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Matched Legal Cases: ['Application No. 93', 'Application No. 96943576', 'Application No. 96943576', 'Application No. 96943576', 'Application No. 96943576', 'Application No. 96943576', 'Application No. 03018949', 'Application No. 96943576', 'Application No. 03018949', 'Application No. 96943576', 'Application No. 96943576', 'Application No. 96943576', 'Application No. 03', 'Application No. 12784', 'Application No. 12784', 'Application No. 96943576', 'Art. 101', 'Application No. 96943576', 'Application No. 2', 'Application No. 2', 'Application No. 03018949', 'Application No. 96943576', 'Application No. 96943576', 'Application No. 12784', 'Application No. 2', 'Application No. 2', 'Application No. 03018949', 'Application No. 03018949', 'Application No. 96943576', 'Application No. 03018949', 'Application No. 96943576', 'Application No. 96943576', 'Application No. 96943576', 'Application No. 96943576', 'Application No. 96943576', 'Application No. 96943576']

Patent US7811566 - Antibody-induced apoptosis - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsAnti-Her2 antibodies which induce apoptosis in Her2 expressing cells are disclosed. The antibodies are used to “tag” Her2 overexpressing tumors for elimination by the host immune system. Also disclosed are hybridoma cell lines producing the antibodies, methods for treating cancer using the antibodies,...http://www.google.com/patents/US7811566?utm_source=gb-gplus-sharePatent US7811566 - Antibody-induced apoptosisAdvanced Patent SearchPublication numberUS7811566 B2Publication typeGrantApplication numberUS 11/981,562Publication dateOct 12, 2010Priority dateDec 5, 1995Fee statusPaidAlso published asCA2236913A1, DE69630313D1, DE69630313T2, DE69630313T3, EP0865448A1, EP0865448B1, EP0865448B2, EP1375520A1, EP1375520B1, EP2298816A2, EP2298816A3, US5783186, US6458356, US7354583, US8444990, US20020164334, US20090142359, US20110189168, WO1997020858A1Publication number11981562, 981562, US 7811566 B2, US 7811566B2, US-B2-7811566, US7811566 B2, US7811566B2InventorsTsutomu Arakawa, Yoshiko KitaOriginal AssigneeAmgen, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (21), Non-Patent Citations (139), Referenced by (5), Classifications (25), Legal Events (1) External Links: USPTO, USPTO Assignment, EspacenetAntibody-induced apoptosis
US 7811566 B2Abstract
1. A method for treating cancer characterized by overexpression of Her2, in a patient, comprising administering to the patient an antibody or fragment thereof that binds an eptiope on Her2 which is recognized by a monoclonal antibody produced by hybridoma cell line ATCC No. HB 12078, wherein the antibody or fragment thereof is conjugated to a cytotoxic agent.
2. The method of claim 1, wherein the cytotoxic agent is selected from an A chain toxin, a ribosome inactivating protein, and a ribonuclease.
4. The method of claim 1, wherein the antibody or fragment thereof is a humanized antibody.
5. The method of claim 1, wherein the antibody or fragment thereof is a human antibody.
6. The method of claim 1, wherein the antibody or fragment thereof is a F(ab) or Fab′ fragment.
7. The method of claim 1, wherein the antibody or fragment thereof is produced by the hybridoma cell line ATCC No. HB 12078.
8. The method of claim 1, wherein the cancer is selected from breast cancer, ovarian cancer, prostate cancer, gastric cancer, and colorectal cancer.
9. The method of claim 1, wherein the overexpression of Her2 is at least 10% higher than a normal basal level.
10. The method of claim 1, wherein the overexpression of Her2 is at least 20% higher than a normal basal level.
11. The method of claim 1, wherein the overexpression of Her2 is at least 30% higher than a normal basal level.
12. The method of claim 1, wherein the antibody or fragment thereof is administered parenterally, subcutaneously, intravenously, or intramuscularly.
This application is a divisional of U.S. patent application Ser. No. 09/994,068, filed Nov. 27, 2001, now U.S. Pat. No. 7,354,583, which is a continuation of U.S. patent application Ser. No. 09/046,785, filed Mar. 23, 1998, now U.S. Pat. No. 6,458,356, which is a continuation of U.S. patent application Ser. No. 08/568,072, filed Dec. 5, 1995, now U.S. Pat. No. 5,783,186, all of which are incorporated herein by reference in their entirety.
Apoptosis, or programmed cell death, is a form of cell death characterized by cell shrinkage and DNA fragmentation. Collapse of the cell nucleus is apparent as chromation is fragmented into single or multiple mononucleosomal units, a process mediated by an endogenous endonuclease. Apoptosis is distinct from necrotic cell death which results in cell swelling and release of intracellular components (Kerr et al. Br. J. Cancer 26, 239-257 (1972); Wyllie et al. Int. Rev. Cytol. 68, 251-306 (1980); Wyllie Nature 284, 555-556 (1980)). Apoptotic cells, without releasing such components, are phagocytosed and hence degraded (Savill et al. Nature 343, 170-173 (1990)). Therefore, apoptosis results in an efficient process for elimination of nonviable cells by the host's own defense mechanisms.
It is an object of the invention to generate antibodies to Her2 which induce apoptosis in Her2 expressing cells and thereby “tag” such cells for removal from the host. The antibodies are useful for inducing apoptosis in tumors. This represents a substantial improvement over currently available antibody therapy for cancer which typically involves killing tumor cells by antibody in conjunction with a cytotoxic agent. Cytotoxic agents generally produce undesirable side effects which, if severe, can lead to a reduction or interruption of treatment. The present approach allows for killing of tumor cells by the host immune system, thereby avoiding the effects of cytotoxic agents and tumor cell necrosis induced by such agents.
FIG. 2. Her2 and Her3 tyrosine phosphorylation induced by mAb stimulation in SKBR3. SKBR3 cells were seeded in a 48-well plate for 5 min at 37� C. for 18 hours before mAb stimulation. Cells were solubilized with SDS sample buffer. Solubilized samples were electrophoresed on 6% polyacrylamide gels, followed by Western blotting and probing with anti-phosphotyrosine antibody. (a) All mAb concentrations were 250 nM in DMEM. 2 nM neu differentiation factor-α (NDFα) was used as a positive control. (b) mAb dose dependence of tyrosine phosphorylation.
FIG. 5. Cell morphologic change induced by mAbs. Cells (a-d, Her2/MCF7; e, f, MDAMB453) were grown in 1% FBS in culture media with or without mAb. After 5 days, cells were observed and photographed. (a, e) control (without mAb). (b) 250 nM mAb74. (c) 250 nM mAb83. (d) 250 nM mAb42b. (f) 100 nM mAb74.
FIG. 6. Detection of apoptotic cells with a modified TUNEL method. MDAMB453 (a-d) cells or Her2/MCF7 (e, f) cells were incubated with or without mAbs in 1% FBS culture media for one day followed by an apoptosis assay. (a, e) control (without mAb). (b) 50 nM mAb74. (c, f) 500 nM mAb74. (d) 500 nM mAb42b.
Monoclonal antibodies (mAbs) which bind to Her2 have been generated by immunizing mice with purified soluble Her2. Soluble Her2 was expressed and purified as described in Example 1. Mabs which bound to soluble Her2 in enzyme-linked immunosorbent assays (EIA) were subjected to dilution cloning and rescreening by EIA and BIAcore for binding to Her2 (Example 2). Ten clones were selected for further analysis. Purified antibodies from these clones were found to preferentially bind soluble Her2 and showed little or no binding to soluble Her3 and Her4. The biological effects of selected antibodies were studied for receptor dimerization, receptor phosphorylation and changes in cell physiology. All the antibodies tested formed 2:1 (receptor:antibody) complexes with Her2 (Example 4). Three different antibodies stimulated phosphorylation of Her2 and Her3 receptors on SKBR3 cells and Her2, Her3 and Her4 receptors on MDAMB453 cells. Phosphorylation of all receptors was inhibited by soluble Her2, suggesting that the ligand-like effects of the mabs are mediated directly through Her2.
Antibodies of the invention may be polyclonal or monoclonal or fragments thereof. Murine polyclonal and monoclonal antibodies are produced by standard immunological techniques. Antibody fragments encompass those antibodies which interact specifically with Her2 and induce apoptosis in cells and tissues expressing Her2. As indicated below in the examples, there is a correlation between apoptotic activity of mAb74 and Her2 receptor phosphorylation and dimerization. Therefore, it is preferred that the antibody fragments of the invention-retain their bivalent structure which is likely to promote receptor dimerization and activation. Also encompassed are antibodies made by recombinant means such as chimeric antibodies (variable region and constant region derived from different species) and CDR-grafted antibodies (complementary determining region derived from a different species) as described in U.S. Pat. Nos. 4,816,567 and 5,225,539. Preferably, the antibodies are at least partly of human origin. These include humanized antibodies, typically produced by recombinant methods, wherein the human sequences comprise part or all of the antibody. Also included are fully human antibodies produced in genetically-altered mice (see PCT Application No. 93/12227).
Thr Ser Asp Tyr Lys Asp Asp Asp Asp Asp Lys STOP (SEQ ID NO: 1)
This construct was transfected into CHOd-cells. Single cell clones were derived from the selected population and assayed for soluble Her2 production by both anti-FLAG and anti-Her2 Western blot analysis.
5′CCACCCGGGTTAGAGGAAGA 3′(SEQ ID NO: 2) and 5′-AGTTACGTTCTCTGGGCATTA-3′(SEQ ID NO: 3) were synthesized and used to screen the SKBR3 cDNA library filters. The hybridization was done in 6�SSC, 50 mM sodium-phosphate (pH 6.8), 0.1% sodium-pyrophosphate, 0.2% SDS, 2 mM EDTA, 2�Denhardt's solution and 50 mg/ml salmon sperm DNA at 42� C. for 16 hours. The filters were then washed at 42� C. with 2�SSC, 0.2% SDS, 2 mM EDTA for 30 minutes and exposed to X-ray films at −80� C. for 2 days.
5′CATGAGGGCGAACGACGCTCTG 3′(SEQ ID NO: 4) and 5′CTTGGTCAATGTCTGGCAGTC 3′(SEQ ID NO: 5) PCR was carried out for 40 cycles; with each cycle at 94� C., 30 seconds; 50� C., 30 seconds; and 72� C., 30 seconds. Three of the ten pools contained a full length Her3 cDNA. The three pools were rescreened by the colony hybridization procedure of Lin et al (Gene 44, 201-209. (1986)) until single clones were obtained from each pool. cDNA sequencing revealed a sequence identical to that published (Kraus et al., supra).
(SEQ ID NO: 6) Sense 5′CGCTCTAGACCACCATGAGGGCGAACGACGCTCTGCA 3′ (SEQ ID NO: 7) Antisense 5′CGCGGATCCGTCGACTCACTATGTCAGATGGGTTT TGCCGAT 3′ After digestion with the restriction enzymes XbaI and SalI, the 1.9 kb PCR fragment was subcloned into pDSRα2 (PCT Application No. WO91/05795) which had been cleaved with XbaI and SalI. The Her3 sequences in the resulting plasmid were confirmed by DNA sequencing. Plasmid pDSRα2/Her3 was used to transfect CHOd-cells for expression of soluble Her3.
A full-length Her4 cDNA clone was obtained by screening a human fetal brain cDNA library (Stratagene, San Diego, Calif.). Two Her4 cDNA probes were prepared by PCR amplification of human brain cDNA (Clontech Laboratories, Inc., Palo Alto, Calif.). cDNA probe-1 corresponds to the Her4 5′-end sequences encoding amino acid residues 32 to 177 and cDNA probe-2 corresponds to the Her4 3′-end sequences encoding amino acid residues 1137 to 1254. (Plowman et al., supra) Approximately 4�106 pfu of the human fetal brain cDNA library were screened sequentially with the Her4 5′-end probe and the Her4 3′-end probe. The hybridization solution contained 6�SSC, 50 mM sodium-phosphate (pH 6.8), 0.2% SDS, 2 mM EDTA, 0.1% sodium-pyrophosphate, 2�Denhardt's solution, 50 mg/ml salmon sperm DNA and 50% formamide. Hybridization was at 42� C. for 16 hours. The filters were washed at 67� C. with 2�SSC, 0.2% SDS, 2 mM EDTA for 60 minutes and then exposed to x-ray films at −8� C. over night. Autoradiography of the filters showed that 12 clones hybridized to the 5′-end probe and another 5 clones hybridized to the 3′-end probe. Single clones were purified by re-plating, screened by probe hybridizations as described above and positive clones sequenced.
All positive cDNA clones which were sequenced were found to be partial Her4 cDNA clones. The sequences were found to be identical to the published Her4 sequence (Plowman et al. supra) except for a short deletion/replacement in the extracellular domain. Amino acids 626 to 648 of the published Her3 sequence (NGPTSHDCIYYPWTGHSTLPQHA (SEQ ID NO: 10)) were replaced by the peptide sequence IGSSIEDCIGLMD (SEQ ID NO: 11). Also, G at amino acid position 573 of Plowman's sequence was replaced by D.
(SEQ ID NO: 8) 5′CCAAACATGACTGACTTCAGTG 3′ and (SEQ ID NO: 9) 5′GGCCAATTGCGGCCGCTTACTAATCCATCAGGCCGATGCAG TCTTC 3′ PCR was carried out for 25 cycles; with each cycle at 94� C., 30 seconds; 55� C., 30 seconds; and 72� C., 30 seconds. This 700 bp PCR product was purified by agarose gel electrophoresis. Plasmid pGEM4/Her4 was digested with Not I and BstE II to produce two fragments: one containing plasmid pGEM4 and the Her4 5′-end cDNA encoding the extracellular domain of the receptor from amino acid 1 to 420; and a second fragment spanning amino acid 421 of Her4 to the end of the Her4 molecule These two DNA fragments were separated in agarose gel and the pGEM4/HER45′-end fragment was recovered. The 700 bp Her4 PCR fragment was digested with BstE II and Not I and was ligated with the pGEM4/HER45′-end fragment. The resulting cDNA encodes the Her4 receptor extracellular domain spanning amino acid residues from 1 to 639. The PCR amplified portion was sequenced to confirm that no PCR errors has occurred.
The soluble Her4 cDNA construct was released from plasmid pGEM4, inserted into plasmid pDSRa2 and transfected into CHOd− cells using standard techniques (Maniatis et al., supra). Single cell clones were derived from the selected population and assayed for soluble Her4 production by BIAcore analysis.
Enzyme-Linked Immunosorbant Assay (EIA) 96-well plates were coated with 2 μg/ml sHer2, 2 μg/ml sHer3 or 2 μg/ml sHer4 in a carbonate-bicarbonate buffer. After blocking, hybridoma conditioned medium was added to the plate and incubated for 2 hours. The medium was aspirated and the plates were washed before addition of rabbit-anti-mouse IgG antibody conjugated with horseradish peroxidase (Boehringer Mannheim). After a one hour incubation, the plates were aspirated and washed five times. Bound antibody was detected with ABTS color reagent (Kirkegaard and Perry Labs., Inc.). The extent of antibody binding was determined by monitoring the increase in absorbance at 405 nm.
Cloning and IgG subtype determination. Single cell cloning was done in a 96-well plate using a limiting dilution method. Conditioned media of single cell clones were screened for antibody production using the EIA described above. The strongest antibody producing clones were chosen for cell growth expansion, subsequent subtype determination and competition studies.
BIAcore analysis. Purified sHer2, sHer3 or sHer4 were covalently coupled to a sensor chip CM5 via the primary amine group using 40 μl of the receptor in 10 mM Na acetate, pH 4.0 (10 μg receptor per ml). The unreacted groups on the sensor chip were blocked with an injection of 50 μl of 1 M ethanolamine hydrochloride (Pharmacia Biosensor AB). Each analysis cycle consisted of an injection of 40 μl of hybridoma supernatant (or purified mAbs), followed by injection of 10 μl of 10 mM HCl to regenerate the chip. Binding of the mAbs was detected by a change in SPR, measured in resonance units (RU). For most proteins, 1000 RU corresponds to a surface concentration of approximately 1 ng/mm2.
Preparation and Screening of Hybridoma Cell Lines. 7 balb/C mice were injected subcutaneously three times at three week intervals with 10 μg of soluble Her2. The protein was emulsified with RIBI adjuvant. Serum titers to Her2 were evaluated at 8 weeks, and the two mice with the highest titers were selected and given a final IV injection of 10 μg of soluble Her2. Three days later, the two mice were euthanized, and spleens removed, disrupted in a Stomacher tissue disintegrator, and filtered, and single cells were recovered. After three washes, the spleen cells were counted, mixed with mouse myeloma cells (SP2/0) in a ratio of 3:1 (spleen: SP2/0) and fused in the presence of 50% PEG (MW 1500). The fused cells were plated in a total of 10 96-well plates at a spleen cell concentration of 1.25�105 per well in a medium consisting of DMEM:RPMI (1:1), 10% FBS and 10% ORIGEN. Selection of fused cells was carried out in HAT selection medium. Culture media were screened by EIA for antibodies to Her2 after viable cell colonies occupied approximately 30% of the well. Sixty eight positives were identified from 960 wells. Cells from 43 wells were cloned by limiting dilution to produce single-cell colonies. Wells containing single colonies were marked and, when grown to 30% of well area, were assayed for anti-Her2 antibodies by EIA and BIAcore. The final number of single cell clones was 26, representing 20 original masterwells.
Binding of mAbs to sHer2, sHer3 and sHer4 Binding of mabs to sHer2 on a BIAcore chip was investigated using 10 μg/ml mAbs, and evaluated as resonance units (RU). As shown in Table I, two clones (52 and 58) showed greater than 1000 RU, 2 clones (35 and 42B) showed around 700 RU, 2 clones (43A and 74) showed around 300 RU, 2 clones (83 and 97) showed around 100 RU, and 2 clones (29 and 86) were less than 100 RU. The results indicated a wide range of affinity among the ten clones. No detectable binding of anti-sHer2 mAbs to sHer3 and sHer4 was observed. These results, along with the EIA data, confirm that the mAbs generated against sHer2 bind specifically to sHER2 with little or no binding to sHer3 and sHer4.
sHER2 BIA
sHER3 BIA
sHER4 BIA
sHER2 EIA
sHER3 EIA
sHER4 EIA
CORE RU OF
PLATE O.D. OF
Epitope Competition assay. The epitope specificity of anti-sHer2 mAbs was determined by binding pairs of monoclonal antibodies simultaneously to sHer2 immobilized on a BIAcore chip. mAbs directed against different epitopes should bind independently of each other, whereas mabs directed against closely related epitopes should interfere sterically with each other's binding. The first mAb was injected three times in a volume of 40 μl at a concentration of 10 μg/ml onto the immobilized sHer2 surface. A 40 μl of the second mAb was then injected and the ability to simultaneously bind to the sHer2 was evaluated. The biosensor surface was regenerated by the injection of 10 μl of 50 mM HCl. Binding was also analyzed when the injection sequence of each pair of mAbs was reversed. This analysis divided the mAbs into 4 different groups of epitope specificity, as shown in Table I. No correlation between epitope grouping and phosphorylation activity was apparent except for mAb74, which appears to have a unique epitope from the other mAbs.
Typically, antibodies have two binding sites for antigens, so it may be expected that antibodies which bind receptors can promote receptor dimerization. Size exclusion chromatography (SEC) with light scattering detection was used to determine the stoichiometry of anti-Her2 antibody binding to sHer2. The use of SEC with on-line light scattering has advantages over SEC alone for determining the molecular weight or stoichiometry of a protein complex. While the elution position of a protein or complex is indicative of molecular weight using conventional SEC, a light scattering measurement is independent of the elution position of a protein or a complex. In addition, the molecular weight from light scattering reflects only the polypeptide if the extinction coefficient of the polypeptide alone is used in the analysis. The on-line light scattering/size exclusion chromatography system uses three detectors in series: a light scattering detector (Wyatt Minidawn), a refractive index detector (Polymer Laboratories PL-RI), and a UV absorbance monitor at 280 nm (Knauer A293). A Superdex 200 (Pharmacia) SEC column equilibrated with Dulbecco's phosphate-buffered saline (PBS) and a 100 μl sample loop were used. The system was operated at a flow rate of 0.5 ml/min. The complexes of anti-sHer2 mAb and sHer2 were made by mixing 55 μl of 1.5 mg/ml mAb35, 0.8 mg/ml mAb52, 1.2 mg/ml mAb58, 1.6 mg/ml mAb42, 0.84 mg/ml mAb74, and 0.89 mg/ml mAb83 with 55 μl of 2.0, 2.0, 1.3, 2.0, 2.0, and 2.0 mg/ml sHer2, respectively. The complexes of the above mAbs and sHer3 were made in a similar way. 100 μl samples of each complex were injected onto a Superdex 200 column and the elution was monitored by light scattering, refractive index and UV absorbance detectors.
For a glycoprotein complex, the molecular weight of its polypeptide is proportional to (uv)(LS)/[ep(RI)2] (Takagi J. Chromatogr. 506, 409-446 (1990); Arakawa et al. Arch. Biochem. Biophs. 308, 267-273 (1994); Philo et al. J. Biol. Chem. 269, 27840-27846 (1994) where uv, LS, and RI are the signals from the absorbance, light scattering, and refractive index detectors, respectively, and ep is the extinction coefficient (the absorbance of a 1 mg/ml solution for 1 cm pathlength) of the polypeptide. For a complex with a known stoichiometry (AmBn), its extinction coefficient can be calculated with the equation εp=(m�εA�MA+n�εB�MB)/(m�MA+n�MB) where εA, εB, MA and MB are the polypeptide extinction coefficient and molecular weight of either protein A or B.
TABLE II Binding of mAb to sHer2 determined by SEC/light scattering ε Proteins or L Experimental Theoretical Correct Complexes g � cm MW � 10−3 MW � 10−3 Assumption? sHer2 0.85 69 mAb35 1.4 139 mAb52 1.4 151 mAb58 1.4 142 mAb42b 1.4 136 mAb74 1.4 145 mAb83 1.4 141 Assumption of sHer2-mAb35 Complex Stoichiometry: 1sHer2:1mAb35 1.24 237 208 No 2:1 1.14 261 277 Yes 3:1 1.08 275 346 No 1:2 1.31 226 347 No 1:3 1.41 208 486 No Assumption of sHer2-mAb52 Complex Stoichiometry: 1sHer2:1mAb52 1.24 252 220 No 2:1 1.14 275 289 Yes 3:1 1.08 289 358 No 1:2 1.31 240 371 No 1:3 1.41 223 522 No Assumption of sHer2-mAb58 Complex Stoichiometry 1sHer2:1mAb58 1.24 252 211 No 2:1 1.14 272 280 Yes 3:1 1.08 289 348 No 1:2 1.31 237 353 No 1:3 1.41 220 522 No Assumption of sHer2-mAb42b Complex Stoichiometry 1sHer2:1mAb42b 1.24 246 205 No 2:1 1.14 266 274 Yes 3:1 1.08 281 343 No 1:2 1.31 232 341 No 1:3 1.41 214 477 NO Assumption of sHer2-mAb74 Complex Stoichiometry 1sHer2:1mAb74 1.24 258 214 No 2:1 1.14 281 283 Yes 3:1 1.08 298 352 No 1:2 1.31 245 359 No 1:3 1.41 228 504 No *The molecular weights (MW) in the table reflect polypeptide only. The experimental molecular weights (excluding carbohydrate) for the complexes are most consistent with the theoretical values assuming 2 sHer2 per 1 mAb for each of the 5 mAbs tested. This proves that these antibodies could dimerize Her2 expressed on the cell surface. However, since the sHer2 and mAbs were mixed at 2:1, the observed results do not exclude the possibility of 1 sHer2:1 mAb complex formation when the mAb is present in excess. No complex was observed for sHer2 and mAb83 mixture. This may be caused by weak binding and complex dissociation during the chromatographic procedure. The samples containing sHer2 mAb at a 2:1 molar ratio eluted as a single peak, suggesting formation of 2 sHer2:1 mAb complex without dissociation during elution.
Receptor Phosphorylation by Anti-Her2 Antibodies
Cell morphologic change. Cells were seeded in 5 cm dishes to about 20% confluency and mAbs added after 18 hr. After 5 days, cells were observed with light microscopy, photographed, and counted.
Cell apoptosis. Cells were seeded in 8-well Chamber Slide (Nunc) to about 60-70% confluency and after 18 hr, culture media was changed to 1% FBS-containing media with or without mAb. On day one, cells were fixed with 4% neutral-buffered formalin (NBF) followed by three washes with PBS. After cells were dried, apoptosis was detected using a modified TUNEL method. TUNEL detects 3′-OH DNA ends generated by DNA fragmentation by labeling the ends with digoxigenin-conjugated dUTP using terminal deoxynucleotidyl transferal and then incubating with horseradish peroxidase (HRP)-conjugated anti-digoxigenin. Bound HRP was detected with the substrate, 3-amino-9-ethylcarbazole (Sigma). Most of the reagents were used from Apop Tag in situ apoptosis detection kit (Oncor). HRP-conjugated antibodies were from Boehringer Mannheim.
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USA, 87: 2569-2573 (1990).* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS8444990Oct 8, 2010May 21, 2013Amgen Inc.Antibody-induced apoptosisUS8637026 *Dec 23, 2008Jan 28, 2014Vaccinex, Inc.Anti-C35 antibody combination therapies and methodsUS9328024Apr 28, 2011May 3, 2016The Regents Of The University Of CaliforniaApplication of high toughness, low viscosity nano-molecular resin for reinforcing pothole patching materials in asphalt and concrete base pavementUS20110008322 *Dec 23, 2008Jan 13, 2011Vaccinex, Inc.Anti-c35 antibody combination therapies and methodsUS20110189168 *Oct 8, 2010Aug 4, 2011Tsutomu ArakawaAntibody-induced apoptosis* Cited by examinerClassifications U.S. Classification424/143.1, 424/142.1, 424/130.1, 424/141.1, 424/138.1International ClassificationC12N5/20, A61K39/00, C12R1/91, A61K38/00, A61P35/00, C07K14/82, A61P43/00, C12N15/02, A61K39/395, C12P21/08, C07K16/32, C12N5/10, C07K16/46Cooperative ClassificationC07K2319/00, C07K14/82, C07K16/32, A61K2039/505, A61K38/00European ClassificationC07K16/32, C07K14/82Legal EventsDateCodeEventDescriptionMar 12, 2014FPAYFee paymentYear of fee payment: 4RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services