastuzumab and anti-kappa-ETA'-conjugated pertuzumab
(TH1014-pert). Moreover, the percentage of killed AU565 cells was
higher for anti-kappa-ETA'-conjugated HER2 antibodies (70.3-49.9%),
compared to anti-kappa-ETA'-conjugated trastuzumab (31.9%) and
anti-kappa-ETA'-conjugated pertuzumab (47.51%), and the EC.sub.50
values were increased. EC.sub.50 values for
anti-kappa-ETA'-conjugated HER2 antibodies ranged between 12.12
ng/mL and 46.49 ng/mL) compared to 78.49 ng/mL for
anti-kappa-ETA'-conjugated trastuzumab and 117.8 ng/mL for
anti-kappa-ETA'-conjugated pertuzumab. Antibody 159 had the highest
percentage of cell-kill, and 098 the lowest EC.sub.50.
TABLE-US-00008 TABLE 6 Data shown are EC.sub.50 values and maximal
percentage cell kill of AU565 cells treated with anti-kappa-ETA'
-conjugated HER2 antibodies, measured in one representative
experiment. Cell-kill induced by Staurosporin was set as 100% and
MFI of untreated cells was set as 0%. Ndet = not detected. % cells
EC50 antibody killed ng/mL PC1014-159 70.3 34.93 PC1014-127 69.0
34.46 PC1014-132 61.6 39.35 PC1014-129 60.8 30.85 PC1014-153 60.3
32.26 PC1014-025 60.0 16.71 PC1014-098 58.7 12.12 PC1014-084 58.1
26.97 PC1014-050 52.4 12.71 PC1014-091 50.6 46.49 PC1014-169 49.9
35.62 TH1014-pert 47.5 117.8 trastuzumab 31.9 78.49 isotype control
Ndet Ndet
[0538] As shown in FIG. 8C,D and Table 7, antibodies 025, 091, 098,
129 and 153 were able to induce effective killing of A431 cells
(.gtoreq.75%). The highest percentage of cell-kill, and lowest
EC.sub.50 was shown by antibody 098. When conjugated to
anti-kappa-ETA', trastuzumab and isotype control antibody did not
induce killing of A431 cells. Antibodies 169, 084 and pertuzumab
induced percentages of cell kill of no more than about 50%. No cell
kill was observed with non-conjugated HER2 antibodies.
TABLE-US-00009 TABLE 7 Data shown are EC.sub.50 values and maximal
percentage cell kill of A431 cells treated with anti-kappa-ETA'-
conjugated HER2 antibodies, measured in one representative
experiment. Cell kill induced by Staurosporin was setas 100% and
MFI of untreated cells was set as 0%. "NDet" means not detected. %
cells EC50 antibody killed ng/mL PC1014-025 86.7 ~9.77 PC1014-084
50.5 ND PC1014-091 83.3 ~9.86 PC1014-098 87.2 1.65 PC1014-129 75.9
~10.60 PC1014-153 82.4 ~10.11 PC1014-169 34.0 ND TH1014-pert 37.0
61.58 trastuzumab Ndet Ndet isotype control NDet NDet
Example 19--Internalization of HER2 Antibodies Measured with an
FMAT-Based Fab-CypHer5E Assay
[0539] To investigate whether the enhanced killing of AU565 cells
observed in the kappa-toxin-ETA' assay described in the previous
Example correlated with enhanced internalization of HER2
antibodies, a fab-CypHer5E-based internalization assay was
performed. CypHer5E is a pH sensitive dye which is non-fluorescent
at basic pH (extracellular: culture medium) and fluorescent at
acidic pH (intracellular: lysosomes), with an acid dissociation
constant (pKa) of 7.3.
[0540] AU565 cells were seeded in 384-well tissue culture plates
(Greiner bio-one), at a density of 3000 cells/well in normal cell
culture medium supplemented with 240 ng/mL fab-CypHer5E
(conjugation of Goat-fab-anti-Human IgG [Jackson] with CypHer5E [GE
Healthcare, Eindhoven, The Netherlands] was made according to
manufacturer's instructions). Next, HER2 antibodies were serially
diluted in normal cell culture medium, added to the cells and left
at room temperature for 9 hours. Mean fluorescent intensities (MFI)
of intracellular CypHer5E were measured using the 8200 FMAT
(Applied Biosystems, Nieuwerkerk A/D IJssel, The Netherlands) and
`counts.times.fluorescence` was used as read-out. An isotype
control antibody was used as negative control antibody. EC.sub.50
values and maximal MFI were determined by means of non-linear
regression (sigmoidal dose-response with variable slope) using
GraphPad Prism V4.03 software (GraphPad Software, San Diego,
Calif., USA).
[0541] The results are shown in Table 8, depicting the EC.sub.50
and maximal MFI values for all tested HER2 antibodies in the
CypHer5E internalization assay with AU565 cells. The maximal MFI
values indicate how many HER2 receptors are internalized upon
antibody binding. All HER2 antibodies showed higher maximal MFI
values (137,904-38,801) compared to trastuzumab (35,000) and
pertuzumab (TH1014-pert) (32,366), indicating that the tested HER2
antibodies induced enhanced receptor internalization. Notably,
antibodies that did not compete with trastuzumab or TH1014-pert
induced more receptor internalization compared to antibodies that
did compete with trastuzumab and TH1014-pert, with the highest MFI
achieved by antibodies 098 and 127. Without being limited to
theory, this might be inherent to an inability to inhibit HER2
heterodimerization.
TABLE-US-00010 TABLE 8 Cypher-5-based internalization assay of HER2
antibodies. Data shown are MFI and EC.sub.50 values of one
representative experiment of two experiments with AU565 cells
treated with fab-CypHer5E-labeled HER2 antibodies. Some EC.sub.50
values could not be calculated (ND). Cypher 5 Antibody EC.sub.50
ng/mL Maximal MFI PC1014-025 30.05 63428 PC1014-091 32.99 50711
mAbs that compete PC1014-129 7.15 60302 {close oversize bracket}
with Herceptin TH1014-pert 530 32366 PC1014-169 ND 38801 mAbs that
compete PC1014-084 30.51 71059 {close oversize bracket} with
TH1014-pert trastuzumab 21.70 35000 PC1014-098 13.77 134575
PC1014-127 ~9.68 137904 mAbs that compete PC1014-159 ND 92427
{close oversize bracket} wtih TH1014-F5 TH1014-F5 22.65 113116
PC1014-132 11.42 112270 {close oversize bracket} Non-competing mAbs
PC1014-153 ~14.91 87531
Example 20: Generation of Bispecific Antibodies by 2-MEA-Induced
Fab-Arm Exchange
[0542] An in vitro method for producing bispecific antibodies is
described in WO 2008119353 (Genmab) and reported van der
Neut-Kolfschoten et al. (Science. 2007 Sep. 14; 317(5844):1554-7).
Herein, a bispecific antibody is formed by "Fab-arm" or
"half-molecule" exchange (swapping of a heavy chain and attached
light chain) between two monospecific IgG4- or IgG4-like antibodies
upon incubation under mildly reducing conditions. This Fab-arm
exchange reaction is the result of a disulfide-bond isomerization
reaction wherein the inter heavy-chain disulfide bonds in the hinge
regions of monospecific antibodies are reduced and the resulting
free cysteines form a new inter heavy-chain disulfide bond with
cysteine residues of another antibody molecule with a different
specificity. The resulting product is a bispecific antibody having
two Fab arms with different sequences.
[0543] In a novel invention the knowledge of this natural IgG4
Fab-arm exchange is adapted to generate a method to produce stable
IgG1-based bispecific antibodies. The bispecific antibody product
generated by this method described below will no longer participate
in IgG4 Fab-arm exchange. The basis for this method is the use of
complimentary CH3 domains, which promote the formation of
heterodimers under specific assay conditions. To enable the
production of bispecific antibodies by this method, IgG1 molecules
carrying certain mutations in the CH3 domain were generated: in one
of the parental IgG1 antibody T350I, K370T and F405L mutations in
the other parental IgG1 antibody the K409R mutation.
[0544] To generate bispecific antibodies, these two parental
antibodies, each antibody at a final concentration of 0.5 mg/mL
(equimolar concentration), were incubated with 25 mM
2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 .mu.L TE
at 37.degree. C. for 90 min. The reduction reaction is stopped when
the reducing agent 2-MEA is removed by using spin columns (Microcon
centrifugal filters, 30 k, Millipore) according to the
manufacturer's protocol.
Example 21--HER2.times.HER2 Bispecific Antibodies Tested in an In
Vitro Kappa-Directed ETA' Killing Assay
[0545] The example shows that HER2.times.HER2 bispecific antibodies
can deliver a cytotoxic agent into tumor cells after
internalization in a generic in vitro cell-based killing assay
using kappa-directed pseudomonas-exotoxin A (anti-kappa-ETA'). This
assay makes use of a high affinity anti-kappa domain antibody
conjugated to a truncated form of the pseudomonas-exotoxin A.
Similar fusion proteins of antibody binding proteins (IgG-binding
motif from Streptococcal protein A or protein G) and diphtheria
toxin or Pseudomonas exotoxin A have previously been (Mazor Y. et
al., J. Immunol. Methods 2007; 321:41-59); Kuo S R. et al., 2009
Bioconjugate Chem. 2009; 20:1975-1982). These molecules in contrast
to anti-kappa-ETA' bound the Fc part of complete antibodies. Upon
internalization and endocytic sorting the anti-kappa-ETA' domain
antibody undergoes proteolysis and disulfide-bond reduction,
separating the catalytic from the binding domain. The catalytic
domain is then transported from the Golgi to the endoplasmic
reticulum via a KDEL retention motif, and subsequently translocated
to the cytosol where it inhibits protein synthesis and induces
apoptosis (Kreitman R J. et. al., BioDrugs 2009; 23:1-13).
[0546] The anti-HER2 antibodies used in this example are 025, 153
and 169. In addition a fully human monoclonal IgG1,.kappa. antibody
005 of the following sequence was used:
TABLE-US-00011 005: SEQ ID VH 005 EVQLVQSGAEVKKPGESLKI NO: 164
SCKASGYSFHFYWIGWVRQM PGKGLEWMGSIYPGDSDTRY RPSFQGQVTISADKSISTAY
LQWTSLKASDTAIYYCARQR GDYYYFYGMDVWGQGTTVTV SS SEQ ID VL 005
EIVLTQSPGTLSLSPGERAT NO: 165 LSCRASQSVSSSYLAWYQQK
PGQVPRLLIYGASSRATGIP DRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSLTFGG
GTKVEIK SEQ ID VH GYSFHFYW NO: 166 CDR1 SEQ ID VH YPGDSDT NO: 167
CDR2 SEQ ID VH ARQRGDYYYFYGMDV NO: 168 CDR3 SEQ ID VL QSVSSSY NO:
169 CDR1 VL GAS CDR2 SEQ ID VL QQYGSSLT NO: 170 CDR3
[0547] The following antibodies were used as starting
materials:
IgG1-005-ITL=005 IgG1,.kappa. having Ile at position 350, Thr at
position 370, and Leu at position 405 IgG1-005-K409R=005
IgG1,.kappa. having an Arg at position 409 IgG1-025-ITL=025
IgG1,.kappa. having Ile at position 350, Thr at position 370, and
Leu at position 405 IgG1-153-ITL=153 IgG1,.kappa. having contains
Ile at position 350, Thr at position 370, and Leu at position 405
IgG1-153-K409R=153 IgG1,.kappa. having an Arg at position 409
IgG1-169-K409R=169 IgG1,.kappa. having an Arg at position 409
[0548] The following bispecific antibodies were generated in
analogy with the below procedure:
IgG1-005-ITL.times.IgG1-169-K409R
IgG1-025-ITL.times.IgG1-005-K409R
IgG1-025-ITL.times.IgG1-153-K409R
IgG1-025-ITL.times.IgG1-169-K409R
IgG1-153-ITL.times.IgG1-005-K409R
IgG1-153-ITL.times.IgG1-169-K409R
[0549] The bispecific antibodies were produced according to the
procedure described in example 20.
[0550] The antibody mixtures, containing each antibody at a final
concentration of 0.5 mg/mL, was incubated with 25 mM
2-mercaptoethylamine HCl (2-MEA) in a total volume of 100 .mu.L TE
at 37.degree. C. for 90 min. To stop the reduction reaction, the
reducing agent 2-MEA was removed by desalting the samples using
spin columns (Microcon centrifugal filters, 30 k, Millipore)
according to the manufacturer's recommendations.
[0551] The HER2.times.HER2 bispecific antibodies were pre-incubated
with the anti-kappa-ETA' before incubation with A431 cells. A431
cells express .about.15,000 HER2 antibodies per cell (determined
via Qifi analysis) and are not sensitive to treatment with `naked`
HER2-antibodies.
[0552] First, the optimal concentration of anti-kappa-ETA' was
determined for each cell line, i.e. the maximally tolerated dose
that does not lead to induction of non-specific cell death. A431
cells (2500 cells/well) were seeded in normal cell culture medium
in a 96-wells tissue culture plate (Greiner bio-one) and allowed to
adhere for at least 4 hours. These cells were incubated with an
anti-kappa-ETA' dilution series, 100, 10, 1, 0.1, 0.01, 0.001 and 0
.mu.g/mL in normal cell culture medium. After 3 days, the amount of
viable cells was quantified with Alamarblue (BioSource
International, San Francisco, US) according to the manufacturer's
instruction. Fluorescence was monitored using the EnVision 2101
Multilabel reader (PerkinElmer, Turku, Finland) with standard
Alamarblue settings. The highest concentration anti-kappa-ETA' that
did not kill the cells by itself (1 .mu.g/mL for A431 cells) was
used for following experiments.
[0553] Next, the effect of HER2.times.HER2 bispecific antibodies
and HER2 monospecific antibodies pre-incubated with anti-kappa-ETA'
was tested for their ability to induce cell kill. A431 cells were
seeded as described above. A dilution series of the HER2 specific
antibodies (monospecific and bispecific antibodies) was made and
pre-incubated for 30 min with the predetermined concentration of
anti-kappa-ETA' before adding them to the cells. After 3 days
incubation at 37.degree. C., the amount of viable cells was
quantified as described above. The Alamarblue signal of cells
treated with anti-kappa-ETA' pre-incubated with the antibodies was
plotted compared to cells treated without antibody treatment.
EC.sub.50 values and maximal cell death were calculated using
GraphPad Prism 5 software. Staurosporin (23.4 .mu.g/mL) was used as
positive control for cell killing. An isotype control antibody
(IgG1/kappa; IgG1-3G8-QITL) was used as negative control.
[0554] FIG. 9 and table 9 shows that all anti-kappa-ETA'
pre-incubated HER2 bispecific antibodies were able to kill A431
cells in a dose-dependent manner. These results demonstrate that
most HER2 bispecific antibodies tested were more effective than the
monospecific antibody present in the combination in this
anti-kappa-ETA' assay. In addition, the efficacy of bispecific
antibody 005X169, 025X169 and 153X169 showed that the efficacy of a
monospecific antibody which lacks activity in this in vitro
kappa-directed ETA' killing, HER2 specific antibody (169), can be
increased through bispecific combination with another HER2 specific
antibody.
TABLE-US-00012 TABLE 9 EC.sub.50 values and maximal percentage cell
kill of AU565 cells treated with anti-kappa-ETA'-conjugated HER2
.times. HER2 bispecific antibodies. "Ndet" means not detected.
percentage EC50 antibody kill [ng/mL] Herceptin 2.79 Ndet
IgG1-005-ITL 79.34 2.57 IgG1-005-K409R 79.83 2.87 IgG1-025-ITL
69.81 3.76 IgG1-153-ITL 70.66 12.45 IgG1-153-K409R 72.84 15.47
IgG1-169-K409R 16.45 3.45 IgG1-005-ITL .times. IgG1-169-K409R 59.94
4.28 IgG1-025-ITL .times. IgG1-005-K409R 63.45 4.27 IgG1-025-ITL
.times. IgG1-153-K409R 80.82 7.66 IgG1-025-ITL .times.
IgG1-169-K409R 45.88 7.97 IgG1-153-ITL .times. IgG1-005-K409R 80.05
4.51 IgG1-153-ITL .times. IgG1-169-K409R 84.68 29.14
Example 22--HER2 Receptor Downmodulation by Incubation with
Bispecific Antibodies Targeting Different HER2 Epitopes
[0555] HER2.times.HER2 bispecific antibodies may bind two different
epitopes on two spatially different HER2 receptors. This may allow
other HER2.times.HER2 bispecific antibodies to bind to the
remaining epitopes on these receptors. This could result in
multivalent receptor cross-linking (compared to dimerization
induced by monovalent antibodies) and consequently enhance receptor
downmodulation. To investigate whether HER2.times.HER2 bispecific
antibodies induce enhanced downmodulation of HER2, AU565 cells were
incubated with antibodies and bispecific antibodies for three days.
Total levels of HER2 and levels of antibody bound HER2 were
determined.
[0556] AU565 cells were seeded in a 24-well tissue culture plate
(100.000 cells/well) in normal cell culture medium and cultured for
three days at 37.degree. C. in the presence of 10 .mu.g/mL HER2
antibody with either the ITL or the K409R mutation or
HER2.times.HER2 bispecific antibodies. As a control, the
combination of two monospecific HER2 antibodies, with unmodified
IgG1 backbones, was also tested (1:1), at a final concentration of
10 .mu.g/mL. After washing with PBS, cells were lysed by incubating
them for 30 min at room temperature with 25 .mu.L Surefire Lysis
buffer (Perkin Elmer, Turku, Finland). Total protein levels were
quantified using bicinchoninic acid (BCA) protein assay reagent
(Pierce) following manufacturer's protocol. HER2 protein levels in
the lysates were analyzed using a HER2-specific sandwich ELISA.
Rabbit-anti-human HER2 intracellular domain antibody (Cell
Signaling) was used to capture HER2 and biotinylated
goat-anti-human HER2 polyclonal antibody R&D systems,
Minneapolis, USA), followed by streptavidin-poly-HRP, were used to
detect bound HER2. The reaction was visualized using 2,2'-azino-bis
3-ethylbenzothiazoline-6-sulfonic acid (one ABTS tablet diluted in
50 mL ABTS buffer [Roche Diagnostics, Almere, The Netherlands]) and
stopped with oxalic acid (Sigma-Aldrich, Zwijndrecht, The
Netherlands). Fluorescence at 405 nm was measured on a microtiter
plate reader (Biotek Instruments, Winooski, USA) and the amount of
HER2 was expressed as a percentage relative to untreated cells.
[0557] The results are shown in FIG. 10 and Table 10 which
demonstrates that all the tested HER2.times.HER2 bispecific
antibodies induced .gtoreq.40% HER2 downmodulation. Interestingly,
all HER2.times.HER2 bispecific antibodies demonstrated increased
HER2 downmodulation compared to both of their monospecific
counterparts.
TABLE-US-00013 TABLE 10 HER2 .times. HER2 bispecific induced
downmodulation of HER2 depicted as percentage HER2 compared to
untreated cells % HER2 compared to antibody untreated cells
Herceptin 71 IgG1-005-ITL 54 IgG1-005-K409R 50 IgG1-025-ITL 64
IgG1-153-ITL 43 IgG1-153-K409R 40 IgG1-169-K409R 64 IgG1-005-ITL
.times. IgG1-169-K409R 29 IgG1-025-ITL .times. IgG1-005-K409R 38
IgG1-025-ITL .times. IgG1-153-K409R 29 IgG1-025-ITL .times.
IgG1-169-K409R 34 IgG1-153-ITL .times. IgG1-005-K409R 23
IgG1-153-ITL .times. IgG1-169-K409R 28 IgG1-005 + IgG1-169 28
IgG1-025 + IgG1-005 28 IgG1-025 + IgG1-153 23 IgG1-025 + IgG1-169
25 IgG1-153 + IgG1-005 23 IgG1-153 + IgG1-169 23 isotype control
108
Example 23--Colocalization of HER2.times.HER2 Bispecific Antibodies
with Lysosomal Marker LAMP1 Analyzed by Confocal Microscopy
[0558] The HER2 downmodulation assay as described in Example 22
indicated that HER2.times.HER2 bispecific antibodies were able to
increase lysosomal degradation of HER2. To confirm these findings,
confocal microscopy technology was applied. AU565 cells were grown
on glass coverslips (thickness 1.5 micron, Thermo Fisher
Scientific, Braunschweig, Germany) in standard tissue culture
medium at 37.degree. C. for 3 days. Cells were pre-incubated for 1
hour with 50 .mu.g/mL leupeptin (Sigma) to block lysosomal activity
after which 10 .mu.g/mL HER2 monospecific antibodies or
HER2.times.HER2 bispecific antibodies were added. Also the
combination of two monospecific IgG1 antibodies (1:1) was tested at
a final concentration of 10 .mu.g/mL. The cells were incubated for
an additional 3 or 18 hours at 37.degree. C. Hereafter the cells
were washed with PBS and incubated for 30 min. at room temperature
with 4% formaldehyde (Klinipath). Slides were washed with blocking
buffer (PBS supplemented with 0.1% saponin [Roche] and 2% BSA
[Roche]) and incubated for 20 min with blocking buffer containing
20 mM NH.sub.4Cl to quench formaldehyde. Slides were washed again
with blocking buffer and incubated for 45 min at room temperature
with mouse-anti-human CD107a (LAMP1) (BD Pharmingen) to
stain/identify lysosomes. Following washing with blocking buffer,
the slides were incubated 30 min at room temperature with a
cocktail of secondary antibodies; goat-anti-mouse IgG-Cy5 (Jackson)
and goat-anti-human IgG-FITC (Jackson). Slides were washed again
with blocking buffer and mounted overnight on microscope slides
using 20 .mu.L mounting medium (6 gram Glycerol [Sigma] and 2.4
gram Mowiol 4-88 [Omnilabo] was dissolved in 6 mL distilled water
to which 12 mL 0.2M Tris [Sigma] pH8.5 was added followed by
incubation for 10 min at 50-60.degree. C. Mounting medium was
aliquoted and stored at -20.degree. C.). Slides were imaged with a
Leica SPE-II confocal microscope (Leica Microsystems) equipped with
a 63.times.1.32-0.6 oil immersion objective lens and LAS-AF
software. To allow for quantification of overlapping pixel
intensities, saturation of pixels should be avoided. Therefore the
FITC laser intensity was decreased to 10%, smart gain was set at
830 V and smart offset was set at -9.48%. By using these settings,
the bispecific antibodies were clearly visualized without pixel
saturation, but the monospecific antibodies were sometimes
difficult to detect. To compare lysosomal colocalization between
monospecific and bispecific antibodies, these settings were kept
the same for all analyzed confocal slides.
[0559] 12-bit grayscale TIFF images were analyzed for
colocalisation using MetaMorph.COPYRGT. software (version Meta
Series 6.1, Molecular Devices Inc, Sunnyvale Calif., USA). FITC and
Cy5 images were imported as stacks and background was subtracted.
Identical thresholds settings were used (manually set) for all FITC
images and all Cy5 images. Colocalisation was depicted as the pixel
intensity of FITC in the region of overlap (ROI), were the ROI is
composed of all Cy5 positive regions. To compare different slides
stained with several HER2 antibodies, HER2.times.HER2 bispecific
antibodies or the combination of two different monospecific
antibodies the images were normalized using the pixel intensity of
Cy5. Goat-anti-mouse IgG-Cy5 was used to stain the lysosomal marker
LAMP1 (CD107a). The pixel intensity of LAMP1 should not differ
between various HER2 antibodies or the HER2.times.HER2 bispecific
antibodies tested (one cell had a pixel intensity of Cy5 of roughly
200.000).
Normalized values for colocalization of FITC and
Cy5=[(TPI-FITC.times.percentage FITC-Cy5
colocalization)/100].times.[200.000/TPI-Cy5]
[0560] In this formula, TPI stands for Total Pixel Intensity.
[0561] FIG. 11 and Table 11 present percentage of viable cells, as
measured by the FITC pixel intensity overlapping with Cy5 for
various monospecific HER2 antibodies and HER2.times.HER2 bispecific
antibodies. For each antibody or bispecific molecule depicted,
three different images were analyzed from one slide containing
.about.1, 3 or >5 cells. Significant variation was observed
between the different images within each slide. However, it was
evident that all HER2.times.HER2 bispecific antibodies demonstrate
increased colocalisation with the lysosomal marker LAMP1, when
compared with their monospecific counterparts. These results
indicate that once internalized, HER2.times.HER2 bispecific
antibodies are efficiently sorted towards lysosomal compartments,
making them suitable for a bispecific antibody drug conjugate
approach.
TABLE-US-00014 TABLE 11 Mean FITC pixel intensities overlapping
with Cy5 depicted as arbitrary units FITC pixel intensity in
lysosomes antibody [arbitrary units] Herceptin 0.218 IgG1-005-ITL
0.070 IgG1-025-ITL 0.268 IgG1-153-ITL 0.102 IgG1-169-K409R 0.220
IgG1-005-ITL .times. IgG1-169-K409R 0.531 IgG1-025-ITL .times.
IgG1-005-K409R 0.347 IgG1-025-ITL .times. IgG1-153-K409R 0.582
IgG1-025-ITL .times. IgG1-169-K409R 0.439 IgG1-153-ITL .times.
IgG1-005-K409R 0.494 IgG1-153-ITL .times. IgG1-169-K409R 0.604
IgG1-025 + IgG1-169 0.576 IgG1-153 + IgG1-005 0.636 IgG1-153 +
IgG1-169 0.626
Example 24--Inhibition of Proliferation of AU565 Cells Upon
Incubation with HER2 Monospecific or HER2.times.HER2 Bispecific
Antibodies
[0562] The HER2.times.HER2 bispecific antibodies were tested for
their ability to inhibit proliferation of AU565 cells in vitro. Due
to the high HER2 expression levels on AU565 cells (.about.1,000,000
copies per cell as determined with Qifi-kit), HER2 is
constitutively active in these cells and thus not dependent on
ligand-induced heterodimerization. In a 96-wells tissue culture
plate (Greiner bio-one, Frickenhausen, Germany), 9.000 AU565 cells
were seeded per well in the presence of 10 .mu.g/mL HER2 antibody
or HER2.times.HER2 bispecific antibodies in serum-free cell culture
medium. As a control, cells were seeded in serum-free medium
without antibody or bispecific antibodies. After three days, the
amount of viable cells was quantified with Alamarblue (BioSource
International, San Francisco, US) according to the manufacturer's
instructions. Fluorescence was monitored using the EnVision 2101
Multilabel reader (PerkinElmer, Turku, Finland) with standard
Alamarblue settings. The Alamarblue signal of antibody-treated
cells was plotted as a percentage relative to untreated cells.
[0563] FIG. 12 and table 12 depicts the fluorescent intensity of
Alamarblue of AU565 cells after incubation with HER2 antibodies and
HER2.times.HER2 bispecific antibodies. Herceptin.RTM. (trastuzumab)
was included as positive control and demonstrated inhibition of
proliferation as described by Juntilla T T. et al., Cancer Cell
2009; 15: 429-440. All HER2.times.HER2 bispecific antibodies were
able to inhibit proliferation of AU565 cells. Bispecific
antibodies: IgG1-005-ITL.times.IgG1-169-K409R and
IgG1-025-ITL.times.IgG1-005-K409R were more effective compared to
their monospecific antibody counterparts in this assay.
TABLE-US-00015 TABLE 12 Percentage viable AU565 cells after
treatment with HER2 .times. HER2 bispecific antibodies. percentage
viable antibody cells Herceptin 62 IgG1-005-ITL 91 IgG1-005-K409R
96 IgG1-025-ITL 79 IgG1-153-ITL 98 IgG1-153-K409R 97 IgG1-169-K409R
63 IgG1-005-ITL .times. IgG1-169-K409R 49 IgG1-025-ITL .times.
IgG1-005-K409R 61 IgG1-025-ITL .times. IgG1-153-K409R 74
IgG1-025-ITL .times. IgG1-169-K409R 76 IgG1-153-ITL .times.
IgG1-005-K409R 71 IgG1-153-ITL .times. IgG1-169-K409R 77 isotype
control 95
Example 25--Her2.times.CD3 Bispecific Antibodies Tested in an In
Vitro Cytotoxicity Assay
[0564] CD3 is a co-receptor in the T cell receptor complex
expressed on mature T cells. Combination of a CD3 specific antibody
Fab-arm with a tumor antigen specific antibody Fab-arm in a
bispecific antibody would result in the specific targeting of T
cells to tumor cells, leading to T cell mediated tumor cell lysis.
Likewise, CD3 positive T cells could be targeted to other derailed
cells in the body, to infected cells or directly to pathogens.
[0565] HER2.times.CD3 bispecific antibodies were generated. Heavy
and light chain variable region sequences for the HER2 specific
Fab-arm were as indicated for antibody 153 and 169 in Example 21.
The following heavy and light chain variable region sequences for
the CD3 specific Fab-arm were used:
TABLE-US-00016 YTH12.5 (Sequence as described by Routledge et al.,
Eur J Immunol. 1991, 21(11):2717-25.) SEQ VH EVQLLESGGGLVQPGGSL ID
YTH12.5 RLSCAASGFTFSSFPMAW NO: VRQAPGKGLEWVSTISTS 171
GGRTYYRDSVKGRFTISR DNSKNTLYLQMNSLRAED TAVYYCAKFRQYSGGFDY
WGQGTLVTVSS SEQ VL DIQLTQPNSVSTSLGSTV ID YTH12.5 KLSCTLSSGNIENNYVHW
NO: YQLYEGRSPTTMIYDDDK 172 RPDGVPDRFSGSIDRSSN SAFLTIHNVAIEDEAIYF
CHSYVSSFNVFGGGTKLT VL
TABLE-US-00017 huCLB-T3/4 (Sequence as described by Parren et al.,
Res Immunol. 1991, 142(9):749-63. Minor amino acid substitutions
were introduced to make the sequence resemble the closest human
germline.) SEQ VH EVQLVESGGGLVKPGGSLRLSCA ID huCLB-
ASGFTFSSYGMFWVRQAPGKGLE NO: T3/4 WVATISRYSRYIYYPDSVKGRFT 173
ISRDNAKNSLYLQMNSLRAEDTA VYYCARRPLYGSSPDYWGQGTLV TVSS SEQ VL
EIVLTQSPATLSLSPGERATLSCS ID huCLB- ASSSVTYVHWYQQKPGQAPRLLIY NO:
T3/4 DTSKLASGIPARFSGSGSGTDFTL 174 TISSLEPEDFAVYYCFQGSGYPLT
FGSGTKLEMR
[0566] All antibodies were expressed as IgG1,.kappa. being modified
in their Fc regions as follows: IgG1-HER2-153-K409R and
IgG1-HER2-153-N297Q-K409R, IgG1-HER2-169-K409R,
IgG1-hu-CLB-T3/4-F405L and IgG1-hu-CLB-T3/4-N297Q-F405L,
IgG1-YTH12.5-F405L and IgG1-YTH12.5-N297Q-F405L.
[0567] Bispecific antibodies from these HER2 and CD3 specific
antibodies were generated as described in Example 20 and tested in
an in vitro cytotoxicity assay using AU565 cells. AU565 cells were
cultured to near confluency. Cells were washed twice with PBS, and
trypsinized for 5 minutes at 37.degree. C. 12 mL culture medium was
added to inactivate trypsin and cells were spun down for 5 min, 800
rpm. Cells were resuspended in 10 mL culture medium and a single
cell suspension was made by passing the cells through a
cellstrainer. 100 .mu.L of a 5.times.10.sup.5 cells/mL suspension
was added to each well of a 96-well culture plate, and cells were
incubated at least 3 hrs at 37.degree. C., 5% CO2 to allow
adherence to the plate. Peripheral blood mononuclear cells (PBMC)
were isolated from blood from healthy volunteers using Leucosep 30
mL tubes, according to the manufacturer's protocol (Greiner
Bio-one). T cells were isolated from PBMC preparations by negative
selection using the Untouched Human T-cells Dynabead kit (Dynal).
Isolated cells were resuspended in culture medium to a final
concentration op 7.times.10.sup.6 cells/mL.
[0568] Culture medium was removed from the adhered AU565 cells, and
replaced with 50 .mu.l/well 2.times. concentrated antibody-dilution
and 50 .mu.l/well 7.times.10.sup.6 T cells/mL (ratio
effector:target=7:1). Plates were incubated for 3 days at
37.degree. C., 5% CO.sub.2. Supernatants were removed and plates
were washed twice with PBS. To each well 150 .mu.L culture medium
and 15 .mu.L Alamar blue was added. Plates were incubate for 4
hours at 37.degree. C., 5% CO.sub.2, and absorbance was measured
(Envision, Perkin Elmer).
[0569] FIG. 13 and table 13 shows that whereas control antibodies
(Her2 monospecific IgG1-Herceptin.COPYRGT., CD3 monospecific
IgG1-YTH12.5 and monospecific IgG1-huCLB-T3/4, irrelevant antigen
monospecific IgG1-b12, and CD3.times.b12 bispecific antibodies) did
not induce T cell mediated cytotoxicity, bispecific (Duo)
Her2.times.CD3 antibodies huCLB/Her2-153, huCLB/Her2-169,
YTH12.5/Her2-153 and YTH12.5/Her2-169 induced dose dependent T cell
mediated cytotoxicity of AU565 cells. Bispecific antibodies
containing Her2-169 were more potent than those containing
Her2-153.
[0570] Mutants of IgG1-hu-CLB-T3/4, IgG1-YTH12.5 and Her2-153 were
made containing a N297Q mutation to remove a glycosylation site;
glycosylation at this site is critical for IgG-Fcgamma receptor
interactions (Bolt S et al., Eur J Immunol 1993, 23:403-411). FIG.
13 shows that N297Q mutation and therefore absence of Fc
glycosylation of Her2.times.CD3 bispecific antibodies
YTH12.5/Her2-153 and huCLB/Her2-153 did not impact the potential to
induce dose dependent T cell mediated cytotoxicity of AU565
cells.
TABLE-US-00018 TABLE 13 EC.sub.50 values of cell kill induced by
HER2 .times. CD3 bispecific antibodies. "Ndet" means not detected.
EC50 antibody [ng/mL] Herceptin Ndet Duo huCLB-Q/153-Q 10.55 Duo
huCLB-Q/B12-Q Ndet huCLB-Q Ndet B12-Q Ndet Duo YTH12.5-Q/153-Q
10.73 Duo YTH12.5-Q/B12-Q Ndet YTH12.5-Q Ndet B12-Q Ndet
Example 26--HER2 Downmodulation
[0571] To investigate if enhanced HER2 internalization induced by
Group 3 antibodies 098 and 153 also results in enhanced receptor
downmodulation, AU565 cells were incubated with HER2 antibodies for
3 days, and analyzed for presence of HER2. AU565 cells were seeded
in a 24-wells tissue culture plate (100.000 cells/well) in normal
cell culture medium and cultured for 3 days at 37.degree. C. in the
presence of 10 .mu.g/mL HER2 antibody. After washing with PBS,
cells were lysed by incubating 30 min at room temperature with 25
.mu.L Surefire Lysis buffer (Perkin Elmer, Turku, Finland). Total
protein levels were quantified using bicinchoninic acid (BCA)
protein assay reagent (Pierce) according to the manufacturer's
protocol. HER2 protein levels in the lysates were analyzed using a
HER2-specific sandwich ELISA. Rabbit-anti-human HER2 intracellular
domain antibody (Cell Signaling) was used to capture HER2 and
biotinylated goat-anti-human HER2 polyclonal antibody (R&D),
followed by streptavidin-poly-HRP, were used to detect bound HER2.
The reaction was visualized using 2,2'-azino-bis
3-ethylbenzothiazoline-6-sulfonic acid (ABTS: dilute one ABTS
tablet in 50 mL ABTS buffer [Roche Diagnostics, Almere, The
Netherlands]) and stopped with oxalic acid (Sigma-Aldrich,
Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured
on a microtiter plate reader (Biotek Instruments, Winooski, USA)
and the amount of HER2 was expressed as a percentage relative to
untreated cells.
[0572] The results shown in FIG. 14 and Table 14 demonstrate that
both Group 3 antibodies (098 and 153) induced more than 50% HER2
downmodulation. In contrast, antibodies 025, 169 and Herceptin
barely induced downmodulation (approximately 20% of untreated
cells). This was in line with enhanced internalization observed by
antibodies 098 and 153.
TABLE-US-00019 TABLE 14 Antibody induced downmodulation of HER2
depicted as percentage HER2 compared to untreated cells % HER2
compared to antibody untreated cells Herceptin 80 IgG1-1014-169 82
IgG1-1014-025 85 IgG1-1014-098 44 IgG1-1014-153 50 isotype control
108
Example 27--Colocalization of HER2 Antibodies with Lysosomal Marker
LAMP1 Analyzed by Confocal Microscopy
[0573] The HER2 downmodulation assay as described in example 26 and
the CypHer-5E based internalization assay as described in example
19 indicated that HER2 antibodies from group 3 were more
efficiently internalized and targeted towards lysosomes compared to
antibodies from Groups 1 and 2. However, in these experiments the
confocal imaging was done with settings that allowed discriminating
between monospecific and bispecific antibodies but not between
different monospecific antibodies, in fact, with these settings
monospecific antibodies could hardly be detected. To be able to
compare between the different monospecific antibodies, the confocal
slides were measured again with increased gain settings, to enhance
fluorescence intensity. All other steps of the procedure were the
same as described in example 23.
[0574] The results are depicted in FIG. 15 and Table 15, and show
that the FITC pixel intensity overlapping with Cy5 for various
monospecific HER2 antibodies. From each slide three different
images were analyzed containing .about.1, 3 or >5 cells.
Significant variation was observed between the different images
within each slide. Still, it was evident that antibodies 098 and
153 were more efficiently targeted towards lysosomal compartments,
compared to 025, pertuzumab, 169 and Herceptin. This correlated
well with the enhanced internalization and receptor degradation
induced by these antibodies.
TABLE-US-00020 TABLE 15 Mean FITC pixel intensities overlapping
with Cy5 depicted as arbitrary units FITC pixel intensity in
lysosomes antibody [arbitrary units] TH1014-098 0.522 TH1014-153
0.409 TH1014-025 0.248 TH1014-pert 0.214 TH1014-169 0.255 Herceptin
0.236
Example 28--HER2 Extracellular Domain Shuffle Human-to-Chicken
[0575] To further define the HER2 binding regions recognized by
antibodies from the four different cross-competition groups, a HER2
extracellular domain shuffle experiment was performed. To this end,
a small gene-synthesis library with five constructs was generated,
swapping the sequences of domain I, II, III or IV of the
extracellular domain of human HER2 to the corresponding sequence of
chicken HER2 (Gallus Gallus isoform B NCBI: NP_001038126.1): 1)
fully human HER2 (Uniprot P04626) hereafter named hu-HER2, 2)
hu-HER2 with chicken domain I (replacing amino acids (aa) 1-203 of
the human Her2 with the corresponding chicken Her2 region)
hereafter named hu-HER2-ch(I), 3) hu-HER2 with chicken domain II
(replacing amino acids (aa) 204-330 of the human Her2 with the
corresponding chicken Her2 region) hereafter named hu-HER2-ch(II),
4) hu-HER2 with chicken domain III (replacing aa 331-507 of the
human Her2 with the corresponding chicken Her2 region) hereafter
named hu-HER2-ch(III) and 5) hu-HER2 with chicken domain IV
(replacing aa 508-651 of the human Her2 with the corresponding
chicken Her2 region) hereafter named hu-HER2-ch(IV). The human and
chicken HER2 orthologs show 67% homology in their extracellular
domain with 62% homology in domain I, 72% homology in domain II,
63% homology in domain III and 68% homology in domain IV. The
constructs were transiently transfected in the Freestyle.TM.
CHO--S(Invitrogen) cell line using Freestyle MAX transfection
reagent (Invitrogen) according to the instructions of the
manufacturer, and transfected cells were cultured for 20 hours.
HER2 antibody binding to the transfected cells was analyzed by
means of flow cytometry: The transfected CHO--S cells were
harvested, washed with FACS buffer and incubated with 10 .mu.g/mL
HER2 antibody (30 minutes on ice). Binding of HER2 antibodies was
detected using a Phycoerythrin (PE)-conjugated goat-anti-human IgG
antibody (Jackson). To check if expression between different
batches was the same, cells were fixed and permeabilized using
Cytofix/Cytoperm solution (BD) according manufacturer's instruction
and stained with a rabbit-anti-human intracellular HER2 antibody
(DAKO) in combination with a secondary PE-conjugated
goat-anti-rabbit antibody (Jackson). An isotype control antibody
was used as negative control. Fluorescence was measured on a
FACSCanto-II (BD) and binding curves were made by means of
non-linear regression (sigmoidal dose-response with variable slope)
using GraphPad Prism V4.03 software (GraphPad Software, San Diego,
Calif., USA). Loss of binding was used as read out to identify
which HER2 domains were recognized by the different antibodies.
[0576] Exemplary binding curves for antibody 153 are shown in FIG.
16. All binding results are shown in Table 16. Group 1 HER2
antibodies 050, 084, 169 and Herceptin showed loss of binding to
Hu-HER2-ch(IV), but not to the proteins with one of the remaining
domains shuffled, demonstrating that the epitopes of Group 1 mAbs
reside in HER2 domain IV. Group 2 antibodies 025, 091, 129 and
pertuzumab showed only loss of binding to Hu-HER2-ch(II),
indicating that the epitope resides in HER2 domain II. Antibodies
098 and 153 were both defined to Group 3 in the cross-competition
assay but showed some variation in the shuffle experiment. Antibody
098 clearly showed loss of binding to Hu-HER2-ch(I) and a minor
decrease in binding to Hu-HER2-ch(II), while 153 showed only loss
of binding to Hu-HER2-ch(II). These data suggest that Group 3 mAbs
098 and 153 can also bind, at least partially, to the HER2 domain
II, with epitopes that possibly extend into HER2 domain I, as is
the case for 098.
TABLE-US-00021 TABLE 16 Summary of HER2 antibody binding to
different HER2ECD receptor constructs. HER2-domain shuffled
Antibody Group FL I II III IV Herceptin 1 +++ +++ +++ +++ - 050 1
+++ +++ +++ +++ - 084 1 +++ +++ +++ +++ - 169 1 +++ +++ +++ +++ +
Pertuzumab 2 +++ +++ + +++ +++ 025 2 +++ +++ - +++ +++ 091 2 +++
+++ - +++ +++ 129 2 +++ +++ - +++ +++ 153 3 +++ +++ - +++ +++ 098 3
+++ - ++ +++ +++ FL; hu-HER2, I; hu-HER2-ch(I), II; hu-HER2-ch(II),
III; hu-HER2-ch(III), IV; hu-HER2-ch(IV). +++indicates normal
binding, ++ indicates reduced EC.sub.50 but the similar maximal
binding compared to binding observed to hu-HER2, +indicates reduced
EC.sub.50 and reduced maximal binding detected compared to binding
observed to hu-HER2, -indicates no binding.
Example 29--In Vivo Efficacy of HER2 HuMabs 091, 084 and 169 in
NCI-N87 Human Gastric Carcinoma Xenografts in SCID Mice
[0577] The in vivo effect of HER2-HuMabs 091 (cross-competition
Group 2), 084 and 169 (both cross-competition Group 1) on tumor
growth and survival in a NCI-N87 human gastric carcinoma xenograft
model in female CB.17 severe combined immunodeficiency (SCID) mice
was determined. 10.times.10.sup.6 NCI-N87 tumor cells in 50%
matrigel were injected s.c. in female SCID mice, 10 mice per group.
Eight days after tumor inoculation, intravenous treatment with
HER2-HuMabs 091, 084, and 169 or control antibody HuMab-HepC was
started. In FIG. 17 (A) this is indicated as day 1, day of
treatment initiation. The first dose was at 40 mg/kg, followed by
10 mg/kg on days 4, 8, 11, 15, 18, 22, and 25 after treatment
initiation.
Tumor volume was determined at least 2 times per week. Volumes
(mm.sup.3) were calculated from caliper (PLEXX) measurements as
(width.sup.2.times.length)/2.
[0578] The results are depicted in FIGS. 17A and 17B which shows
that the mice administered with HuMab 084, 169 and 091 demonstrated
slower tumor growth (A) and better survival (B) than the mice that
received negative control antibody HuMab-HepC. All treatments were
well-tolerated.
Example 30--Therapeutic Treatment of BT-474 Breast Tumor Xenografts
in Balb/C Nude Mice
[0579] The effect of therapeutic treatment of five different HER2
HuMabs on human subcutaneous BT-474 breast tumor xenografts in
Balb/C nude mice was determined. BT-474 tumor cells were injected
24 to 72 hours after a whole body irradiation with a .gamma.-source
(1.8 Gy, Co60, BioMep, France). 2.times.10.sup.7 BT-474 cells in
200 .mu.l of RPMI 1640 containing matrigel (50:50, v:v; BD
Biosciences) were injected subcutaneously into the right flank of
female Balb/C nude mice. Body weight and tumor volume of the mice
was recorded twice a week. Tumor volumes (mm.sup.3) were calculated
from caliper (PLEXX) measurements as:
(width.sup.2.times.length)/2.
[0580] Treatment with HER2 HuMabs was started when the tumors
reached a mean volume of 100-200 mm.sup.3. Tumor bearing mice were
randomized into groups of 8 mice. One group received twice weekly
intravenous (i.v.) injections of the control mAb HuMab-HepC. Four
other groups received twice weekly i.v. injections of HER2 HuMab
025, 129, 153 and 091, with a first dose of 20 mg/kg and following
9 doses of 5 mg/kg.
[0581] The results are depicted in FIGS. 18A and 18B and show that
BT-474 tumor growth was partially inhibited with HuMab 129 and
HuMab 153 treatment (about 30 and 50% of inhibition compared to
HuMab-HepC control treatment). HuMab-025 and HuMab-091 strongly
inhibited the BT-474 tumor growth and the time to reach a tumor
volume of 800 mm.sup.3 was significantly delayed by these
antibodies. Survival was also improved in the HER2 HuMAb receiving
mice.
Sequence CWU 1
1
2081121PRTHomo sapiens 1Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Leu Ser Ala Tyr Ser Gly
Asn Thr Ile Tyr Ala Gln Lys Leu 50 55 60Gln Gly Arg Val Thr Met Thr
Thr Asp Thr Ser Thr Thr Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Arg
Ile Val Val Arg Pro Asp Tyr Phe Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser 115 12028PRTHomo sapiens 2Gly Tyr Thr
Phe Thr Asn Tyr Gly1 538PRTHomo sapiens 3Leu Ser Ala Tyr Ser Gly
Asn Thr1 5414PRTHomo sapiens 4Ala Arg Asp Arg Ile Val Val Arg Pro
Asp Tyr Phe Asp Tyr1 5 105107PRTHomo sapiens 5Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Arg
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10566PRTHomo sapiens 6Gln Ser Val Ser Ser Tyr1 579PRTHomo sapiens
7Gln Gln Arg Ser Asn Trp Pro Arg Thr1 58119PRTHomo sapiens 8Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ala Ile Ser Gly Arg Gly Gly Thr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Ala Arg Ala Asn Trp Asp Tyr Phe Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11598PRTHomo sapiens 9Gly Phe Thr Phe Ser Ser Tyr Ala1 5108PRTHomo
sapiens 10Ile Ser Gly Arg Gly Gly Thr Thr1 51112PRTHomo sapiens
11Ala Lys Ala Arg Ala Asn Trp Asp Tyr Phe Asp Tyr1 5 1012107PRTHomo
sapiens 12Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln His Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ile Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ala Asn Ser Phe Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg
Leu Glu Ile Lys 100 105136PRTHomo sapiens 13Gln Gly Ile Ser Ser
Trp1 5149PRTHomo sapiens 14Gln Gln Ala Asn Ser Phe Pro Ile Thr1
515121PRTHomo sapiens 15Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Arg Thr Tyr 20 25 30Ala Ile Asn Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asn Thr Val Leu Gly
Ile Val Asn His Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Lys Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Asn Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Lys
Gly Val Asp Tyr Tyr Tyr Gly Ile Glu Val Trp Gly 100 105 110Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120168PRTHomo sapiens 16Gly Gly Thr
Phe Arg Thr Tyr Ala1 5178PRTHomo sapiens 17Ile Asn Thr Val Leu Gly
Ile Val1 51814PRTHomo sapiens 18Ala Arg Glu Lys Gly Val Asp Tyr Tyr
Tyr Gly Ile Glu Val1 5 1019107PRTHomo sapiens 19Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Val
Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105206PRTHomo sapiens 20Gln Gly Ile Ser Ser Trp1 5219PRTHomo
sapiens 21Gln Gln Ala Asn Ser Phe Pro Leu Thr1 522120PRTHomo
sapiens 22Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe
Ser Asp Tyr 20 25 30Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly
Leu Glu Trp Ile 35 40 45Gly Glu Ile His His Ser Gly Ser Thr Asn Tyr
Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser
Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Tyr Tyr Asp Ser Gly
Val Tyr Tyr Phe Asp Tyr Trp Ala Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser 115 120238PRTHomo sapiens 23Gly Gly Ser Phe Ser Asp Tyr
Tyr1 5247PRTHomo sapiens 24Ile His His Ser Gly Ser Thr1
52514PRTHomo sapiens 25Ala Arg Gly Tyr Tyr Asp Ser Gly Val Tyr Tyr
Phe Asp Tyr1 5 1026107PRTHomo sapiens 26Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Arg Trp 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser
Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Ile 85 90
95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105276PRTHomo
sapiens 27Gln Gly Ile Ser Arg Trp1 5289PRTHomo sapiens 28Gln Gln
Tyr Asn Ser Tyr Pro Ile Thr1 529120PRTHomo sapiens 29Gln Val Gln
Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu
Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr
Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Glu Ile Tyr His Ser Gly Asp Thr Asn Tyr Asn Pro Ser Leu Lys
50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
Leu65 70 75 80Lys Leu Tyr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Arg Leu Tyr Phe Gly Ser Gly Ile Tyr Tyr Leu Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120308PRTHomo sapiens 30Gly Gly Ser Phe Ser Gly Tyr Tyr1
53114PRTHomo sapiens 31Ala Arg Leu Tyr Phe Gly Ser Gly Ile Tyr Tyr
Leu Asp Tyr1 5 1032107PRTHomo sapiens 32Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Val Trp Tyr Gln
Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro Pro 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105336PRTHomo
sapiens 33Gln Gly Ile Ser Ser Trp1 5349PRTHomo sapiens 34Gln Gln
Tyr Asn Ser Phe Pro Pro Thr1 535119PRTHomo sapiens 35Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe 20 25 30Ala
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Ser Tyr Asp Gly Gly His Lys Phe Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala Arg Gly Leu Gly Val Trp Gly Ala Phe Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115368PRTHomo sapiens 36Gly Phe Thr Phe Ser Thr Phe Ala1
5378PRTHomo sapiens 37Ile Ser Tyr Asp Gly Gly His Lys1 53812PRTHomo
sapiens 38Ala Arg Gly Leu Gly Val Trp Gly Ala Phe Asp Tyr1 5
1039106PRTHomo sapiens 39Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala
Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Trp Thr 85 90 95Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105406PRTHomo sapiens 40Gln Ser Val
Ser Ser Tyr1 5418PRTHomo sapiens 41Gln Gln Arg Ser Asn Trp Trp Thr1
542123PRTHomo sapiens 42Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Glu1 5 10 15Ser Leu Thr Ile Ser Cys Lys Gly Ser Gly
Tyr Ser Phe Ser Ile Tyr 20 25 30Trp Ile Gly Trp Val Arg Gln Met Pro
Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Phe Pro Gly Asp Ser
Asp Ile Arg Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile Ser
Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser
Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Gln Pro
Gly Asp Trp Ser Pro Arg His Trp Tyr Phe Asp Leu 100 105 110Trp Gly
Arg Gly Thr Leu Val Thr Val Ser Ser 115 120438PRTHomo sapiens 43Gly
Tyr Ser Phe Ser Ile Tyr Trp1 5448PRTHomo sapiens 44Ile Phe Pro Gly
Asp Ser Asp Ile1 54516PRTHomo sapiens 45Ala Arg Gln Pro Gly Asp Trp
Ser Pro Arg His Trp Tyr Phe Asp Leu1 5 10 1546107PRTHomo sapiens
46Val Ile Trp Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Thr Gly1
5 10 15Asp Arg Val Thr Ile Ser Cys Arg Met Ser Gln Gly Ile Ser Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Tyr Leu Gln Ser65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Tyr Ser Phe Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105476PRTHomo sapiens 47Gln Gly Ile Ser Ser Tyr1
5489PRTHomo sapiens 48Gln Gln Tyr Tyr Ser Phe Pro Leu Thr1
549126PRTHomo sapiens 49Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly
Tyr Asn Phe Thr Ser Tyr 20 25 30Trp Ile Gly Trp Val Arg Gln Met Pro
Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Tyr Pro Gly Asp Ser
Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile Ser
Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser
Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Trp Gly
Thr Tyr Tyr Asp Ile Leu Thr Gly Tyr Phe Asn Trp 100 105 110Phe Asp
Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
125508PRTHomo sapiens 50Gly Tyr Asn Phe Thr Ser Tyr Trp1
5518PRTHomo sapiens 51Ile Tyr Pro Gly Asp Ser Asp Thr1 55215PRTHomo
sapiens 52Ala Arg Trp Gly Thr Tyr Tyr Asp Ile Leu Thr Gly Tyr Phe
Asn1 5 10 1553107PRTHomo sapiens 53Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Trp 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105546PRTHomo sapiens
54Gln Gly Ile Ser Ser Trp1 5559PRTHomo sapiens 55Gln Gln Tyr Tyr
Ile Tyr Pro Trp Thr1 556124PRTHomo sapiens 56Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Ala Tyr Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Trp65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Ala His Tyr His Gly Ser Gly Ser Tyr Tyr Thr Leu Phe
Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120578PRTHomo sapiens 57Gly Phe Thr Phe Ser Asn Tyr Gly1
5588PRTHomo sapiens 58Ile Ser Gly Ser Ala Tyr Ser Thr1 55917PRTHomo
sapiens 59Ala Lys Ala His Tyr His Gly Ser Gly Ser Tyr Tyr Thr Leu
Phe Asp1 5 10 15Tyr60107PRTHomo sapiens 60Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn
Ser Tyr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105616PRTHomo sapiens 61Gln Gly Ile Ser Ser Trp1 5629PRTHomo
sapiens 62Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr1 563121PRTHomo
sapiens 63Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asp Tyr 20 25 30Val Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Thr Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Ser Ala
Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Ile Thr Gly
Thr Thr Gly Val Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120648PRTHomo sapiens 64Gly Phe Thr Phe Ser Asp
Tyr Val1 5658PRTHomo sapiens 65Ile Ser Tyr Asp Gly Ser Asn Lys1
56614PRTHomo sapiens 66Ala Arg Gly Gly Ile Thr Gly Thr Thr Gly Val
Phe Asp Tyr1 5 1067107PRTHomo sapiens 67Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Asp Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Tyr
Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Tyr Lys Ser Tyr Pro Ile 85 90
95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105686PRTHomo
sapiens 68Gln Gly Ile Ser Ser Trp1 5699PRTHomo sapiens 69Gln Gln
Tyr Lys Ser Tyr Pro Ile Thr1 570125PRTHomo sapiens 70Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Trp Ile Ser Ala Tyr Asn Gly Asn Ser Asn Tyr Val Gln Lys Phe
50 55 60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Thr Thr Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Glu Tyr Ser Tyr Asp Ser Gly Thr Tyr Phe
Tyr Tyr Gly Met 100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser 115 120 125718PRTHomo sapiens 71Gly Tyr Thr Phe Thr Ser
Tyr Gly1 5728PRTHomo sapiens 72Ile Ser Ala Tyr Asn Gly Asn Ser1
57318PRTHomo sapiens 73Ala Arg Glu Tyr Ser Tyr Asp Ser Gly Thr Tyr
Phe Tyr Tyr Gly Met1 5 10 15Asp Val74108PRTHomo sapiens 74Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Met 85 90 95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105756PRTHomo sapiens 75Gln Ser Val Ser Ser Tyr1
57610PRTHomo sapiens 76Gln Gln Arg Ser Asn Trp Pro Met Tyr Thr1 5
1077119PRTHomo sapiens 77Glu Val Gln Leu Leu Glu Ser Gly Gly Asp
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Arg Gly
Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Ser Thr Leu Cys65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ala
Arg Ala Asn Trp Asp Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 11578107PRTHomo sapiens 78Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala
Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Ala Ala Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Arg Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro
Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
10579119PRTHomo sapiens 79Glu Val Gln Leu Leu Glu Ser Gly Gly Asp
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Arg Gly
Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Ser Thr Leu Cys65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ala
Arg Ala Asn Trp Asp Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 11580107PRTHomo sapiens 80Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala
Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Ala Ala Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Arg Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro
Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
10581119PRTHomo sapiens 81Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Arg Gly
Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Ser Thr Leu Cys65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ala
Arg Ala Asn Trp Asp Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 11582107PRTHomo sapiens 82Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala
Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Ala Ala Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Arg Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro
Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
10583121PRTHomo sapiens 83Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ala
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Ala Leu Glu Trp Met 35 40 45Gly Trp Ile Thr Thr Tyr Ser
Ser Asn Thr Ile Tyr Ala Gln Lys Leu 50 55 60Gln Gly Arg Val Thr Met
Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg
Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Arg Val Val Val Arg Pro Asp Tyr Phe Asp Tyr Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser 115 12084107PRTHomo sapiens 84Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45Tyr Asp Thr Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser His Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10585121PRTHomo sapiens 85Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Leu Ser Ala
Tyr Ser Gly Asn Thr Ile Tyr Ala Gln Lys Leu 50 55 60Gln Gly Arg Val
Thr Met Thr Thr Asp Thr Ser Thr Thr Thr Ala Tyr65 70 75 80Met Glu
Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asp Arg Ile Val Val Arg Pro Asp Tyr Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser 115 12086107PRTHomo sapiens
86Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Asn Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 10587121PRTHomo sapiens 87Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ala Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Ile
Thr Tyr Asn Gly Asn Thr Ile Tyr Ala Gln Arg Phe 50 55 60Gln Asp Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Asp Arg Ile Ile Val Arg Pro Asp Tyr Phe Asp Tyr Trp Gly
100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115 12088107PRTHomo
sapiens 88Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser Asn Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 10589120PRTHomo sapiens 89Gln Val Gln Leu Gln
Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Asn
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu
Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Gly Asn Tyr Gly Ser Gly Tyr Tyr Tyr Phe Asp Leu Trp Gly
Arg 100 105 110Gly Thr Gln Val Thr Val Ser Ser 115 12090107PRTHomo
sapiens 90Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Phe Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Ile Ser Phe Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg
Leu Glu Ile Lys 100 10591120PRTHomo sapiens 91Gln Val Gln Leu Gln
Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Asn
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu
Ile His His Ser Gly Ser Ala Asn Tyr Asn Pro Ser Leu Met 50 55 60Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Gln Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Gly Tyr Tyr Gly Ser Gly Tyr Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12092107PRTHomo
sapiens 92Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile
35 40 45Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn
Ser Tyr Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 10593120PRTHomo sapiens 93Gln Val Gln Leu Gln Gln Trp Gly Ala
Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val
Tyr Gly Gly Ser Phe Ser Asp Tyr 20 25 30Tyr Trp Asn Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile His His Val
Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile
Ser Val Asp Thr Ser Lys Ser Gln Phe Ser Leu65 70 75 80Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly
Tyr Tyr Asp Ser Gly Val Tyr Tyr Phe Asp Tyr Trp Ala Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 12094107PRTHomo sapiens
94Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Asn Ser Tyr Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu
Ile Lys 100 10595120PRTHomo sapiens 95Gln Val Gln Leu Gln Gln Trp
Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys
Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr 20 25 30Tyr Trp Asn Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile His
His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95Arg Gly Tyr Tyr Ala Ser Gly Val Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12096107PRTHomo
sapiens 96Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Tyr Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg
Leu Glu Ile Lys 100 10597120PRTHomo sapiens 97Gln Val Gln Leu Gln
Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr 20 25 30Phe Trp Asn
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu
Ile His His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Asn Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Gly Leu Ile Gly Ser Gly Tyr Tyr Tyr Phe Asp Tyr Trp Asp
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12098107PRTHomo
sapiens 98Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Tyr Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg
Leu Glu Ile Lys 100 10599120PRTHomo sapiens 99Gln Val Gln Leu Gln
Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu
Ile Asn His Ser Gly Asp Thr Asn Tyr Asn Pro Ser Leu Thr 50 55 60Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Leu Phe Tyr Gly Ser Gly Ile Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 120100107PRTHomo
sapiens 100Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Thr Phe Arg Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Phe Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105101120PRTHomo sapiens 101Gln Val Gln Leu Gln
Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Ile Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu
Ile Asn His Ser Gly Asp Thr Asn Tyr Asn Pro Ser Leu Thr 50 55 60Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Leu Phe Tyr Gly Ser Gly Ile Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 120102107PRTHomo
sapiens 102Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Thr Phe Arg Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Phe Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105103120PRTHomo sapiens 103Gln Val Gln Leu Gln
Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr 20 25 30Tyr Trp Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu
Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Leu Tyr Tyr Gly Ser Gly Thr Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 120104107PRTHomo
sapiens 104Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Thr Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Phe Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105105120PRTHomo sapiens 105Gln Val Gln Leu Gln
Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu
Ile His His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Leu Trp Tyr Gly Ser Gly Ser Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 120106107PRTHomo
sapiens 106Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Phe Pro Pro 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105107120PRTHomo sapiens 107Gln Val Gln Leu Gln
Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Val Ser Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Thr
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu
Ile Tyr His Ser Gly Asp Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Tyr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Leu Tyr Phe Gly Ser Gly Ile Tyr Tyr Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 120108107PRTHomo
sapiens 108Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Val Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Phe Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105109124PRTHomo sapiens 109Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Ala Tyr Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Trp65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Ala His Tyr His Gly Ser Gly Ser Tyr Tyr Thr Leu Phe
Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120110107PRTHomo sapiens 110Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105111124PRTHomo sapiens 111Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr
20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Ala Ile Ser Gly Thr Gly Tyr Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ala His Tyr Phe Gly Ser Gly Ser
Tyr Tyr Thr Leu Phe Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120112107PRTHomo sapiens 112Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105113124PRTHomo sapiens 113Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Thr Asp Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Ile Ser Gly Ser Gly
Tyr Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Thr Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Lys Gly His Thr Leu Gly Ser Gly Ser Tyr Tyr Thr Leu
Phe Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120114107PRTHomo sapiens 114Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Asn Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105115124PRTHomo sapiens
115Glu Val Gln Leu Trp Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Gly Ile Ser Gly Ser Gly Tyr Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Tyr Tyr His Gly Ser Gly
Ser Tyr Tyr Thr Ser Phe Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120116107PRTHomo sapiens 116Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro
Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105117121PRTHomo sapiens 117Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Thr Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser His 20 25 30Ala Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ala Ile Ser Tyr Asp Gly
Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Asp Tyr Ile Ser Ser Ser Gly Val Phe Asp Tyr Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120118107PRTHomo sapiens 118Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu
Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn Ser Tyr Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile
Lys 100 105119121PRTHomo sapiens 119Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ala Ile Ser Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Met Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Cys Tyr Cys 85 90 95Ala
Arg Gly Ser Ile Thr Gly Ser Thr Gly Val Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser 115 120120107PRTHomo sapiens
120Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Asn Ser Tyr Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu
Ile Lys 100 105121121PRTHomo sapiens 121Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser
Tyr Asp Gly Ser Asn Glu Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gly Ser Ile Ile Gly Ser Thr Gly Val Phe Asp Tyr Trp Gly
100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115 120122107PRTHomo
sapiens 122Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Asn Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Tyr Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg
Leu Glu Ile Lys 100 105123121PRTHomo sapiens 123Gln Val Gln Val Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val
Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Ile Thr Gly Ser Thr Gly Val Phe Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
120124107PRTHomo sapiens 124Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Asn Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asn Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Ile 85 90 95Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys 100 105125121PRTHomo sapiens 125Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Ile Thr Gly Ser Thr Gly
Val Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120126107PRTHomo sapiens 126Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly Ile Ser Asn Trp 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Asp Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Ile 85 90 95Thr
Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 1051278PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa is Arg or SerMISC_FEATURE(7)..(7)Xaa
is Thr or Ser 127Ile Ser Gly Xaa Gly Gly Xaa Thr1 51288PRTHomo
sapiensMISC_FEATURE(5)..(5)Xaa is Arg or SerMISC_FEATURE(6)..(6)Xaa
is Thr or Ser 128Gly Gly Thr Phe Xaa Xaa Tyr Ala1 51298PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa is Asn or IleMISC_FEATURE(3)..(3)Xaa
is Thr or ProMISC_FEATURE(4)..(4)Xaa is Val or
IleMISC_FEATURE(8)..(8)Xaa is Val or Ala 129Ile Xaa Xaa Xaa Leu Gly
Ile Xaa1 513013PRTHomo sapiensMISC_FEATURE(12)..(12)Xaa is Ile or
MetMISC_FEATURE(13)..(13)Xaa is Glu or Asp 130Ala Arg Glu Lys Gly
Val Asp Tyr Tyr Tyr Gly Xaa Xaa1 5 101318PRTHomo
sapiensMISC_FEATURE(6)..(6)Xaa is Asn or Ser 131Gly Tyr Thr Phe Thr
Xaa Tyr Gly1 51328PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa is Ser,
Thr or IleMISC_FEATURE(3)..(3)Xaa is Ala or
ThrMISC_FEATURE(5)..(5)Xaa is Ser or Asn 132Ile Xaa Xaa Tyr Xaa Gly
Asn Thr1 513314PRTHomo sapiensMISC_FEATURE(5)..(5)Xaa is Ile or
ValMISC_FEATURE(6)..(6)Xaa is Ile or Val 133Ala Arg Asp Arg Xaa Xaa
Val Arg Pro Asp Tyr Phe Asp Tyr1 5 101348PRTHomo
sapiensMISC_FEATURE(6)..(6)Xaa is Asp or GlyMISC_FEATURE(8)..(8)Xaa
isTyr or Phe 134Gly Gly Ser Phe Ser Xaa Tyr Xaa1 51357PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa is His or AsnMISC_FEATURE(4)..(4)Xaa
is Ser or ValMISC_FEATURE(7)..(7)Xaa is Thr or Ala 135Ile Xaa His
Xaa Gly Ser Xaa1 513614PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa is
Tyr, Asn or LeuMISC_FEATURE(5)..(5)Xaa is Tyr or
IleMISC_FEATURE(6)..(6)Xaa is Asp, Gly or
AlaMISC_FEATURE(9)..(9)Xaa is Val or TyrMISC_FEATURE(14)..(14)Xaa
is Tyr or Leu 136Ala Arg Gly Xaa Xaa Xaa Ser Gly Xaa Tyr Tyr Phe
Asp Xaa1 5 101378PRTHomo sapiensMISC_FEATURE(6)..(6)Xaa is Gly or
Asp 137Gly Gly Ser Phe Ser Xaa Tyr Tyr1 51387PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa is Tyr, Asn or
HisMISC_FEATURE(6)..(6)Xaa is Asp or Ser 138Ile Xaa His Ser Gly Xaa
Thr1 513914PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa is Tyr, Phe or
TrpMISC_FEATURE(5)..(5)Xaa is Tyr or PheMISC_FEATURE(9)..(9)Xaa is
Ile, Thr or SerMISC_FEATURE(12)..(12)Xaa is Leu or Phe 139Ala Arg
Leu Xaa Xaa Gly Ser Gly Xaa Tyr Tyr Xaa Asp Tyr1 5 101408PRTHomo
sapiensMISC_FEATURE(6)..(6)Xaa is Thr or PheMISC_FEATURE(7)..(7)Xaa
is Phe or Tyr 140Gly Phe Thr Phe Ser Xaa Xaa Ala1 51418PRTHomo
sapiensMISC_FEATURE(6)..(6)Xaa is Gly or SerMISC_FEATURE(7)..(7)Xaa
is His or Asn 141Ile Ser Tyr Asp Gly Xaa Xaa Lys1 514212PRTHomo
sapiensMISC_FEATURE(9)..(9)Xaa is Ala or Tyr 142Ala Arg Gly Leu Gly
Val Trp Gly Xaa Phe Asp Tyr1 5 101438PRTHomo
sapiensMISC_FEATURE(5)..(5)Xaa is Ser, Asn or
ThrMISC_FEATURE(6)..(6)Xaa is Asn, Asp or
SerMISC_FEATURE(8)..(8)Xaa is Gly or Ala 143Gly Phe Thr Phe Xaa Xaa
Tyr Xaa1 51448PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa is Ser or
ThrMISC_FEATURE(5)..(5)Xaa is Ala or GlyMISC_FEATURE(6)..(6)Xaa is
Tyr or GlyMISC_FEATURE(7)..(7)Xaa is Ser or Ala 144Ile Ser Gly Xaa
Xaa Xaa Xaa Thr1 514517PRTHomo sapiensMISC_FEATURE(3)..(3)Xaa is
Ala or GlyMISC_FEATURE(4)..(4)Xaa is His or
TyrMISC_FEATURE(5)..(5)Xaa is Tyr or ThrMISC_FEATURE(6)..(6)Xaa is
His, Phe or LeuMISC_FEATURE(14)..(14)Xaa is Leu or Ser 145Ala Lys
Xaa Xaa Xaa Xaa Gly Ser Gly Ser Tyr Tyr Thr Xaa Phe Asp1 5 10
15Tyr1468PRTHomo sapiensMISC_FEATURE(5)..(5)Xaa is Ser or
ThrMISC_FEATURE(6)..(6)Xaa is Ile or Ser 146Gly Tyr Ser Phe Xaa Xaa
Tyr Trp1 51478PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa is Phe or
TyrMISC_FEATURE(8)..(8)Xaa is Ile or Thr 147Ile Xaa Pro Gly Asp Ser
Asp Xaa1 514816PRTHomo sapiens 148Ala Arg Gln Pro Gly Asp Trp Ser
Pro Arg His Trp Tyr Phe Asp Leu1 5 10 151498PRTHomo
sapiensMISC_FEATURE(3)..(3)Xaa is Asn or Ser 149Gly Tyr Xaa Phe Thr
Ser Tyr Trp1 51508PRTHomo sapiensMISC_FEATURE(8)..(8)Xaa is Ser or
Thr 150Ile Ser Ala Tyr Asn Gly Asn Xaa1 515118PRTHomo sapiens
151Ala Arg Glu Tyr Ser Tyr Asp Ser Gly Thr Tyr Phe Tyr Tyr Gly Met1
5 10 15Asp Val1528PRTHomo sapiensMISC_FEATURE(6)..(6)Xaa is Asp or
SerMISC_FEATURE(7)..(7)Xaa is Tyr or HisMISC_FEATURE(8)..(8)Xaa is
Val or Ala 152Gly Phe Thr Phe Ser Xaa Xaa Xaa1 51538PRTHomo
sapiensMISC_FEATURE(7)..(7)Xaa is Asn or TyrMISC_FEATURE(8)..(8)Xaa
is Lys or Glu 153Ile Ser Tyr Asp Gly Ser Xaa Xaa1 515414PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa is Gly, Asp or
SerMISC_FEATURE(5)..(5)Xaa is Ile or TyrMISC_FEATURE(6)..(6)Xaa is
Thr or IleMISC_FEATURE(7)..(7)Xaa is Gly or
SerMISC_FEATURE(8)..(8)Xaa is Thr or SerMISC_FEATURE(9)..(9)Xaa is
Thr or SerMISC_FEATURE(11)..(11)Xaa is Tyr or Val 154Ala Arg Gly
Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Phe Asp Tyr1 5 101559PRTHomo
sapiensMISC_FEATURE(8)..(8)Xaa is Ile or Leu 155Gln Gln Ala Asn Ser
Phe Pro Xaa Thr1 51569PRTHomo sapiensMISC_FEATURE(5)..(5)Xaa is Asn
or His 156Gln Gln Arg Ser Xaa Trp Pro Arg Thr1 51576PRTHomo
sapiensMISC_FEATURE(5)..(5)Xaa is Arg or Ser 157Gln Gly Ile Ser Xaa
Trp1 51589PRTHomo sapiens 158Gln Gln Tyr Asn Ser Phe Pro Pro Thr1
51596PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa is Ser or Asn 159Gln
Gly Ile Xaa Ser Trp1 51609PRTHomo sapiensMISC_FEATURE(8)..(8)Xaa is
Tyr or Leu 160Gln Gln Tyr Asn Ser Tyr Pro Xaa Thr1 51616PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa is Ser or AsnMISC_FEATURE(5)..(5)Xaa
is Ser or Asn 161Gln Gly Ile Xaa Xaa Trp1 51629PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa is Lys or Asn 162Gln Gln Tyr Xaa Ser
Tyr Pro Ile Thr1 51637PRTHomo sapiens 163Ile Tyr His Ser Gly Asp
Thr1 5164122PRTHomo sapiens 164Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Ser Phe His Phe Tyr 20 25 30Trp Ile Gly Trp Val Arg Gln
Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Ser Ile Tyr Pro Gly
Asp Ser Asp Thr Arg Tyr Arg Pro Ser Phe 50 55
60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65
70 75 80Leu Gln Trp Thr Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr
Cys 85 90 95Ala Arg Gln Arg Gly Asp Tyr Tyr Tyr Phe Tyr Gly Met Asp
Val Trp 100 105 110Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120165107PRTHomo sapiens 165Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Val Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg
Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 1051668PRTHomo sapiens 166Gly
Tyr Ser Phe His Phe Tyr Trp1 51678PRTHomo sapiens 167Ile Tyr Pro
Gly Asp Ser Asp Thr1 516815PRTHomo sapiens 168Ala Arg Gln Arg Gly
Asp Tyr Tyr Tyr Phe Tyr Gly Met Asp Val1 5 10 151697PRTHomo sapiens
169Gln Ser Val Ser Ser Ser Tyr1 51708PRTHomo sapiens 170Gln Gln Tyr
Gly Ser Ser Leu Thr1 5171119PRTHomo sapiens 171Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Pro Met Ala
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr
Ile Ser Thr Ser Gly Gly Arg Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Phe Arg Gln Tyr Ser Gly Gly Phe Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115172110PRTHomo sapiens
172Asp Ile Gln Leu Thr Gln Pro Asn Ser Val Ser Thr Ser Leu Gly Ser1
5 10 15Thr Val Lys Leu Ser Cys Thr Leu Ser Ser Gly Asn Ile Glu Asn
Asn 20 25 30Tyr Val His Trp Tyr Gln Leu Tyr Glu Gly Arg Ser Pro Thr
Thr Met 35 40 45Ile Tyr Asp Asp Asp Lys Arg Pro Asp Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala Phe Leu
Thr Ile His Asn65 70 75 80Val Ala Ile Glu Asp Glu Ala Ile Tyr Phe
Cys His Ser Tyr Val Ser 85 90 95Ser Phe Asn Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 110173119PRTHomo sapiens 173Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Thr Ile Ser Arg Tyr Ser Arg Tyr Ile Tyr Tyr Pro Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Arg Pro Leu Tyr Gly Ser Ser Pro Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115174106PRTHomo sapiens 174Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Ser Ala
Ser Ser Ser Val Thr Tyr Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser
Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu65 70 75 80Asp Phe Ala Val
Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Leu Thr 85 90 95Phe Gly Ser
Gly Thr Lys Leu Glu Met Arg 100 105175113PRTHomo sapiens 175Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser 100 105 110Ser176119PRTArtificial
SequenceConsensus sequence derived from human
sequencesmisc_feature(10)..(10)X can be G or
Dmisc_feature(35)..(35)X can be N or Smisc_feature(54)..(54)X can
be R or Smisc_feature(57)..(57)X can be S or
Tmisc_feature(77)..(77)X can be S or Nmisc_feature(80)..(80)X can
be C or Ymisc_feature(84)..(84)X can be N or S 176Glu Val Gln Leu
Leu Glu Ser Gly Gly Xaa Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met
Xaa Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Ala Ile Ser Gly Xaa Gly Gly Xaa Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Xaa Thr Leu Xaa65
70 75 80Leu Gln Met Xaa Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Lys Ala Arg Ala Asn Trp Asp Tyr Phe Asp Tyr Trp Gly
Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115177113PRTHomo
sapiens 177Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 100 105
110Ser178121PRTArtificial SequenceConsensus sequence derived from
human sequencesmisc_feature(30)..(30)X can be S or
Rmisc_feature(31)..(31)X can be S or Tmisc_feature(52)..(52)X can
be I or Nmisc_feature(53)..(53)X can be P or
Tmisc_feature(54)..(54)X can be I or Vmisc_feature(58)..(58)X can
be A or Vmisc_feature(60)..(60)X can be Y or
Hmisc_feature(77)..(77)X can be S or Nmisc_feature(84)..(84)X can
be S or Nmisc_feature(108)..(108)X can be M or
Imisc_feature(109)..(109)X can be D or E 178Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Gly Thr Phe Xaa Xaa Tyr 20 25 30Ala Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile
Xaa Xaa Xaa Leu Gly Ile Xaa Asn Xaa Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Xaa Thr Ala Tyr65 70 75
80Met Glu Leu Xaa Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Lys Gly Val Asp Tyr Tyr Tyr Gly Xaa Xaa Val Trp
Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
120179113PRTHomo sapiens 179Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Ser Ala Tyr Asn
Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60Gln Gly Arg Val Thr Met
Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg
Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr
Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105
110Ser180121PRTArtificial SequenceConsensus sequence derived from
human sequencesmisc_feature(31)..(31)X can be S or
Nmisc_feature(44)..(44)X can be G or Amisc_feature(52)..(52)X can
be S, T or Imisc_feature(53)..(53)X can be A or
Tmisc_feature(55)..(55)X can be S or Nmisc_feature(59)..(59)X can
be N or Imisc_feature(63)..(63)X can be K or
Rmisc_feature(64)..(64)X can be L or Fmisc_feature(77)..(77)X can
be S or Tmisc_feature(101)..(101)X can be I or
Vmisc_feature(102)..(102)X can be I or V 180Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Xaa Tyr 20 25 30Gly Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Xaa Leu Glu Trp Met 35 40 45Gly Trp Ile
Xaa Xaa Tyr Xaa Gly Asn Thr Xaa Tyr Ala Gln Xaa Xaa 50 55 60Gln Gly
Arg Val Thr Met Thr Thr Asp Thr Ser Thr Xaa Thr Ala Tyr65 70 75
80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Arg Xaa Xaa Val Arg Pro Asp Tyr Phe Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
120181112PRTHomo sapiens 181Gln Val Gln Leu Gln Gln Trp Gly Ala Gly
Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr
Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn His Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105
110182120PRTArtificial SequenceConsensus sequence derived from
human sequencesmisc_feature(31)..(31)X can be G or
Dmisc_feature(33)..(33)X can be Y or Fmisc_feature(35)..(35)X can
be S or Nmisc_feature(52)..(52)X can be H or
Nmisc_feature(54)..(54)X can be S or Vmisc_feature(57)..(57)X can
be T or Amisc_feature(64)..(64)X can be K or
Mmisc_feature(76)..(76)X can be N or Smisc_feature(81)..(81)X can
be K, Q or Nmisc_feature(99)..(99)X can be Y, N or
Lmisc_feature(100)..(100)X can be Y or Imisc_feature(101)..(101)X
can be D, G or Amisc_feature(104)..(104)X can be V or
Ymisc_feature(109)..(109)X can be Y or Lmisc_feature(111)..(111)X
can be G, A or Dmisc_feature(112)..(112)X can be Q or
Rmisc_feature(115)..(115)X can be L or Q 182Gln Val Gln Leu Gln Gln
Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Ala Val Tyr Gly Gly Ser Phe Ser Xaa Tyr 20 25 30Xaa Trp Xaa Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile
Xaa His Xaa Gly Ser Xaa Asn Tyr Asn Pro Ser Leu Xaa 50 55 60Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Xaa Gln Phe Ser Leu65 70 75
80Xaa Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Gly Xaa Xaa Xaa Ser Gly Xaa Tyr Tyr Phe Asp Xaa Trp Xaa
Xaa 100 105 110Gly Thr Xaa Val Thr Val Ser Ser 115 120183112PRTHomo
sapiens 183Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe
Ser Gly Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly
Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr
Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser
Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110184120PRTArtificial
SequenceConsensus sequence derived from human
sequencesmisc_feature(24)..(24)X can be V or
Imisc_feature(25)..(25)X can be S or Ymisc_feature(31)..(31)X can
be G or Dmisc_feature(35)..(35)X can be S or
Tmisc_feature(52)..(52)X can be Y, N, or Hmisc_feature(56)..(56)X
can be S or Dmisc_feature(64)..(64)X can be K or
Tmisc_feature(83)..(83)X can be S or Ymisc_feature(99)..(99)X can
be Y, F or Wmisc_feature(100)..(100)X can be F or
Ymisc_feature(104)..(104)X can be I, T or
Smisc_feature(107)..(107)X can be F or L 184Gln Val Gln Leu Gln Gln
Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Ala Xaa Xaa Gly Gly Ser Phe Ser Xaa Tyr 20 25 30Tyr Trp Xaa Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile
Xaa His Ser Gly Xaa Thr Asn Tyr Asn Pro Ser Leu Xaa 50 55 60Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Xaa Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Leu Xaa Xaa Gly Ser Gly Xaa Tyr Tyr Xaa Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 120185113PRTHomo
sapiens 185Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser 100 105
110Ser186119PRTArtificial SequenceConsensus sequence derived from
human sequencesmisc_feature(31)..(31)X can be S or
Tmisc_feature(32)..(32)X can be Y or Fmisc_feature(34)..(34)X can
be M or Imisc_feature(56)..(56)X can be S or
Gmisc_feature(57)..(57)X can be N or Hmisc_feature(59)..(59)X can
be Y or Fmisc_feature(93)..(93)X can be V or
Mmisc_feature(105)..(105)X can be Y or A 186Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Xaa Xaa 20 25 30Ala Xaa His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile
Ser Tyr Asp Gly Xaa Xaa Lys Xaa Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Xaa Tyr Tyr Cys
85 90 95Ala Arg Gly Leu Gly Val Trp Gly Xaa Phe Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115187113PRTHomo sapiens
187Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser 100 105 110Ser188124PRTArtificial
SequenceConsensus sequence derived from human
sequencesmisc_feature(5)..(5)X can be L or Wmisc_feature(11)..(11)X
can be L or Smisc_feature(30)..(30)X can be S, N or
Tmisc_feature(31)..(31)X can be S, N or Dmisc_feature(33)..(33)X
can be A or Gmisc_feature(35)..(35)X can be S or
Nmisc_feature(50)..(50)X can be A, T or Gmisc_feature(54)..(54)X
can be S or Imisc_feature(55)..(55)X can be G or
Amisc_feature(56)..(56)X can be G or Ymisc_feature(57)..(57)X can
be S or Amisc_feature(77)..(77)X can be N or
Tmisc_feature(80)..(80)X can be Y or Wmisc_feature(99)..(99)X can
be A or Gmisc_feature(100)..(100)X can be H or
Ymisc_feature(101)..(101)X can be Y or Tmisc_feature(102)..(102)X
can be H, F or Lmisc_feature(110)..(110)X can be Y, L or S 188Glu
Val Gln Leu Xaa Glu Ser Gly Gly Gly Xaa Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Xaa Xaa Tyr
20 25 30Xaa Met Xaa Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Xaa Ile Ser Gly Xaa Xaa Xaa Xaa Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Xaa
Thr Leu Xaa65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Xaa Xaa Xaa Xaa Gly Ser Gly Ser
Tyr Tyr Thr Xaa Phe Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120189113PRTHomo sapiens 189Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile
Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30Trp Ile Gly
Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60Gln
Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75
80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val
Ser 100 105 110Ser190123PRTArtificial SequenceConsensus sequence
derived from human sequencesmisc_feature(19)..(19)X can be K or
Tmisc_feature(30)..(30)X can be T or Smisc_feature(31)..(31)X can
be S or Imisc_feature(52)..(52)X can be Y or
Fmisc_feature(58)..(58)X can be T or I 190Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Xaa Ile Ser
Cys Lys Gly Ser Gly Tyr Ser Phe Xaa Xaa Tyr 20 25 30Trp Ile Gly Trp
Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Ile Ile
Xaa Pro Gly Asp Ser Asp Xaa Arg Tyr Ser Pro Ser Phe 50 55 60Gln Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75
80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Gln Pro Gly Asp Trp Ser Pro Arg His Trp Tyr Phe Asp
Leu 100 105 110Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115
120191126PRTHomo sapiens 191Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser
Gly Tyr Ser Phe Thr Ser Tyr 20 25 30Trp Ile Gly Trp Val Arg Gln Met
Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Tyr Pro Gly Asp
Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile
Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser
Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Trp
Gly Thr Tyr Tyr Asp Ile Leu Thr Gly Tyr Phe Asn Trp 100 105 110Phe
Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
125192126PRTArtificial SequenceConsensus sequence derived from
human sequencesmisc_feature(28)..(28)X can be S or N 192Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu
Lys Ile Ser Cys Lys Gly Ser Gly Tyr Xaa Phe Thr Ser Tyr 20 25 30Trp
Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40
45Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
50 55 60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala
Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala Arg Trp Gly Thr Tyr Tyr Asp Ile Leu Thr Gly
Tyr Phe Asn Trp 100 105 110Phe Asp Pro Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 125193113PRTHomo sapiens 193Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55
60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
Val Ser 100 105 110Ser194125PRTArtificial SequenceConsensus
sequence derived from human sequencesmisc_feature(58)..(58)X can be
T or Smisc_feature(61)..(61)X can be A or Vmisc_feature(64)..(64)X
can be L or F 194Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Ser Ala Tyr Asn Gly Asn
Xaa Asn Tyr Xaa Gln Lys Xaa 50 55 60Gln Gly Arg Val Thr Met Thr Thr
Asp Thr Thr Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Tyr Ser
Tyr Asp Ser Gly Thr Tyr Phe Tyr Tyr Gly Met 100 105 110Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125195113PRTHomo
sapiens 195Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser 100 105
110Ser196121PRTArtificial SequenceConsensus sequence derived from
human sequencesmisc_feature(14)..(14)X can be P or
Tmisc_feature(31)..(31)X can be S or Dmisc_feature(32)..(32)X can
be Y or Hmisc_feature(33)..(33)X can be A or
Vmisc_feature(34)..(34)X can be M or Imisc_feature(49)..(49)X can
be A or Tmisc_feature(50)..(50)X can be V or
Amisc_feature(57)..(57)X can be N or Ymisc_feature(58)..(58)X can
be K or Emisc_feature(79)..(79)X can be L or
Mmisc_feature(87)..(87)X can be R or Smisc_feature(93)..(93)X can
be V or Mmisc_feature(94)..(94)X can be Y or
Cmisc_feature(100)..(100)X can be G, D or
Smisc_feature(101)..(101)X can be I or Ymisc_feature(102)..(102)X
can be T or Imisc_feature(103)..(103)X can be G or
Smisc_feature(104)..(104)X can be T or Smisc_feature(105)..(105)X
can be T or Smisc_feature(107)..(107)X can be Y or V 196Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Xaa Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Xaa Xaa 20 25 30Xaa
Xaa His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Xaa Xaa Ile Ser Tyr Asp Gly Ser Xaa Xaa Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Xaa
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Xaa Ala Glu Asp Thr Ala Xaa
Xaa Tyr Cys 85 90 95Ala Arg Gly Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Phe
Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
120197107PRTHomo sapiens 197Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile 85 90 95Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys 100 105198107PRTArtificial
SequenceConsensus sequence derived from human
sequencesmisc_feature(38)..(38)X can be Q or
Hmisc_feature(50)..(50)X can be A or Vmisc_feature(53)..(53)X can
be S, I or Tmisc_feature(79)..(79)X can be Q or
Rmisc_feature(96)..(96)X can be I or Lmisc_feature(100)..(100)X can
be Q or Gmisc_feature(104)..(104)X can be L or V 198Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala
Trp Tyr Gln Xaa Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Xaa Ala Ser Xaa Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Xaa Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro
Xaa 85 90 95Thr Phe Gly Xaa Gly Thr Arg Xaa Glu Ile Lys 100
105199107PRTHomo sapiens 199Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala
Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105200107PRTArtificial
SequenceConsensus sequence derived from human
sequencesmisc_feature(51)..(51)X can be A or
Tmisc_feature(93)..(93)X can be N or H 200Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Xaa
Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Xaa Trp Pro Arg
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105201107PRTHomo sapiens 201Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Ile 85 90 95Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys 100 105202107PRTArtificial
SequenceConsensus sequence derived from human
sequencesmisc_feature(31)..(31)X can be S or
Rmisc_feature(49)..(49)X can be Y or Fmisc_feature(53)..(53)X can
be S or Rmisc_feature(55)..(55)X can be Q or
Rmisc_feature(94)..(94)X can be Y or F 202Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Ser Xaa Trp 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Xaa Ala Ala
Ser Xaa Leu Xaa Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Xaa Pro Ile
85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
105203107PRTHomo sapiens 203Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro Pro 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105204107PRTArtificial
SequenceConsensus sequence derived from human
sequencesmisc_feature(34)..(34)X can be A, V or
Tmisc_feature(51)..(51)X can be A or Tmisc_feature(52)..(52)X can
be S or Fmisc_feature(53)..(53)X can be S or
Rmisc_feature(100)..(100)X can be Q or G 204Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20
25 30Leu Xaa Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu
Ile 35 40 45Tyr Ala Xaa Xaa Xaa Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn Ser Phe Pro Pro 85 90 95Thr Phe Gly Xaa Gly Thr Lys Val Glu Ile
Lys 100 105205107PRTHomo sapiens 205Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105206107PRTArtificial
SequenceConsensus sequence derived from human
sequencesmisc_feature(30)..(30)X can be S or
Nmisc_feature(96)..(96)X can be Y or Lmisc_feature(100)..(100)X can
be Q or Gmisc_feature(104)..(104)X can be L or V 206Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Xaa Ser Trp 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro
Xaa 85 90 95Thr Phe Gly Xaa Gly Thr Lys Xaa Glu Ile Lys 100
105207107PRTHomo sapiens 207Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Ile 85 90 95Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys 100 105208107PRTArtificial
SequenceConsensus sequence derived from human
sequencesmisc_feature(30)..(30)X can be S or
Nmisc_feature(31)..(31)X can be S or Nmisc_feature(50)..(50)X can
be A or Dmisc_feature(67)..(67)X can be S or
Ymisc_feature(72)..(72)X can be T or Smisc_feature(82)..(82)X can
be D or Nmisc_feature(85)..(85)X can be T or
Imisc_feature(92)..(92)X can be N or K 208Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Xaa Xaa Trp 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Xaa Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Xaa Gly Thr Asp Phe Xaa Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Xaa Phe Ala Xaa Tyr Tyr Cys Gln Gln Tyr Xaa Ser Tyr Pro Ile
85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
* * * * *