U.S. patent application number 15/346532 was filed with the patent office on 2017-06-15 for cysteine engineered anti-tenb2 antibodies and antibody drug conjugates.
The applicant listed for this patent is GENENTECH, INC.. Invention is credited to Jagath Reddy Junutula, Weiguang Mao, Paul Polakis.
Application Number | 20170166635 15/346532 |
Document ID | / |
Family ID | 40242684 |
Filed Date | 2017-06-15 |
United States Patent
Application |
20170166635 |
Kind Code |
A1 |
Mao; Weiguang ; et
al. |
June 15, 2017 |
CYSTEINE ENGINEERED ANTI-TENB2 ANTIBODIES AND ANTIBODY DRUG
CONJUGATES
Abstract
Cysteine engineered anti-TENB2 antibodies are engineered by
replacing one or more amino acids of a parent anti-TENB2 antibody
with non cross-linked, reactive cysteine amino acids. Methods of
design, preparation, screening, and selection of the cysteine
engineered anti-TENB2 antibodies are provided. Cysteine engineered
anti-TENB2 antibodies (Ab) are conjugated with one or more drug
moieties (D) through a linker (L) to form cysteine engineered
anti-TENB2 antibody-drug conjugates having Formula I:
Ab-(L-D).sub.p (I) where p is 1 to 4. Diagnostic and therapeutic
uses for cysteine engineered antibody drug compounds and
compositions are disclosed.
Inventors: |
Mao; Weiguang; (San Mateo,
CA) ; Junutula; Jagath Reddy; (Fremont, CA) ;
Polakis; Paul; (Mill Valley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENENTECH, INC. |
South San Francisco |
CA |
US |
|
|
Family ID: |
40242684 |
Appl. No.: |
15/346532 |
Filed: |
November 8, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14552244 |
Nov 24, 2014 |
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15346532 |
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12288181 |
Oct 16, 2008 |
8937161 |
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14552244 |
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60981411 |
Oct 19, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/3069 20130101;
A61P 13/02 20180101; A61P 1/04 20180101; A61K 47/6869 20170801;
A61K 47/6889 20170801; A61K 2039/505 20130101; C07K 2317/14
20130101; C07K 2317/515 20130101; A61P 35/00 20180101; A61K 47/6851
20170801; A61K 38/05 20130101; C07K 2317/56 20130101; A61P 13/08
20180101; C07K 2317/40 20130101; A61K 47/6849 20170801; A61P 15/00
20180101; A61P 1/18 20180101; C07K 2317/73 20130101; A61K 45/06
20130101; A61K 39/39558 20130101; C07K 16/28 20130101; G01N
33/57492 20130101; C07K 16/30 20130101; G01N 33/574 20130101; A61P
43/00 20180101; C07K 2317/24 20130101; A61P 11/00 20180101; C07K
2317/77 20130101; C07K 2317/51 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30; G01N 33/574 20060101
G01N033/574; A61K 38/05 20060101 A61K038/05; A61K 39/395 20060101
A61K039/395 |
Claims
1-60. (canceled)
61. A cysteine engineered anti-TENB2 antibody comprising one or
more free cysteine amino acids, wherein the cysteine engineered
anti-TENB2 antibody comprises a heavy chain sequence comprising:
TABLE-US-00003 (SEO ID NO: 3)
MAVLGLLLCLVTFPSCVLSDVQLQESGPGLVKPSETLSLTCAVSGYS
ITSGYYWSWIRQPPGKGLEWMGFISYDGSNKYNPSLKNRITISRDTSKN
QFSLKLSSVTAADTAVYYCARGLRRGDYSMDYWGQGTLVTVSSCSTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
and a light chain sequence comprising: TABLE-US-00004 (SEQ ID NO:
2) MDFQVQIFSFLLISASVIMSRGDIQMTQSPSSLSASVGDRVTITCKAS
QNVVTAVAWYQQKPGKAPKLLIYSASNRHTGVPSRFSGSGSGTDFTLTI
SSLQPEDFATYYCQQYSSYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
62. The cysteine engineered anti-TENB2 antibody according claim 61
which is produced in bacteria or CHO cells.
63. The cysteine engineered anti-TENB2 antibody according to claim
61 wherein the antibody is covalently attached to an auristatin
drug moiety whereby an antibody drug conjugate is formed.
64. The antibody-drug conjugate of claim 63 comprising a cysteine
engineered anti-TENB2 antibody (Ab), and an auristatin drug moiety
(D) wherein the cysteine engineered anti-TENB2 antibody is attached
through one or more free cysteine amino acids by a linker moiety
(L) to D; the compound having Formula I: Ab-(L-D).sub.p I where p
is 1, 3, 4, or preferably 2.
65. The antibody-drug conjugate compound of claim 64 wherein L has
the formula: -A.sub.a-W.sub.w--Y.sub.y-- where: A is a Stretcher
unit covalently attached to a cysteine thiol of the cysteine
engineered antibody (Ab); a is 0 or 1; each W is independently an
Amino Acid unit; w is an integer ranging from 0 to 12; Y is a
Spacer unit covalently attached to the drug moiety; and y is 0, 1
or 2.
66. The antibody-drug conjugate compound of claim 65 having the
formula: ##STR00025## where PAB is para-aminobenzylcarbamoyl, and
R.sup.17 is a divalent radical selected from (CH.sub.2).sub.r,
C.sub.3-C.sub.8 carbocyclyl, O--(CH.sub.2).sub.r, arylene,
(CH.sub.2).sub.r-arylene, -arylene-(CH.sub.2).sub.r--,
(CH.sub.2).sub.r--(C.sub.3-C.sub.8 carbocyclyl), (C.sub.3-C.sub.8
carbocyclyl)-(CH.sub.2).sub.r, C.sub.3-C.sub.8 heterocyclyl,
(CH.sub.2).sub.r--(C.sub.3-C.sub.8 heterocyclyl),
--(C.sub.3-C.sub.8 heterocyclyl)-(CH.sub.2).sub.r--,
--(CH.sub.2).sub.rC(O)NR.sup.b(CH.sub.2).sub.r--,
--(CH.sub.2CH.sub.2O).sub.r--,
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--,
--(CH.sub.2).sub.rC(O)NR.sup.b(CH.sub.2CH.sub.2O).sub.r--,
--(CH.sub.2).sub.rC(O)NR.sup.b(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--,
--(CH.sub.2CH.sub.2O).sub.rC(O)NR.sup.b(CH.sub.2CH.sub.2O).sub.r--,
--(CH.sub.2CH.sub.2O).sub.rC(O)NR.sup.b(CH.sub.2CH.sub.2O).sub.r--CH.sub.-
2--, and --(CH.sub.2CH.sub.2O).sub.rC(O)NR.sup.b(CH.sub.2).sub.r--;
where R.sup.b is H, C.sub.1-C.sub.6 alkyl, phenyl, or benzyl; and r
is independently an integer ranging from 1 to 10.
67. The antibody-drug conjugate compound of claim 65 wherein
W.sub.w is valine-citrulline.
68. The antibody-drug conjugate compound of claim 65 having the
formula: ##STR00026##
69. The antibody-drug conjugate compound of either claim 66 or 68
wherein R.sup.17 is (CH.sub.2).sub.5 or (CH.sub.2).sub.2.
70. The antibody-drug conjugate compound of claim 65 having the
formula: ##STR00027##
71. The antibody-drug conjugate compound of claim 64 wherein L is
SMCC or BMPEO.
72. The antibody-drug conjugate compound of claim 64 wherein D is
either MMAE, preferably having the structure: ##STR00028## where
the wavy line indicates the attachment site to the linker L; or
MMAF, preferably having the structure: ##STR00029## where the wavy
line indicates the attachment site to the linker L.
73. The cysteine engineered anti-TENB2 antibody of claim 61 or the
antibody-drug conjugate compound of any one of claims 63 to 72
wherein the parent anti-TENB2 antibody is selected from a
monoclonal antibody, a bispecific antibody, a chimeric antibody, a
human antibody, and a humanized antibody.
74. The cysteine engineered anti-TENB2 antibody of claim 61 or
antibody-drug conjugate compound of any one of claims 63 to 72
wherein the parent anti-TENB2 antibody is an antibody fragment,
preferably a Fab fragment.
75. The antibody drug conjugate of claim 63 wherein L is
MC-val-cit-PAB or MC, SMCC, SPP, or BMPEO.
76. An antibody-drug conjugate compound selected from the
structures: ##STR00030## wherein Val is valine; Cit is citrulline;
p is 1, 2, 3, or 4; and Ab is a cysteine engineered anti-TENB2
antibody of claim 61.
77. A pharmaceutical formulation comprising the cysteine engineered
anti-TENB2 antibody of claim 61 or the antibody drug conjugate of
claim 63, and a pharmaceutically acceptable diluent, carrier or
excipient.
78. The pharmaceutical formulation comprising an antibody drug
conjugate according to claim 77, further comprising a
therapeutically effective amount of a chemotherapeutic agent
selected from letrozole, oxaliplatin, doxetaxel, 5-FU, lapatinib,
capecitabine, leucovorin, erlotinib, pertuzumab, bevacizumab, and
gemcitabine.
79. An article of manufacture comprising the pharmaceutical
formulation comprising an antibody drug conjugate according to
claim 77; a container; and a package insert or label indicating
that the compound can be used to treat cancer characterized by the
overexpression of a TENB2 polypeptide, wherein the cancer is
preferably selected from ovarian cancer, prostate cancer, cancer of
the urinary tract, pancreatic cancer, lung cancer, breast cancer,
or colon cancer.
80. A method of determining the presence of a TENB2 protein in a
sample suspected of containing said protein, said method comprising
exposing said sample to a cysteine engineered anti-TENB2 antibody
of claim 61 and determining binding of said antibody to said TENB2
protein in said sample, wherein binding of the antibody to said
protein is indicative of the presence of said protein in said
sample.
81. The method of claim 80, wherein the antibody is covalently
attached to a label selected from a fluorescent dye, a
radioisotope, biotin, or a metal-complexing ligand; or wherein said
sample comprises a cell suspected of expressing said TENB2 protein;
or wherein said cell is a prostate, ovarian, breast, lung, or
pancreatic cancer cell.
82. An assay for detecting cancer cells comprising: (a) exposing
cells to an antibody-drug conjugate compound of claim 63; and (b)
determining the extent of binding of the antibody-drug conjugate
compound to the cells; wherein preferably the cells are prostate,
pancreatic, lung, breast, colon or ovarian tumor cells.
83. A method of inhibiting cellular proliferation comprising
treating mammalian tumor cells in a cell culture medium with an
antibody-drug conjugate compound of claim 63, whereby proliferation
of the tumor cells is inhibited, wherein the mammalian tumor cells
are preferably ovarian tumor cells.
84. A method of treating cancer comprising administering the
pharmaceutical formulation of claim 77 to the patient, wherein the
cancer is selected from the group consisting of prostate cancer,
cancer of the urinary tract, pancreatic cancer, lung cancer, breast
cancer, colon cancer and ovarian cancer.
85. The method of claim 84,wherein the method comprises
administering a chemotherapeutic agent to the patient in
combination with the antibody-drug conjugate compound, where the
chemotherapeutic agent is selected from letrozole, cisplatin,
carboplatin, taxol, paclitaxel, oxaliplatin, doxetaxel, 5-FU,
leucovorin, erlotinib, pertuzumab, bevacizumab, lapatinib, and
gemcitabine.
86. A method for making an antibody drug conjugate compound
comprising a cysteine engineered anti-TENB2 antibody (Ab) of claim
61, and an auristatin drug moiety (D) wherein the cysteine
engineered antibody is attached through the one or more engineered
cysteine amino acids by a linker moiety (L) to D; the compound
having Formula I: Ab-(L-D).sub.p (I) where p is 1, 2, 3, or 4; the
method comprising the steps of: (a) reacting an engineered cysteine
group of the cysteine engineered antibody with a linker reagent to
form antibody-linker intermediate Ab-L; and (b) reacting Ab-L with
an activated drug moiety D; whereby the antibody-drug conjugate is
formed; or comprising the steps of: (c) reacting a nucleophilic
group of a drug moiety with a linker reagent to form drug-linker
intermediate D-L; and (d) reacting D-L with an engineered cysteine
group of the cysteine engineered antibody; whereby the
antibody-drug conjugate is formed.
87. The method of claim 86, wherein the method comprises the step
of expressing the cysteine engineered antibody in chinese hamster
ovary (CHO) cells.
88. The method of claim 86 further comprising the step of treating
the expressed cysteine engineered antibody with a reducing agent,
wherein the reducing agent is selected from TCEP and DTT.
89. The method of claim 87 further comprising the step of treating
the expressed cysteine engineered antibody with an oxidizing agent,
after treating with the reducing agent, wherein the oxidizing agent
is selected from copper sulfate, dehydroascorbic acid, and air.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a non-provisional application under 37
CFR 1.53(b)(1), claiming priority under 35 USC 119(e) to
provisional application No. 60/981,411 filed Oct. 19, 2007, the
contents of which are incorporated herein by reference
FIELD OF THE INVENTION
[0002] The invention relates generally to antibodies engineered
with reactive cysteine residues and more specifically to antibodies
with therapeutic or diagnostic applications. The cysteine
engineered antibodies may be conjugated with chemotherapeutic
drugs, toxins, affinity ligands such as biotin, and detection
labels such as fluorophores. The invention also relates to methods
of using antibodies and antibody-drug conjugate compounds for in
vitro, in situ, and in vivo diagnosis or treatment of mammalian
cells, or associated pathological conditions.
BACKGROUND OF THE INVENTION
[0003] Antibody therapy has been established for the targeted
treatment of patients with cancer, immunological and angiogenic
disorders. Transmembrane or otherwise tumor-associated polypeptides
specifically expressed on the surface of cancer cells as compared
to normal, non-cancerous cell(s) have been identified as cellular
targets for cancer diagnosis and therapy with antibodies.
Identification of such tumor-associated cell surface antigen
polypeptides, i.e. tumor associated antigens (TAA), allows specific
targeting of cancer cells for destruction via antibody-based
therapies.
[0004] The use of antibody-drug conjugates (ADC), i.e.
immunoconjugates, for the local delivery of cytotoxic or cytostatic
agents, i.e. drugs to kill or inhibit tumor cells in the treatment
of cancer (Lambert, J. (2005) Curr. Opinion in Pharmacology
5:543-549; Wu et al (2005) Nature Biotechnology 23(9):1137-1146;
Payne, G. (2003) Cancer Cell 3:207-212; Syrigos and Epenetos (1999)
Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997)
Adv. Drug Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows
targeted delivery of the drug moiety to tumors, and intracellular
accumulation therein, where systemic administration of these
unconjugated drug agents may result in unacceptable levels of
toxicity to normal cells as well as the tumor cells sought to be
eliminated (Baldwin et al (1986) Lancet pp. (Mar. 15, 1986):603-05;
Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer
Therapy: A Review," in Monoclonal Antibodies '84: Biological And
Clinical Applications, A. Pinchera et al (ed.s), pp. 475-506).
Efforts to improve the therapeutic index, i.e. maximal efficacy and
minimal toxicity of ADC have focused on the selectivity of
polyclonal (Rowland et al (1986) Cancer Immunol. Immunother.,
21:183-87) and monoclonal antibodies (mAbs) as well as drug-linking
and drug-releasing properties (Lambert, J. (2005) Curr. Opinion in
Pharmacology 5:543-549). Drug moieties used in antibody drug
conjugates include bacterial protein toxins such as diphtheria
toxin, plant protein toxins such as ricin, small molecules such as
auristatins, geldanamycin (Mandler et al (2000) J. of the Nat.
Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic
& Med. Chem. Letters 10:1025-1028; Mandler et al (2002)
Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et
al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), calicheamicin
(Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer
Res. 53:3336-3342), daunomycin, doxorubicin, methotrexate, and
vindesine (Rowland et al (1986) supra). The drug moieties may
affect cytotoxic and cytostatic mechanisms including tubulin
binding, DNA binding, or topoisomerase inhibition. Some cytotoxic
drugs tend to be inactive or less active when conjugated to large
antibodies or protein receptor ligands.
[0005] The auristatin peptides, auristatin E (AE) and
monomethylauristatin (MMAE), synthetic analogs of dolastatin (WO
02/088172), have been conjugated as drug moieties to: (i) chimeric
monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas);
(ii) cAC10 which is specific to CD30 on hematological malignancies
(Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773;
Doronina et al (2003) Nature Biotechnology 21(7):778-784; Francisco
et al (2003) Blood 102(4):1458-1465; US 2004/0018194; (iii)
anti-CD20 antibodies such as rituxan (WO 04/032828) for the
treatment of CD20-expressing cancers and immune disorders; (iv)
anti-EphB2R antibody 2H9 for treatment of colorectal cancer (Mao et
al (2004) Cancer Research 64(3):781-788); (v) E-selectin antibody
(Bhaskar et al (2003) Cancer Res. 63:6387-6394); (vi) trastuzumab
(HERCEPTIN.RTM., US 2005/0238649), and (vi) anti-CD30 antibodies
(WO 03/043583). Variants of auristatin E are disclosed in U.S. Pat.
No. 5,767,237 and U.S. Pat. No. 6,124,431. Monomethyl auristatin E
conjugated to monoclonal antibodies are disclosed in Senter et al,
Proceedings of the American Association for Cancer Research, Volume
45, Abstract Number 623, presented Mar. 28, 2004. Auristatin
analogs MMAE and MMAF have been conjugated to various antibodies
(US 2005/0238649).
[0006] Conventional means of attaching, i.e. linking through
covalent bonds, a drug moiety to an antibody generally leads to a
heterogeneous mixture of molecules where the drug moieties are
attached at a number of sites on the antibody. For example,
cytotoxic drugs have typically been conjugated to antibodies
through the often-numerous lysine residues of an antibody,
generating a heterogeneous antibody-drug conjugate mixture.
Depending on reaction conditions, the heterogeneous mixture
typically contains a distribution of antibodies with from 0 to
about 8, or more, attached drug moieties. In addition, within each
subgroup of conjugates with a particular integer ratio of drug
moieties to antibody, is a potentially heterogeneous mixture where
the drug moiety is attached at various sites on the antibody.
Analytical and preparative methods may be inadequate to separate
and characterize the antibody-drug conjugate species molecules
within the heterogeneous mixture resulting from a conjugation
reaction. Antibodies are large, complex and structurally diverse
biomolecules, often with many reactive functional groups. Their
reactivities with linker reagents and drug-linker intermediates are
dependent on factors such as pH, concentration, salt concentration,
and co-solvents. Furthermore, the multistep conjugation process may
be nonreproducible due to difficulties in controlling the reaction
conditions and characterizing reactants and intermediates.
[0007] Cysteine thiols are reactive at neutral pH, unlike most
amines which are protonated and less nucleophilic near pH 7. Since
free thiol (RSH, sulfhydryl) groups are relatively reactive,
proteins with cysteine residues often exist in their oxidized form
as disulfide-linked oligomers or have internally bridged disulfide
groups. Extracellular proteins generally do not have free thiols
(Garman, 1997, Non-Radioactive Labelling: A Practical Approach,
Academic Press, London, at page 55). Antibody cysteine thiol groups
are generally more reactive, i.e. more nucleophilic, towards
electrophilic conjugation reagents than antibody amine or hydroxyl
groups. Cysteine residues have been introduced into proteins by
genetic engineering techniques to form covalent attachments to
ligands or to form new intramolecular disulfide bonds (Better et al
(1994) J. Biol. Chem. 13:9644-9650; Bernhard et al (1994)
Bioconjugate Chem. 5:126-132; Greenwood et al (1994) Therapeutic
Immunology 1:247-255; Tu et al (1999) Proc. Natl. Acad. Sci USA
96:4862-4867; Kanno et al (2000) J. of Biotechnology, 76:207-214;
Chmura et al (2001) Proc. Nat. Acad. Sci. USA 98(15):8480-8484;
U.S. Pat. No. 6,248,564). However, engineering in cysteine thiol
groups by the mutation of various amino acid residues of a protein
to cysteine amino acids is potentially problematic, particularly in
the case of unpaired (free Cys) residues or those which are
relatively accessible for reaction or oxidation. In concentrated
solutions of the protein, whether in the periplasm of E. coli,
culture supernatants, or partially or completely purified protein,
unpaired Cys residues on the surface of the protein can pair and
oxidize to form intermolecular disulfides, and hence protein dimers
or multimers. Disulfide dimer formation renders the new Cys
unreactive for conjugation to a drug, ligand, or other label.
Furthermore, if the protein oxidatively forms an intramolecular
disulfide bond between the newly engineered Cys and an existing Cys
residue, both Cys thiol groups are unavailable for active site
participation and interactions. Furthermore, the protein may be
rendered inactive or non-specific, by misfolding or loss of
tertiary structure (Zhang et al (2002) Anal. Biochem. 311:1-9).
[0008] Cysteine-engineered antibodies have been designed as FAB
antibody fragments (thioFab) and expressed as full-length, IgG
monoclonal (thioMab) antibodies (US 2007/0092940, the contents of
which are incorporated by reference). ThioFab and ThioMab
antibodies have been conjugated through linkers at the newly
introduced cysteine thiols with thiol-reactive linker reagents and
drug-linker reagents to prepare antibody drug conjugates (Thio
ADC).
[0009] TENB2 is a tumor associated antigen polypeptide (also known
as PR1), and the TENB2 protein contains 2 follistatin-like domains
and a conserved EGF-like domain. The gene encoding the protein was
first characterized from a human brain cDNA library (see Uchida, et
al. (1999) Biochem. Biophys. Res. Commun. 266:593-602), and later
isolated from a human fetal brain cDNA library (see Horie, et al.
(2000) Genomics 67:146-152). See also, e.g., Online Mendelian
Inheritance in Man, number 605734; Unigene Cluster Hs.22791;
LocusLink 23671; and other linked sites. TENB2 has been referred to
as PR1, tomoregulin, TR, hyperplastic polyposis gene 1, HPP1, and
TMEFF2. It's nucleic acid sequence can be identified by ATCC
Accession Nos. AF264150, AB004064, AB017269, and AF179274; and it's
amino acid sequence can be identified by ATCC Accession Nos.
AAF91397, BAA90820, BAA87897, and AAD55776. TENB2's UniGene Cluster
identification number is hs.22791, Locuslink identification number
is 23671, and OMIM identification number is 605734.
[0010] The gene has also been implicated in certain cancerous
conditions. Young, et al. (2001) Proc. Nat'l Acad. Sci. USA
98:265-270 reported expression in colorectal polyps. Glynne-Jones,
et al. (2001) Int. J. Cancer 94:178-184 reported it as a marker for
prostate cancer.
[0011] Due to its overexpression in certain human tumors, the TENB2
polypeptide and the nucleic acid encoding that polypeptide are
targets for quantitative and qualitative comparisons among various
mammalian tissue samples. The unique expression profiles of TENB2
polypeptide, and the nucleic acid encoding that polypeptide, can be
exploited for the diagnosis and therapeutic treatment of certain
types of cancerous tumors in mammals.
[0012] Recently, certain anti-TENB2 antibodies, including
anti-TMEFF2 antibody #19, were disclosed and shown to be
internalized and useful for the treatment of proliferative
conditions of the prostate, including, e.g., benign prostate
hyperplasia and prostate cancer (PCT/US03/07209; U.S. Ser. No.
10/383,447, filed Mar. 7, 2003, now U.S. Pat. No. 7,288,248; Vinay
et al., "Antibodies Against Cancer Antigen TMEFF2 and Uses Thereof"
the contents of which are incorporated by reference).
SUMMARY
[0013] In one aspect, the invention includes a cysteine engineered
anti-TENB2 antibody comprising one or more free cysteine amino
acids and a sequence selected from SEQ ID NOS:8-23. The cysteine
engineered anti-TENB2 antibody may bind to a TENB2 polypeptide.
Tumor-associated antigens (TAA) such as TENB2 polypeptides can be
prepared for use in generating cysteine engineered antibodies using
methods and information which are well known in the art, and for
example in PCT/US03/07209 (U.S. Pat. No. 7,288,248). The cysteine
engineered anti-TENB2 antibody may be prepared by a process
comprising replacing one or more amino acid residues of a parent
anti-TENB2 antibody by cysteine.
[0014] The one or more free cysteine amino acid residues of the
cysteine engineered anti-TENB2 antibody are located in a light
chain or a heavy chain.
[0015] In one aspect, the invention includes a method of
determining the presence of a TENB2 protein in a sample suspected
of containing said protein, said method comprising exposing said
sample to a cysteine engineered anti-TENB2 antibody and determining
binding of said antibody to said TENB2 protein in said sample,
wherein binding of the antibody to said protein is indicative of
the presence of said protein in said sample.
[0016] Cysteine engineered anti-TENB2 antibodies may be used as
naked antibodies (unconjugated to a drug or label moiety) or as
antibody-drug conjugates (ADC). The cysteine engineered anti-TENB2
antibody may be covalently attached to an auristatin drug moiety
whereby an antibody drug conjugate is formed. The antibody-drug
conjugate may comprising a cysteine engineered anti-TENB2 antibody
(Ab), and an auristatin drug moiety (D) wherein the cysteine
engineered anti-TENB2 antibody is attached through one or more free
cysteine amino acids by a linker moiety (L) to D; the compound
having Formula I:
Ab-(L-D).sub.p (I)
where p is 1, 2, 3, or 4. Auristatin drug moieties include MMAE and
MMAF.
[0017] An aspect of the invention is an assay for detecting cancer
cells comprising: (a) exposing cells to an antibody-drug conjugate
compound; and (b) determining the extent of binding of the
antibody-drug conjugate compound to the cells.
[0018] An aspect of the invention is a pharmaceutical formulation
comprising the antibody drug conjugate, and a pharmaceutically
acceptable diluent, carrier or excipient.
[0019] An aspect of the invention is a method of inhibiting
cellular proliferation comprising treating mammalian tumor cells in
a cell culture medium with an antibody-drug conjugate compound,
whereby proliferation of the tumor cells is inhibited.
[0020] An aspect of the invention is a method of treating cancer
comprising administering to a patient the pharmaceutical
formulation. The patient may be administered a chemotherapeutic
agent in combination with the antibody-drug conjugate compound.
[0021] An aspect of the invention is an article of manufacture
comprising the pharmaceutical formulation, a container; and a
package insert or label indicating that the compound can be used to
treat cancer characterized by the overexpression of a TENB2
polypeptide.
[0022] An aspect of the invention is a method for making a Formula
I antibody drug conjugate compound comprising the steps of: (a)
reacting an engineered cysteine group of the cysteine engineered
antibody with a linker reagent to form antibody-linker intermediate
Ab-L; and (b) reacting Ab-L with an activated drug moiety D;
whereby the antibody-drug conjugate is formed; or comprising the
steps of: (c) reacting a nucleophilic group of a drug moiety with a
linker reagent to form drug-linker intermediate D-L; and (d)
reacting D-L with an engineered cysteine group of the cysteine
engineered antibody; whereby the antibody-drug conjugate is
formed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows the Heavy Chain sequence: SEQ ID NO:1, and
Light Chain sequence: SEQ ID NO:2 of humanized anti-TENB2 antibody,
hu TMEFF2#19.
[0024] FIG. 2 shows the Heavy Chain sequence: SEQ ID NO:3, and
Light Chain sequence: SEQ ID NO:2 of a humanized cysteine
engineered anti-TENB2 antibody, A121C thio hu TMEFF2#19. Signal
sequence is not included in the sequential numbering of anti-TENB2
antibody.
[0025] FIG. 3 shows alignment of humanized trastuzumab light chain
(HuTMAb-LC, SEQ ID NO:4) and hu TMEFF2#19 light chain (SEQ ID
NO:5). The numbering follows the sequential numbering
convention.
[0026] FIG. 4 shows alignment of humanized trastuzumab heavy chain
(HuTMAb-HC, SEQ ID NO:6), and hu TMEFF2#19 heavy chain (SEQ ID
NO:7). The numbering follows the sequential numbering
convention.
[0027] FIG. 5 shows depictions of cysteine engineered anti-TENB2
antibody drug conjugates (ADC) where a drug moiety is attached to
an engineered cysteine group in: the light chain (LC-ADC); the
heavy chain (HC-ADC); and the Fc region (Fc-ADC).
[0028] FIG. 6 shows the steps of: (i) reducing cysteine disulfide
adducts and interchain and intrachain disulfides in a cysteine
engineered anti-TENB2 antibody (ThioMab); (ii) partially oxidizing,
i.e. reoxidation to reform interchain and intrachain disulfides;
and (iii) conjugation of the reoxidized antibody with a drug-linker
intermediate to form a cysteine engineered anti-TENB2 antibody drug
conjugate (ADC).
[0029] FIG. 7 shows expression of TENB2 in cancer and normal human
tissues: oligonucleotide microarray analysis was performed on RNA
extracted from 4841 human tissue samples. Each box in the plot
provides signal intensity (average difference scaled to 100) for
TENB2 for a sample of the indicated tissue. Green boxes are normal
tissue, red boxes are tumors, and blue boxes represent other
diseased tissues.
[0030] FIG. 8 shows TENB2 expression in human prostate tumors: Top
and bottom panels are from human prostate explant models, PC3TENB2
medium stable cell line with vector control and prostate tumor,
respectively.
[0031] FIG. 9 shows internalization of TENB2 monoclonal antibody
(Mab) on PC3TENB2 Medium cell line and LuCaP 70 tumor.
[0032] FIGS. 10A and B shows FACS data on PC3 TENB2 Medium cells
with thio (FIG. 10B) or conventional anti-TENB2 ADC treatment (FIG.
10A).
[0033] FIGS. 11A and B shows a cell killing assay on PC3 TENB2
Medium cells with conventional anti-TENB2 (FIG. 11A) and
thio-anti-TENB2 ADCs (FIG. 11B).
[0034] FIG. 12 shows an efficacy study on PC3 TENB2 Medium cells
using anti-TENB2 and thio-anti-TENB2 ADCs (conjugated with vc-MMAE
or MC-MMAF).
[0035] FIG. 13 shows a Western Blot with various LuCaP explant
tumor tissues using humanized anti-TENB2 Ab (hu TMEFF2#19).
[0036] FIG. 14 shows xenograft experiments using human prostate
cancer LuCaP 70, 77 and 96.1.
[0037] FIG. 15 shows pharmacokinetic evaluation of rats using
thio-anti-TENB2 and conventional ADCs.
[0038] FIG. 16 shows a safety assessment on rats with
anti-TENB2-vc-MMAE vs. MC-MMAF.
[0039] FIGS. 17A and B shows a safety assessment on cynomolgus
monkeys with anti-TENB2- vc-MMAE vs. anti-TENB2-MC-MMAF.
[0040] FIGS. 18A and B shows a safety assessment on rats with
thio-anti-TENB2-vc-MMAE vs. anti-TENB2-vc-MMAE.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0041] Reference will now be made in detail to certain embodiments
of the invention, examples of which are illustrated in the
accompanying structures and formulas. While the invention will be
described in conjunction with the enumerated embodiments, it will
be understood that they are not intended to limit the invention to
those embodiments. On the contrary, the invention is intended to
cover all alternatives, modifications, and equivalents, which may
be included within the scope of the present invention as defined by
the claims.
[0042] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. The present
invention is in no way limited to the methods and materials
described.
Definitions
[0043] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs, and are
consistent with: Singleton et al (1994) Dictionary of Microbiology
and Molecular Biology, 2nd Ed., J. Wiley & Sons, New York,
N.Y.; and Janeway, C., Travers, P., Walport, M., Shlomchik (2001)
Immunobiology, 5th Ed., Garland Publishing, New York.
[0044] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
dimers, multimers, multispecific antibodies (e.g., bispecific
antibodies), and antibody fragments, so long as they exhibit the
desired biological activity (Miller et al (2003) Jour. of
Immunology 170:4854-4861). Antibodies may be murine, human,
humanized, chimeric, or derived from other species. An antibody is
a protein that is capable of recognizing and binding to a specific
antigen (Janeway, C., Travers, P., Walport, M., Shlomchik (2001)
Immuno Biology, 5th Ed., Garland Publishing, New York). A target
antigen generally has numerous binding sites, also called epitopes,
recognized by CDRs on multiple antibodies. Each antibody that
specifically binds to a different epitope has a different
structure. Thus, one antigen may have more than one corresponding
antibody. An antibody includes a full-length immunoglobulin
molecule or an immunologically active portion of a full-length
immunoglobulin molecule, i.e., a molecule that contains an antigen
binding site that immunospecifically binds an antigen of a target
of interest or part thereof, such targets including but not limited
to, cancer cell or cells that produce autoimmune antibodies
associated with an autoimmune disease. The immunoglobulin disclosed
herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA),
class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of
immunoglobulin molecule. The immunoglobulins can be derived from
any species such as human, murine, or rabbit. For the structure and
properties of the different classes of antibodies, see, e.g., Basic
and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I.
Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk,
Conn., 1994, page 71 and Chapter 6.
[0045] "Antibody fragments" comprise a portion of a full length
antibody, generally the antigen binding or variable region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; minibodies (U.S. Pat.
No. 5,641,870, Example 2; Zapata et al (1995) Protein Eng. 8(10):
1057-1062); Olafsen et al (2004) Protein Eng. Design & Sel.
17(4):315-323), fragments produced by a Fab expression library,
anti-idiotypic (anti-Id) antibodies, CDR (complementary determining
region), and epitope-binding fragments of any of the above which
immunospecifically bind to cancer cell antigens, viral antigens or
microbial antigens, single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0046] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al
(1975) Nature 256:495, or may be made by recombinant DNA methods
(see for example: U.S. Pat. No. 4,816,567; U.S. Pat. No.
5,807,715). In the hybridoma method, a mouse or other appropriate
host animal, such as a hamster, is immunized as described above to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
After immunization, lymphocytes are isolated and then fused with a
myeloma cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, (1986)
Monoclonal Antibodies: Principles and Practice, pp.59-103 Academic
Press). The monoclonal antibodies may also be isolated from phage
antibody libraries using the techniques described in Clackson et al
(1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol.,
222:581-597.
[0047] The DNA that encodes the antibody may be modified to produce
chimeric or fusion antibody polypeptides, for example, by
substituting human heavy chain and light chain constant domain
(C.sub.H and C.sub.L) sequences for the homologous murine sequences
(U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl Acad.
Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding
sequence with all or part of the coding sequence for a
non-immunoglobulin polypeptide (heterologous polypeptide). The
non-immunoglobulin polypeptide sequences can substitute for the
constant domains of an antibody, or they are substituted for the
variable domains of one antigen-combining site of an antibody to
create a chimeric bivalent antibody comprising one
antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0048] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end.
The constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light-chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light chain and heavy chain variable domains.
[0049] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al (1984) Proc. Natl.
Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest
herein include "primatized" antibodies comprising variable domain
antigen-binding sequences derived from a non-human primate (e.g.,
Old World Monkey, Ape etc) and human constant region sequences.
[0050] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from the
non-human antibody. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
antibody specificity, affinity, and capability. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all, or substantially all, of the
FRs are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. The Fc fragment comprises the carboxy-terminal
portions of both H chains held together by disulfides. The effector
functions of antibodies are determined by sequences in the Fc
region, which region is also the part recognized by Fc receptors
(FcR) found on certain types of cells (Jones et al (1986) Nature
321:522-525; Riechmann et al (1988) Nature 332:323-329; Presta,
(1992) Curr. Op. Struct. Biol. 2:593-596; Verhoeyen et al (1988)
Science, 239:1534-1536; Sims et al (1993) J. Immunol. 151:2296;
Chothia et al (1987) J. Mol. Biol., 196:901). Other methods use a
particular framework region derived from the consensus sequence of
all human antibodies of a particular subgroup of light or heavy
chains (Carter et al (1992) Proc. Natl. Acad. Sci. USA, 89:4285;
Presta et al (1993) J. Immunol. 151:2623).
[0051] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues. Transgenic animals (e.g., mice)
are available that are capable, upon immunization, of producing a
full repertoire of human antibodies in the absence of endogenous
immunoglobulin production. For example, it has been described that
the homozygous deletion of the antibody heavy-chain joining region
(J.sub.H) gene in chimeric and germ-line mutant mice results in
complete inhibition of endogenous antibody production. Transfer of
the human germ-line immunoglobulin gene array into such germ-line
mutant mice will result in the production of human antibodies upon
antigen challenge (Jakobovits et al (1993) Proc. Natl. Acad. Sci.
USA, 90:2551; Jakobovits et al (1993) Nature, 362:255-258;
Bruggemann et al (1993) Year in Immuno. 7:33; U.S. Pat. No.
5,545,806; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,591,669; U.S.
Pat. No. 5,545,807; and WO 97/17852.
[0052] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result in an
improvement in the affinity of the antibody for antigen, compared
to an antibody which does not possess those alteration(s).
Preferred affinity matured antibodies will have nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies are produced affinity maturation by VH and VL domain
shuffling (Marks et al (1992) Bio/Technology 10:779-783), or random
mutagenesis of CDR and/or framework residues (Barbas et al (1994)
Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al (1995) Gene
169:147-155; Yelton et al (1995) J. Immunol. 155:1994-2004; Jackson
et al (1995) J. Immunol. 154(7):3310-9; and Hawkins et al (1992) J.
Mol. Biol. 226:889-896).
[0053] An "intact antibody" herein is one comprising VL and VH
domains, as well as a light chain constant domain (CL) and heavy
chain constant domains, CH1, CH2 and CH3. The constant domains may
be native sequence constant domains (e.g., human native sequence
constant domains) or amino acid sequence variant thereof. The
intact antibody may have one or more "effector functions" which
refer to those biological activities attributable to the Fc
constant region (a native sequence Fc region or amino acid sequence
variant Fc region) of an antibody. Examples of antibody effector
functions include C1q binding; complement dependent cytotoxicity;
Fc receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; and down regulation of cell surface receptors
such as B cell receptor and BCR.
[0054] The term "amino acid sequence variant" refers to
polypeptides having amino acid sequences that differ to some extent
from a native sequence polypeptide. Ordinarily, amino acid sequence
variants will possess at least about 70% sequence identity with at
least one receptor binding domain of a native sequence polypeptide
or with at least one ligand binding domain of a native receptor,
and preferably, they will be at least about 80%, more preferably,
at least about 90% homologous by sequence with such receptor or
ligand binding domains. The amino acid sequence variants possess
substitutions, deletions, and/or insertions at certain positions
within the amino acid sequence of the native amino acid sequence.
Amino acids are designated by the conventional names, one-letter
and three-letter codes.
[0055] "Sequence identity" is defined as the percentage of residues
in the amino acid sequence variant that are identical after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. Methods and computer
programs for the alignment are well known in the art. One such
computer program is "Align 2," authored by Genentech, Inc., which
was filed with user documentation in the United States Copyright
Office, Washington, D.C. 20559, on Dec. 10, 1991, and which code is
found in PCT/US03/07209 (U.S. Pat. No. 7,288,248).
[0056] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells in summarized
is Table 3 on page 464 of Ravetch and Kinet, (1991) "Annu. Rev.
Immunol." 9:457-92. To assess ADCC activity of a molecule of
interest, an in vitro ADCC assay, such as that described in U.S.
Pat. No. 5,500,362 and U.S. Pat. No. 5,821,337 may be performed.
Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in Clynes et al (1998) Proc. Nat. Acad. Sci. (USA)
95:652-656.
[0057] "Human effector cells" are leukocytes which express one or
more constant region receptors (FcRs) and perform effector
functions. Preferably, the cells express at least Fc.gamma.RIII and
perform ADCC effector function. Examples of human leukocytes which
mediate ADCC include peripheral blood mononuclear cells (PBMC),
natural killer (NK) cells, monocytes, cytotoxic T cells and
neutrophils; with PBMCs and NK cells being preferred. The effector
cells may be isolated from a native source thereof, e.g., from
blood or PBMCs as described herein.
[0058] The terms "Fc receptor" or "FcR" mean a receptor that binds
to the Fc constant region of an antibody. The preferred FcR is a
native sequence human FcR. Moreover, a preferred FcR is one which
binds an IgG antibody (a gamma receptor) and includes receptors of
the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses,
including allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (See review M. in Daeron,
(1997) "Annu. Rev. Immunol." 15:203-234). FcRs are reviewed in
Ravetch and Kinet, (1991) "Annu. Rev. Immunol"., 9:457-92; Capel et
al (1994) Immunomethods 4:25-34; and de Haas et al (1995) J. Lab.
Clin. Med. 126:330-41. Other FcRs, including those to be identified
in the future, are encompassed by the term "FcR" herein. The term
also includes the neonatal receptor, FcRn, which is responsible for
the transfer of maternal IgGs to the fetus (Guyer et al (1976) J.
Immunol., 117:587 and Kim et al (1994) J. Immunol. 24:249).
[0059] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen
(Gazzano-Santoro et al (1996) J. Immunol. Methods 202:163).
[0060] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a .beta.-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions from the
other chain, contribute to the formation of the antigen-binding
site of antibodies (see Kabat et al (1991) Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md.). The constant domains are not
involved directly in binding an antibody to an antigen, but exhibit
various effector functions, such as participation of the antibody
in antibody dependent cellular cytotoxicity (ADCC).
[0061] The term "hypervariable region", "HVR", or "HV", when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six hypervariable regions;
three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A
number of hypervariable region delineations are in use and are
encompassed herein. The Kabat Complementarity Determining Regions
(CDRs) are based on sequence variability and are the most commonly
used (Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991)). Chothia refers instead to the
location of the structural loops (Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)). The "contact" hypervariable regions are based
on an analysis of the available complex crystal structures. The
residues from each of these hypervariable regions are noted below.
Unless otherwise denoted, Kabat numbering according to the Kabat
Database of aligned sequences of proteins will be employed (Wu and
Kabat (1970) J. Exp. Med. 132:211-250; Johnson and Wu (2000) Nuc.
Acids Res. 28(1):214-218). Hypervariable region locations are
generally as follows: amino acids 24-34 (HVR-L1), amino acids 49-56
(HVR-L2), amino acids 89-97 (HVR-L3), amino acids 26-35A (HVR-H1),
amino acids 49-65 (HVR-H2), and amino acids 93-102 (HVR-H3).
Hypervariable regions may also comprise "extended hypervariable
regions" as follows: amino acids 24-36 (L1), and amino acids 46-56
(L2) in the VL. The variable domain residues are numbered according
to Kabat et al., supra for each of these definitions. An "altered
hypervariable region" for the purposes herein is a hypervariable
region comprising one or more (e.g. one to about 16) amino acid
substitution(s) therein. An "un-modified hypervariable region" for
the purposes herein is a hypervariable region having the same amino
acid sequence as a non-human antibody from which it was derived,
i.e. one which lacks one or more amino acid substitutions
therein.
[0062] The terms "variable domain residue numbering as in Kabat",
"amino acid position numbering as in Kabat", and variations
thereof, refer to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). Using this numbering
system, the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, an FR or CDR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0063] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present invention. Specific illustrative
embodiments are described in the following.
[0064] An "antigen" is a predetermined polypeptide, carbohydrate,
nucleic acid, lipid, hapten or other naturally occurring or
synthetic compound to which an antibody can selectively bind.
[0065] "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined. A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residue in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al, Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991). In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al. A "VH subgroup III consensus
framework" comprises the consensus sequence obtained from the amino
acid sequences in variable heavy subgroup III of Kabat et al. A "VL
subgroup I consensus framework" comprises the consensus sequence
obtained from the amino acid sequences in variable light kappa
subgroup I of Kabat et al.
[0066] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and antigen-binding site. This region
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the V.sub.H-V.sub.L dimer. Collectively, the six hypervariable
regions confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three hypervariable regions specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0067] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear at least one free thiol
group. F(ab')2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0068] The "light chains" of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequences
of their constant domains.
[0069] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the VH
and VL domains which enables the scFv to form the desired structure
for antigen binding (Pluckthun in The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag,
New York, pp. 269-315 (1994).
[0070] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a variable
heavy domain (VH) connected to a variable light domain (VL) in the
same polypeptide chain (VH-VL). By using a linker that is too short
to allow pairing between the two domains on the same chain, the
domains are forced to pair with the complementary domains of
another chain and create two antigen-binding sites (EP 404,097; WO
93/11161; Hollinger et al (1993) Proc. Natl. Acad. Sci. USA
90:6444-6448).
[0071] A "free cysteine amino acid" refers to a cysteine amino acid
residue which has been engineered into a parent antibody, has a
thiol functional group (--SH), and is not paired as, or otherwise
part of, an intramolecular or intermolecular disulfide bridge.
[0072] The term "thiol reactivity value" is a quantitative
characterization of the reactivity of free cysteine amino acids.
The thiol reactivity value is the percentage of a free cysteine
amino acid in a cysteine engineered antibody which reacts with a
thiol-reactive reagent, and converted to a maximum value of 1. For
example, a free cysteine amino acid on a cysteine engineered
antibody which reacts in 100% yield with a thiol-reactive reagent,
such as a biotin-maleimide reagent, to form a biotin-labelled
antibody has a thiol reactivity value of 1.0. Another cysteine
amino acid engineered into the same or different parent antibody
which reacts in 80% yield with a thiol-reactive reagent has a thiol
reactivity value of 0.8. Another cysteine amino acid engineered
into the same or different parent antibody which fails totally to
react with a thiol-reactive reagent has a thiol reactivity value of
0. Determination of the thiol reactivity value of a particular
cysteine may be conducted by ELISA assay, mass spectroscopy, liquid
chromatography, autoradiography, or other quantitative analytical
tests. Thiol-reactive reagents which allow capture of the cysteine
engineered antibody and comparison and quantitation of the cysteine
reactivity include biotin-PEO-maleimide
((+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctainediamine, Oda
et al (2001) Nature Biotechnology 19:379-382, Pierce Biotechnology,
Inc.) Biotin-BMCC, PEO-Iodoacetyl Biotin, Iodoacetyl-LC-Biotin, and
Biotin-HPDP (Pierce Biotechnology, Inc.), and
N.alpha.-(3-maleimidylpropionyl)biocytin (MPB, Molecular Probes,
Eugene, Oreg.). Other commercial sources for biotinylation,
bifunctional and multifunctional linker reagents include Molecular
Probes, Eugene, Oreg., and Sigma, St. Louis, Mo.
[0073] A "parent antibody" is an antibody comprising an amino acid
sequence from which one or more amino acid residues are replaced by
one or more cysteine residues. The parent antibody may comprise a
native or wild type sequence. The parent antibody may have
pre-existing amino acid sequence modifications (such as additions,
deletions and/or substitutions) relative to other native, wild
type, or modified forms of an antibody. A parent antibody may be
directed against a target antigen of interest, e.g. a biologically
important polypeptide. Antibodies directed against nonpolypeptide
antigens (such as tumor-associated glycolipid antigens; see U.S.
Pat. No. 5,091,178) are also contemplated.
[0074] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0075] An antibody "which binds" a molecular target or an antigen
of interest, e.g., TENB2 or CA125 antigens, is one capable of
binding that antigen with sufficient affinity such that the
antibody is useful in targeting a cell expressing the antigen.
Where the antibody is one which binds TENB2, it will usually
preferentially bind TENB2, and may be one which does not
significantly cross-react with other proteins. In such embodiments,
the extent of binding of the antibody to these non-TENB2 proteins
(e.g., cell surface binding to endogenous receptor) will be less
than 10% as determined by fluorescence activated cell sorting
(FACS) analysis or radioimmunoprecipitation (RIA).
[0076] "Treating" or "treatment" or "alleviation" refers to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent or slow down (lessen) the targeted
pathologic condition or disorder. Those in need of treatment
include those already with the disorder as well as those prone to
have the disorder or those in whom the disorder is to be prevented.
A subject or mammal is successfully "treated" for a CA125/O772P
polypeptide-expressing cancer if, after receiving a therapeutic
amount of an anti-CA125/O772P antibody, such as a cysteine
engineered anti-TENB2 antibody, or antibody drug conjugate thereof,
according to the methods of the present invention, the patient
shows observable and/or measurable reduction in or absence of one
or more of the following: reduction in the number of cancer cells
or absence of the cancer cells; reduction in the tumor size;
inhibition (i.e., slow to some extent and preferably stop) of
cancer cell infiltration into peripheral organs including the
spread of cancer into soft tissue and bone; inhibition (i.e., slow
to some extent and preferably stop) of tumor metastasis;
inhibition, to some extent, of tumor growth; and/or relief to some
extent, one or more of the symptoms associated with the specific
cancer; reduced morbidity and mortality, and improvement in quality
of life issues. To the extent the cysteine engineered anti-TENB2
antibody, or antibody drug conjugate thereof, may prevent growth
and/or kill existing cancer cells, it may be cytostatic and/or
cytotoxic. Reduction of these signs or symptoms may also be felt by
the patient. The above parameters for assessing successful
treatment and improvement in the disease are readily measurable by
routine procedures familiar to a physician. For cancer therapy,
efficacy can be measured, for example, by assessing the time to
disease progression (TTP) and/or determining the response rate
(RR). Metastasis can be determined by staging tests and by bone
scan and tests for calcium level and other enzymes to determine
spread to the bone. CT scans can also be done to look for spread to
the pelvis and lymph nodes in the area. Chest X-rays and
measurement of liver enzyme levels by known methods are used to
look for metastasis to the lungs and liver, respectively. Other
routine methods for monitoring the disease include transrectal
ultrasonography (TRUS) and transrectal needle biopsy (TRNB).
[0077] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. A "tumor" comprises one or more
cancerous cells, and refers to all neoplastic cell growth and
proliferation, whether malignant or benign, and all pre-cancerous
and cancerous cells and tissues. Examples of cancer include, but
are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and
leukemia or lymphoid malignancies. More particular examples of such
cancers include squamous cell cancer (e.g., epithelial squamous
cell cancer), lung cancer including small-cell lung cancer,
non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung
and squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer including
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
breast cancer, colon cancer, rectal cancer, colorectal cancer,
endometrial or uterine carcinoma, salivary gland carcinoma, kidney
or renal cancer, prostate cancer, vulval cancer, thyroid cancer,
hepatic carcinoma, anal carcinoma, penile carcinoma, as well as
head and neck cancer.
[0078] A cancer which "overexpresses" an antigenic receptor is one
which has significantly higher levels of the receptor, such as
TENB2, at the cell surface thereof, compared to a noncancerous cell
of the same tissue type. Such overexpression may be caused by gene
amplification or by increased transcription or translation.
Receptor overexpression may be determined in a diagnostic or
prognostic assay by evaluating increased levels of the receptor
protein present on the surface of a cell (e.g., via an
immunohistochemistry assay; IHC). Alternatively, or additionally,
one may measure levels of receptor-encoding nucleic acid in the
cell, e.g., via fluorescent in situ hybridization (FISH; see WO
98/45479), southern blotting, or polymerase chain reaction (PCR)
techniques, such as real time quantitative reverse-transcriptase
PCR (qRT-PCR).
[0079] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source, e.g., from blood.
[0080] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0081] The term "therapeutically effective amount" refers to an
amount of a drug, e.g. a cysteine engineered anti-TENB2 antibody
drug conjugate or chemotherapeutic agent, effective to treat a
disease or disorder in a mammal. In the case of cancer, the
therapeutically effective amount of the drug may reduce the number
of cancer cells; reduce the tumor size; inhibit (i.e., slow to some
extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the cancer. To the extent the drug may prevent
growth and/or kill existing cancer cells, it may be cytostatic
and/or cytotoxic. The term "cytostatic" refers to the effect of
limiting the function of cells, such as limiting cellular growth or
proliferation of cells. For cancer therapy, efficacy can, for
example, be measured by assessing the time to disease progression
(TTP) and/or determining the response rate (RR).
[0082] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include erlotinib (TARCEVA.RTM., Genentech/OSI Pharm.), bortezomib
(VELCADE.RTM., Millenium Pharm.), fulvestrant (FASLODEX.RTM.,
AstraZeneca), sutent (SU11248, Pfizer), letrozole (FEMARA.RTM.,
Novartis), imatinib mesylate (GLEEVEC.RTM., Novartis), PTK787/ZK
222584 (Novartis), oxaliplatin (Eloxatin.RTM., Sanofi), 5-FU
(5-fluorouracil), leucovorin, Rapamycin (Sirolimus, RAPAMUNE.RTM.,
Wyeth), lapatinib (TYKERB.RTM., GSK572016, GlaxoSmithKline),
lonafarnib (SCH 66336), sorafenib (BAY43-9006, Bayer Labs.), and
gefitinib (IRESSA.RTM., Astrazeneca), AG1478, AG1571 (SU 5271;
Sugen), alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosoureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e. g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegaI1 (Angew Chem Intl.
Ed. Engl. (1994) 33:183-186); dynemicin, including dynemicin A;
bisphosphonates, such as clodronate; an esperamicin; as well as
neocarzinostatin chromophore and related chromoprotein enediyne
antibiotic chromophores), aclacinomysins, actinomycin, anthramycin,
azaserine, bleomycins, cactinomycin, carabicin, carminomycin,
carzinophilin, chromomycinis, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM.
doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., paclitaxel (TAXOL.RTM., Bristol- Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.TM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin and carboplatin;
vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide;
edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;
topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0083] Also included in this definition of "chemotherapeutic agent"
are: (i) anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens and selective
estrogen receptor modulators (SERMs), including, for example,
tamoxifen (including NOLVADEX.RTM. tamoxifen), raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON. toremifene; (ii) aromatase inhibitors
that inhibit the enzyme aromatase, which regulates estrogen
production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, MEGASE.RTM. megestrol acetate,
AROMASIN.RTM. exemestane, formestanie, fadrozole, RIVISOR.RTM.
vorozole, FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole;
(iii) anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; as well as troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); (iv) aromatase
inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase
inhibitors; (vii) antisense oligonucleotides, particularly those
which inhibit expression of genes in signaling pathways implicated
in abherant cell proliferation, such as, for example, PKC-alpha,
Ralf and H-Ras; (viii) ribozymes such as a VEGF expression
inhibitor (e.g., ANGIOZYME.RTM. ribozyme) and a HER2 expression
inhibitor; (ix) vaccines such as gene therapy vaccines, for
example, ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and
VAXID.RTM. vaccine; PROLEUKIN.RTM. rIL-2; LURTOTECAN.RTM.
topoisomerase 1 inhibitor; ABARELIX.RTM. rmRH; (x) anti-angiogenic
agents such as bevacizumab (AVASTIN.RTM., Genentech); and (xi)
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0084] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.;
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis
factor such as TNF-.alpha. or TNF-.beta.3; and other polypeptide
factors including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0085] The term "label" means any moiety which can be covalently
attached to an antibody and that functions to: (i) provide a
detectable signal; (ii) interact with a second label to modify the
detectable signal provided by the first or second label, e.g. FRET
(fluorescence resonance energy transfer); (iii) stabilize
interactions or increase affinity of binding, with antigen or
ligand; (iv) affect mobility, e.g. electrophoretic mobility, or
cell-permeability, by charge, hydrophobicity, shape, or other
physical parameters, or (v) provide a capture moiety, to modulate
ligand affinity, antibody/antigen binding, or ionic
complexation.
[0086] The phrase "pharmaceutically acceptable salt," as used
herein, refers to pharmaceutically acceptable organic or inorganic
salts of an ADC. Exemplary salts include, but are not limited, to
sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,
nitrate, bisulfate, phosphate, acid phosphate, isonicotinate,
lactate, salicylate, acid citrate, tartrate, oleate, tannate,
pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucuronate, saccharate, formate,
benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A
pharmaceutically acceptable salt may involve the inclusion of
another molecule such as an acetate ion, a succinate ion or other
counterion. The counterion may be any organic or inorganic moiety
that stabilizes the charge on the compound. Furthermore, a
pharmaceutically acceptable salt may have more than one charged
atom in its structure. Instances where multiple charged atoms are
part of the pharmaceutically acceptable salt can have multiple
counter ions. Hence, a pharmaceutically acceptable salt can have
one or more charged atoms and/or one or more counterion.
[0087] "Pharmaceutically acceptable solvate" refers to an
association of one or more solvent molecules and an ADC. Examples
of solvents that form pharmaceutically acceptable solvates include,
but are not limited to, water, isopropanol, ethanol, methanol,
DMSO, ethyl acetate, acetic acid, and ethanolamine.
[0088] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.RTM., polyethylene glycol (PEG), and PLURONICS.RTM..
[0089] Stereochemical definitions and conventions used herein
generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of
Chemical Terms (1984) McGraw-Hill Book Company, New York; and
Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds
(1994) John Wiley & Sons, Inc., New York. Many organic
compounds exist in optically active forms, i.e., they have the
ability to rotate the plane of plane-polarized light. In describing
an optically active compound, the prefixes D and L, or R and S, are
used to denote the absolute configuration of the molecule about its
chiral center(s). The prefixes d and 1 or (+) and (-) are employed
to designate the sign of rotation of plane-polarized light by the
compound, with (-) or 1 meaning that the compound is levorotatory.
A compound prefixed with (+) or d is dextrorotatory. For a given
chemical structure, these stereoisomers are identical except that
they are mirror images of one another. A specific stereoisomer may
also be referred to as an enantiomer, and a mixture of such isomers
is often called an enantiomeric mixture. A 50:50 mixture of
enantiomers is referred to as a racemic mixture or a racemate,
which may occur where there has been no stereoselection or
stereospecificity in a chemical reaction or process. The terms
"racemic mixture" and "racemate" refer to an equimolar mixture of
two enantiomeric species, devoid of optical activity.
[0090] The following abbreviations are used herein and have the
indicated definitions: BME is beta-mercaptoethanol, Boc is
N-(t-butoxycarbonyl), cit is citrulline (2-amino-5-ureido pentanoic
acid), dap is dolaproine, DCC is 1,3-dicyclohexylcarbodiimide, DCM
is dichloromethane, DEA is diethylamine, DEAD is
diethylazodicarboxylate, DEPC is diethylphosphorylcyanidate, DIAD
is diisopropylazodicarboxylate, DIEA is N,N-diisopropylethylamine,
dil is dolaisoleucine, DMA is dimethylacetamide, DMAP is
4-dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or
1,2-dimethoxyethane), DMF is N,N-dimethylformamide, DMSO is
dimethylsulfoxide, doe is dolaphenine, dov is N,N-dimethylvaline,
DTNB is 5,5'-dithiobis(2-nitrobenzoic acid), DTPA is
diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ
is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is
electrospray mass spectrometry, EtOAc is ethyl acetate, Fmoc is
N-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU is
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is high
pressure liquid chromatography, ile is isoleucine, lys is lysine,
MeCN (CH.sub.3CN) is acetonitrile, MeOH is methanol, Mtr is
4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is (1S,
2R)-(+)-norephedrine, PAB is p-aminobenzylcarbamoyl, PBS is
phosphate-buffered saline (pH 7), PEG is polyethylene glycol, Ph is
phenyl, Pnp is p-nitrophenyl, MC is 6-maleimidocaproyl, phe is
L-phenylalanine, PyBrop is bromo tris-pyrrolidino phosphonium
hexafluorophosphate, SEC is size-exclusion chromatography, Su is
succinimide, TFA is trifluoroacetic acid, TLC is thin layer
chromatography, UV is ultraviolet, and val is valine.
[0091] Cysteine Engineered Anti-TENB2 Antibodies
[0092] The compounds of the invention include cysteine engineered
anti-TENB2 antibodies where one or more amino acids of any form of
wild-type or parent anti-TENB2 antibody is replaced with a cysteine
amino acid. The engineered cysteine amino acid is a free cysteine
acid and not part of an intrachain or interchain disulfide unit.
Any form of anti-TENB2 antibody may be so engineered, i.e. mutated.
For example, a parent Fab antibody fragment may be engineered to
form a cysteine engineered Fab, referred to herein as "ThioFab."
Similarly, a parent monoclonal antibody may be engineered to form a
"ThioMab." It should be noted that a single site mutation yields a
single engineered cysteine residue in a ThioFab, while a single
site mutation yields two engineered cysteine residues in a ThioMab,
due to the dimeric nature of the IgG antibody. The cysteine
engineered anti-TENB2 antibodies of the invention include
monoclonal antibodies, humanized or chimeric monoclonal antibodies,
antigen-binding fragments of antibodies, fusion polypeptides and
analogs that preferentially bind cell-associated TENB2
polypeptides.
[0093] Cysteine engineered anti-TENB2 antibodies retain the antigen
binding capability of their wild type, parent anti-TENB2 antibody
counterparts. Thus, cysteine engineered anti-TENB2 antibodies are
capable of binding to TENB2 antigens.
[0094] A cysteine engineered anti-TENB2 antibody comprises one or
more free cysteine amino acids with reduced sulfhydryl (thiol)
groups wherein the cysteine engineered anti-TENB2 antibody binds to
a TENB2 polypeptide.
[0095] In one embodiment, the cysteine engineered anti-TENB2
antibody is prepared by a process comprising replacing one or more
amino acid residues of a parent anti-TENB2 antibody by
cysteine.
[0096] Mutants with replaced ("engineered") cysteine (Cys) residues
may be evaluated for the reactivity of the newly introduced,
engineered cysteine thiol groups. The thiol reactivity value is a
relative, numerical term in the range of 0 to 1.0 and can be
measured for any cysteine engineered antibody. Thiol reactivity
values of cysteine engineered antibodies of the invention may be in
the ranges of 0.6 to 1.0; 0.7 to 1.0; or 0.8 to 1.0.
[0097] In one aspect, the invention concerns an isolated cysteine
engineered anti-TENB2 antibody comprising an amino acid sequence
that is encoded by a nucleotide sequence that hybridizes to the
complement of a DNA molecule encoding (a) a cysteine engineered
antibody having a full-length amino acid sequence as disclosed
herein, (b) a cysteine engineered antibody amino acid sequence
lacking the signal peptide as disclosed herein, (c) an
extracellular domain of a transmembrane cysteine engineered
antibody protein, with or without the signal peptide, as disclosed
herein, (d) an amino acid sequence encoded by any of the nucleic
acid sequences disclosed herein or (e) any other specifically
defined fragment of a full-length cysteine engineered antibody
amino acid sequence as disclosed herein.
[0098] In one aspect, the invention provides an isolated cysteine
engineered anti-TENB2 antibody without the N-terminal signal
sequence and/or without the initiating methionine and is encoded by
a nucleotide sequence that encodes such an amino acid sequence as
described in. Processes for producing the same are also herein
described, wherein those processes comprise culturing a host cell
comprising a vector which comprises the appropriate encoding
nucleic acid molecule under conditions suitable for expression of
the cysteine engineered antibody and recovering the cysteine
engineered antibody from the cell culture.
[0099] Another aspect of the invention provides an isolated
cysteine engineered anti-TENB2 antibody which is either
transmembrane domain-deleted or transmembrane domain-inactivated.
Processes for producing the same are also herein described, wherein
those processes comprise culturing a host cell comprising a vector
which comprises the appropriate encoding nucleic acid molecule
under conditions suitable for expression of the cysteine engineered
antibody and recovering the cysteine engineered antibody from the
cell culture.
[0100] In other embodiments, the invention provides isolated
anti-TENB2 chimeric cysteine engineered antibodies comprising any
of the herein described cysteine engineered antibody fused to a
heterologous (non-TENB2) polypeptide. Examples of such chimeric
molecules comprise any of the herein described cysteine engineered
antibodies fused to a heterologous polypeptide such as, for
example, an epitope tag sequence or an Fc region of an
immunoglobulin.
[0101] The cysteine engineered anti-TENB2 antibody may be a
monoclonal antibody, antibody fragment, chimeric antibody,
humanized antibody, single-chain antibody or antibody that
competitively inhibits the binding of an anti-TENB2 polypeptide
antibody to its respective antigenic epitope. Antibodies of the
present invention may optionally be conjugated to a growth
inhibitory agent or cytotoxic agent such as a toxin, including, for
example, an auristatin, an antibiotic, a radioactive isotope, a
nucleolytic enzyme, or the like. The antibodies of the present
invention may optionally be produced in CHO cells or bacterial
cells and preferably inhibit the growth or proliferation of or
induce the death of a cell to which they bind. For diagnostic
purposes, the antibodies of the present invention may be detectably
labeled, attached to a solid support, or the like.
[0102] In other embodiments of the present invention, the invention
provides vectors comprising DNA encoding any of the herein
described cysteine engineered anti-TENB2 antibodies. Host cells
comprising any such vector are also provided. By way of example,
the host cells may be CHO cells, E. coli cells, or yeast cells. A
process for producing any of the herein described polypeptides is
further provided and comprises culturing host cells under
conditions suitable for expression of the desired polypeptide and
recovering the desired polypeptide from the cell culture.
[0103] Parent and cysteine engineered anti-TENB2 antibodies bind to
a TENB2 polypeptide or TENB2 polypeptide variant described in
PCT/US03/07209 U.S. Pat. No. 7,288,248).
[0104] A TENB2 polypeptide variant is a TENB2 polypeptide having at
least about 80% amino acid sequence identity with a TENB2 which is
a: (i) full-length native sequence; (ii) a polypeptide sequence
lacking the signal peptide; (iii) an extracellular domain, with or
without the signal peptide; (iv) or any other fragment of a
full-length TENB2 polypeptide sequence. Such TENB2 polypeptide
variants include, for instance, polypeptides wherein one or more
amino acid residues are added, or deleted, at the N- or C-terminus
of the full-length native amino acid sequence. Ordinarily, a TENB2
polypeptide variant will have at least about 80% amino acid
sequence identity, alternatively at least about 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% amino acid sequence identity, to a full-length native
sequence TENB2 polypeptide sequence, a TENB2 polypeptide sequence
lacking the signal peptide, an extracellular domain of a TENB2
polypeptide, with or without the signal peptide, or any other
specifically defined fragment of a full-length TENB2 polypeptide
sequence. Ordinarily, TENB2 polypeptide variants are at least about
10 amino acids in length, alternatively at least about 20, 30, 40,
50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,
190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,
320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,
450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,
580, 590, 600 amino acids in length, or more. Optionally, TENB2
variant polypeptides will have no more than one conservative amino
acid substitution as compared to the native TENB2 polypeptide
sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10
conservative amino acid substitution as compared to the native
TENB2 polypeptide sequence.
[0105] TENB2 polypeptides may be prepared by recombinant expression
in: (i) E. coli with pBR322 vector; (ii) mammalian cells such as
human HEK293 cells (ATCC CCL 1573), COS (simian fibroblast, SV-40)
cells, Chinese Hamster Ovary (CHO) cells with the pRK5 vector;
(iii) yeast, such as yeast strain AB110; or (iv)
baculovirus-infected insect cells (PCT/US03/07209; U.S. Pat. No.
7,288,248). Native or recombinant TENB2 polypeptides may be
purified by a variety of standard techniques in the art of protein
purification. For example, pro-TENB2 polypeptide, mature TENB2
polypeptide, or pre-TENB2 polypeptide is purified by immunoaffinity
chromatography using antibodies specific for the TENB2 polypeptide
of interest. In general, an immunoaffinity column is constructed by
covalently coupling the anti-TENB2 polypeptide antibody to an
activated chromatographic resin. TENB2 polypeptides may be produced
recombinantly as a fusion polypeptide with a heterologous
polypeptide, which may be a signal sequence or other polypeptide
having a specific cleavage site at the N-terminus of the mature
protein or polypeptide. Alternatively, TENB2 polypeptides may be
produced as fusion polypeptides with a signal sequence and a
heterologous polypeptide sequence that allows purification of the
TENB2 fusion polypeptide; examples of such polypeptides are
polyhistidine (His.sub.6 (SEQ ID NO: 24) or His.sub.8 (SEQ ID NO:
25)), human IgG Fc, the FLAG epitope (KDYKDDDDK (SEQ ID NO: 26)),
and the gD epitope (KYALADASLKMADPNRFRGKDLPVL (SEQ ID NO: 27)). The
signal sequence may be a component of the vector, or it may be a
part of the anti-TENB2 antibody- or TENB2 polypeptide-encoding DNA
that is inserted into the vector. The signal sequence may be a
prokaryotic signal sequence selected, for example, from the group
of the alkaline phosphatase, penicillinase, lpp, or heat-stable
enterotoxin II leaders. For yeast secretion the signal sequence may
be, e.g., the yeast invertase leader, alpha factor leader
(including Saccharomyces and Kluyveromyces .alpha.-factor leaders
(U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C.
albicans glucoamylase leader (EP 0362179), or the signal described
in WO 90/13646. In mammalian cell expression, mammalian signal
sequences may be used to direct secretion of the protein, such as
signal sequences from secreted polypeptides of the same or related
species, as well as viral secretory leaders.
[0106] A TENB2-expressing cell expresses an endogenous or
transfected TENB2 polypeptide antigen either on the cell surface or
in a secreted form. A TENB2-expressing cancer comprises cells that
have a TENB2 polypeptide present on the cell surface or that
produce and secrete a TENB2 antigenic polypeptide. A
TENB2-expressing cancer optionally produces sufficient levels of
TENB2 polypeptide on the surface of cells thereof, such that an
anti-TENB2 antibody, or antibody drug conjugate thereof, can bind
thereto and may exert a therapeutic effect with respect to the
cancer. A cancer which overexpresses a TENB2 polypeptide is one
which has significantly higher levels of TENB2 polypeptide at the
cell surface thereof, or produces and secretes, compared to a
noncancerous cell of the same tissue type. Such overexpression may
be caused by gene amplification or by increased transcription or
translation. TENB2 polypeptide overexpression may be determined in
a clinical setting by evaluating increased levels of the TENB2
protein present on the surface of a cell, or secreted by the cell
(e.g., via an immunohistochemistry assay using anti-TENB2
antibodies prepared against an isolated TENB2 polypeptide which may
be prepared using recombinant DNA technology from an isolated
nucleic acid encoding the TENB2 polypeptide; FACS analysis, etc.).
Alternatively, or additionally, one may measure levels of TENB2
polypeptide-encoding nucleic acid or mRNA in the cell, e.g., via
fluorescent in situ hybridization (FISH) using a nucleic acid based
probe corresponding to a TENB2-encoding nucleic acid or the
complement thereof; (WO 98/45479), Southern blotting, Northern
blotting, or polymerase chain reaction (PCR) techniques, such as
real time quantitative reverse-transcriptase PCR (qRT-PCR). One may
also detect TENB2 polypeptide overexpression by measuring shed
antigen in a biological fluid such as serum, e.g., using
antibody-based assays (U.S. Pat. No. 4,933,294; WO 91/05264; U.S.
Pat. No. 5,401,638; Sias et al (1990) J. Immunol. Methods
132:73-80). Various other in vivo assays may be contemplated.
Alternatively, cells within the body of the patient may be exposed
to an antibody which is optionally labeled with a detectable label,
e.g., a radioactive isotope, and binding of the antibody to cells
in the patient can be evaluated, e.g., by external scanning for
radioactivity or by analyzing a biopsy taken from a patient
previously exposed to the antibody.
[0107] Parent and cysteine engineered anti-TENB2 antibodies are
capable of binding, preferably specifically, to a TENB2 polypeptide
as described herein. TENB2 binding oligopeptides may be identified
without undue experimentation using well known techniques. In this
regard, it is noted that techniques for screening oligopeptide
libraries for oligopeptides that are capable of specifically
binding to a polypeptide target are well known in the art (U.S.
Pat. No. 5,556,762; U.S. Pat. No. 5,750,373; U.S. Pat. No.
4,708,871; U.S. Pat. No. 4,833,092; U.S. Pat. No. 5,223,409; U.S.
Pat. No. 5,403,484; U.S. Pat. No. 5,571,689; U.S. Pat. No.
5,663,143; WO 84/03506; WO84/03564; Geysen et al (1984) Proc. Natl.
Acad. Sci. USA, 81:3998-4002; Geysen et al (1985) Proc. Natl. Acad.
Sci. USA, 82:178-182; Geysen et al., in Synthetic Peptides as
Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth.,
102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616
(1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA,
87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832;
Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al.
(1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc.
Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current
Opin. Biotechnol., 2:668).
[0108] The parent and cysteine engineered anti-TENB2 antibodies of
the invention include polyclonal, monoclonal, humanized, human,
bispecific, and heteroconjugate antibodies. Various forms of a
humanized anti-TENB2 antibody are contemplated. For example, the
humanized antibody may be an antibody fragment, such as a Fab.
Alternatively, the humanized antibody may be an intact antibody,
such as an intact IgG1 antibody.
[0109] Bispecific anti-TENB2 antibodies are antibodies that have
binding specificities for at least two different epitopes.
Exemplary bispecific anti-TENB2 antibodies may bind to two
different epitopes of a TENB2 protein as described herein. Other
such antibodies may combine a TENB2 binding site with a binding
site for another protein. Alternatively, an anti-TENB2 arm may be
combined with an arm which binds to a triggering molecule on a
leukocyte such as a T-cell receptor molecule (e.g. CD3), or Fc
receptors for IgG (Fc.gamma.R), such as Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16), so as to focus and
localize cellular defense mechanisms to the TENB2-expressing cell.
Bispecific antibodies may also be used to localize cytotoxic agents
to cells which express TENB2. These antibodies possess a
TENB2-binding arm and an arm which binds the cytotoxic agent (e.g.,
saporin, anti-interferon-.alpha., vinca alkaloid, ricin A chain,
methotrexate or radioactive isotope hapten). Bispecific antibodies
can be prepared as full length antibodies or antibody fragments
(e.g., F(ab').sub.2 bispecific antibodies). Traditional production
of full length bispecific antibodies is based on the co-expression
of two immunoglobulin heavy chain-light chain pairs, where the two
chains have different specificities (Millstein et al (1983) Nature
305:537-539).
[0110] Heteroconjugate anti-TENB2 antibodies are also within the
scope of the present invention. Heteroconjugate antibodies are
composed of two covalently joined antibodies. Such antibodies have,
for example, been proposed to target immune system cells to
unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV
infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated
that the antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents.
[0111] The anti-TENB2 antibodies of the present invention can be
multivalent antibodies with three or more antigen binding sites
(e.g. tetravalent antibodies), which can be readily produced by
recombinant expression of nucleic acid encoding the polypeptide
chains of the antibody. The multivalent antibody can comprise a
dimerization domain and three or more antigen binding sites. The
preferred dimerization domain comprises (or consists of) an Fc
region or a hinge region. In this scenario, the antibody will
comprise an Fc region and three or more antigen binding sites
amino-terminal to the Fc region. The preferred multivalent antibody
herein comprises (or consists of) three to about eight, but
preferably four, antigen binding sites. The multivalent antibody
comprises at least one polypeptide chain (and preferably two
polypeptide chains), wherein the polypeptide chain(s) comprise two
or more variable domains. For instance, the polypeptide chain(s)
may comprise VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a
first variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino
acid or polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region
chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody
herein preferably further comprises at least two (and preferably
four) light chain variable domain polypeptides. The multivalent
antibody herein may, for instance, comprise from about two to about
eight light chain variable domain polypeptides. The light chain
variable domain polypeptides contemplated here comprise a light
chain variable domain and, optionally, further comprise a CL
domain.
[0112] The effector function of an anti-TENB2 antibody may be
modified by introducing one or more amino acid substitutions in an
Fc region. Such modification may enhance antigen-dependent
cell-mediated cyotoxicity (ADCC) and/or complement dependent
cytotoxicity (CDC) of the anti-TENB2 antibody. The homodimeric
antibody thus generated may have improved internalization
capability and/or increased complement-mediated cell killing and
antibody-dependent cellular cytotoxicity (ADCC). See Caron et al
(1992) J. Exp Med. 176:1191-1195 and Shopes, B. J. (1992) Immunol.
148:2918-2922. Homodimeric anti-TENB2 antibodies with enhanced
anti-tumor activity may also be prepared using heterobifunctional
cross-linkers as described in Wolff et al (1993) Cancer Research
53:2560-2565. Alternatively, an antibody can be engineered which
has dual Fc regions and may thereby have enhanced complement lysis
and ADCC capabilities (Stevenson et al (1989) Anti-Cancer Drug
Design 3:219-230).
[0113] The serum half life of an anti-TENB2 antibody may be
modulated by incorporating a salvage receptor binding epitope, e.g.
an antibody fragment (U.S. Pat. No. 5,739,277). As used herein, the
term "salvage receptor binding epitope" refers to an epitope of the
Fc region of an IgG molecule (e.g., IgG.sub.1, IgG.sub.2,
IgG.sub.3, or IgG.sub.4) that is responsible for increasing the in
vivo serum half-life of the IgG molecule.
[0114] Monoclonal antibodies binding to TENB2 epitopes, including
TMEFF2#19, are determined by standard competitive binding analysis
and epitope mapping (PCT/US03/07209; U.S. Pat. No. 7,288,248).
[0115] Immunohistochemistry analysis was performed using TMEFF2#19
monoclonal antibodies (PCT/US03/07209; Sambrook et al Molecular
Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press,
1989; Ausubel et al., Current Protocols of Molecular Biology, Unit
3.16, John Wiley and Sons, 1997). Monoclonal antibody TMEFF2#19
demonstarted weak to strong binding in 176 of 241 human prostate
cancer specimans.
[0116] Monoclonal antibody TMEFF2#19 becomes internalized into
cells to which it binds TENB2 polypeptide on the cell surface at a
rapid rate.
[0117] Modifications of Anti-TENB2 Antibodies
[0118] Modifications and variations in the anti-TENB2 antibodies
described herein, can be made, for example, using any of the
techniques and guidelines known in the art for conservative and
non-conservative mutations, for example, those in U.S. Pat. No.
5,364,934. Variations may be a substitution, deletion or insertion
of one or more codons encoding the antibody or polypeptide that
results in a change in the amino acid sequence as compared with the
native sequence anti-TENB2 antibody. Optionally the variation is by
substitution of at least one amino acid with any other amino acid
in one or more of the domains of the anti-TEMB2 antibody. The
variations can be made using methods known in the art such as
oligonucleotide-mediated (site-directed) mutagenesis, alanine
scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et
al (1986) Nucl. Acids Res., 13:4331; Zoller et al (1987) Nucl.
Acids Res., 10:6487), cassette mutagenesis (Wells et al (1985)
Gene, 34:315), restriction selection mutagenesis (Wells et al
(1986) Philos. Trans. R. Soc. London SerA, 317:415) or other known
techniques can be performed on the cloned DNA to produce the
anti-TENB2 antibody variant DNA. Amino acid changes may alter
post-translational processes of the anti-TENB2 antibody, such as
changing the number or position of glycosylation sites or altering
the membrane anchoring characteristics. Other modifications include
deamidation of glutaminyl and asparaginyl residues to the
corresponding glutamyl and aspartyl residues, respectively,
hydroxylation of proline and lysine, phosphorylation of hydroxyl
groups of seryl or threonyl residues, methylation of the
.alpha.-amino groups of lysine, arginine, and histidine side chains
(T. E. Creighton, Proteins: Structure and Molecular Properties,
(1983) W.H. Freeman & Co., San Francisco, pp. 79-86),
acetylation of the N-terminal amine, and amidation of any
C-terminal carboxyl group. Anti-TENB2 antibodies can be prepared by
introducing appropriate nucleotide changes into the encoding DNA,
and/or by chemical synthesis.
[0119] Anti-TENB2 antibody fragments may be truncated at the
N-terminus or C-terminus, or may lack internal residues, for
example, when compared with a full length anti-TENB2 antibody.
Certain fragments lack amino acid residues that are not essential
for a desired biological activity of the anti-TENB2 antibody.
Anti-TENB2 antibody fragments may be prepared by any of a number of
conventional techniques. Desired peptide fragments may be
chemically synthesized. An alternative approach involves generating
antibody fragments by enzymatic digestion, e.g., by treating the
protein with an enzyme known to cleave proteins at sites defined by
particular amino acid residues, or by digesting the DNA with
suitable restriction enzymes and isolating the desired fragment.
Yet another suitable technique involves isolating and amplifying a
DNA fragment encoding a desired antibody or fragment, by polymerase
chain reaction (PCR). Oligonucleotides that define the desired
termini of the DNA fragment are employed at the 5' and 3' primers
in the PCR. Preferably, anti-TENB2 antibody fragments share at
least one biological and/or immunological activity with the native
anti-TENB2 antibody disclosed herein.
[0120] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a humanized or human antibody. Generally, the resulting variant(s)
selected for further development will have improved biological
properties relative to the antibody from which they are generated.
A convenient way for generating such substitutional variants
involves affinity maturation using phage display. Briefly, several
hypervariable region sites (e.g., 6-7 sites) are mutated to
generate all possible amino substitutions at each site. The
antibody variants thus generated are displayed in a monovalent
fashion from filamentous phage particles as fusions to the gene III
product of M13 packaged within each particle. The phage-displayed
variants are then screened for their biological activity (e.g.,
binding affinity) as herein disclosed. In order to identify
candidate hypervariable region sites for modification, alanine
scanning mutagenesis can be performed to identify hypervariable
region residues contributing significantly to antigen binding.
Alternatively, or additionally, it may be beneficial to analyze a
crystal structure of the antigen-antibody complex to identify
contact points between the antibody and human TENB2 polypeptide.
Such contact residues and neighboring residues are candidates for
substitution according to the techniques elaborated herein. Once
such variants are generated, the panel of variants is subjected to
screening as described herein and antibodies with superior
properties in one or more relevant assays may be selected for
further development.
[0121] Another type of covalent modification of the anti-TENB2
antibody included within the scope of this invention comprises
altering the native glycosylation pattern of the antibody or
polypeptide by deleting one or more carbohydrate moieties found in
native sequence anti-TENB2 antibody (either by removing the
underlying glycosylation site or by deleting the glycosylation by
chemical and/or enzymatic means), and/or adding one or more
glycosylation sites that are not present in the native sequence
anti-TENB2 antibody. In addition, the modification includes
qualitative changes in the glycosylation of the native proteins,
involving a change in the nature and proportions of the various
carbohydrate moieties present. Glycosylation of antibodies and
other polypeptides is typically either N-linked or O-linked.
N-linked refers to the attachment of the carbohydrate moiety to the
side chain of an asparagine residue. The tripeptide sequences
asparagine-X-serine and asparagine-X-threonine, where X is any
amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-Linked glycosylation refers to the attachment of one of the
sugars N-acetylgalactosamine, galactose, or xylose to a
hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used. Addition of
glycosylation sites to the anti-TENB2 antibody is conveniently
accomplished by altering the amino acid sequence such that it
contains one or more of the above-described tripeptide sequences
(for N-linked glycosylation sites). The alteration may also be made
by the addition of, or substitution by, one or more serine or
threonine residues to the sequence of the anti-TENB2 antibody (for
O-linked glycosylation sites). The anti-TENB2 antibody amino acid
sequence may optionally be altered through changes at the DNA
level, particularly by mutating the DNA encoding the anti-TENB2
antibody at preselected bases such that codons are generated that
will translate into the desired amino acids.
[0122] Another means of increasing the number of carbohydrate
moieties on the anti-TENB2 antibody is by chemical or enzymatic
coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO 87/05330 published 11 Sep. 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0123] Removal of carbohydrate moieties present on the anti-TENB2
antibody may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding for amino acid residues
that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by
Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties can be achieved by the use of a variety of
endo- and exo-glycosidases as described by Thotakura et al (1987)
Meth. Enzymol. 138:350.
[0124] Another type of covalent modification of anti-TENB2 antibody
comprises linking the antibody or polypeptide to one of a variety
of nonproteinaceous polymers, e.g., polyethylene glycol (PEG),
polypropylene glycol, or polyoxyalkylenes, in the manner set forth
in U.S. Pat. No. 4,640,835; U.S. Pat. No. 4,496,689; U.S. Pat. No.
4,301,144; U.S. Pat. No. 4,670,417; U.S. Pat. No. 4,791,192 or U.S.
Pat. No. 4,179,337. The antibody or polypeptide also may be
entrapped in microcapsules prepared, for example, by coacervation
techniques or by interfacial polymerization (for example,
hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively), in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules), or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).
[0125] The anti-TENB2 antibody of the present invention may also be
modified in a way to form chimeric molecules comprising an
anti-TENB2 antibody fused to another, heterologous polypeptide or
amino acid sequence. In one embodiment, such a chimeric molecule
comprises a fusion of the anti-TENB2 antibody with a tag
polypeptide which provides an epitope to which an anti-tag antibody
can selectively bind. The epitope tag is generally placed at the
amino- or carboxyl- terminus of the anti-TENB2 antibody. The
presence of such epitope-tagged forms of the anti-TENB2 antibody
can be detected using an antibody against the tag polypeptide.
Also, provision of the epitope tag enables the anti-TENB2 antibody
to be readily purified by affinity purification using an anti-tag
antibody or another type of affinity matrix that binds to the
epitope tag. Various tag polypeptides and their respective
antibodies are well known in the art. Examples include
poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its antibody 12CA5 (Field et
al (1988) Mol. Cell. Biol., 8:2159-2165); the c-myc tag and the
8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al
(1985) Molecular and Cellular Biology, 5:3610-3616); and the Herpes
Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et
al (1990) Protein Engineering, 3(6):547-553). Other tag
polypeptides include the Flag-peptide (Hopp et al (1988)
BioTechnology 6:1204-1210); the KT3 epitope peptide (Martin et al
(1992) Science, 255:192-194); an .alpha.-tubulin epitope peptide
(Skinner et al (1991) J. Biol. Chem., 266:15163-15166); and the T7
gene 10 protein peptide tag (Lutz-Freyermuth et al (1990) Proc.
Natl. Acad. Sci. USA, 87:6393-6397).
[0126] In an alternative embodiment, the chimeric molecule may
comprise a fusion of the anti-TENB2 antibody with an immunoglobulin
or a particular region of an immunoglobulin. For a bivalent form of
the chimeric molecule (also referred to as an "immunoadhesin"),
such a fusion could be to the Fc region of an IgG molecule. The Ig
fusions preferably include the substitution of a soluble
(transmembrane domain deleted or inactivated) form of an anti-TENB2
antibody in place of at least one variable region within an Ig
molecule. In a particularly preferred embodiment, the
immunoglobulin fusion includes the hinge, CH.sub.2 and CH.sub.3, or
the hinge, CH.sub.1, CH.sub.2 and CH.sub.3 regions of an IgG1
molecule (U.S. Pat. No. 5,428,130).
[0127] Preparation of Anti-TENB2 Antibodies
[0128] DNA encoding an amino acid sequence variant of the cysteine
engineered anti-TENB2 antibodies and parent anti-TENB2 antibodies
of the invention is prepared by a variety of methods which include,
but are not limited to, isolation from a natural source (in the
case of naturally occurring amino acid sequence variants),
preparation by site-directed (or oligonucleotide-mediated)
mutagenesis (Carter (1985) et al Nucleic Acids Res. 13:4431-4443;
Ho et al (1989) Gene (Amst.) 77:51-59; Kunkel et al (1987) Proc.
Natl. Acad. Sci. USA 82:488; Liu et al (1998) J. Biol. Chem.
273:20252-20260), PCR mutagenesis (Higuchi, (1990) in PCR
Protocols, pp.177-183, Academic Press; Ito et al (1991) Gene
102:67-70; Bernhard et al (1994) Bioconjugate Chem. 5:126-132; and
Vallette et al (1989) Nuc. Acids Res. 17:723-733), and cassette
mutagenesis (Wells et al (1985) Gene 34:315-323) of an earlier
prepared DNA encoding the polypeptide. Mutagenesis protocols, kits,
and reagents are commercially available, e.g. QuikChange.RTM. Multi
Site-Direct Mutagenesis Kit (Stratagene, La Jolla, Calif.). Single
mutations are also generated by oligonucleotide directed
mutagenesis using double stranded plasmid DNA as template by PCR
based mutagenesis (Sambrook and Russel, (2001) Molecular Cloning: A
Laboratory Manual, 3rd edition; Zoller et al (1983) Methods
Enzymol. 100:468-500; Zoller, M. J. and Smith, M. (1982) Nucl.
Acids Res. 10:6487-6500). Variants of recombinant antibodies may be
constructed also by restriction fragment manipulation or by overlap
extension PCR with synthetic oligonucleotides. Mutagenic primers
encode the cysteine codon replacement(s). Standard mutagenesis
techniques can be employed to generate DNA encoding such mutant
cysteine engineered antibodies (Sambrook et al Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989; and Ausubel et al Current Protocols in
Molecular Biology, Greene Publishing and Wiley-Interscience, New
York, N.Y., 1993).
[0129] Phage display technology (McCafferty et al (1990) Nature
348:552-553) can be used to produce anti-TENB2 human antibodies and
antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell (Johnson et al (1993) Current Opinion in Structural
Biology 3:564-571; Clackson et al (1991) Nature, 352:624-628; Marks
et al (1991) J. Mol. Biol. 222:581-597; Griffith et al (1993) EMBO
J. 12:725-734; U.S. Pat. No. 5,565,332; U.S. Pat. No. 5,573,905;
U.S. Pat. No. 5,567,610; U.S. Pat. No. 5,229,275).
[0130] Anti-TENB2 antibodies may be chemically synthesized using
known oligopeptide synthesis methodology or may be prepared and
purified using recombinant technology. The appropriate amino acid
sequence, or portions thereof, may be produced by direct peptide
synthesis using solid-phase techniques (Stewart et al., Solid-Phase
Peptide Synthesis, (1969) W.H. Freeman Co., San Francisco, Calif.;
Merrifield, (1963) J. Am. Chem. Soc., 85:2149-2154). In vitro
protein synthesis may be performed using manual techniques or by
automation. Automated solid phase synthesis may be accomplished,
for instance, employing t-BOC or Fmoc protected amino acids and
using an Applied Biosystems Peptide Synthesizer (Foster City,
Calif.) using manufacturer's instructions. Various portions of the
anti-TENB2 antibody or TENB2 polypeptide may be chemically
synthesized separately and combined using chemical or enzymatic
methods to produce the desired anti-TENB2 antibody or TENB2
polypeptide.
[0131] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (Morimoto et al (1992)
Journal of Biochemical and Biophysical Methods 24:107-117; and
Brennan et al (1985) Science, 229:81), or produced directly by
recombinant host cells. Fab, Fv and ScFv anti-TENB2 antibody
fragments can all be expressed in and secreted from E. coli, thus
allowing the facile production of large amounts of these fragments.
Antibody fragments can be isolated from the antibody phage
libraries discussed herein. Alternatively, Fab'-SH fragments can be
directly recovered from E. coli and chemically coupled to form
F(ab').sub.2 fragments (Carter et al (1992) Bio/Technology
10:163-167), or isolated directly from recombinant host cell
culture. The anti-TENB2 antibody may be a (scFv) single chain Fv
fragment (WO 93/16185; U.S. Pat. No. 5,571,894; U.S. Pat. No.
5,587,458). The anti-TENB2 antibody fragment may also be a "linear
antibody" (U.S. Pat. No. 5,641,870). Such linear antibody fragments
may be monospecific or bispecific.
[0132] The description below relates primarily to production of
anti-TENB2 antibodies by culturing cells transformed or transfected
with a vector containing anti-TENB2 antibody-encoding nucleic acid.
DNA encoding anti-TENB2 antibodies may be obtained from a cDNA
library prepared from tissue believed to possess the anti-TENB2
antibody mRNA and to express it at a detectable level. Accordingly,
human anti-TENB2 antibody or TENB2 polypeptide DNA can be
conveniently obtained from a cDNA library prepared from human
tissue. The anti-TENB2 antibody-encoding gene may also be obtained
from a genomic library or by known synthetic procedures (e.g.,
automated nucleic acid synthesis).
[0133] Libraries can be screened with probes (such as
oligonucleotides of at least about 20-80 bases) designed to
identify the gene of interest or the protein encoded by it.
Screening the cDNA or genomic library with the selected probe may
be conducted using standard procedures, such as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual (New York:
Cold Spring Harbor Laboratory Press, 1989). An alternative means to
isolate the gene encoding anti-TENB2 antibody or TENB2 polypeptide
is PCR methodology (Sambrook et al., supra; Dieffenbach et al., PCR
Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
1995).
[0134] Host cells are transfected or transformed with expression or
cloning vectors described herein for anti-TENB2 antibody or TENB2
polypeptide production and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences. The culture conditions, such as media, temperature, pH
and the like, can be selected by the skilled artisan without undue
experimentation. In general, principles, protocols, and practical
techniques for maximizing the productivity of cell cultures can be
found in Mammalian Cell Biotechnology: a Practical Approach, M.
Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
[0135] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic
host cells include Enterobacteriaceae such as Escherichia, e.g., E.
coli, Enterobacter, Envinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than limiting.
Strain W3110 is an exemplary host strain for recombinant DNA
product fermentations. Preferably, the host cell secretes minimal
amounts of proteolytic enzymes. For example, strain W3110 may be
modified to effect a genetic mutation in the genes encoding
proteins endogenous to the host, with examples of such hosts
including E. coli W3110 strain 1A2, which has the complete genotype
tonA ; E. coli W3110 strain 9E4, which has the complete genotype
tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the
complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT
kan.sup.r; E. coli W3110 strain 37D6, which has the complete
genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG
kan.sup.r; E. coli W3110 strain 40B4, which is strain 37D6 with a
non-kanamycin resistant degP deletion mutation; and an E. coli
strain having mutant periplasmic protease (U.S. Pat. No.
4,946,783). Alternatively, in vitro methods of cloning, e.g., PCR
or other nucleic acid polymerase reactions, are suitable.
[0136] Full length antibody, antibody fragments, and antibody
fusion proteins can be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) and the immunoconjugate by itself shows effectiveness in
tumor cell destruction. Full length antibodies have greater half
life in circulation. Production in E. coli may be faster and more
cost efficient using, for example, expression of antibody fragments
and polypeptides in bacteria with translation initiation regio
(TIR) and signal sequences for optimizing expression and secretion
(U.S. Pat. No. 5,648,237; U.S. Pat. No. 5,789,199; U.S. Pat. No.
5,840,523). After expression, the antibody is isolated from the E.
coli cell paste in a soluble fraction and can be purified through,
e.g., a protein A or G column depending on the isotype. Final
purification can be carried out similar to the process for
purifying antibody expressed e.g., in CHO cells.
[0137] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-TENB2 antibody- or TENB2 polypeptide-encoding vectors.
Saccharomyces cerevisiae is a commonly used lower eukaryotic host
microorganism. Others include Schizosaccharomyces pombe (Beach and
Nurse, (1981) Nature, 290: 140; EP 139,383); Kluyveromyces hosts
(U.S. Pat. No. 4,943,529; Fleer et al (1991) Bio/Technology,
9:968-975) such as, e.g., K lactis (MW98-8C, CBS683, CBS4574;
Louvencourt et al (1983) J. Bacteriol., 154(2):737-742), K.
fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii
(ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC
36,906; Van den Berg et al (1990) Bio/Technology, 8:135), K.
thermotolerans, and K. marxianus; yarrowia (EP 402226); Pichia
pastoris (EP 183070; Sreekrishna et al (1988) J. Basic Microbiol.,
28:265-278); Candida; Trichoderma reesia (EP 244234); Neurospora
crassa (Case et al (1979) Proc. Natl. Acad. Sci. USA,
76:5259-5263); Schwanniomyces such as Schwanniomyces occidentalis
(EP 394538); and filamentous fungi such as, e.g., Neurospora,
Penicillium, Tolypocladium (WO 91/00357), and Aspergillus hosts
such as A. nidulans (Ballance et al (1983) Biochem. Biophys. Res.
Commun., 112:284-289; Tilburn et al (1983) Gene, 26:205-221; Yelton
et al (1984) Proc. Natl. Acad. Sci. USA, 81: 1470-1474) and A.
niger (Kelly and Hynes, (1985) EMBO J., 4:475-479). Methylotropic
yeasts are suitable herein and include, but are not limited to,
yeast capable of growth on methanol selected from the genera
consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,
Torulopsis, and Rhodotorula.
[0138] Suitable host cells for the expression of glycosylated
anti-TENB2 antibody or TENB2 polypeptide may also be derived from
multicellular organisms. Examples of invertebrate cells include
insect cells such as Drosophila S2 and Spodoptera Sf9, as well as
plant cells, such as cell cultures of cotton, corn, potato,
soybean, petunia, tomato, and tobacco. Numerous baculoviral strains
and variants and corresponding permissive insect host cells from
hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti
(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster
(fruitfly), and Bombyx mori have been identified. A variety of
viral strains for transfection are publicly available, e.g., the
L-1 variant of Autographa californica NPV and the Bm-5 strain of
Bombyx mori NPV, and such viruses may be used as the virus herein
according to the present invention, particularly for transfection
of Spodoptera frugiperda cells.
[0139] Examples of useful mammalian host cell lines are monkey
kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney line (293 or 293 cells subcloned for growth in
suspension culture, Graham et al (1977) J. Gen Virol. 36:59); baby
hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary
cells/-DHFR (CHO, Urlaub et al (1980) Proc. Natl. Acad. Sci. USA
77:4216); mouse sertoli cells (TM4, Mather (1980) Biol. Reprod.
23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green
monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical
carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells
(Mather et al (1982) Annals N.Y. Acad. Sci. 383:44-68); MRC 5
cells; FS4 cells; and a human hepatoma line (Hep G2).
[0140] Host cells are transformed with the above-described
expression or cloning vectors for anti-TENB2 antibody production
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences. The nucleic acid (e.g., cDNA
or genomic DNA) encoding anti-TENB2 antibody or TENB2 polypeptide
may be inserted into a replicable vector for cloning (amplification
of the DNA) or for expression. The vector may, for example, be in
the form of a plasmid, cosmid, viral particle, or phage. The
appropriate nucleic acid sequence may be inserted into the vector
by a variety of procedures.
[0141] Growth inhibition of tumor cells in vitro or in vivo can be
determined in various ways known in the art, such as inhibiting
cell proliferation of a TENB2-expressing tumor cell in vitro or in
vivo by about 25-100% compared to the untreated tumor cell, or by
about 30-100%, or by about 50-100% or 70-100%, in one embodiment,
at an antibody concentration of about 0.5 to 30 .mu.g/ml. The
growth inhibitory effects of an anti-TENB2 antibody in vitro may be
assessed by methods known in the art, e.g., using cells which
express a TENB2 polypeptide either endogenously or following
transfection with the TENB2 gene. For example, appropriate tumor
cell lines and TENB2-transfected cells may treated with an
anti-TENB2 monoclonal antibody at various concentrations for a few
days (e.g., 2-7) days and stained with crystal violet or MTT or
analyzed by some other colorimetric assay. A reduced signal
indicates growth inhibition. Another method of measuring
proliferation would be by comparing .sup.3H-thymidine uptake by the
cells treated in the presence or absence an anti-TENB2 antibody.
After treatment, the cells are harvested and the amount of
radioactivity incorporated into the DNA quantitated in a
scintillation counter. Inhibition of proliferation would be
demonstrated by a reduction of radioactivity. To select for an
anti-TENB2 antibody which induces cell death, loss of membrane
integrity as indicated by, e.g., propidium iodide (PI), trypan blue
or 7AAD uptake, may be assessed relative to control. Appropriate
positive controls include treatment of a selected cell line with a
growth inhibitory antibody known to inhibit growth of that cell
line. Growth inhibition can be measured at an antibody
concentration of about 0.5 to 30 .mu.g/ml or about 0.5 nM to 200 nM
in cell culture, where the growth inhibition is determined 1-10
days after exposure of the tumor cells to the antibody. The
antibody is growth inhibitory in vivo if administration of the
anti-TENB2 antibody at about 1 .mu.g/kg to about 100 mg/kg body
weight results in reduction in tumor size or reduction of tumor
cell proliferation within about 5 days to 3 months from the first
administration of the antibody, preferably within about 5 to 30
days.
[0142] Preparation of Cysteine Engineered Anti-TENB2 Antibodies
[0143] The design, selection, and preparation methods of the
invention enable cysteine engineered anti-TENB2 antibodies which
are reactive with electrophilic functionality. These methods
further enable antibody conjugate compounds such as antibody-drug
conjugate (ADC) compounds with drug molecules at designated,
designed, selective sites. Reactive cysteine residues on an
antibody surface allow specifically conjugating a drug moiety
through a thiol reactive group such as maleimide or haloacetyl. The
nucleophilic reactivity of the thiol functionality of a Cys residue
to a maleimide group is about 1000 times higher compared to any
other amino acid functionality in a protein, such as amino group of
lysine residues or the N-terminal amino group. Thiol specific
functionality in iodoacetyl and maleimide reagents may react with
amine groups, but higher pH (>9.0) and longer reaction times are
required (Garman, 1997, Non-Radioactive Labelling: A Practical
Approach, Academic Press, London). The amount of free thiol in a
protein may be estimated by the standard Ellman's assay.
Immunoglobulin M is an example of a disulfide-linked pentamer,
while immunoglobulin G is an example of a protein with internal
disulfide bridges bonding the subunits together. In proteins such
as this, reduction of the disulfide bonds with a reagent such as
dithiothreitol (DTT) or selenol (Singh et al (2002) Anal. Biochem.
304:147-156) is required to generate the reactive free thiol. This
approach may result in loss of antibody tertiary structure and
antigen binding specificity.
[0144] The PHESELECTOR (Phage ELISA for Selection of Reactive
Thiols) Assay allows for detection of reactive cysteine groups in
antibody-Fabs in an ELISA phage format thereby assisting in the
design of cysteine engineered antibodies (US 2007/0092940). The
antigen that binds to cysteine engineered antibody is coated on
well surfaces, followed by incubation with phage particles
displaying cysteine engineered Fabs, addition of HRP labeled
secondary antibody, and absorbance detection. Mutant proteins
displayed on phage may be screened in a rapid, robust, and
high-throughput manner. Libraries of cysteine engineered antibodies
can be produced and subjected to binding selection using the same
approach to identify appropriately reactive sites of free Cys
incorporation from random protein-phage libraries of antibodies or
other proteins. This technique includes reacting cysteine mutant
proteins displayed on phage with an affinity reagent or reporter
group which is also thiol-reactive.
[0145] The PHESELECTOR assay allows screening of reactive thiol
groups in antibodies. Identification of the A121C variant by this
method is exemplary. The entire Fab molecule may be effectively
searched to identify more ThioFab variants with reactive thiol
groups. A parameter, fractional surface accessibility, was employed
to identify and quantitate the accessibility of solvent to the
amino acid residues in a polypeptide. The surface accessibility can
be expressed as the surface area (.ANG..sup.2) that can be
contacted by a solvent molecule, e.g. water. The occupied space of
water is approximated as a 1.4 .ANG. radius sphere. Software is
freely available or licensable (Secretary to CCP4, Daresbury
Laboratory, Warrington, WA4 4AD, United Kingdom, Fax: (+44) 1925
603825) as the CCP4 Suite of crystallography programs which employ
algorithms to calculate the surface accessibility of each amino
acid of a protein with known x-ray crystallography derived
coordinates ("The CCP4 Suite: Programs for Protein Crystallography"
(1994) Acta. Cryst. D50:760-763). Two exemplary software modules
that perform surface accessibility calculations are "AREAIMOL" and
"SURFACE", based on the algorithms of B. Lee and F. M. Richards
(1971) J.Mol.Biol. 55:379-400. AREAIMOL defines the solvent
accessible surface of a protein as the locus of the centre of a
probe sphere (representing a solvent molecule) as it rolls over the
Van der Waals surface of the protein. AREAIMOL calculates the
solvent accessible surface area by generating surface points on an
extended sphere about each atom (at a distance from the atom centre
equal to the sum of the atom and probe radii), and eliminating
those that lie within equivalent spheres associated with
neighboring atoms. AREAIMOL finds the solvent accessible area of
atoms in a PDB coordinate file, and summarizes the accessible area
by residue, by chain and for the whole molecule. Accessible areas
(or area differences) for individual atoms can be written to a
pseudo-PDB output file. AREAIMOL assumes a single radius for each
element, and only recognizes a limited number of different
elements.
[0146] AREAIMOL and SURFACE report absolute accessibilities, i.e.
the number of square Angstroms (.ANG.). Fractional surface
accessibility is calculated by reference to a standard state
relevant for an amino acid within a polypeptide. The reference
state is tripeptide Gly-X-Gly, where X is the amino acid of
interest, and the reference state should be an `extended`
conformation, i.e. like those in beta-strands. The extended
conformation maximizes the accessibility of X. A calculated
accessible area is divided by the accessible area in a Gly-X-Gly
tripeptide reference state and reports the quotient, which is the
fractional accessibility. Percent accessibility is fractional
accessibility multiplied by 100. Another exemplary algorithm for
calculating surface accessibility is based on the SOLV module of
the program xsae (Broger, C., F. Hoffman-LaRoche, Basel) which
calculates fractional accessibility of an amino acid residue to a
water sphere based on the X-ray coordinates of the polypeptide. The
fractional surface accessibility for every amino acid in an
antibody may be calculated using available crystal structure
information (Eigenbrot et al. (1993) J Mol Biol. 229:969-995).
[0147] DNA encoding the cysteine engineered antibodies is readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells serve as a source of such DNA.
Once isolated, the DNA may be placed into expression vectors, which
are then transfected into host cells such as E. coli cells, simian
COS cells, Chinese Hamster Ovary (CHO) cells, or other mammalian
host cells, such as myeloma cells (U.S. Pat. No. 5,807,715; US
2005/0048572; US 2004/0229310) that do not otherwise produce the
antibody protein, to obtain the synthesis of monoclonal antibodies
in the recombinant host cells.
[0148] After design and selection, cysteine engineered antibodies,
e.g. ThioFabs, with the engineered, highly reactive unpaired Cys
residues, may be produced by: (i) expression in a bacterial, e.g.
E. coli, system (Skerra et al (1993) Curr. Opinion in Immunol.
5:256-262; Pluckthun (1992) Immunol. Revs. 130:151-188) or a
mammalian cell culture system (WO 01/00245), e.g. Chinese Hamster
Ovary cells (CHO); and (ii) purification using common protein
purification techniques (Lowman et al (1991) J. Biol. Chem.
266(17): 10982-10988).
[0149] The engineered Cys thiol groups react with electrophilic
linker reagents and drug-linker intermediates to form cysteine
engineered antibody drug conjugates and other labelled cysteine
engineered antibodies. Cys residues of cysteine engineered
antibodies, and present in the parent antibodies, which are paired
and form interchain and intrachain disulfide bonds do not have any
reactive thiol groups (unless treated with a reducing agent) and do
not react with electrophilic linker reagents or drug-linker
intermediates. The newly engineered Cys residue, can remain
unpaired, and able to react with, i.e. conjugate to, an
electrophilic linker reagent or drug-linker intermediate, such as a
drug-maleimide. Exemplary drug-linker intermediates include:
MC-MMAE, MC-MMAF, MC-vc-PAB-MMAE, and MC-vc-PAB-MMAF. The structure
positions of the engineered Cys residues of the heavy and light
chains are numbered according to a sequential numbering system.
This sequential numbering system is correlated to the Kabat
numbering system (Kabat et al., (1991) Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md.) starting at the N-terminus,
differs from the Kabat numbering scheme (bottom row) by insertions
noted by a,b,c. Using the Kabat numbering system, the actual linear
amino acid sequence may contain fewer or additional amino acids
corresponding to a shortening of, or insertion into, a FR or CDR of
the variable domain. The cysteine engineered heavy chain variant
sites are identified by the sequential numbering and Kabat
numbering schemes.
[0150] In one embodiment, the cysteine engineered anti-TENB2
antibody is prepared by a process comprising:
[0151] (a) replacing one or more amino acid residues of a parent
anti-TENB2 antibody by cysteine; and (b) determining the thiol
reactivity of the cysteine engineered anti-TENB2 antibody by
reacting the cysteine engineered antibody with a thiol-reactive
reagent.
[0152] The cysteine engineered antibody may be more reactive than
the parent antibody with the thiol-reactive reagent.
[0153] The free cysteine amino acid residues may be located in the
heavy or light chains, or in the constant or variable domains.
Antibody fragments, e.g. Fab, may also be engineered with one or
more cysteine amino acids replacing amino acids of the antibody
fragment, to form cysteine engineered antibody fragments.
[0154] Another embodiment of the invention provides a method of
preparing (making) a cysteine engineered anti-TENB2 antibody,
comprising:
[0155] (a) introducing one or more cysteine amino acids into a
parent anti-TENB2 antibody in order to generate the cysteine
engineered anti-TENB2 antibody; and (b) determining the thiol
reactivity of the cysteine engineered antibody with a
thiol-reactive reagent;
[0156] wherein the cysteine engineered antibody is more reactive
than the parent antibody with the thiol-reactive reagent.
[0157] Step (a) of the method of preparing a cysteine engineered
antibody may comprise:
[0158] mutagenizing a nucleic acid sequence encoding the cysteine
engineered antibody;
[0159] (ii) expressing the cysteine engineered antibody; and
[0160] (iii) isolating and purifying the cysteine engineered
antibody.
[0161] Step (b) of the method of preparing a cysteine engineered
antibody may comprise expressing the cysteine engineered antibody
on a viral particle selected from a phage or a phagemid
particle.
[0162] Step (b) of the method of preparing a cysteine engineered
antibody may also comprise:
[0163] (i) reacting the cysteine engineered antibody with a
thiol-reactive affinity reagent to generate an affinity labelled,
cysteine engineered antibody; and
[0164] (ii) measuring the binding of the affinity labelled,
cysteine engineered antibody to a capture media.
[0165] Another embodiment of the invention is a method of screening
cysteine engineered antibodies with highly reactive, unpaired
cysteine amino acids for thiol reactivity comprising:
[0166] (a) introducing one or more cysteine amino acids into a
parent antibody in order to generate a cysteine engineered
antibody;
[0167] (b) reacting the cysteine engineered antibody with a
thiol-reactive affinity reagent to generate an affinity labelled,
cysteine engineered antibody; and
[0168] (c) measuring the binding of the affinity labelled, cysteine
engineered antibody to a capture media; and
[0169] (d) determining the thiol reactivity of the cysteine
engineered antibody with the thiol-reactive reagent.
[0170] Step (a) of the method of screening cysteine engineered
antibodies may comprise:
[0171] (i) mutagenizing a nucleic acid sequence encoding the
cysteine engineered antibody;
[0172] (ii) expressing the cysteine engineered antibody; and
[0173] (iii) isolating and purifying the cysteine engineered
antibody.
[0174] Step (b) of the method of screening cysteine engineered
antibodies may comprise expressing the cysteine engineered antibody
on a viral particle selected from a phage or a phagemid
particle.
[0175] Step (b) of the method of screening cysteine engineered
antibodies may also comprise:
[0176] (i) reacting the cysteine engineered antibody with a
thiol-reactive affinity reagent to generate an affinity labelled,
cysteine engineered antibody; and
[0177] (ii) measuring the binding of the affinity labelled,
cysteine engineered antibody to a capture media.
[0178] Cysteine Engineering of TMEFF2#19 IgG Variants
[0179] Cysteine was introduced at the heavy chain 121 (sequential
numbering excluding the signal sequence) site into full-length,
humanized parent monoclonal anti-TENB2 TMEFF2#19 antibodies by the
cysteine engineering methods described herein to give A121C thio hu
anti-TENB2 TMEFF2#19 humanized variant with heavy chain sequence:
SEQ ID NO:1, and light chain sequence: SEQ ID NO:2, FIG. 1. These
cysteine engineered monoclonal antibodies were expressed in CHO
(Chinese Hamster Ovary) cells by transient fermentation in media
containing 1 mM cysteine.
[0180] According to one embodiment, humanized TMEFF2#19 cysteine
engineered anti-TENB2 antibodies comprise one or more of the
following variable region heavy chain sequences with a free
cysteine amino acid (Table 1).
TABLE-US-00001 TABLE 1 Comparison of heavy chain Sequential, Kabat
and Eu numbering for hu TMEFF2#19 cysteine engineered ant-TENB2
antibody variants Sequence near Sequential Kabat Eu Seq Cys
mutation Numbering Numbering Numbering I.D. DVQLCESGPG Q5C Q5C 8
LSLTCCVSGYS A23C A23C 9 LSSVTCADTAV A88C A84C 10 TLVTVCSASTK S119C
S112C 11 VTVSSCSTKGP A121C A114C A118C 12 VSSASCKGPSV T123C T116C
T120C 13 WYVDGCEVHNA V285C V278C V282C 14 KGFYPCDIAVE S378C S371C
S375C 15 PPVLDCDGSFF S403C S396C S400C 16
[0181] According to one embodiment, humanized TMEFF2#19 cysteine
engineered anti-TENB2 antibodies comprise one or more of the
following variable region light chain sequences with a free
cysteine amino acid (Table 2).
TABLE-US-00002 TABLE 2 Comparison of light chain Sequential and
Kabat numbering for hu TMEFF2#19 cysteine engineered ant-TENB2
antibody variants Sequence near Sequential Kabat Seq. Cys mutation
Numbering Numbering I.D. SLSASCGDRVT V15C V15C 17 EIKRTCAAPSV V110C
V110C 18 TVAAPCVFIFP S114C S114C 19 FIFPPCDEQLK S121C S121C 20
DEQLKCGTASV S127C S127C 21 VTEQDCKDSTY S168C S168C 22 GLSSPCTKSFN
V205C V205C 23
[0182] Labelled Cysteine Engineered Anti-TENB2 Antibodies
[0183] Cysteine engineered anti-TENB2 antibodies may be
site-specifically and efficiently coupled with a thiol-reactive
reagent. The thiol-reactive reagent may be a multifunctional linker
reagent, a capture, i.e. affinity, label reagent (e.g. a
biotin-linker reagent), a detection label (e.g. a fluorophore
reagent), a solid phase immobilization reagent (e.g. SEPHAROSE.TM.,
polystyrene, or glass), or a drug-linker intermediate. One example
of a thiol-reactive reagent is N-ethyl maleimide (NEM). In an
exemplary embodiment, reaction of a ThioFab with a biotin-linker
reagent provides a biotinylated ThioFab by which the presence and
reactivity of the engineered cysteine residue may be detected and
measured. Reaction of a ThioFab with a multifunctional linker
reagent provides a ThioFab with a functionalized linker which may
be further reacted with a drug moiety reagent or other label.
Reaction of a ThioFab with a drug-linker intermediate provides a
ThioFab drug conjugate.
[0184] The exemplary methods described here may be applied
generally to the identification and production of antibodies, and
more generally, to other proteins through application of the design
and screening steps described herein.
[0185] Such an approach may be applied to the conjugation of other
thiol-reactive reagents in which the reactive group is, for
example, a maleimide, an iodoacetamide, a pyridyl disulfide, or
other thiol-reactive conjugation partner (Haugland, 2003, Molecular
Probes Handbook of Fluorescent Probes and Research Chemicals,
Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2;
Garman, 1997, Non-Radioactive Labelling: A Practical Approach,
Academic Press, London; Means (1990) Bioconjugate Chem. 1:2;
Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San
Diego, pp. 40-55, 643-671). The thiol-reactive reagent may be a
drug moiety, a fluorophore such as a fluorescent dye like
fluorescein or rhodamine, a chelating agent for an imaging or
radiotherapeutic metal, a peptidyl or non-peptidyl label or
detection tag, or a clearance-modifying agent such as various
isomers of polyethylene glycol, a peptide that binds to a third
component, or another carbohydrate or lipophilic agent.
[0186] Uses of Cysteine Engineered Anti-TENB2 Antibodies
[0187] Cysteine engineered anti-TENB2 antibodies, and conjugates
thereof may find use as therapeutic and/or diagnostic agents. The
present invention further provides methods of preventing, managing,
treating or ameliorating one or more symptoms associated with a
TENB2 related disorder. In particular, the present invention
provides methods of preventing, managing, treating, or ameliorating
one or more symptoms associated with a cell proliferative disorder,
such as cancer, e.g., ovarian cancer, cervical cancer, uterine
cancer, pancreatic cancer, lung cancer and breast cancer. The
present invention still further provides methods for diagnosing a
TENB2 related disorder or predisposition to developing such a
disorder, as well as methods for identifying antibodies, and
antigen-binding fragments of antibodies, that preferentially bind
cell-associated TENB2 polypeptides.
[0188] Another embodiment of the present invention is directed to
the use of a cysteine engineered anti-TENB2 antibody for the
preparation of a medicament useful in the treatment of a condition
which is responsive to a TENB2 related disorder.
[0189] Cysteine Engineered Anti-TENB2 Antibody Drug Conjugates
[0190] Another aspect of the invention is an antibody-drug
conjugate compound comprising a cysteine engineered anti-TENB2
antibody (Ab), and an auristatin drug moiety (D) wherein the
cysteine engineered antibody is attached through one or more free
cysteine amino acids by a linker moiety (L) to D; the compound
having Formula I:
Ab-(L-D).sub.p (I)
where p is 1, 2, 3, or 4; and wherein the cysteine engineered
antibody is prepared by a process comprising replacing one or more
amino acid residues of a parent anti-TENB2 antibody by one or more
free cysteine amino acids.
[0191] FIG. 5 shows embodiments of cysteine engineered anti-TENB2
antibody drug conjugates (ADC) where an auristatin drug moiety is
attached to an engineered cysteine group in: the light chain
(LC-ADC); the heavy chain (HC-ADC); and the Fc region (Fc-ADC).
[0192] Potential advantages of cysteine engineered anti-TENB2
antibody drug conjugates include improved safety (larger
therapeutic index), improved PK parameters, the antibody interchain
disulfide bonds are retained which may stabilize the conjugate and
retain its active binding conformation, the sites of drug
conjugation are defined, and the preparation of cysteine engineered
antibody drug conjugates from conjugation of cysteine engineered
antibodies to drug-linker reagents results in a more homogeneous
product.
[0193] Drug Moieties
[0194] Auristatin drug moieties of the antibody-drug conjugates
(ADC) of Formula I include dolastatins, auristatins (U.S. Pat. No.
5,635,483; U.S. Pat. No. 5,780,588; U.S. Pat. No. 5,767,237; U.S.
Pat. No. 6,124,431), and analogs and derivatives thereof.
Dolastatins and auristatins have been shown to interfere with
microtubule dynamics, GTP hydrolysis, and nuclear and cellular
division (Woyke et al (2001) Antimicrob. Agents and Chemother.
45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and
antifungal activity (Pettit et al (1998) Antimicrob. Agents
Chemother. 42:2961-2965). Various forms of a dolastatin or
auristatin drug moiety may be covalently attached to an antibody
through the N (amino) terminus or the C (carboxyl) terminus of the
peptidic drug moiety (WO 02/088172; Doronina et al (2003) Nature
Biotechnology 21(7):778-784; Francisco et al (2003) Blood
102(4):1458-1465).
[0195] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, disclosed in:
WO 2005/081711; Senter et al, Proceedings of the American
Association for Cancer Research, Volume 45, Abstract Number 623,
presented Mar. 28, 2004, the disclosure of each which are expressly
incorporated by reference in their entirety. Exemplary auristatin
drug moieties include MMAE, and MMAF.
[0196] The auristatin drug moiety (D) of the antibody-drug
conjugates (ADC) of Formula I include the monomethylauristatin drug
moieties MMAE and MMAF. The N-terminus of the MMAE or MMAF drug
moiety is covalently attached via a linker to a engineered cysteine
of the antibody.
##STR00001##
[0197] Other exemplary auristatin drug moieties include
monomethylvaline compounds having phenylalanine carboxy
modifications at the C-terminus of the pentapeptide auristatin drug
moiety (WO 2007/008848) and monomethylvaline compounds having
phenylalanine sidechain modifications at the C-terminus of the
pentapeptide auristatin drug moiety (WO 2007/008603).
[0198] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry.
[0199] Linkers
[0200] "Linker", "Linker Unit", or "link" means a chemical moiety
comprising a covalent bond or a chain of atoms that covalently
attaches an antibody to a drug moiety. In various embodiments, a
linker is specified as L. A "Linker" (L) is a bifunctional or
multifunctional moiety which can be used to link one or more Drug
moieties (D) and an antibody unit (Ab) to form antibody-drug
conjugates (ADC) of Formula I. Antibody-drug conjugates (ADC) can
be conveniently prepared using a Linker having reactive
functionality for binding to the Drug and to the Antibody. A
cysteine thiol of a cysteine engineered antibody (Ab) can form a
bond with an electrophilic functional group of a linker reagent, a
drug moiety or drug-linker intermediate.
[0201] In one aspect, a Linker has a reactive site which has an
electrophilic group that is reactive to a nucleophilic cysteine
present on an antibody. The cysteine thiol of the antibody is
reactive with an electrophilic group on a Linker and forms a
covalent bond to a Linker. Useful electrophilic groups include, but
are not limited to, maleimide and haloacetamide groups.
[0202] Linkers include a divalent radical such as an alkyldiyl, an
arylene, a heteroarylene, moieties such as:
--(CR.sub.2).sub.nO(CR.sub.2).sub.n--, repeating units of alkyloxy
(e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g.
polyethyleneamino, Jeffamine.TM.); and diacid ester and amides
including succinate, succinamide, diglycolate, malonate, and
caproamide.
[0203] Cysteine engineered antibodies react with linker reagents or
drug-linker intermediates, with electrophilic functional groups
such as maleimide or .alpha.-halo carbonyl, according to the
conjugation method at page 766 of Klussman, et al (2004),
Bioconjugate Chemistry 15(4):765-773, and according to the protocol
of Example 3.
[0204] The linker may be composed of one or more linker components.
Exemplary linker components include 6-maleimidocaproyl ("MC"),
maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"),
alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxycarbonyl
("PAB"), N-succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"),
N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate
("SMCC"), N-Succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"),
ethyleneoxy --CH.sub.2CH.sub.2O-- as one or more repeating units
("EO" or "PEO"). Additional linker components are known in the art
and some are described herein.
[0205] In one embodiment, linker L of an ADC has the formula:
-A.sub.a-W.sub.w--Y.sub.y--
[0206] wherein:
[0207] -A- is a Stretcher unit covalently attached to a cysteine
thiol of the antibody (Ab);
[0208] a is 0 or 1;
[0209] each --W-- is independently an Amino Acid unit;
[0210] w is independently an integer ranging from 0 to 12;
[0211] --Y-- is a Spacer unit covalently attached to the drug
moiety; and
[0212] y is 0, 1 or 2.
[0213] Stretcher Unit
[0214] The Stretcher unit (-A-), when present, is capable of
linking an antibody unit to an amino acid unit (--W--). In this
regard an antibody (Ab) has a free cysteine thiol group that can
form a bond with an electrophilic functional group of a Stretcher
Unit. Exemplary stretcher units in Formula I conjugates are
depicted by Formulas II and III, wherein Ab-, --W--, --Y--, -D, w
and y are as defined above, and R.sup.17 is a divalent radical
selected from (CH.sub.2).sub.r, C.sub.3-C.sub.8 carbocyclyl,
O--(CH.sub.2).sub.r, arylene, (CH.sub.2).sub.r-arylene,
-arylene-(CH.sub.2).sub.r--, (CH.sub.2).sub.r--(C.sub.3-C.sub.8
carbocyclyl), (C.sub.3-C.sub.8 carbocyclyl)-(CH.sub.2).sub.r,
C.sub.3-C.sub.8 heterocyclyl, (CH.sub.2).sub.r--(C.sub.3-C.sub.8
heterocyclyl), --(C.sub.3-C.sub.8 heterocyclyl)-(CH.sub.2).sub.r--,
--(CH.sub.2).sub.rC(O)NR.sup.b(CH.sub.2).sub.r--,
--(CH.sub.2CH.sub.2O).sub.r--,
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--,
--(CH.sub.2).sub.rC(O)NR.sup.b(CH.sub.2CH.sub.2O).sub.r--,
--(CH.sub.2).sub.rC(O)NR.sup.b(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--,
--(CH.sub.2CH.sub.2O).sub.rC(O)NR.sup.b(CH.sub.2CH.sub.2O).sub.r--,
--(CH.sub.2CH.sub.2O).sub.rC(O)NR.sup.b(CH.sub.2CH.sub.2O).sub.r--CH.sub.-
2--, and --(CH.sub.2CH.sub.2O).sub.rC(O)NR.sup.b(CH.sub.2).sub.r--;
where R.sup.b is H, C.sub.1-C.sub.6 alkyl, phenyl, or benzyl; and r
is independently an integer ranging from 1-10.
[0215] Arylene includes divalent aromatic hydrocarbon radicals of
6-20 carbon atoms derived by the removal of two hydrogen atoms from
the aromatic ring system. Typical arylene groups include, but are
not limited to, radicals derived from benzene, substituted benzene,
naphthalene, anthracene, biphenyl, and the like.
[0216] Heterocyclyl groups include a ring system in which one or
more ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur.
The heterocycle radical comprises 1 to 20 carbon atoms and 1 to 3
heteroatoms selected from N, O, P, and S. A heterocycle may be a
monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to
3 heteroatoms selected from N, O, P, and S) or a bicycle having 7
to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms
selected from N, O, P, and S), for example: a bicyclo [4,5], [5,5],
[5,6], or [6,6] system. Heterocycles are described in Paquette, Leo
A.; "Principles of Modern Heterocyclic Chemistry" (W. A. Benjamin,
New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The
Chemistry of Heterocyclic Compounds, A series of Monographs" (John
Wiley & Sons, New York, 1950 to present), in particular Volumes
13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.
[0217] Examples of heterocycles include by way of example and not
limitation pyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl),
thiazolyl, tetrahydrothiophenyl, sulfur oxidized
tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl,
pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl,
indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl,
piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl,
pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,
tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,
tetrahydroisoquinolinyl, decahydroquinolinyl,
octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,
2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,
isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,
isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl,
phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,
cinnolinyl, pteridinyl, 4Ah-carbazolyl, carbazolyl,
.beta.-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,
phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl,
imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl,
isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl,
benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and
isatinoyl.
[0218] Carbocyclyl groups include a saturated or unsaturated ring
having 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms
as a bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, still
more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12
ring atoms, e.g. arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6]
system, or 9 or 10 ring atoms arranged as a bicyclo [5,6] or [6,6]
system. Examples of monocyclic carbocycles include cyclopropyl,
cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,
1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,
1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl, and
cyclooctyl.
[0219] It is to be understood from all the exemplary embodiments of
Formula I ADC such as II-V, that even where not denoted expressly,
from 1 to 4 drug moieties are linked to an antibody (p=1-4),
depending on the number of engineered cysteine residues.
##STR00002##
[0220] An illustrative Formula II Stretcher unit is derived from
maleimido-caproyl (MC) wherein R.sup.17 is
--(CH.sub.2).sub.5--:
##STR00003##
[0221] An illustrative Stretcher unit of Formula II, and is derived
from maleimido-propanoyl (MP) wherein R.sup.17 is
--(CH.sub.2).sub.2--:
##STR00004##
[0222] Another illustrative Stretcher unit of Formula II wherein
R.sup.17 is --(CH.sub.2CH.sub.2O).sub.r--CH.sub.2-- and r is 2:
##STR00005##
[0223] Another illustrative Stretcher unit of Formula II wherein
R.sup.17 is
--(CH.sub.2).sub.rC(O)NR.sup.b(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--
where R.sup.b is H and each r is 2:
##STR00006##
[0224] An illustrative Stretcher unit of Formula III wherein
R.sup.17 is --(CH.sub.2).sub.5--:
##STR00007##
[0225] In another embodiment, the Stretcher unit is linked to the
cysteine engineered anti-TENB2 antibody via a disulfide bond
between the engineered cysteine sulfur atom of the antibody and a
sulfur atom of the Stretcher unit. A representative Stretcher unit
of this embodiment is depicted by Formula IV, wherein R.sup.17,
Ab-, --W--, --Y--, -D, w and y are as defined above.
Ab-S S--R.sup.17--C(O)--W.sub.w--Y.sub.y-D).sub.p IV
[0226] In yet another embodiment, the reactive group of the
Stretcher contains a thiol-reactive functional group that can form
a bond with a free cysteine thiol of an antibody. Examples of
thiol-reaction functional groups include, but are not limited to,
maleimide, .alpha.-haloacetyl, activated esters such as succinimide
esters, 4-nitrophenyl esters, pentafluorophenyl esters,
tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl
chlorides, isocyanates and isothiocyanates. Representative
Stretcher units of this embodiment are depicted by Formulas Va and
Vb, wherein --R.sup.17--, Ab-, --W--, --Y--, -D, w and y are as
defined above;
Ab-S C(O)NH--R.sup.17--C(O)--W.sub.w--Y.sub.y-D).sub.p Va
Ab-S C(S)NH--R.sup.17--C(O)--W.sub.w--Y.sub.y-D).sub.p Vb
[0227] In another embodiment, the linker may be a dendritic type
linker for covalent attachment of more than one drug moiety through
a branching, multifunctional linker moiety to an antibody (Sun et
al (2002) Bioorganic & Medicinal Chemistry Letters
12:2213-2215; Sun et al (2003) Bioorganic & Medicinal Chemistry
11:1761-1768; King (2002) Tetrahedron Letters 43:1987-1990).
Dendritic linkers can increase the molar ratio of drug to antibody,
i.e. loading, which is related to the potency of the ADC. Thus,
where a cysteine engineered antibody bears only one reactive
cysteine thiol group, a multitude of drug moieties may be attached
through a dendritic linker.
[0228] Amino Acid Unit
[0229] The linker may comprise amino acid residues. The Amino Acid
unit (--W.sub.w--), when present, links the antibody (Ab) to the
drug moiety (D) of the cysteine engineered antibody-drug conjugate
(ADC) of the invention.
[0230] --W.sub.w-- is a dipeptide, tripeptide, tetrapeptide,
pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,
decapeptide, undecapeptide or dodecapeptide unit. Amino acid
residues which comprise the Amino Acid unit include those occurring
naturally, as well as minor amino acids and non-naturally occurring
amino acid analogs, such as citrulline. Each --W-- unit
independently has the formula denoted below in the square brackets,
and w is an integer ranging from 0 to 12:
##STR00008##
[0231] wherein R.sup.19 is hydrogen, methyl, isopropyl, isobutyl,
sec-butyl, benzyl, p-hydroxybenzyl, --CH.sub.2OH, --CH(OH)CH.sub.3,
--CH.sub.2CH.sub.2SCH.sub.3, --CH.sub.2CONH.sub.2, --CH.sub.2COOH,
--CH.sub.2CH.sub.2CONH.sub.2, --CH.sub.2CH.sub.2COOH,
--(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.3NH.sub.2,
--(CH.sub.2).sub.3NHCOCH.sub.3, --(CH.sub.2).sub.3NHCHO,
--(CH.sub.2).sub.4NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.4NH.sub.2,
--(CH.sub.2).sub.4NHCOCH.sub.3, --(CH.sub.2).sub.4NHCHO,
--(CH.sub.2).sub.3NHCONH.sub.2, --(CH.sub.2).sub.4NHCONH.sub.2,
--CH.sub.2CH.sub.2CH(OH)CH.sub.2NH.sub.2, 2-pyridylmethyl-,
3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,
##STR00009##
[0232] When R.sup.19 is other than hydrogen, the carbon atom to
which R.sup.19 is attached is chiral. Each carbon atom to which
R.sup.19 is attached is independently in the (S) or (R)
configuration, or a racemic mixture. Amino acid units may thus be
enantiomerically pure, racemic, or diastereomeric.
[0233] Exemplary --W.sub.w-- Amino Acid units include a dipeptide,
a tripeptide, a tetrapeptide or a pentapeptide. Exemplary
dipeptides include: valine-citrulline (vc or val-cit),
alanine-phenylalanine (af or ala-phe). Exemplary tripeptides
include: glycine-valine-citrulline (gly-val-cit) and
glycine-glycine-glycine (gly-gly-gly). Amino acid residues which
comprise an amino acid linker component include those occurring
naturally, as well as minor amino acids and non-naturally occurring
amino acid analogs, such as citrulline.
[0234] The Amino Acid unit can be enzymatically cleaved by one or
more enzymes, including a tumor-associated protease, to liberate
the Drug moiety (-D), which in one embodiment is protonated in vivo
upon release to provide a Drug (D). Amino acid linker components
can be designed and optimized in their selectivity for enzymatic
cleavage by a particular enzymes, for example, a tumor-associated
protease, cathepsin B, C and D, or a plasmin protease.
[0235] Spacer Unit
[0236] The Spacer unit (--Y.sub.y--), when present (y=1 or 2),
links an Amino Acid unit (--W.sub.w--) to the drug moiety (D) when
an Amino Acid unit is present (w=1-12). Alternately, the Spacer
unit links the Stretcher unit to the Drug moiety when the Amino
Acid unit is absent. The Spacer unit also links the drug moiety to
the antibody unit when both the Amino Acid unit and Stretcher unit
are absent (w, y=0). Spacer units are of two general types:
self-immolative and non self-immolative. A non self-immolative
Spacer unit is one in which part or all of the Spacer unit remains
bound to the Drug moiety after cleavage, particularly enzymatic, of
an Amino Acid unit from the antibody-drug conjugate or the Drug
moiety-linker. When an ADC containing a glycine-glycine Spacer unit
or a glycine Spacer unit undergoes enzymatic cleavage via a
tumor-cell associated-protease, a cancer-cell-associated protease
or a lymphocyte-associated protease, a glycine-glycine-Drug moiety
or a glycine-Drug moiety is cleaved from Ab-A.sub.a-Ww-. In one
embodiment, an independent hydrolysis reaction takes place within
the target cell, cleaving the glycine-Drug moiety bond and
liberating the Drug.
[0237] In another embodiment, --Y.sub.y-- is a
p-aminobenzylcarbamoyl (PAB) unit whose phenylene portion is
substituted with Q.sub.m wherein Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; and m is
an integer ranging from 0-4.
[0238] Exemplary embodiments of a non self-immolative Spacer unit
(--Y--) are: -Gly-Gly-; -Gly-; -Ala-Phe-; -Val-Cit-.
[0239] In one embodiment, a Drug moiety-linker or an ADC is
provided in which the Spacer unit is absent (y=0), or a
pharmaceutically acceptable salt or solvate thereof
[0240] Alternatively, an ADC containing a self-immolative Spacer
unit can release -D. In one embodiment, --Y-- is a PAB group that
is linked to --W.sub.w-- via the amino nitrogen atom of the PAB
group, and connected directly to -D via a carbonate, carbamate or
ether group, where the ADC has the exemplary structure:
##STR00010##
[0241] wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -halogen, -nitro or -cyano; m is an integer ranging from
0-4; and p ranges from 1 to 4.
[0242] Other examples of self-immolative spacers include, but are
not limited to, aromatic compounds that are electronically similar
to the PAB group such as 2-aminoimidazol-5-methanol derivatives
(Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237), heterocyclic
PAB analogs (US 2005/0256030), beta-glucuronide (WO 2007/011968),
and ortho or para-aminobenzylacetals. Spacers can be used that
undergo cyclization upon amide bond hydrolysis, such as substituted
and unsubstituted 4-aminobutyric acid amides (Rodrigues et al
(1995) Chemistry Biology 2:223), appropriately substituted
bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al (1972)
J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides
(Amsberry, et al (1990) J. Org. Chem. 55:5867). Elimination of
amine-containing drugs that are substituted at glycine (Kingsbury
et al (1984) J. Med. Chem. 27:1447) are also examples of
self-immolative spacer useful in ADCs.
[0243] Exemplary Spacer units (--Y.sub.y--) are represented by
Formulas X-XII:
##STR00011##
[0244] Dendritic Linkers
[0245] In another embodiment, linker L may be a dendritic type
linker for covalent attachment of more than one drug moiety through
a branching, multifunctional linker moiety to an antibody (Sun et
al (2002) Bioorganic & Medicinal Chemistry Letters
12:2213-2215; Sun et al (2003) Bioorganic & Medicinal Chemistry
11:1761-1768). Dendritic linkers can increase the molar ratio of
drug to antibody, i.e. loading, which is related to the potency of
the ADC. Thus, where a cysteine engineered antibody bears only one
reactive cysteine thiol group, a multitude of drug moieties may be
attached through a dendritic linker. Exemplary embodiments of
branched, dendritic linkers include 2,6-bis(hydroxymethyl)-p-cresol
and 2,4,6-tris(hydroxymethyl)-phenol dendrimer units (WO
2004/01993; Szalai et al (2003) J. Amer. Chem. Soc.
125:15688-15689; Shamis et al (2004) J. Amer. Chem. Soc.
126:1726-1731; Amir et al (2003) Angew. Chem. Int. Ed.
42:4494-4499).
[0246] In one embodiment, the Spacer unit is a branched
bis(hydroxymethyl)styrene (BHMS), which can be used to incorporate
and release multiple drugs, having the structure:
##STR00012##
[0247] comprising a 2-(4-aminobenzylidene)propane-1,3-diol
dendrimer unit (WO 2004/043493; de Groot et al (2003) Angew. Chem.
Int. Ed. 42:4490-4494), wherein Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; m is an
integer ranging from 0-4; n is 0 or 1; and p ranges ranging from 1
to 4.
[0248] Exemplary embodiments of the Formula I antibody-drug
conjugate compounds include XIIIa (MC), XIIIb (val-cit), XIIIc
(MC-val-cit), and XIIId (MC-val-cit-PAB):
##STR00013##
[0249] Other exemplary embodiments of the Formula Ia antibody-drug
conjugate compounds include XIVa-e:
##STR00014##
[0250] where R is independently H or C.sub.1-C.sub.6 alkyl; and n
is 1 to 12.
[0251] In another embodiment, a Linker has a reactive functional
group which has a nucleophilic group that is reactive to an
electrophilic group present on an antibody. Useful electrophilic
groups on an antibody include, but are not limited to, aldehyde and
ketone carbonyl groups. The heteroatom of a nucleophilic group of a
Linker can react with an electrophilic group on an antibody and
form a covalent bond to an antibody unit. Useful nucleophilic
groups on a Linker include, but are not limited to, hydrazide,
oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate,
and arylhydrazide. The electrophilic group on an antibody provides
a convenient site for attachment to a Linker.
[0252] Typically, peptide-type Linkers can be prepared by forming a
peptide bond between two or more amino acids and/or peptide
fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (E. Schroder and K.
Lubke (1965) "The Peptides", volume 1, pp 76-136, Academic Press)
which is well known in the field of peptide chemistry. Linker
intermediates may be assembled with any combination or sequence of
reactions including Spacer, Stretcher, and Amino Acid units. The
Spacer, Stretcher, and Amino Acid units may employ reactive
functional groups which are electrophilic, nucleophilic, or free
radical in nature. Reactive functional groups include, but are not
limited to carboxyls, hydroxyls, para-nitrophenylcarbonate,
isothiocyanate, and leaving groups, such as O-mesyl, O-tosyl, --Cl,
--Br, --I; or maleimide.
[0253] In another embodiment, the Linker may be substituted with
groups which modulated solubility or reactivity. For example, a
charged substituent such as sulfonate (--SO.sub.3.sup.-) or
ammonium, may increase water solubility of the reagent and
facilitate the coupling reaction of the linker reagent with the
antibody or the drug moiety, or facilitate the coupling reaction of
Ab-L (antibody-linker intermediate) with D, or D-L (drug-linker
intermediate) with Ab, depending on the synthetic route employed to
prepare the ADC.
[0254] Linker Reagents
[0255] Conjugates of the antibody and auristatin may be made using
a variety of bifunctional linker reagents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene).
[0256] The antibody drug conjugates may also be prepared with
linker reagents: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH,
SBAP, SIA, SIAB, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS,
sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate), and including
bis-maleimide reagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO).sub.3,
and BM(PEO).sub.4, which are commercially available from Pierce
Biotechnology, Inc., P.O. Box 117, Rockford, Ill. 61105, USA.
Bis-maleimide reagents allow the attachment of the thiol group of a
cysteine engineered antibody to a thiol-containing drug moiety,
label, or linker intermediate, in a sequential or concurrent
fashion. Other functional groups besides maleimide, which are
reactive with a thiol group of a cysteine engineered antibody, drug
moiety, label, or linker intermediate include iodoacetamide,
bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide,
isocyanate, and isothiocyanate.
##STR00015##
[0257] Useful linker reagents can also be obtained via other
commercial sources, such as Molecular Biosciences Inc. (Boulder,
Colo.), or synthesized in accordance with procedures described in
Toki et al (2002) J. Org. Chem. 67:1866-1872; Walker, M. A. (1995)
J. Org. Chem. 60:5352-5355; Frisch et al (1996) Bioconjugate Chem.
7:180-186; U.S. Pat. No. 6,214,345; WO 02/088172; US 2003130189;
US2003096743; WO 03/026577; WO 03/043583; and WO 04/032828.
[0258] Stretchers of formula (IIIa) can be introduced into a Linker
by reacting the following linker reagents with the N-terminus of an
Amino Acid unit:
##STR00016##
[0259] where n is an integer ranging from 1-10 and T is --H or
--SO.sub.3Na;
##STR00017##
[0260] where n is an integer ranging from 0-3;
##STR00018##
[0261] Stretcher units of can be introduced into a Linker by
reacting the following bifunctional reagents with the N-terminus of
an Amino Acid unit:
##STR00019##
[0262] where X is Br or I.
[0263] Stretcher units of formula can also be introduced into a
Linker by reacting the following bifunctional reagents with the
N-terminus of an Amino Acid unit:
##STR00020##
[0264] An exemplary valine-citrulline (val-cit or vc) dipeptide
linker reagent having a maleimide Stretcher and a
para-aminobenzylcarbamoyl (PAB) self-immolative Spacer has the
structure:
##STR00021##
[0265] An exemplary phe-lys(Mtr, mono-4-methoxytrityl) dipeptide
linker reagent having a maleimide Stretcher unit and a PAB
self-immolative Spacer unit can be prepared according to Dubowchik,
et al. (1997) Tetrahedron Letters, 38:5257-60, and has the
structure:
##STR00022##
[0266] Exemplary drug-linker intermediates include:
##STR00023##
[0267] Exemplary antibody-drug conjugate compounds of the invention
include:
##STR00024##
[0268] where Val is valine; Cit is citrulline; p is 1, 2, 3, or 4;
and Ab is a cysteine engineered anti-TENB2 antibody.
[0269] Preparation of Cysteine Engineered Anti-TENB2 Antibody-Drug
Conjugates
[0270] The ADC of Formula I may be prepared by several routes,
employing organic chemistry reactions, conditions, and reagents
known to those skilled in the art, including: (1) reaction of a
cysteine group of a cysteine engineered antibody with a linker
reagent, to form antibody-linker intermediate Ab-L, via a covalent
bond, followed by reaction with an activated drug moiety D; and (2)
reaction of a nucleophilic group of a drug moiety with a linker
reagent, to form drug-linker intermediate D-L, via a covalent bond,
followed by reaction with a cysteine group of a cysteine engineered
antibody. Conjugation methods (1) and (2) may be employed with a
variety of cysteine engineered antibodies, drug moieties, and
linkers to prepare the antibody-drug conjugates of Formula I.
[0271] Antibody cysteine thiol groups are nucleophilic and capable
of reacting to form covalent bonds with electrophilic groups on
linker reagents and drug-linker intermediates including: (i) active
esters such as NHS esters, HOBt esters, haloformates, and acid
halides; (ii) alkyl and benzyl halides, such as haloacetamides;
(iii) aldehydes, ketones, carboxyl, and maleimide groups; and (iv)
disulfides, including pyridyl disulfides, via sulfide exchange.
Nucleophilic groups on a drug moiety include, but are not limited
to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,
thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups
capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents.
[0272] Cysteine engineered antibodies may be made reactive for
conjugation with linker reagents by treatment with a reducing agent
such as DTT (Cleland's reagent, dithiothreitol) or TCEP
(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999)
Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.),
followed by reoxidation to reform interchain and intrachain
disulfide bonds (Example 2). For example, full length, cysteine
engineered monoclonal antibodies (ThioMabs) expressed in CHO cells
are reduced with about a 50 fold excess of TCEP for 3 hrs at
37.degree. C. to reduce disulfide bonds in cysteine adducts which
may form between the newly introduced cysteine residues and the
cysteine present in the culture media. The reduced ThioMab is
diluted and loaded onto HiTrap S column in 10 mM sodium acetate, pH
5, and eluted with PBS containing 0.3M sodium chloride. Disulfide
bonds were reestablished between cysteine residues present in the
parent Mab with dilute (200 nM) aqueous copper sulfate (CuSO.sub.4)
at room temperature, overnight. Alternatively, dehydroascorbic acid
(DHAA) is an effective oxidant to reestablish the intrachain
disulfide groups of the cysteine engineered antibody after
reductive cleavage of the cysteine adducts. Other oxidants, i.e.
oxidizing agents, and oxidizing conditions, which are known in the
art may be used. Ambient air oxidation is also effective. This
mild, partial reoxidation step forms intrachain disulfides
efficiently with high fidelity and preserves the thiol groups of
the newly introduced cysteine residues. An approximate 3 fold
excess of drug-linker intermediate, e.g. MC-vc-PAB-MMAE, relative
to antibody (about 1.5 fold excess relative to newly introduced
cysteine residues) was added, mixed, and let stand for about an
hour at room temperature to effect conjugation and form the
TMEFF2#19 anti-TENB2 antibody-drug conjugate. The conjugation
mixture was gel filtered and loaded and eluted through a HiTrap S
column to remove excess drug-linker intermediate and other
impurities.
[0273] FIG. 6 shows the general process to prepare a cysteine
engineered antibody expressed from cell culture for conjugation.
When the cell culture media contains cysteine, disulfide adducts
can form between the newly introduced cysteine amino acid and
cysteine from media. These cysteine adducts, depicted as a circle
in the exemplary ThioMab (left) in FIG. 6, must be reduced to
generate cysteine engineered antibodies reactive for conjugation.
Cysteine adducts, presumably along with various interchain
disulfide bonds, are reductively cleaved to give a reduced form of
the antibody with reducing agents such as TCEP. The interchain
disulfide bonds between paired cysteine residues are reformed under
partial oxidation conditions with copper sulfate, DHAA, or exposure
to ambient oxygen. The newly introduced, engineered, and unpaired
cysteine residues remain available for reaction with linker
reagents or drug-linker intermediates to form the antibody
conjugates of the invention. The ThioMabs expressed in mammalian
cell lines result in externally conjugated Cys adduct to an
engineered Cys through --S--S-- bond formation. Hence the purified
ThioMabs are treated with the reduction and reoxidation procedures
as described in Example 2 to produce reactive ThioMabs. These
ThioMabs are used to conjugate with maleimide containing cytotoxic
drugs, fluorophores, and other labels.
[0274] Analysis of cysteine engineered antibody drug conjugate
reactions show decreased heterogeneity relative to antibody drug
conjugates prepared by reduction of interchain or intrachain
disulfide bonds followed by conjugation (standard ADC) with a thiol
reactive drug linker intermediate.
[0275] Methods of Screening
[0276] Yet another embodiment of the present invention is directed
to a method of determining the presence of a TENB2 polypeptide in a
sample suspected of containing the TENB2 polypeptide, wherein the
method comprises exposing the sample to a cysteine engineered
anti-TENB2 antibody, or antibody drug conjugate thereof, that binds
to the TENB2 polypeptide and determining binding of the cysteine
engineered anti-TENB2 antibody, or antibody drug conjugate thereof,
to the TENB2 polypeptide in the sample, wherein the presence of
such binding is indicative of the presence of the TENB2 polypeptide
in the sample. Optionally, the sample may contain cells (which may
be cancer cells) suspected of expressing the TENB2 polypeptide. The
cysteine engineered anti-TENB2 antibody, or antibody drug conjugate
thereof, employed in the method may optionally be detectably
labeled, attached to a solid support, or the like.
[0277] Another embodiment of the present invention is directed to a
method of diagnosing the presence of a tumor in a mammal, wherein
the method comprises (a) contacting a test sample comprising tissue
cells obtained from the mammal with a cysteine engineered
anti-TENB2 antibody, or antibody drug conjugate thereof, that binds
to a TENB2 polypeptide and (b) detecting the formation of a complex
between the cysteine engineered anti-TENB2 antibody, or antibody
drug conjugate thereof, and the TENB2 polypeptide in the test
sample, wherein the formation of a complex is indicative of the
presence of a tumor in the mammal. Optionally, the cysteine
engineered anti-TENB2 antibody, or antibody drug conjugate thereof,
is detectably labeled, attached to a solid support, or the like,
and/or the test sample of tissue cells is obtained from an
individual suspected of having a cancerous tumor.
[0278] In Vitro Cell Proliferation Assays
[0279] One embodiment of the present invention is directed to a
method for inhibiting the growth of a cell that expresses a TENB2
polypeptide, wherein the method comprises contacting the cell with
a cysteine engineered anti-TENB2 antibody, or antibody drug
conjugate thereof, to the TENB2 polypeptide causes inhibition of
the growth of the cell expressing the TENB2. The cell may be a
cancer cell and binding of the cysteine engineered antibody, or
antibody drug conjugate thereof, to the TENB2 polypeptide causes
death of the cell expressing the TENB2 polypeptide.
[0280] Generally, the cytotoxic or cytostatic activity of an
antibody-drug conjugate (ADC) is measured by: exposing mammalian
cells expressing TENB2 polypeptide to ADC in a cell culture medium;
culturing the cells for a period from about 6 hours to about 5
days; and measuring cell viability. Mammalian cells useful for cell
proliferation assays include: (1) a TENB2 polypeptide-expressing
LuCaP77 tumor xenograft; (2) a PC3-derived cell line engineered to
stably express a portion of the TENB2 polypeptide on its cell
surface (PC3/TENB2); and (3) a PC3 cell line that does not express
TENB2 polypeptide (PC3/neo). Cell-based in vitro assays are used to
measure viability (proliferation), cytotoxicity, and induction of
apoptosis (caspase activation) of the ADC of the invention.
[0281] Pharmacokinetics--Serum Clearance and Stability
[0282] The disposition of the anti-TENB2 antibody-drug conjugates
in vivo was analyzed by measuring the serum concentrations of
antibody and of drug conjugate after a single intravenous bolus
dose into Sprague-Dawley rats. Concentrations of antibody-drug
conjugates bearing at least one cytotoxic drug were measured with
an ELISA that used anti-MMAE for the capture and biotinylated TENB2
extra-cellular domain (ECD) and streptavidin-horseradish peroxidase
(HRP) for detection. Total TMEFF2#19 and ThioTMEFF2#19
concentrations in serum were measured with an ELISA that used TENB2
ECD for capture and anti-.quadrature.human-Fc HRP as the secondary
antibody. This assay measured any anti-TENB2 antibody, both with
and without conjugated MMAE. The assays have lower limits of
quantitation of 16.4 ng/mL with a minimum dilution of 1:100. The
serum concentration-time data from each animal was analyzed using a
two-compartment model with IV bolus input, first-order elimination,
and macro-rate constants (Model 8, WinNonlin Pro v.5.0.1, Pharsight
Corporation, Mountain View, Calif.). Overall goodness of fit was
based on the predicted estimate, standard error for the prediction,
and percentage of coefficient of variation for primary and
secondary parameters, as well as inspection of residual plots
between observed and predicted concentration.quadrature.-time data.
Individual primary PK parameters comprised the zero-time intercepts
(A and B) associated with the alpha and beta phases, respectively,
and the micro-rate constants (alpha and beta). The following
modeling options were used: Initial estimates were determined using
WinNonlin; Concentrations were weighted by the reciprocal of the
predicted concentration squared (1/y.sup.2); Nelder-Mead
minimization algorithm was used. The following PK parameters were
reported: AUC.sub.0.quadrature.INF, CL, C.sub.max, MRT,
t.sub.1/2,a, t.sub.1/2,b, V.sub.1 and V.sub.ss.
[0283] Results of 28-day pharmacokinetics analyses in rats are
shown in FIG. 15. Rats were dosed with 5 mg/kg body weight of thio
TMEFF2#19-VC-MMAE or 5 mg/kg TMEFF2#19-VC-MMAE. Serum from rats was
collected at 5 minutes, 1 hour, 6 hours, 24 hours, and 2, 3, 4, 8,
11, 15, 21, and 28 days after dosing. Dose linearity of kinetics
was observed for ch TMEFF2#19-VC-MMAE between 0.5 and 5 mg/kg dose,
so the 5 mg/kg dose data have been arithmetically converted to
reflect the predicted data at 5 mg/kg for comparison with thio
TMEFF2#19-VC-MMAE.
[0284] Rodent Toxicity
[0285] The toxicity of cysteine engineered anti-TENB2 antibody-drug
conjugates was evaluated in an acute toxicity rat and cynomolgus
models. Toxicity of ADC was investigated by treatment of female
Sprague-Dawley rats and cynomolgus monkeys with the ADC and
subsequent inspection and analysis of the effects on various
organs. Based on gross observations (body weights), clinical
pathology parameters (serum chemistry and hematology) and
histopathology, the toxicity of ADC may be observed, characterized,
and measured. It was found that at equivalent dose levels, no
target-dependant effects appeared. Target-independent toxicities
were observed at doe that exceeded the efficacious doses in animal
tumor models.
Methods of Treatment
[0286] Another embodiment of the present invention is directed to a
method of therapeutically treating a mammal having a cancerous
tumor comprising cells that express a TENB2 polypeptide, wherein
the method comprises administering to the mammal a therapeutically
effective amount of a cysteine engineered antibody, or antibody
drug conjugate thereof, that binds to the TENB2 polypeptide,
thereby resulting in the effective therapeutic treatment of the
tumor.
[0287] Another embodiment of the present invention is directed to a
method for treating or preventing a cell proliferative disorder
associated with altered, preferably increased, expression or
activity of a TENB2 polypeptide, the method comprising
administering to a subject in need of such treatment an effective
amount of a cysteine engineered anti-TENB2 antibody, or antibody
drug conjugate thereof. An exemplary cell proliferative disorder is
cancer. Effective treatment or prevention of the cell proliferative
disorder may be a result of direct killing or growth inhibition of
cells that express a TENB2 polypeptide or by antagonizing the cell
growth potentiating activity of a TENB2 polypeptide with the
cysteine engineered anti-TENB2 antibody, or antibody drug conjugate
thereof.
[0288] Yet another embodiment of the present invention is directed
to a method of binding a cysteine engineered anti-TENB2 antibody,
or antibody drug conjugate thereof, to a cell that expresses a
TENB2 polypeptide, wherein the method comprises contacting a cell
that expresses a TENB2 polypeptide with said cysteine engineered
anti-TENB2 antibody, or antibody drug conjugate thereof, under
conditions which are suitable for binding of the cysteine
engineered anti-TENB2 antibody, or antibody drug conjugate thereof,
to said TENB2 polypeptide and allowing binding therebetween. In
preferred embodiments, the cysteine engineered anti-TENB2 antibody,
or antibody drug conjugate thereof, is labeled with a molecule or
compound that is useful for qualitatively and/or quantitatively
determining the location and/or amount of binding of the cysteine
engineered anti-TENB2 antibody, or antibody drug conjugate thereof,
to the cell.
[0289] Other embodiments of the present invention are directed to
the use of a cysteine engineered anti-TENB2 antibody, or antibody
drug conjugate thereof, in the preparation of a medicament useful
for (i) the therapeutic treatment or diagnostic detection of a
cancer or tumor, or (ii) the therapeutic treatment or prevention of
a cell proliferative disorder.
[0290] Another embodiment of the present invention is directed to a
method for inhibiting the growth of a cancer cell, wherein the
growth of said cancer cell is at least in part dependent upon the
growth potentiating effect(s) of a TENB2 polypeptide, wherein the
method comprises contacting the TENB2 polypeptide with a cysteine
engineered anti-TENB2 antibody, or antibody drug conjugate thereof,
thereby antagonizing the growth-potentiating activity of the TENB2
polypeptide and, in turn, inhibiting the growth of the cancer cell,
whereby the growth of the cancer cell is inhibited.
[0291] Another embodiment of the present invention is directed to a
method of therapeutically treating a tumor in a mammal, wherein the
growth of said tumor is at least in part dependent upon the growth
potentiating effect(s) of a TENB2 polypeptide, wherein the method
comprises administering to the mammal a therapeutically effective
amount of an anti-TENB2 cysteine engineered antibody, or antibody
drug conjugate thereof, that binds to the TENB2 polypeptide,
thereby antagonizing the growth potentiating activity of said TENB2
polypeptide and resulting in the effective therapeutic treatment of
the tumor.
[0292] The antibodies, antibody fragments, and conjugates thereof
recognize extracellular epitopes of plasma membrane TENB2 proteins
that are released into the extracellular fluid. The invention
further provides methods for the detection, monitoring and
treatment of malignancies such as breast cancer and ovarian cancer
using the antibodies, antibody fragments and conjugates.
[0293] Antibody-drug conjugates (ADC) of the present invention may
be used to treat various diseases or disorders, e.g. characterized
by the overexpression of a TENB2 tumor antigen. Exemplary
conditions or hyperproliferative disorders include benign or
malignant tumors including prostate cancer.
[0294] The ADC compounds which are identified in the animal models
and cell-based assays can be further tested in tumor-bearing higher
primates and human clinical trials. The clinical trial may be
designed to evaluate the efficacy of an ADC in combinations with
known therapeutic regimens, such as radiation and/or chemotherapy
involving known chemotherapeutic and/or cytotoxic agents.
[0295] Generally, the disease or disorder to be treated is a
hyperproliferative disease such as cancer. Examples of cancer to be
treated herein include, but are not limited to, carcinoma,
lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
More particular examples of such cancers include squamous cell
cancer (e.g. epithelial squamous cell cancer), lung cancer
including small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung and squamous carcinoma of the lung,
cancer of the peritoneum, hepatocellular cancer, gastric or stomach
cancer including gastrointestinal cancer, pancreatic cancer,
glioblastoma, cervical cancer, ovarian cancer, liver cancer,
bladder cancer, hepatoma, breast cancer, colon cancer, rectal
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney or renal cancer, prostate cancer,
vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma,
penile carcinoma, as well as head and neck cancer.
[0296] For the prevention or treatment of disease, the appropriate
dosage of an ADC will depend on the type of disease to be treated,
as defined above, the severity and course of the disease, whether
the molecule is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the antibody, and the discretion of the attending
physician. The molecule is suitably administered to the patient at
one time or over a series of treatments. Depending on the type and
severity of the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1-20
mg/kg) of molecule is an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. A typical
daily dosage might range from about 1 .mu.g/kg to 100 mg/kg or
more, depending on the factors mentioned above. An exemplary dosage
of ADC to be administered to a patient is in the range of about 0.1
to about 10 mg/kg of patient weight.
[0297] For repeated administrations over several days or longer,
depending on the condition, the treatment is sustained until a
desired suppression of disease symptoms occurs. An exemplary dosing
regimen comprises administering an initial loading dose of about 4
mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of an
anti-TENB2 antibody. Other dosage regimens may be useful. The
progress of this therapy is easily monitored by conventional
techniques and assays including ultrasound imaging.
[0298] Administration of Antibody-Drug Conjugates
[0299] The antibody-drug conjugates (ADC) of the invention may be
administered by any route appropriate to the condition to be
treated. The ADC will typically be administered parenterally, i.e.
infusion, subcutaneous, intraperitoneal, intramuscular,
intravenous, intradermal, intrathecal and epidural.
[0300] Pharmaceutical Formulations
[0301] Pharmaceutical formulations of therapeutic antibody-drug
conjugates (ADC) of the invention are typically prepared for
parenteral administration, i.e. bolus, intravenous, intratumor
injection with a pharmaceutically acceptable parenteral vehicle and
in a unit dosage, sterile injectable form. An antibody-drug
conjugate (ADC) having the desired degree of purity is optionally
mixed with pharmaceutically acceptable diluents, carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences
(1980) 16th edition, Osol, A. Ed.), in the form of a lyophilized
formulation or an aqueous solution.
[0302] Acceptable diluents, carriers, excipients, and stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0303] The active pharmaceutical ingredients may also be entrapped
in microcapsules prepared, for example, by coacervation techniques
or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[0304] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi permeable
matrices of solid hydrophobic polymers containing the ADC, which
matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0305] The formulations include those suitable for the foregoing
administration routes. The formulations may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well known in the art of pharmacy. Techniques and
formulations generally are found in Remington's Pharmaceutical
Sciences (Mack Publishing Co., Easton, Pa.). Such methods include
the step of bringing into association the active ingredient with
the carrier which constitutes one or more accessory ingredients. In
general the formulations are prepared by uniformly and intimately
bringing into association the active ingredient with liquid
carriers or finely divided solid carriers or both, and then, if
necessary, shaping the product.
[0306] Aqueous suspensions of the invention contain the active
materials in admixture with excipients suitable for the manufacture
of aqueous suspensions. Such excipients include a suspending agent,
such as sodium carboxymethylcellulose, croscarmellose, povidone,
methylcellulose, hydroxypropyl methylcelluose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethyleneoxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous
suspension may also contain one or more preservatives such as ethyl
or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or
more flavoring agents and one or more sweetening agents, such as
sucrose or saccharin.
[0307] The pharmaceutical compositions of ADC may be in the form of
a sterile injectable preparation, such as a sterile injectable
aqueous or oleaginous suspension. This suspension may be formulated
according to the known art using those suitable dispersing or
wetting agents and suspending agents which have been mentioned
above. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic parenterally
acceptable diluent or solvent, such as a solution in
1,3-butane-diol or prepared as a lyophilized powder. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile fixed oils may conventionally be employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid may likewise be used in
the preparation of injectables.
[0308] The amount of active ingredient that may be combined with
the carrier material to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. For example, an aqueous solution intended for
intravenous infusion may contain from about 3 to 500 .mu.g of the
active ingredient per milliliter of solution in order that infusion
of a suitable volume at a rate of about 30 mL/hr can occur.
[0309] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents.
[0310] Although oral administration of protein therapeutics are
disfavored due to hydrolysis or denaturation in the gut,
formulations of ADC suitable for oral administration may be
prepared as discrete units such as capsules, cachets or tablets
each containing a predetermined amount of the ADC.
[0311] The formulations may be packaged in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water, for
injection immediately prior to use. Extemporaneous injection
solutions and suspensions are prepared from sterile powders,
granules and tablets of the kind previously described. Preferred
unit dosage formulations are those containing a daily dose or unit
daily sub-dose, as herein above recited, or an appropriate fraction
thereof, of the active ingredient.
[0312] The compositions of the invention may also be formulated as
immunoliposomes. A "liposome" is a small vesicle composed of
various types of lipids, phospholipids and/or surfactant which is
useful for delivery of a drug to a mammal. The components of the
liposome are commonly arranged in a bilayer formation, similar to
the lipid arrangement of biological membranes. Liposomes containing
the antibody are prepared by methods known in the art, such as
described in Epstein et al (1985) Proc. Natl. Acad. Sci. USA
82:3688; Hwang et al (1980) Proc. Natl Acad. Sci. USA 77:4030; U.S.
Pat. No. 4,485,045; U.S. Pat. No. 4,544,545; U.S. Pat. No.
5,013,556; WO 97/38731. Liposomes can be generated by the reverse
phase evaporation method with a lipid composition comprising
phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes may be extruded
through filters of defined pore size to yield liposomes with the
desired diameter. Fab' fragments of the compositions of the present
invention can be conjugated to liposomes (Martin et al (1982) J.
Biol. Chem. 257:286-288), via a disulfide interchange reaction. A
chemotherapeutic agent is optionally contained within the liposome
(Gabizon et al (1989) J. National Cancer Inst. 81(19):1484.
[0313] Combination Therapy
[0314] An antibody-drug conjugate (ADC) of the invention may be
combined in a pharmaceutical combination formulation, or dosing
regimen as combination therapy, with a second compound having
anti-cancer properties. The second compound of the pharmaceutical
combination formulation or dosing regimen preferably has
complementary activities to the ADC of the combination such that
they do not adversely affect each other.
[0315] The second compound may be a chemotherapeutic agent,
cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal
agent, and/or cardioprotectant. Such molecules are suitably present
in combination in amounts that are effective for the purpose
intended. A pharmaceutical composition containing an ADC of the
invention may also have a therapeutically effective amount of a
chemotherapeutic agent such as a tubulin-forming inhibitor, a
topoisomerase inhibitor, a DNA intercalator, or a DNA binder.
[0316] Other therapeutic regimens may be combined with the
administration of an anticancer agent identified in accordance with
this invention. The combination therapy may be administered as a
simultaneous or sequential regimen. When administered sequentially,
the combination may be administered in two or more administrations.
The combined administration includes coadministration, using
separate formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities.
[0317] In one embodiment, treatment with an ADC involves the
combined administration of a cysteine engineered anti-TENB2
antibody or antibody-drug conjugate thereof, and one or more
chemotherapeutic agents, therapeutic biological, or growth
inhibitory agents, including coadministration of cocktails of
different chemotherapeutic agents. Chemotherapeutic agents include,
but are not limited to: taxanes (such as paclitaxel and docetaxel);
platinum-containing compounds, such as carboplatin; EGFR inhibitors
such as erlotinib, and gefitinib; tyrosine kinase inhibitors such
as imatinib; and anthracycline antibiotics (such as doxorubicin or
doxil). Therapeutic biological agents to be used in combination
with a cysteine engineered anti-TENB2 antibody or antibody-drug
conjugate thereof include bevacizumab (Avastin.RTM.) or pertuzumab
(Omnitarg.TM., Genentech Inc). Preparation and dosing schedules for
such chemotherapeutic agents may be used according to
manufacturer's instructions or as determined empirically by the
skilled practitioner. Preparation and dosing schedules for such
chemotherapy are also described in "Chemotherapy Service", (1992)
Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md.
[0318] The ADC may be combined with an anti-hormonal compound;
e.g., an anti-estrogen compound such as tamoxifen; an
anti-progesterone such as onapristone (EP 616812); or an
anti-androgen such as flutamide, in dosages known for such
molecules. Where the cancer to be treated is hormone independent
cancer, the patient may previously have been subjected to
anti-hormonal therapy and, after the cancer becomes hormone
independent, the ADC (and optionally other agents as described
herein) may be administered to the patient. It may be beneficial to
also coadminister a cardioprotectant (to prevent or reduce
myocardial dysfunction associated with the therapy) or one or more
cytokines to the patient. In addition to the above therapeutic
regimes, the patient may be subjected to surgical removal of cancer
cells and/or radiation therapy.
[0319] Suitable dosages for any of the above coadministered agents
are those presently used and may be lowered due to the combined
action (synergy) of the newly identified agent and other
chemotherapeutic agents or treatments.
[0320] The combination therapy may provide "synergy" and prove
"synergistic", i.e. the effect achieved when the active ingredients
used together is greater than the sum of the effects that results
from using the compounds separately. A synergistic effect may be
attained when the active ingredients are: (1) co-formulated and
administered or delivered simultaneously in a combined, unit dosage
formulation; (2) delivered by alternation or in parallel as
separate formulations; or (3) by some other regimen. When delivered
in alternation therapy, a synergistic effect may be attained when
the compounds are administered or delivered sequentially, e.g. by
different injections in separate syringes. In general, during
alternation therapy, an effective dosage of each active ingredient
is administered sequentially, i.e. serially, whereas in combination
therapy, effective dosages of two or more active ingredients are
administered together.
[0321] Metabolites of the Antibody-Drug Conjugates
[0322] Also falling within the scope of this invention are the in
vivo metabolic products of the ADC compounds described herein, to
the extent such products are novel and unobvious over the prior
art. Such products may result for example from the oxidation,
reduction, hydrolysis, amidation, esterification, enzymatic
cleavage, and the like, of the administered compound. Accordingly,
the invention includes novel and unobvious compounds produced by a
process comprising contacting a compound of this invention with a
mammal for a period of time sufficient to yield a metabolic product
thereof.
[0323] Metabolite products typically are identified by preparing a
radiolabelled (e.g. .sup.14C or .sup.3H) ADC, administering it
parenterally in a detectable dose (e.g. greater than about 0.5
mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to
man, allowing sufficient time for metabolism to occur (typically
about 30 seconds to 30 hours) and isolating its conversion products
from the urine, blood or other biological samples. These products
are easily isolated since they are labeled (others are isolated by
the use of antibodies capable of binding epitopes surviving in the
metabolite). The metabolite structures are determined in
conventional fashion, e.g. by MS, LC/MS or NMR analysis. In
general, analysis of metabolites is done in the same way as
conventional drug metabolism studies well-known to those skilled in
the art. The conversion products, so long as they are not otherwise
found in vivo, are useful in diagnostic assays for therapeutic
dosing of the ADC compounds of the invention.
Articles of Manufacture
[0324] In another embodiment of the invention, an article of
manufacture, or "kit", containing materials useful for the
treatment of the disorders described above is provided. The article
of manufacture comprises a container and a label or package insert
on or associated with the container. The package insert may refer
to instructions customarily included in commercial packages of
therapeutic products and that contain information about the
indications, usage, dosage, administration, contraindications
and/or warnings concerning the use of such therapeutic products.
Suitable containers include, for example, bottles, vials, syringes,
blister pack, etc. The containers may be formed from a variety of
materials such as glass or plastic.
[0325] In one embodiment, the article of manufacture comprises a
container and a formulation of a cysteine engineered anti-TENB2
antibody, or antibody-drug conjugate thereof, contained within the
container. The article may further optionally comprise a label
affixed to the container, or a package insert included with the
container, that refers to the use of the composition of matter for
the therapeutic treatment or diagnostic detection of a tumor. The
container holding the formulation is effective for storing and
delivering the therapeutic and may have a sterile access port (for
example the container may be an intravenous solution bag or a vial
having a stopper pierceable by a hypodermic injection needle). The
label or package insert indicates that the formulation is used for
treating the condition of choice, such as cancer. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0326] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0327] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0328] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Example 1
Preparation of Anti-TMEFF2#19 Antibodies
[0329] Humanized TMEFF2#19 antibodies were prepared according to
PCT/US03/07209 (U.S. Pat. No. 7,288,248). FIG. 1 shows the heavy
chain amino acid sequence (SEQ ID NO:1) and the ligh chain amino
acid sequence (SEQ ID NO:2).
Example 2
Preparation of Cysteine Engineered Anti-TENB2 Antibodies for
Conjugation by Reduction and Reoxidation
[0330] Full length, cysteine engineered anti-TENB2 monoclonal
antibodies (ThioMabs) expressed in CHO cells bear cysteine adducts
(cystines) on the engineered cysteines due to cell culture
conditions. To liberate the reactive thiol groups of the engineered
cysteines, the ThioMabs are dissolved in 500 mM sodium borate and
500 mM sodium chloride at about pH 8.0 and reduced with about a
50-100 fold excess of 1 mM TCEP (tris(2-carboxyethyl)phosphine
hydrochloride; Getz et al (1999) Anal. Biochem. Vol 273:73-80;
Soltec Ventures, Beverly, Mass.) for about 1-2 hrs at 37.degree. C.
The reduced ThioMab (FIG. 6) is diluted and loaded onto a HiTrap S
column in 10 mM sodium acetate, pH 5, and eluted with PBS
containing 0.3M sodium chloride. The eluted reduced ThioMab is
treated with 2 mM dehydroascorbic acid (dhAA) at pH 7 for 3 hours,
or 2 mM aqueous copper sulfate (CuSO.sub.4) at room temperature
overnight. Ambient air oxidation may also be effective. The buffer
is exchanged by elution over Sephadex G25 resin and eluted with PBS
with 1 mM DTPA. The thiol/Ab value is checked by determining the
reduced antibody concentration from the absorbance at 280 nm of the
solution and the thiol concentration by reaction with DTNB
(Aldrich, Milwaukee, Wis.) and determination of the absorbance at
412 nm.
Example 3
Conjugation of Cysteine Engineered Anti-TENB2 Antibodies and
Drug-Linker Intermediates
[0331] After the reduction and reoxidation procedures of Example 2,
the cysteine engineered anti-TENB2 antibody is dissolved in PBS
(phosphate buffered saline) buffer and chilled on ice. About 1.5
molar equivalents relative to engineered cysteines per antibody of
an auristatin drug linker intermediate, such as MC-MMAE
(maleimidocaproyl-monomethyl auristatin E), MC-MMAF,
MC-val-cit-PAB-MMAE, or MC-val-cit-PAB-MMAF, with a thiol-reactive
functional group such as maleimido, is dissolved in DMSO, diluted
in acetonitrile and water, and added to the chilled reduced,
reoxidized antibody in PBS. After about one hour, an excess of
maleimide is added to quench the reaction and cap any unreacted
antibody thiol groups. The reaction mixture is concentrated by
centrifugal ultrafiltration and the cysteine engineered anti-TENB2
antibody drug conjugate is purified and desalted by elution through
G25 resin in PBS, filtered through 0.2 .mu.m filters under sterile
conditions, and frozen for storage.
[0332] By the procedure above, the following cysteine engineered
anti-TENB2 antibody drug conjugates were prepared:
[0333] thio hu TMEFF2#19-MC-MMAF by conjugation of A114C (Kabat)
thio hu TMEFF2#19 and MC-MMAF; and
[0334] thio hu TMEFF2#19-MC-val-cit-PAB-MMAE by conjugation of
A114C (Kabat) thio hu TMEFF2#19 and MC-val-cit-PAB-MMAE.
Example 4
Materials and Methods for IHC, Internalization Studies, FACS, Cell
Killing Assays, Western Blots, Xenograft Studies, Pharmacokinetic
Studies and Safety Assessments
[0335] Antibodies and Recombinant Proteins: Humanized anti-tenb2
Mab PR1 was obtained from PDL. ThioMab anti-tenB2 PR1(HC-A121C;
sequential numbering) and tenB2ECD Flag protein were produced as
discussed above.
[0336] Cell Lines and Human Tumor Xenografts: PC3 is a human
prostate carcinoma cell line (ATCC). PC3TenB2 Medium stable cell
line was generated by Genentech. Human prostate explant models,
LuCap70, 77 and 96.1 were obtained from the University of
Washington.
[0337] RNA and Protein Expression: RNA expression analysis,
Immunological procedures (IHC, Western), antibody binding (FACS)
and internalization followed previously published methods (Cancer
research 64,781-788 (2004)).
[0338] Preparation of Conventional or ThioMab
Anti-TenB2-Valine-Citrulline(vc)-Monomethylauristatine E(MMAE) and
MC-MMAF Armed Drug Conjugated(ADC): The conjugation of
conventional, thio-mab and control mab with vc-MMAE, MC-MMAF ADC
was performed as described above.
[0339] In vitro Cell Killing and in vivo Studies: The cell killing
assay was done similar to as described in Cancer research
64,781-788 (2004). Each prostate explant model tumor cell line was
maintained by serial transplantation in castrated (androgen
independent model, LuCap70) or uncastrated (androgen dependent
model, LuCAP77 and LuCAP96.1), male SCID-beige mice from Charles
River lab. Tumors were measured once to twice per week for duration
of the study.
[0340] Rats and Cynomolgus Monkeys Models for Safety Assessment:
Anti-tenb2 Mab specifically recognized human, monkey and rats tenb2
target (FS I domain).
[0341] Pharmacokinetic Study: Standard protocol and assay methods
were used.
[0342] The data demonstrate that human TenB2 (TMEFF2) is generally
restricted to expression in the prostate and CNS, with
significantly elevated levels in cancerous prostate. Anti-TenB2
antibodies were also demonstrated to be rapidly internalized. These
antibodies, when conjugated to MMAE and MMAF were shown to kill
prostate tumor cells in vitro and in vivo in various cell killing
assays. Furthermore, efficacious doses of TENB2-ADCs were
significantly lower than those that are required to elicit toxic
effects in rodents and primates.
[0343] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
271469PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Met Ala Val Leu Gly Leu Leu Leu Cys Leu Val
Thr Phe Pro Ser Cys 1 5 10 15 Val Leu Ser Asp Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Leu
Thr Cys Ala Val Ser Gly Tyr Ser Ile 35 40 45 Thr Ser Gly Tyr Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly 50 55 60 Leu Glu Trp
Met Gly Phe Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Asn 65 70 75 80 Pro
Ser Leu Lys Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn 85 90
95 Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
100 105 110 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Asp Tyr Ser Met
Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215
220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val 275 280 285 Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340
345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460
Leu Ser Pro Gly Lys 465 2236PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 2Met Asp Phe Gln Val Gln
Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser
Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45
Gln Asn Val Val Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50
55 60 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg His Thr Gly
Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln 100 105 110 Tyr Ser Ser Tyr Pro Phe Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180
185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 225 230 235 3469PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 3Met Ala Val Leu Gly Leu Leu Leu Cys
Leu Val Thr Phe Pro Ser Cys 1 5 10 15 Val Leu Ser Asp Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu
Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile 35 40 45 Thr Ser Gly
Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly 50 55 60 Leu
Glu Trp Met Gly Phe Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Asn 65 70
75 80 Pro Ser Leu Lys Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys
Asn 85 90 95 Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Asp
Tyr Ser Met Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Cys Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195
200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val 210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315
320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala 340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440
445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460 Leu Ser Pro Gly Lys 465 4214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
4Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
5214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Gln Asn Val Val Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn
Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Phe 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe
Asn Arg Gly Glu Cys 210 6450PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 6Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met
Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305
310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425
430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445 Gly Lys 450 7450PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 7Asp Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu
Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45
Met Gly Phe Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Asn Pro Ser Leu 50
55 60 Lys Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe
Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Arg Arg Gly Asp Tyr Ser Met
Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405
410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro 435 440 445 Gly Lys 450 810PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Asp
Val Gln Leu Cys Glu Ser Gly Pro Gly 1 5 10 911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Leu
Ser Leu Thr Cys Cys Val Ser Gly Tyr Ser 1 5 10 1011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Leu
Ser Ser Val Thr Cys Ala Asp Thr Ala Val 1 5 10 1111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Thr
Leu Val Thr Val Cys Ser Ala Ser Thr Lys 1 5 10 1211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Val
Thr Val Ser Ser Cys Ser Thr Lys Gly Pro 1 5 10 1311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Val
Ser Ser Ala Ser Cys Lys Gly Pro Ser Val 1 5 10 1411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Trp
Tyr Val Asp Gly Cys Glu Val His Asn Ala 1 5 10 1511PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Lys
Gly Phe Tyr Pro Cys Asp Ile Ala Val Glu 1 5 10 1611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Pro
Pro Val Leu Asp Cys Asp Gly Ser Phe Phe 1 5 10 1711PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Ser
Leu Ser Ala Ser Cys Gly Asp Arg Val Thr 1 5 10 1811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Glu
Ile Lys Arg Thr Cys Ala Ala Pro Ser Val 1 5 10 1911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Thr
Val Ala Ala Pro Cys Val Phe Ile Phe Pro 1 5 10 2011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Phe
Ile Phe Pro Pro Cys Asp Glu Gln Leu Lys 1 5 10 2111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 21Asp
Glu Gln Leu Lys Cys Gly Thr Ala Ser Val 1 5 10 2211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 22Val
Thr Glu Gln Asp Cys Lys Asp Ser Thr Tyr 1 5 10 2311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Gly
Leu Ser Ser Pro Cys Thr Lys Ser Phe Asn 1 5 10 246PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6x His tag
24His His His His His His 1 5 258PRTArtificial SequenceDescription
of Artificial Sequence Synthetic 8x His tag 25His His His His His
His His His 1 5 269PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 26Lys Asp Tyr Lys Asp Asp Asp Asp Lys 1
5 2725PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Lys Tyr Ala Leu Ala Asp Ala Ser Leu Lys Met Ala
Asp Pro Asn Arg 1 5 10 15 Phe Arg Gly Lys Asp Leu Pro Val Leu 20
25
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