Anti-CD43 antibody and use thereof for cancer treatment
11542339 · 2023-01-03
Assignee
Inventors
- Kwon Pyo Hong (Cheongju-si, KR)
- Sangsoon Yoon (Seoul, KR)
- Irene Koukoulas (Watsonia North, AU)
- Vincent Batori (Munich, DE)
- Briony Cristiano (Gaithersburg, MA, US)
- David S Wilson, Jr. (Redwood City, CA, US)
- George Kopsidas (Preston, AU)
Cpc classification
A61K39/395
HUMAN NECESSITIES
C07K2317/41
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
A61K38/16
HUMAN NECESSITIES
C07K2317/72
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
C07K2319/55
CHEMISTRY; METALLURGY
C07K2317/732
CHEMISTRY; METALLURGY
G01N2333/70596
PHYSICS
G01N2500/04
PHYSICS
C07K2317/24
CHEMISTRY; METALLURGY
G01N33/50
PHYSICS
A61K47/6803
HUMAN NECESSITIES
G01N33/57492
PHYSICS
C07K2317/34
CHEMISTRY; METALLURGY
A61K2300/00
HUMAN NECESSITIES
A61K47/6849
HUMAN NECESSITIES
C07K16/2896
CHEMISTRY; METALLURGY
A61K47/6825
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K39/395
HUMAN NECESSITIES
G01N33/50
PHYSICS
A61K47/68
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
Abstract
Provided is an antibody for treating a cancer, more specifically, an anti-CD43 antibody binding to an extracellular domain of CD43, compositions for treating a cancer or inhibiting a cancer stem cell comprising the antibody as an active ingredient, and methods for screening an agent of inhibiting a cancer stem cell.
Claims
1. A method of treating a cancer or inhibiting a cancer stem cell, comprising administering a pharmaceutically effective amount of an anti-CD43 antibody or an antigen-binding fragment thereof to a subject in need of treating the cancer or inhibiting a cancer stem cell, wherein the anti-CD43 antibody or the antigen-binding fragment thereof comprises: a CDR1H of SEQ ID NO: 111, a CDR2H of SEQ ID NO: 114, a CDR3H of SEQ ID NO: 118, a CDR1L of SEQ ID NO: 119, a CDR2L of SEQ ID NO: 122, 123, or 124, and a CDR3L of SEQ ID NO: 125.
2. The method of claim 1, wherein the anti-CD43 antibody or an antigen-binding fragment thereof comprises: a CDR1H of SEQ ID NO: 111, a CDR2H of SEQ ID NO: 114, a CDR3H of SEQ ID NO: 118, a CDR1L of SEQ ID NO: 119, a CDR2L of SEQ ID NO: 124, and a CDR3L of SEQ ID NO: 125.
3. The method of claim 1, wherein the cancer is a hematopoietic malignancy.
4. The method of claim 3, wherein the hematopoietic malignancy is acute myeloid leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.
5. The method of claim 1, wherein the cancer is a solid cancer.
6. The method of claim 5, wherein the solid cancer is gastric cancer, breast cancer, lung cancer, colon cancer, liver cancer, gallbladder cancer, renal cancer, pancreatic cancer, thyroid cancer, prostatic cancer, ovarian cancer, cervical cancer, or bladder cancer.
7. The method of claim 1, wherein the cancer stem cell is a cancer stem cell in hematopoietic malignancy or solid cancer.
8. The method of claim 1, wherein the anti-CD43 antibody or the antigen-binding fragment thereof is administered together with a cytotoxic material.
9. The method of claim 8, wherein the cytotoxic material is at least one selected from the group consisting of ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, pseudomonas toxin, radioisotopes, duocarmycin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)maytansine (DM1), and pyrrolobenzodiazepine (PBD) dimer.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION OF THE EMBODIMENTS
(47) Hereinafter, the present invention will be described in more detail with examples, but these examples are only for illustrative purpose and are not intended to limit the scope of the invention. It is obvious to a person skilled in the art that the examples described below may be modified without departing from the spirit of the invention.
Example 1. Preparation of Anti-CD43 Antibody
(48) 1-1. Preparation of Mouse Antibody
(49) 1-1-1. Preparation of Cell Producing Monoclonal Antibody
(50) It was prepared by the fusion of splenocyte of Balb/c white mouse in which a human thymocyte was injected as an antigen and myeloma cell line SP2/0-Ag14 (ATCC, CRL-1581) of 8-azaguanine resistant mouse.
(51) 10.sup.7 of human thymocytes (Seoul National University Hospital) were intraperitoneally injected into Balb/c white mouse per 2 weeks for 6 weeks to induce an immune response, and the spleen was extracted at 3 days after the last additional inoculation to prepare cell suspension. According to the method of Koeler & Milstein (1975), 10.sup.8 of splenocytes and 10.sup.7 of myeloma cells were under cell fusion by using 400 of polyethylene glycol. The fused cells were washed and then suspended in DMEM culture solution supplemented with 100 uM hypoxanthine, 0.44 uM animopterin and 16 uM thymidine (HAT culture solution), and cells were aliquoted in a 96-well plate, and cultured in a culture medium in which 37° C., 5% CO.sub.2 were supplied.
(52) When the formation of colony was observed after 2 weeks, the supernatant was collected and the antibody titer was measured by using immunohistochemistry and flowcytometry.
(53) The positive group meant the case in which 10.sup.5 or more of cells were formed per well. The monoclonal cells with high antibody titer were harvested by collecting cells in the well in which the colony with high antibody titer of supernatant, was formed and subcloned according to the limiting dilution assay method. The culture solution of monoclonal cells was stored by collecting the supernatant for the later experiments.
(54) 1-1-2. Screening of Monoclonal Cell Producing Antibody to Cell Surface Protein of Thymocyte
(55) Frozen tissues of thymus (Seoul National University Hospital) and paraffin embedded tissues (Seoul National University Hospital) were sectioned in 4 micrometer thickness to use. After passing the process of removing paraffin and then adding a normal goat serum (BioGenex company product), the paraffin embedded tissues were left for 1 hr at the room temperature. After administering each primary antibody (Dinona) to the tissues and then leaving them in 4° C. cold chamber overnight to react, they were washed 3 times with phosphate-buffered saline the next day. They were incubated for 1 hr at the room temperature with biotinylated goat anti-mouse immunoglobulin (2 drops, DAKO) as the second antibody, and then washed 3 times with phosphate-buffered saline, and the streptavidin-HRP conjugate was treated. After administering H.sub.2O.sub.2-aminoethyl carbazole solution for 20 min, they were washed 3 times with phosphate-buffered saline, and the color development was observed in the optical microscope.
(56) As a result, the monoclonal cell line H-JL1 producing an antibody which specifically reacts only to a human thymocyte could be sorted. The obtained cell line was donated to Korean Cell Line Research Foundation (KCLRF) located in Yeongeon-dong, Jongno-gu, Seoul, Korea in Jan. 13, 1997, and the accession number KCLRF-BP-00010 was given.
(57) The thymus tissues were immunohistochemically stained with the supernatant of the selected monoclonal cell line, and it was confirmed that the thymocyte was stained as positive (
(58) In order to confirm the reaction of the antibody in the development stage of thymocyte, the flow cytometry was carried out. The thymus of human extracted for heart surgery was cut into small fragments and grounded with a glass slide, and the thymocyte was collected. After reacting the antibody (1*10{circumflex over ( )}5 cell/antibody 10 μg/mL) for 30 min at 4° C. in the isolated thymocyte, it was washed with cold phosphate-buffered saline, and FITC (Fluorescein Isothiocyanate)-linked goat anti-mouse immunoglobulin antibody (Jackson ImmunoResearch) was reacted for 30 min at 4° C. It was then washed with cold phosphate-buffered saline and 5 μL of each PE (phycoerythrin)-linked anti-CD8 antibody (BD Bioscience) and APC-linked anti-CD4 antibody (BD Bioscience) were added and reacted for 30 min at 4° C. It was washed with cold phosphate-buffered saline and the flow cytometry was conducted by using FACSCalibur (Becton Dickinson, Mountain View, Calif.). The thymocytes were classified to CD4-CD8-thymocyte.fwdarw.CD4+CD8+ thymocyte.fwdarw.CD4+CD8- or CD4-CD8+ thymocyte according to the expression aspect of CD4 and CD8, and the antibody reacted to all 4 kinds of thymocytes, but in particular, it reacted to CD4+CD8+ thymocyte highly (refer to
(59) 1-1-3. Production of Monoclonal Antibody from Sorted Monoclonal Cell Line
(60) of Balb/c mouse was intraperitoneally injected with 0.5 mL of pristane before 3 weeks, and 10.sup.7 of monoclonal cells were cultured in DMEM medium comprising 10% fetal bovine serum and injected in peritoneal cavity of those mice, and then hydrops abdominis of mouse was collected after 2-3 weeks. 5-10 mg/mL of high concentration antibodies were obtained from the hydrops abdominis.
(61) To purity antibodies from the hydrops abdominis, Q-sepharose (Pharmacia product) chromatography and hydroxylapatite (Bio-gel HTP Gel, Pharmacia product) chromatography were conducted. 3.14 g of ammonium sulfate ((NH4)2SO4) per 10 mL of hydrops abdominis was added and dissolved on ice slowly (50% (NH4)2SO4 precipitation). This mixture was centrifuged at 15,000 rpm for 30 min, and the precipitate was dissolved in deionized water, and then was dialyzed in 1 L of buffer solution (20 mM phosphate, pH 7.4).
(62) The solution passed Q-sepharose column equilibrated with a buffer solution (20 mM phosphate, pH 7.4) in advance and was adsorbed, and then the concentration gradient of NaCl was flowed to a linear gradient from 0 M to 0.8 M by using buffer solution I (20 mM phosphate, pH 7.4) and buffer solution II (20 mM phosphate, 0.5M NaCl, pH 7.4), to obtain eluates. Each fraction was the collected fraction containing plenty of antibodies by 15% SDS-PAGE. The fraction was dialyzed with buffer solution (20 mM phosphate, pH 6.8), and adsorbed by passing the hydroxylapatite column equilibrated with buffer solution (20 mM phosphate, pH 6.8) in advance, and then the concentration gradient of phosphate was flowed to a linear gradient from 0 M to 0.3 M by using buffer solution III (20 mM phosphate, pH 6.8) and buffer solution IV (300 mM phosphate, pH 6.8), to obtain eluates. The fraction was collected only in the fraction having 95% or more of purity of antibody by 15% SDS-PAGE. By the experiment, 5-10 mg of monoclonal antibody per 1 ml of hydrops abdominis could be collected.
(63) The obtained antibody was called YG5.
(64) 1-1-4. Analysis of Epitope of CD43
(65) <Construction of Structure Comprising CD43 Partial Fragment>
(66) As shown in
(67) After constructing a DNA structure in order that each deletion mutant was expressed at the C-terminus of Glutathione-S-transferase (GST), it was inserted to pGEX-2T (Pharmacia Biotech Inc., Piscataway, N.J.) vector. Hereinafter, the vector comprising the amino acid sequence from no. 1 to no. 253 of CD43 protein was called pGEX1-253, and the vector comprising the amino acid sequence from no. 1 to no. 87 was called pGEX1-87, and the vector comprising the amino acid sequence from no. 1 to no. 87 of CD43 protein was called pGEX1-81, and the vector comprising the amino acid sequence from no. 1 to no. 75 of CD43 protein was called pGEX1-75, and the vector comprising the amino acid sequence from no. 1 to no. 70 of CD43 protein was called pGEX1-70, and the vector comprising the amino acid sequence from no. 70 to no. 98 of CD43 protein was called pGEX70-98, and pGEX71-81, pGEX76-81, pGEX73-81, and pGEX73-80 were named under the same principle as above.
(68) The sequence encoding the deletion mutant was amplified from human CD43 cDNA, and PCR primers were constructed from the sequence on Genebank, and BamHI/EcoRI or BamHI/BglII restriction enzyme site was included. PCR products were cut with BamHI/EcoRI or BamHI/BglII and linked to pGEX-2T of same restriction enzyme site, and then transformed into E. coli competent TOP10F′ cell [F′ [laclq, Tn10(TetR)], mcrA, D(mrr-hsdRMS-mcrBC), 80lacZDM15, lacX74, deoR, recA1, araD139 D(ara-leu)7697, galK, rpsL(StrR), endA1, nupG]. The sequence of transformant was analyzed, thereby reconfirmed the sequence of deletion mutant.
(69) <Expression of GST-CD43 Deletion Mutant Fusion Protein>
(70) The transformed E. coli TOP10 cell was cultured at 37° C. in LB medium in which 50 μg/mL of ampicillin was added overnight and the cultured cell was diluted 20-fold with LB medium, and then it was cultured for 3 to 4 hours to be OD 0.6. IPTG (Sigma Chemical Co., St. Louis, Mo.) was added to cultures at the final concentration 1 mM and it was cultured for additional 4 hours, and then it was centrifuged at 6,000 g for 15 min. After collecting cells only cells and which was suspended with 3 ml of lysis buffer solution (50 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl) per 1 g cell, the final concentration 0.2 mM of phenylmethylsulfonyl fluoride (Sigma Chemical Co.) was added and then placed on ice for 30 min.
(71) <CD43 Epitope Analysis>
(72) After the lysates of each transformants expressing the total 11 kinds of deletion mutants were subjected for 10% SDS-PAGE, western blot was performed with YG5 antibody and anti-GST antibody, respectively.
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(74) To sum up the examples, it was demonstrated that YG5 directly recognized the amino acid sequence from no. 73 to no. 81, not glycocomponent of CD43 glycoprotein, that is different from conventional other antibodies. This sequence was exposed mainly in lymphocyte progenitor cell and thymocyte in the development stages of hemoblast, and thereby YG5 antibody recognized it, and it was covered by glycosylation or structural changes around the amino acid sequence from no. 73 to no. 81 in hematopoietic stem cell, but mature white blood cell and thrombocyte, and thus YG5 antibody could not recognize it.
(75) 1-2. Chimeric Antibody Preparation
(76) Based on the amino acid sequence of the constructed anti-CD43 mouse antibody YG5, the anti-CD43 chimeric antibody was prepared.
(77) 1-2-1. Plasmid Preparation
(78) For the expression of anti-CD43 chimeric antibody, the plasmid for heavy chain expression and light chain expression were prepared, respectively. The pOptiVEC (Invitrogen Company) vector was used for the plasmid for heavy chain expression, and pcDNA3.3 (Invitrogen Company) vector was used for the plasmid for light chain expression. cDNA coding variable regions of heavy chain and light chain for antibody expression was cloned by using Ig-Primer sets (Novagen Company), and it was inserted to pGem-T vector (Promega Company), and then DNA sequence was confirmed by sequencing, and the mouse antibody gene was confirmed by IMGT site.
(79) In order to express cDNA coding variable region and cDNA encoding invariable region of each antibody as a consecutive amino acid sequence without insertion of additional amino acid, the gene fragments in which the coding sequence of the cloned variable region linked with known human IgG1 invariable region (heavy chain) and kappa invariable region (light chain) coding sequence were synthesized (Bioneer Inc), respectively. After the heavy chain and light chain expressed genes synthesized as above were cut with restriction enzymes Xho I and Sal I, the heavy chain gene fragment was ligated into pOptiVec vector, and the light chain gene fragment was ligated into pcDNA3.3 vector, respectively, thereby constructing the complete plasmid for antibody expression (pcDNA3.3-anti-CD43 light chain expression plasmid and pOptiVEC-anti-CD43 heavy chain expression plasmid). The process of construction of the heavy chain and light chain expression plasmids was schematically shown in
(80) 2-1-2. Transformation
(81) The transformation process was performed by transfecting the constructed pcDNA3.3-anti-CD43 light chain expression plasmid and pOptiVEC-anti-CD43 heavy chain expression plasmid into DG44 cell (Invitrogen) derived from CHO.
(82) At first, suspended DG44 cell was adapted to MEMα medium containing 5% FBS at 3 days before transfection, thereby converting it to adsorbed cell to be adapted for increasing the efficiency of transfection. The transfection was conducted in 6 well plate by using Effectene transfection regent (QIAGEN Company). The adapted DG44 cell subcultured at the concentration of 1×10.sup.5 cells/well one day before transfection was prepared, and the amount of DNA used for transfection was used in the same amount of 2 μg each of pcDNA3.3-anti-CD43 light chain expression plasmid and pOptiVEC-anti-CD43 heavy chain expression plasmid. The transfection was performed for 48 hours. To sort the transfected cell group, flow cytometer and Enzyme Linked Immunosorbent Assay (ELIA) were conducted, as a result, two clones of E #4, E #5 were selected. The selected cell group was cultured in MEMα selection medium comprising 5% Dialyzed Fetal Bovine Serum containing 30 nM Methotrexate (MTX) and 400 μg/mL of G418 (Geneticin) and the concentration of MTX and G418 was increased gradually to select the transformed cell group.
(83) 2-1-3. Transformed Cell Culture and Antibody Purification
(84) The transfected cell group selected above (24.0×10.sup.5 cells/mL or more, viability (%) 90% or more) was cultured until the expression level reached by 600 mg/L (according to IPC (in-process control) standard) in power CH02 CDM (Lonza; final medium amount 880 L) under the condition of 37° C. and 5% CO.sub.2.
(85) After cell clarification (using POD filter (1.1/0.2 μm)) process by collecting 800 L of culture solution obtained as above, the antibody was purified by 3 stages column process (Protein A affinity chromatography (stationary phase: ProteinA, equilibrium buffer solution: 50 mM Sodium phosphate, 50 mM sodium chloride, pH 7.5, elution buffer solution: 20 mM sodium citrate pH3.0); cation exchange chromatography (stationary phase: SP FF, equilibrium buffer solution: 20 mM sodium citrate pH 5.5, elution buffer solution: 20 mM Sodium citrate, 150 mM, sodium chloride, pH 6.1); anion exchange chromatography (stationary phase: Q FF, equilibrium buffer solution: 20 mM Sodium citrate, pH 6.5)).
(86) The chromatography condition was as follows:
(87) TABLE-US-00001 Protein A Cation exchange Anion exchange Colum type Mabselect sure SP FF Q FF Colum controller 6 mm 10 mm 10 mm Bioprocess Bioprocess Bioprocess (CL-3271) (CL-3201) (CL-3201) Column size BPG 200 BPG 300 BPG 300 Column volume 6.5 L 14.0 L 14.0 L Column Height 20 cm 20 cm 20 cm Flow rate 62.8 L/hr 141 L/hr 141 L/hr
(88) The formulation of the final crude liquid was completed by simultaneously performing buffer change and concentration processes through ultrafiltration/diafiltration (UF/DF) process, and the concentration of the final protein was adjusted to 11.5 mg/mL.
(89) The anti-CD43 chimeric antibody was obtained by the process as above, and named DNP001.
(90) 2-1-4. Confirmation of Binding Capacity of Epitope Region of Chimeric Antibody
(91) In order to confirm that the prepared chimeric antibody DNP001 (having the same CDR region as mouse antibody) bound to the same epitope as the mouse antibody, the synthesized epitope peptide was chemically combined to bovine serum albumin (BSA) protein, and then ELISA was conducted. epitope peptide synthesis sequence (named DN2)
(92) EGSPLWTSIGASTGSC (SEQ ID NO: 129; epitope was represented by underlining)
(93) The DN2 peptide-BSA conjugate was prepared by conjugating the synthesized DN2 peptide into BSA protein through EDC linker. Then, the molar ratio of peptide:BSA protein was 15:1.
(94) After coating the prepared DN2 peptide-BSA conjugate at 50 μg/mL per well, the chimeric antibody DNP001 was incubated at various concentration gradients. Next, the antibody linked to the conjugate was detected by measuring the reactivity of the chimeric antibody to the DN2 peptide-BSA conjugate. The linked antibody was detected by anti-human antibody-HRP (anti-human Ig-HRP), and the OD values at 450 nm were measured and shown in the following table and
(95) TABLE-US-00002 Chi. Ab (ug/ml) OD value 100 1.878 50 1.398 25 0.803 12.5 0.5 6.25 0.327 3.13 0.204 1.56 0.137 0 0.007
(96) As shown in the result of the table and
Example 2: Investigation of Expression Level of CD43 Epitope in Human Solid Cancer Cell Lines
(97) In order to investigate the expression level of CD43 in various solid cancer cell lines, immunostaining and flow cytometry were conducted.
(98) The information of cell lines used for analysis was as follows:
(99) TABLE-US-00003 Name Origin Histopathology Accession NO. SNU-1 stomach, adenocarcinoma ATCC, CRL-5971 gastric SNU-719 stomach adenocarcinoma, primary KCLB, No. 00719 NCI-N87 stomach carcinoma; metastatic ATCC, CRL-5822 to liver AGS stomach adenocarcinoma ATCC, CRL-1739 HT29 colon adenocarcinoma ATCC, HTB-38 LS174T colon Dukes' type B, ATCC, CL-188 colorectal adenocarcinoma HCT116 colon colorectal carcinoma ATCC, CCL-247 C3A liver hepatocellular carcinoma ATCC, CRL-10741 HepG2 liver hepatoblastoma ATCC, HB-8065 PLC/PRF/5 liver hepatoma ATCC, CRL-8024
(100) Specifically, each cell line was inoculated and cultured in 100 mm of cell culture container, and when 70˜80% of surface was concentrated with the culture cell, the culture cell was washed with phosphate-buffered solution and then treated with Trypsin-EDTA (Invitrogen), and dissociated, and then centrifuged. The precipitated cell was suspended in buffer solution again and aliquoted 1×10.sup.5 each, and 1.5 μL of the anti-CD43 antibody (YG5)-phycoerythrin (PE) prepared in the example 1-1 was added and reacted in a 4° C. refrigerator for 20 min. After reaction at 4° C. for 20 min, cell was washed with 4 ml of buffer solution (1× Phosphate Buffered Saline, PBS buffer) again, then it was analyzed with Flow Cytometer (Becton, Dickinson and Company, Franklin Lakes, N.J., USA). For comparison, the same test using DFT-1 antibody (2 μL, Ancell corporation) instead of the anti-CD43 antibody was performed.
(101) The obtained results were shown in
Example 3: Test of Cytotoxicity of Anti-CD43 Antibody to Cancer Cell (In Vitro)
(102) 3-1. Preparation of Antibody-Toxin Conjugate
(103) The saporin (Sigma, St. Louis, Mo.) conjugation of monoclonal antibody was conducted according to the conventional method (Polito et al., 2004). After dissolving the antibody (DNP001; prepared in example 1-2) and saporin at the concentration of 2 mg/mL (antibody concentration) and 8 mg/mL (saporin concentration), respectively in 50 mM sodium borate buffer (pH 9.0), 2-iminothiolane (Sigma) was treated at the concentration of 0.4 mM and 1.0 mM, respectively. Afterward, the antibody and saporin were mixed at the ratio of 10:1 and reacted at the room temperature for 16 hours, and the antibody-saporin conjugate was purified by gel filtration. Hereinafter, the prepared conjugate was described as anti-CD43-saporin conjugate.
(104) Referring the method above, anti-CD43-MMAE conjugate in which anti-CD43 antibody (DNP001; prepared in example 1-2) and monomethyl auristatin E (MMAE; Creative Biolabs) were conjugated, anti-CD43-DM1 conjugate in which anti-CD43 antibody (DNP001) and N2′-diacetyl-N2′-(3-mercapto-1-oxopropyl) maytansine (DM1; The Chemistry Research Solution LLC) were conjugated, and anti-CD43-Duocarmycin conjugate in which anti-CD43 antibody (DNP001) and Duocarmycin (The Chemistry Research Solution LLC) were conjugated, and anti-CD43 antibody (DNP001)-DM1 conjugate were prepared, respectively.
(105) 3-2. Cytotoxicity of Anti-CD43 Antibody-Toxin Conjugate to Stomach Cancer Cell
(106) The cytotoxicity of the antibody-toxin conjugates prepared in the example 3-1 (anti-CD43-saporin conjugate, anti-CD43-DM1 conjugate, anti-CD43-MMAE conjugate, and anti-CD43-Duocarmycin conjugate) to the stomach cancer cell was tested.
(107) The day before the test, stomach cancer cell lines NCI-N87, AGS, and SNU719 were respectively plated at 4×10.sup.3 per well. The each antibody-toxin conjugate was treated to each stomach cell line at the concentration of 10000 ng/mL (in case of (anti-CD43 antibody)-MMAE conjugate and (anti-CD43)-DM1 conjugate) or 1000 ng/mL (in case of (anti-CD43)-Duocarmycin conjugate). Afterward, 10 μL of EzCytox (Daeil lab, Korea) was added to each well after 24 hours, 48 hours, and 72 hours, and cells were cultured in a 37° C. CO.sub.2 container, and then their viability were measured by microspectrophotometry.
(108) The cytotoxicity of each conjugate obtained was shown in
Cytotoxicity (%)=[1−(the number of survived cells/the number of initial cells)]×100
(109) As shown in
Example 4: Test of Anti-Cancer Effect of Anti-CD43 Antibody in Animal Model (In Vivo)
(110) 4-1. Preparation of Stomach Cancer Animal Model (Tumorigenesis)
(111) The stomach cancer model was prepared by using the cell lines in which CD43 expression was confirmed in the result of example 2 (NCI-N87; ATCC, CRL-5822). At first, 2.8×10.sup.7 of NCI-N87 cells were prepared. The prepared cells were subcutaneously inoculated 5×10.sup.6 cells/100 μL (RPMI) into the right side of nude mice. The inoculated nude mice were arranged into the control group (PBS administration) and test group, and the anti-CD43 antibody (DNP001) prepared in the example 1-2 was injected into the tail vein in an amount of 12 mg/kg 2 times per week for 3 weeks at 3 days, or 0.2 mg/kg of the anti-CD43-Duocarmycin conjugate (DNP001-Duocarmycin) prepared in the example 3-1 was intraperitoneally injected once per week for 3 weeks, after inoculating the cancer cell. The size of tumor was measured before administrating the therapeutic agent 2 times per week, and the size of tumor was calculated by the following equation:
Tumor size (mm.sup.3)=(major axis×minor axis.sup.2)/2
(112) The obtained result was shown in
Example 5: Investigation of Distribution of CD43 in Human Solid Cancer Tissue
(113) In addition to stomach cancer, to confirm the expression of CD43 and the possibility to test the therapeutic efficacy in various solid cancer, (stomach cancer, signet ring cell stomach cancer, breast cancer, ductal infiltrating adenocarcinoma among breast cancer, renal cancer, pancreatic cancer, gallbladder cancer, cervical cancer, uterine cervix cancer, bladder cancer, granulocytic sarcoma) targeting CD43, immunohistochemistry was performed in various human origin tumor tissues.
(114) The immunohistochemical staining was progressed in the following order. As the solid cancer tissue, the paraffin embedded solid cancer tissue (Chungbuk National University Hospital) was used. At first, the paraffin solid cancer slide was under the de-paraffin process of 3 times of xylene for 10 min each, twice of 100% alcohol for 5 min each, 80% alcohol for 3 min, 50% alcohol for 1 min, 20% alcohol for 1 min, and then washed with running water twice. Then, it was soaked in 1×TBS (Tris-buffered saline) for 10 min. To regenerate antigen in tissue, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 8.0) buffer was added and proceeded in a microwave for 15 min and rapidly cooled in running water, and the slide was then soaked in 1×TBS for 20 min. The endogenous peroxidase was removed with 0.1% H.sub.2O.sub.2+100% methanol for 10 min and washed with running water twice. Then, to remove the non-specific reaction of biotin and antigen-antibody, the blocking process was proceeded. The blocking process was performed by dropping 4 drops of biotin solution and reacted at the room temperature for 15 min. After dropping 4 drops of avidin solution (VECTOR laboratories) to the tissue, it was reacted at the room temperature for 15 min and then washed with 1×PBS.
(115) 10 μg/mL, 5 μg/mL each of the anti-CD43 (YG5) antibody and DFT-1 antibody (control group; Ancell corporation) prepared in the example 1-1 were suspended in 150 μL of 1×TBS to cover the tissue, and it was reacted at the room temperature for 30 min. After washing it with 1×TBST (1×TBS+0.1% tween 20) 3 times for 15 min each, the second antibody (anti-mouse/rabbit HRP; DAKO) was covered to the tissue 2 drops each, and reacted at the room temperature for 30 min. Then, after washing it with 1×TBST (1×TBS+0.1% tween 20) 3 times for 15 min each, the color development reaction was carried out with DAB (Diaminobenzidine). After washing it with 1×TBS for 5 min twice and counter staining, it was washed with running water. Then, after the dehydration process was proceeded, it was mounted.
(116) The obtained result was shown in
(117) TABLE-US-00004 TABLE 1 Expression of CD43 in various cancer tissue section Origin Total (n) Positive (n) %, positivity Stomach 213 179 84 Breast 231 90 39 Lung 28 13 46 Kidney 54 5 9 Pancreas 30 3 10 Thyroid 21 1 5 Gall bladder 4 1 25 Uterus 2 1 50 Urinary bladder 5 2 40 Cervix 4 1 25
(118) (In the table 1,
(119) Positivity means the numerical value obtained by dividing the number of positive tissue except for the negative tissues in which YG5 was not stained by the number of total tissues of corresponding cancer;
(120) Total means the total number of tissues used for staining;
(121) Positive means the number of tissues showing a positive reaction to YG5 among tissues used, respectively)
(122) As confirmed in table 1,
Example 6: CD43 Expression in Cancer Stem Cell of Stomach Cancer
(123) The level of expression of CD43 in cancer stem cells of various tumors was tested. CD44 and CD133 (Prominin-1) were publicly known cancer stem cell markers. The cancer stem cell markers CD44 and CD133 were triple-stained to confirm the CD43 expression, thereby demonstrating that CD43 was expressed in CD44 or CD44.sup.+CD133.sup.+ positive group. NCI-N87 cell was inoculated and cultured in 100 mm of cell culture container, and when 70˜80% of surface was concentrated with the culture cell, the culture cell was washed with phosphate-buffered solution and then treated with Trypsin-EDTA (Invitrogen), and dissociated, and then centrifuged. The precipitated cell was suspended in buffer solution again. After reacting them with anti-CD44-allophycocyanin (APC) (10 μL, Miltenyi Biotec), anti-CD133-fluorescein isothiocyanate (FITC) (10 μL, Miltenyi Biotec), and anti-CD43 antibody (prepared in example 1; YG5)-phycoerythrin (PE; 1.5 μL, Dinona) in a 4° C. refrigerator for 20 min, the unreacted antibody was washed and fixed with 1% paraformaldehyde. Cells were then analyzed by Flow Cytometer (Becton, Dickinson and Company, Franklin Lakes, N.J., USA). The same test using mouse IgG1 instead of the anti-CD44 antibody and anti-CD133 antibody as a negative control group was performed.
(124) The obtained result was shown in
(125) As shown in
Example 7: Investigation of Expression of CD43 in Various Solid Cancer Stem Cells
(126) The expression of CD43 in cancer stem cells of CD43 positive solid cancer quoted in the example 5 was confirmed by the same method quoted in the example 6.
(127) Cells originated from each CD43 positive tissue disclosed in the example 5 (breast cancer, lung cancer, rectal cancer, liver cancer and gallbladder cancer, renal cancer, pancreatic cancer, thyroid cancer, prostate cancer, cervical cancer, uterine cervix cancer, bladder cancer-originated cell lines) were used. Each cell was inoculated and cultured in 100 mm of cell culture container, and when 70˜80% of surface was concentrated with the culture cell, the culture cell was washed with phosphate-buffered solution and then treated with Trypsin-EDTA (Invitrogen), and dissociated, and then centrifuged. The precipitated cell mass was suspended in buffer solution again and reacted with 100-fold diluted anti-CD44-allophycocyanin (APC), anti-CD44-allophycocyanin (APC), anti-CD326 (EpCAM)-allophycocyanin (APC), anti-CD133-fluorescein isothiocyanate (FITC), anti-CD43 antibody-phycoerythrin (PE) in a 4° C. refrigerator for 20 min. The unreacted antibody was washed and fixed with 1% paraformaldehyde, and then cells were analyzed by Flow Cytometer (Becton, Dickinson and Company, Franklin Lakes, N.J., USA).
(128) As a result, as the result of confirming the CD43 expression in cancer stem cells of CD43 positive tissue originated cells in the example 5, it was confirmed that CD43 was positive in cells in which markers differentiating each cancer stem cell (CD44, CD133, or EpCAM) were positive.
Example 8: Test of CD43 Expression in Cancer Stem Cell of Fresh Cancer Tissue of Patient
(129) Based on the result of the example 5, the expression of CD43 was tested after classification using cancer stem cell markers in cancer tissues of patient.
(130) The fresh cancer tissue of patient was finely monoclonalized. The monoclonalized tumor cell was centrifuged at 1700 rpm for 3 min and then the supernatant was removed, and it was resuspended to 10 mL medium containing 10% FBS and then centrifuged at 1700 rpm for 3 min. The supernatant was removed and it was resuspended with 10 mL 1×PBS, and then counted. Cells were distributed to FACS tubes, and then after reacting them with anti-CD44-allophycocyanin (APC), anti-CD44-allophycocyanin (APC), anti-CD326 (EpCAM)-allophycocyanin (APC), anti-CD133-fluorescein isothiocyanate (FITC), anti-CD43 antibody-phycoerythrin (PE) in a 4° C. refrigerator for 20 min, the unreacted antibody was washed and fixed with 1% paraformaldehyde, and then cells were analyzed by Flow Cytometer (Becton, Dickinson and Company, Franklin Lakes, N.J., USA).
(131) As above, as the result of testing the CD43 expression in cancer stem cells of stomach cancer in cancer stem cells of cancer tissues of patient, it was confirmed that CD43 was positive in cancer stem cells originated from cancer tissues of patient.
Example 9: Inhibition of Colony Formation of Cancer Stem Cell by Anti-CD43 Antibody (In Vitro)
(132) It was confirmed that the colony formation of CD43 positive stem cells in the CD43-positive solid tumor presented in Example 5 was inhibited by the anti-CD43 antibody was confirmed in Example 6. Cells originated from each CD43 positive tissue disclosed in the example 5 (breast cancer, lung cancer, rectal cancer, liver cancer and gallbladder cancer, renal cancer, pancreatic cancer, thyroid cancer, prostate cancer, cervical cancer, uterine cervix cancer, bladder cancer-originated cell lines) were used. Each cell line was inoculated and cultured in 100 mm of cell culture container, and when 70˜80% of surface was concentrated with the culture cell, the culture cell was washed with phosphate-buffered solution and then treated with Trypsin-EDTA (Invitrogen), and dissociated, and then centrifuged. The precipitated cell mass was suspended in buffer solution again and the anti-CD43 antibody was added at the concentration of 20 μg/mL per 10.sup.7 of tumor cells and reacted in a 4° C. refrigerator for 20 min, and then unreacted antibody was washed with 1×PBS. After adding 20 μL of magnetic bead-boung IgG, it was reacted in a 4° C. refrigerator for 15 min and then washed with 1×PBS, and CD43 positive cells were classified by MACS separating system.
(133) After reacting the classified CD43 positive cells or negative cells with anti-CD44-allophycocyanin (APC), anti-CD326(EpCAM)-allophycocyanin (APC), anti-CD133-fluorescein isothiocyanate (FITC), anti-Mignetic Bead antibody-phycoerythrin (PE, 20ul, Miltenyi Biotec) in a 4° C. refrigerator for 15 min, it was washed and fixed with 1% paraformaldehyde, and then analyzed by Flow Cytometer (Becton, Dickinson and Company, Franklin Lakes, N.J., USA).
(134) The CD43 positive cells or negative cells obtained from the examples were added in ultra low attachment 6-well plates (Corning Inc., Corning, N.Y., USA) at the number of 5,000 per well in the well containing serum-free media (100 IU/ml penicillin G, 100 μg/mL streptomycin, 20 ng/mL human recombinant epidermal growth factor (hrEGF), 10 ng/ml human recombinant basic fibroblast growth factor (hrbFGF), 2% B27 supplement without vitamin A, 1% N2 supplement (Invitrogen, Carlsbad, Calif., USA) were comprised). Then, the anti-CD43 antibody and control antibody were added 100 μg/mL each and cultured. Afterward, spheres were observed. The same experiment was conducted by classifying CD43 positive cells in fresh cancer tissues of patient.
(135) As a result, it was confirmed that the tumorigenesis of cancer stem cell was inhibited, compared to the control group, as the result of administering the anti-CD43 antibody to CD43 positive cancer stem cells of various tumors and cancer tissues of patient. This result showed the significant inhibitory effect of oncogenesis of cancer stem cell of anti-CD43 antibody.
Example 10: Test of Anti-Cancer Effect of Anti-CD43 Antibody in Various Cancer Animal Models (In Vivo)
(136) The animal model was prepared by using cell lines in which CD43 expression was confirmed in the results of the examples 7 and 8 and fresh cancer tissues, and the method was same as the example 4. The mice were randomly assigned into the control group (PBS administration), and test group, and the anti-CD43 antibody (DNP001 mAb) prepared in the example 2 was injected into the tail vein in an amount of 8 mg/kg 2 times per week for 3 weeks, or 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 5 mg/kg of the anti-CD43-saporin, DM1, MMAE, Duocarmycin conjugate prepared in the example 3-1 was intraperitoneally injected once per week for 3 weeks, at 3 days after inoculating cancer cells.
Example 11: Test of Change of Binding Capacity to Normal Blood Cell after Neuraminidase Treatment
(137) It was confirmed whether there was change of binding capacity to the normal lymphocyte in which the antibody recognizing CD43 expressed in normal blood (DFT-1) and the anti-CD43 antibody (YG5) recognizing tumor-specific CD43 were treated with neuraminidase.
(138) 10 mL of blood was gathered from health people, and 40 mL of red blood cell lysis solution (RBC lysis solution; NH4Cl, NaHCO.sub.3, EDTA pH8.0) was added into the blood, and lysed at the room temperature for 10 min. The blood in which red blood cells were lysed was centrifuged at 1700 rpm for 5 min and then the supernatant was removed, and it was washed with 10 mL of PBS twice. 3*10.sup.6 of lymphocytes were suspended in 130 μL of the obtained red blood cell lysis solution above, and 50 μL of neuminidase (ELPIS, Korea) and 20 μL of buffer were added. And then, cell was reacted at 37° C. for 50 min, and washed with PBS. In order to confirm whether there was change of epitope of antibody recognizing CD43 by the neuraminidase, FITC and PE-bound DFT-1 and YG5 antibodies were added into the cell and after reacting at 4° C. for 15 min, the cell was then washed with 4 ml of PBS. The cell was then measured with flow cytometry and the titer to the normal lymphocyte was measured.
(139) The result of confirming the titers of two antibodies to CD43 obtained above (DFT-1 and YG5) was shown in
Example 12: Test of Cross-Reactivity to CEACAM5 and CEACAM6
(140) 5F1 clone known to recognize CD43 protein was known to simultaneously recognize CD43 and CEACAM6 and bind to fucosylated position of two proteins. It was tested that the anti-CD43 antibody showed the cross-reactivity with CEACAM5 and CEACAM6, and the change of the binding capacity of anti-CD43 antibody by the glycosylation change of CD43 epitope by kifunensine and fucosidase was tested.
(141) After rCEACAM5-hFC (Sinobiologics, Cat. No: 11077-H03H-50) and rCEACAM6-hFC (DinonA inc.) recombinant proteins were added to maxisorp ELISA plate at 200 μL per well and reacted at 37° C. for 1 hour, thereby blocking. IgG1, YG5, DFT-1, 9A6, or 8F5 monoclonal antibodies (8F5: Biomaterials, 2015 October, 67, 32-41, 9A6: SantaCruz Biotechnology, Cat. No: sc-59899) were added at 100 ng per well, respectively, to the well coated with rCEACAM5-hFC and rCEACAM6-hFC proteins and reacted at 37° C. for 1 hour, and then washed with PBS, to remove unbound antibodies. Then, goat anti-mouse IgG-HRP (Jackson) was diluted and added, and it was reacted for 30 min and then washed with PBS, and TMB solution was added at 50 μL per well and it was reacted for 10 min, and then 50 μL of sulfuric acid was added to ceasing the reaction and the absorbance at 450 nm was measured.
(142) The obtained result was shown in
(143) In addition, there was no the change of binding capacity of anti-CD43 antibody to the fucosidase and kifunensine treated CEM7 cell (cell sorted as having 50% or more increased level of cell surface expression of CD43 compared to the original cell by single cell culturing CCRF-CEM cell obtained from ATCC (CCL-119); same hereinafter). The result showed that the antibody provided in the present invention (for example, (anti-CD43 antibody (YG5)) kept the binding capacity with CD43 even under the condition in which the sugar condition of CD43 was changed, and this means that the epitope of antibody was not independent to sugar. On the other hand, the conventional CD43 antibody, 5F1 was known to show the sugar-dependent epitope binding capacity, and thereby it was demonstrated that the antibody provided in the present invention had discrimination from the conventional antibody.
Example 13: 3-Dimensional Culture Experiment of Stomach Cancer Cell (Tumor Sphere Assay)
(144) The stomach cancer cell line, NCI-N87 cell was prepared to be 80˜90% of 150 mm dish before the experiment. NCI-N87 cell was suspended by treating 1×Trypsin⋅EDTA and then washed. The prepared NCI-N87 cell was resuspended with media (DMEM/F12 (GIBCO), B27 (Invitrogen), EGF & bFGF (Invitrogen)) and then aliquoted with 1*10.sup.5/2 ml in 6 well (ultra-low attached plate), and then cultured in a 37° C. CO.sub.2 incubator for 5 days. After photographing cells of each well with the optical microscope, cells were separated to single cells by 1×TE 200 μL and then washed with PBS. 1/50 of the normal mouse serum was added into cells, and it was blocked at 4° C. for 10 min. Subsequently, cells were aliquoted into flow cytometry tubes at 100 μL each, and the anti-CD44 antibody (eBioscience, Cat. No: 17-0441-82) and anti-CD43 antibody (YG5, DFT-1) were added at 10 μL, 1 μL, respectively, and it was reacted at 4° C. for 25 min. After washing by the same method as above, 1% (w/v) paraform aldehyde was added per sample to fix cells, and then the flow cytometry was carried out.
(145) The obtained result was shown in
Example 14: ELISA Protocol Measuring Modified Cell Binding Capacity by Using Suspended Cells in Suspension
(146) This analysis was designed as a pilot research to confirm binding capacity of scFv or IgG to suspended cells in suspension by using ELISA experiment with reduced the background signal in the poly-D-lysine plate.
(147) Method:
(148) 1. Step of collecting cells.
(149) a. Cells were pelleted by putting cells in 50 mL falcon tube and centrifuging at 500×g for 5 min (pelleting);
(150) b. Cells were pelleted by washing the obtained cell pellets with 10 ml PBS once and centrifuging at 500×g for 5 min (pelleting);
(151) c. Cells were counted after resuspending cells with 1 mL PBS (counting cells);
(152) d. Cells were diluted with blocking buffer (PBS+3% FCS) (cell dilution concentration: 5×10.sup.5 cell/well (10×10.sup.6 cells/mL));
(153) e. Cell stock 50 μL per well was added to the V-bottomed 96-well plate.
(154) 2. 50 μL per well of cytoplasmic extract (anti-CD43 scFv) or IgG1 (desired final concentration×2-fold concentrated stock; for example, when 25 μg/mL of final concentration was desired, preparing 50 μg/mL stock) was added (according to Layout analysis, prepared in duplicate or triplicate). The samples were mixed by pipetting 4 times carefully.
(155) 3. They were cultured at the room temperature for 1 hour.
(156) 4. They were centrifuged at 500×g for 5 min, to pellet cells.
(157) 5. The supernatant was removed by turning the plate inside out or aspiration.
(158) 6. Cells were washed with 200 μL blocking buffer and samples were mixed by pipetting 4 times.
(159) 7. Cells were pelleted by centrifugation at 500×g for 5 min.
(160) 8. The supernatant was removed by turning the plate inside out carefully or aspiration.
(161) 9. 100 μL of anti-Flag HRP-conjugated antibody diluted by 1:1,500 in the blocking buffer was added to cell pellets and resuspended carefully, and cultured at the room temperature for 30 min.
(162) 10. Cells were pelleted by centrifugation at 500×g for 5 min.
(163) 11. The supernatant was removed by turning the plate inside out carefully or aspiration.
(164) 12. Cells were carefully washed by adding 200 μL blocking buffer and the obtained samples were mixed by pipetting 4 times.
(165) 13. Cells were pelleted by centrifugation at 500×g for 5 min.
(166) 14. The supernatant was removed by turning the plate inside out carefully or aspiration.
(167) 15. Cells were carefully washed by adding 200 μL blocking buffer and the obtained samples were mixed by pipetting 4 times.
(168) 16. Cells were pelleted by centrifugation at 500×g for 5 min.
(169) 17. Cells were carefully resuspended to SureBlue™ TMB Microwell Peroxidase substrate 80 μL and cultured at the room temperature for 5 min, and then the reaction was ceased by 1 M HCl.
(170) 18. Samples of 100 μL each were transferred to the standard 96-well plate.
(171) 19. The plate (absorbance) at 450 nm was read.
Example 15: FACS Protocol Measuring Modified Cell Binding Capacity by Using Suspended Cells in Suspension
(172) This analysis was designed as another method for FCAS experiment to reduce the background signal in the poly-D-lysine plate and evaluate the binding capacity of scFv or IgG.
(173) Method:
(174) 1. Flow cytometry:
(175) a. Step of collecting cell:
(176) i. Cells were pelleted in 50 mL falcon tube by centrifuging at 500×g for 5 min (pelleting);
(177) ii. Cells were pelleted by washing the obtained cell pellets with 10 mL PBS once and centrifuging at 500×g for 5 min (pelleting);
(178) iii. Cells were counted after resuspending cells with 1 mL PBS (counting cells);
(179) iv. Cells were diluted with blocking buffer (PBS+3% FCS) (cell dilution concentration: 5×10.sup.5 cell/well (10×10.sup.6 cells/mL));
(180) v. Cell stock 50 μL per well was added to the V-bottomed 96-well plates. 2. According to Layout analysis, cells were added with 50 μL per well of IgG in duplicate or triplicate (desired final concentration×2-fold concentrated stock; for example, when 25 μg/mL of final concentration was desired, preparing 50 μg/ml stock), the obtained samples were mixed by pipetting 4 times.
(181) 3. They were incubated at the room temperature for 30 min. 4. Blocking buffer 200 μL was added.
(182) 5. Cells were pelleted by centrifugation at 500×g for 5 min.
(183) 6. The supernatant was removed by turning the plate inside out carefully or aspiration.
(184) 7. 200 μL blocking buffer was added and cells were softly washed out, and then evenly mixed about 4 times by using the pipet.
(185) 8. Cells were pelleted by centrifugation at 500×g for 5 min.
(186) 9. Medium was removed.
(187) 10. Goat anti-human IgG Alexa Fluor 488 200 μL diluted by 1:20 in the blocking buffer was added to cell pellets and they were carefully resuspended, and left on ice for 1 hour where the light was blocked.
(188) 11. Cells were pelleted by centrifugation at 500×g for 5 min.
(189) 12. Medium was removed.
(190) 13. 200 μL blocking buffer was added and cells were softly washed out, and then evenly mixed by using the pipet about 4 times.
(191) 14. Cells were pelleted by centrifugation at 500×g for 5 min.
(192) 15. Blocking buffer 200 μL was added and cells were softly resuspended.
(193) 16. The plate was read by using flow cytometer.
Example 16: scFv Flow Cytometry Using Soluble scFv Preparations
(194) The present example tested the level of binding of scFv to CEM7 and U937 cell (ATCC® CRL1593.2™), and used soluble scFvs expressed in E. coli periplasm, and designed for scFv cell binding analysis by flow cytometry.
(195) Method:
(196) Day 1: Clone Inoculation
(197) 1. Starter culture plate:
(198) a. 200 μL 2YT (2×yeast extract)+5% (w/v) glucose+amphicilin was filled in the 96-well culture plate.
(199) b. scFvs which could be the comparison group together with desired clones (anti-CD43 (mJL1) scFv coding DNA: SEQ ID NO: 49; Sh741-112 scFv coding DNA: SEQ ID NO: 51; Sh145-112 scFv coding DNA: SEQ ID NO: 53; Sh146-112: SEQ ID NO: 55; or Sh434-112 scFv coding DNA: SEQ ID NO: 57) were inoculated to the well.
(200) 2. They were cultured overnight as shaking them under the condition of 650 rpm, 37° C.
(201) Day 2: Expression of scFvs
(202) Periplasmic Extract Cultures:
(203) 3. According to the final use of inoculate expression plates: periplasmic extract, one or more wells per sample could be inoculated.
(204) a. 96 deep-well plate was filled with 1.0 mL/well of 2YT+Amp (no glucose).
(205) b. After diluting the starter cultures to have 0.1 value at OD.sub.600, they were inoculated to the 96 well plate. They were cultured as shaking under the condition of 650 rpm, 30° C. for 2-4 hours. Periodically, the turbidity at OD.sub.600 was measured by collecting the samples. Cells were raised to have OD.sub.600 value between 0.7 and 1.0.
(206) 4. Induction of scFv expression in expression plates:
(207) a. To induce the expression of periplasmic extract culture, 100 μL 2YT+Amp in which IPTG (diluting stock IPTG by 1:100) was added to the expression plates each.
(208) b. They were cultured over night shaking under the condition of 650 rpm and 22° C.
(209) Day 3: Preparation of Periplasmic Extract and Flow Cytometry
(210) Periplasmic Extractions:
(211) 5. Preparation of periplasmic extract:
(212) a. Cells were pelleted by centrifugation at 2000×g for 10 min.
(213) b. The supernatant in the expression plate was dusted to the container containing a bleaching agent, and the medium left in the plate was removed by putting it up on the paper towel.
(214) c. 75 μL cold PPB (Potassium Phosphate Buffer)+protease inhibitor (1 tablet per 50 mL; complete; Roche, Cat. No: 04693116001) was put into each well, and resuspended by pipetting 4 times, and then cultured under the condition of 1000 rpm and 4° C. for 10 min.
(215) d. was put into each well, and resuspended by pipetting 4 times, and then cultured under the condition of 1000 rpm and 4° C. for 1 hour under shaking.
(216) e. Plate was centrifuged at 3000×g for 10 min.
(217) f. Periplasmic extracts (approximately 270 μL) was transferred and put filter into the stack (ensure A1 orientation corresponds).
(218) i. Top part: 1.2 μm 96 well filter plate
(219) ii. Middle part: 100 K 96 well filter plate
(220) iii. Bottom part: 96-well, flat based standard plate.
(221) g. Plate was centrifuged at 4000 rpm for 20 min.
(222) h. (If necessary) for preparing flow cytometry analysis, samples for each clone were collected.
(223) 6. Flow Cytometry:
(224) a. scFv samples: use of periplasmic extracts
(225) b. collection of cells:
(226) i. Cells were pelleted by putting into 50 mL falcon tube and centrifuging at 500×g for 5 min (pelleting).
(227) ii. Cells were pelted by washing cell pellets once by using 10 mL PBS and centrifuging at 500×g for 5 min.
(228) iii. Cells were counted, after resuspending cells in 1 mL PBS (counting cells).
(229) iv. Cells were diluted with 0.5×10.sup.5 cells/well (2.5×10.sup.6 cells/mL).
(230) v. 20 μL cell stock per well was added to V-bottomed 96-well plates.
(231) c. 20 μL per well of periplasmic extract was added in duplicate (scFv).
(232) And samples were mixed softly by pipetting about 4 times.
(233) d. They were left at the room temperature for 30 min.
(234) e. 180 μL Blocking buffer was added.
(235) f. Cells were pelleted by centrifugation at 500×g for 5 min.
(236) g. The supernatant was removed by turning the plate inside out or aspiration.
(237) h. 200 μL blocking buffer was added and cells were softly washed out, and then evenly mixed by pipetting about 4 times.
(238) i. Cells were pelleted by centrifugation at 500×g for 5 min.
(239) j. Medium was removed.
(240) k. Cells were softly resuspended by adding 50 μL of 5 μg/mL anti-Flag PE-conjugated antibody (BioLegend, Cat. No: 637310) to the readily prepared binding buffer. The plates should be protected from light at maximum, since the antibody was sensitive to light. They were left on ice for 30 min under the condition of protection from light.
(241) l. Cells were pelleted by centrifugation at 500×g for 5 min.
(242) m. Medium was removed.
(243) n. 200 μL blocking buffer was added and cells were softly washed out, and then evenly mixed by pipetting about 4 times.
(244) o. Cells were pelleted by centrifugation at 500×g for 5 min.
(245) p. 200 μL blocking buffer was added and cells were softly resuspended.
(246) q. The plate was read by using Guava flow cytometer (Merckmillipore). The flow cytometry should be prepared in advance to recognize the yellow fluorescence.
Example 17: Functional Properties of Murine Anti-CD43 Antibody being Template
(247) The present experiment was performed to demonstrate the target epitope proper for antibody treatment (JL-1). In the present experiment, the mouse anti-CD43 monoclonal antibody binding the epitope (heavy chain: SEQ ID NO: 34; heavy chain coding DNA: SEQ ID NO: 33; heavy chain expression vector (pTT5 based): SEQ ID NO: 35; light chain: SEQ ID NO: 37, light chain coding DNA: SEQ ID NO: 36; light chain expression vector (pTT5 based): SEQ ID NO: 37) was used.
(248) It was confirmed that the significant amount of antigen was not sheded from the cell, when JL-1 antigen (CD43) was cultured at 37° C. for 4 hours. It was demonstrated that there was no large difference in the aspect of size, when the anti-CD43 antibody mixed in the buffer and the anti-CD43 antibody mixed in the supernatant of the actual Molt-4 (CD43+ acute lymphocytic leukemia cell line; ATCC, CRL-1582) or HL-60 (ATCC) were compared by western blot analysis.
(249) In addition, it was confirmed that the amount of JL-1 antigen (CD43) circulating in the serum of normal human was not sufficient to significantly interrupt the binding of anti-CD43 antibody to the target. The effect of 50% human serum to the anti-CD43 antibody binding to the leukemia cell was evaluated in vitro (measuring IF (immunofluorescence) reactivity). The obtained result was shown in table 2.
(250) TABLE-US-00005 TABLE 2 Effect of normal human serum for binding of JL-1(CD43) to leukemia cell JL-1 (CD43) IF reactivity Cell line ug/ml No human serum 50% human serum Molt-4 50 ++ ND* ″ 25 ++ ND ″ 12.5 ++ ++ ″ 6.25 +/++ +/++ HL-60 50 + ND ″ 25 + ND ″ 12.5 + +/− ″ 6.25 +/− +/− *ND, Not determined
(251) As shown in the table 2, it was confirmed that the serum did not interrupt the binding of the anti-CD43 antibody to Molt-4 cell or HL-60 cell.
(252) In addition, it was tested that the naked anti-CD43 antibody which was not conjugated had no direct cytotoxic effect to the isolated target cell. For this, CD43+ CEM7 cell (represented as CEM7-high antigen) and CCRF-CEM cell (original CEM cell (ATCC); represented as CEM7-medium antigen) were added to 96-well plate in amount of 40,000 cells/well, respectively, and the mouse CD43 antibody (mouse anti-CD43 monoclonal antibody prepared in advance) was serially diluted and treated, and the cytotoxicity was determined by using Cell Titer Glo (Promega™) at the 3rd day of culturing. The same experiment to H9K cell (H9K-low antigen) expressing CD43 at the low concentration was carried out for comparison.
(253) The obtained result was shown in
(254) The cell lines such as CEM7, CCRRF-CEM, Nalm6 (ATCC, CRL-3273) and HL-60 (ATCC, CCL-240), etc. exhibited individually different JL-1 antigen (CD43) expression level (from high expression to low expression in the order described). The saporin-conjugated anti-CD43 antibody (refer to the example 3-1 for construction of conjugate; the anti-CD43 antibody was the mouse anti-CD43 monoclonal antibody prepared in advance) or isotype control was treated at the diluted concentration from 20 μg/mL to each cell (20,000 cells), and the cell viability was measured by using Cell Titer Glom at the 3rd day of culturing. The mouse IgG1 was used as the isotype control used for the comparison.
(255) The obtained result was shown in
(256) The internalization of anti-CD43 antibody was tested. The mouse anti-CD43 antibody (the mouse anti-CD43 monoclonal antibody prepared in advance) was treated to cells (CEM7) in the refrigerated condition for 30 min, and transferred to the condition of 37° C., and then 10.sup.6 cells were collected at the respective time represented in the X axis of
(257) The obtained result was shown in
(258) On the other hand, the homotypic aggregation phenomenon induced by the anti-CD43 antibody in cells expressing antigen was tested. For this, the anti-CD43 antibody (mouse anti-CD43 monoclonal antibody prepared in advance) was treated at the start concentration of 40 μg/mL to 300,000 cells (CEM7, CCRRF-CEM, or HL-60) and cultured in the condition of 37° C., 5% CO.sub.2, and then images were obtained by taking microscopic photographs at the arranged time (2 hours after antibody treatment). The obtained image was shown in
(259) In addition, the CD43 expression in human normal bone marrow cells was tested. The monocytes of normal bone marrow were stained with mouse anti-CD43 antibody (mouse anti-CD43 monoclonal antibody prepared in advance), and then stained with goat-anti-mouse IgG F(ab)2-PE (Jackson, Cat. No: 115-035-072), and was observed by the method disclosed in the example 15.
(260) The obtained result was shown in
(261) On the other hand, CD43 expression was measured in CD34+CD38− cell (hemopoietic cell sorted as CD43 expression and CD38 non-expression cell by the test with FACS; same hereinafter) from various human normal bone marrow samples (Seoul National University Hospital) by flow cytometry, and it was confirmed that the CD43 protein expression was lacked in hematopoietic stem cell and precursor cell that formed colony in bone marrow. The method of confirmation was simply described as follows: The umbilical cord hematopoietic stem cells were inoculated to 30 NSG mice (NOD/SCID×common g chain deficiency) at 0 day. After 12 weeks, PBL (Peripheral Blood Lymphocyte) of all mice was analyzed with the finally differentiated immune cell. The immune system of all mice used in the test was ingrafted, and the anti-CD43 antibody (mouse anti-CD43 monoclonal antibody prepared in advance)-toxin (saporin) conjugate or vehicle (PBS) was administered for 4 weeks. At 4 weeks after the treatment, the presence of human immune cells were analyzed in immunized animal. For comparison, the same test was performed using the mouse IgG1-toxin (saporin) conjugate instead of anti-CD43 antibody.
(262) The obtained result was shown in the following Table 3:
(263) TABLE-US-00006 TABLE 3 % of viable cells (SD) B cells T cells Monocytes PMNs 16 weeks PBL JL1-Toxin 32 (11) 16 (4) 2.9 (2) 1.7 (1.2) Isotype-Toxin 31 (5) 17 (6) 4.5 (4) 2.1 (1.0) Vehicle 28 (8) 20 (9) 2.8 (1) 1.4 (0.4) 16 weeks LN JL1-Toxin 36 (15) 31 (11) 0.3 (0.8) 1.6 (1.0) Isotype-Toxin 38 (10) 39 (10) 0.1 (0.2) 0.9 (0.4) Vehicle 34 (13) 37 (15) 0.2 (0.3) 1.3 (1.6)
(264) (In the table, JL1 represents CD43; PMN: polymorphonuclear leukocyte; LN: lymph node)
(265) As shown in the table 3, when NSG mice were engrafted with normal human Hematopoietic stem cells (HSCs) obtained from the cord blood stem cell and the anti-CD43 antibody-toxin (saporin) conjugate was treated for 4 weeks, any loss of hematopoietic section was not observed. This result showed that the anti-CD43 antibody-toxin (saporin) conjugate did not kill the hematopoietic stem cell or intermediate progenitor cell.
(266) The immunohistochemical staining (IHC) was performed by using the mouse anti-CD43 antibody in various human normal tissues, and the obtained result was shown in the following table 4.
(267) TABLE-US-00007 TABLE 4 Tissue type # positive Intensity Cerebellum 0/3 Adrenal gland 0/3 Ovary 0/3 Pancreas 0 3 Parathyroid gland 0/3 Pituitary gland 0/3 Testis 0/3 Thyroid 0/3 Breast 0/3 Spleen 0/3 Tonsil 0/3 Thymus gland 3/3 +++ Bone marrow 0 3 Lung 0/3 Heart 0/3 Esophagus 0/3 Stomach 0/3 Sm. Intestine* 0/3 Colon* 0/3 Liver 0/3 Salivary gland 0/3 Kidney 0/3 Prostate 0/3 *mucin staining
(268) As shown in the Table 4, CD43-specific staining was not found in any other tissues than the thymus.
(269) The expression rate of CD43 antigen was measured by staining two normal human PBMCs (peripheral blood mononuclear cells) with mouse anti-human CD43 (represented as JL-1: (d)) (refer to example 15 (FACS), and result was shown in
(270) Saporin-conjugated mouse anti-CD43 antibody (10 μL/mL) was treated to normal bone marrow cell in advance (JL1+) and the case of not treating (JL1−), and the colony formation level of these bone marrow subsets was measured. The bone marrow was isolated from human white blood cells, and CD34+ cells were isolated and harvested by dividing into JL-1 (CD43) positive and negative. Harvested cells were then put into Methocult together with the cytokine and the colony formation was measured. The obtained result was shown in
(271) On the other hand, the therapeutic effect of the anti-CD43 antibody itself (naked) and toxin-bound anti-CD43 antibody-toxin (debouganin) conjugate was tested in the leukemia ALL xenograft mouse model using the cell line (acute leukemia mouse model obtained by grafting CEM7 cell to mouse). The mouse anti-CD43 monoclonal antibody prepared in advance as the anti-CD43 antibody was used. The obtained result was shown in
(272) On the other hand, the CD43 expression level in major AML (Acute myeloid leukemia) blast and subsets was measured. The result was shown in
Example 18: Construction of Humanized Anti-CD43 Monoclonal Antibody
(273) Based on the sequence of gremlin maintaining the mouse CD43 antibody (heavy chain: SEQ ID NO: 34; heavy chain coding DNA: SEQ ID NO: 33; heavy chain expression vector: SEQ ID NO: 35, light chain: SEQ ID NO: 37; light chain coding DNA: SEQ ID NO: 36; light chain expression vector (pTT5 based): CDR region sequence of heavy chain and light chain each of SEQ ID NO: 38) (CDRH1: SEQ ID NO: 111 (GYFMN); CDRH2: SEQ ID NO: 114 (RINPNNGDSFYNQKFQG); CDRH3: SEQ ID NO: 118 (EGYYGGRGYALDY); CDRL1: SEQ ID NO: 119 (RTSQDISNYLN); CDRL2: SEQ ID NO: 121 (NTSRLHS); CDRL3: SEQ ID NO: 125 (QQSNMFPY)) and coding human antibody gene, the scFv type of recombinant humanized antibody library, in which the sequence of region to framework region was recombined, was constructed.
(274) The constructed scFv antibody library was expressed and screened by the common phage display method, and the positive clone was constructed as the sub-library expressing variants which were substituted in region except CDR or partial sequence of region in CDR, thereby repeating the screening.
(275) By repeating various cycles of these constructions of library and display method, the humanized antibody variant sequences showing the antigen affinity very similar to parent clone were secured.
(276) The heavy chain sequences of the secured humanized anti-CD43 antibody were shown in SEQ ID NOs: 40, 42, 44, and 46, and the light chain sequence was shown in SEQ ID NO: 48, and the heavy chain variable regions and light chain variable regions were shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, and 83 (heavy chain variable region), and SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, and 83 (light chain variable region), respectively.
(277) In addition, the heavy chain variable regions and light chain variable regions of mutant humanized anti-CD43 antibody from modified SEQ ID NO: 83 (heavy chain variable region) and SEQ ID NO: 95 (light chain variable region) were shown in SEQ ID NOs: 84 to 94 (heavy chain variable region) and SEQ ID NOs: 96 to 106 (light chain variable region), respectively.
(278) The scFv type of antibody was prepared by linking the obtained heavy chain variable region and light chain variable region with GGGASGGGGSGGGGS (SEQ ID NO: 127) or GGGGSGGGGSGGGAS (SEQ ID NO: 128).
Example 19: Antibody-Dependent Cell Cytoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) by Humanized or Chimeric Anti-CD43 Monoclonal Antibody
(279) The apoptosis effect (ADCC and CDC) by the prepared chimeric anti-CD43 antibody (DNP001; Example 2-1-3) was tested. At first, CEM7 cell line effector cell (PBMC; peripheral blood mononuclear cell) was co-cultured with anti-CD43 chimeric or control antibody (human IgG1) for 4 hours, and then the cytotoxicity was measured by using Cell Titer Glo, and the result was shown in
(280)
(281)
(282) The CD43 expression in Leukemia stem cell (LSC) subsets was tested. AML (Acute myeloid leukemia) patient bone marrow was stained with CD45 antibody (BD), CD34 antibody (BD), CD38 antibody (BD), CD43 antibody (DNP001), or mlgG1 (control) with Alexa488, and the CD43 expression level was evaluated by sorting CD34+CD38− leukemia stem cells, and they were compared to the control group. The result was shown in
(283) The mean number of colonies treated with the chimeric anti-CD43 antibody conjugated with toxin (saporin: SAP) (CCC) was measured in vitro and shown in
(284) In addition, the effect of the chimeric anti-CD43 antibody conjugated with toxin (saporin: SAP) (CCC) on the proliferation of antigen-positive, major AML was measured in vitro and shown in
(285) As shown in
(286) The inhibitory effect on major AML cancer cell growth by the toxin-conjugated antibody in NSG mouse (in vivo) was tested. 5×10.sup.7 of bone marrow cells were harvested from CD43+ AML patient, and they were intravenously injected to 30 NSG mice irradiated and after 8 weeks, the chimeric CD43 antibody-debouganin (DB) conjugate (represented as JL1-DB), hIgG1-DB conjugate (represented as Isotype-DB), or PBS (vehicle) was administered into those mice in 45 μg dose every week for 4 weeks. Blood, bone marrow and spleen were then collected form those mice, and engraftment and tumor production were observed. The obtained result was shown in
(287)
(288) As shown in
(289)
(290)
(291)
(292)
Example 20: Preparation of Modified Antibody Removing Amino Acid Residue of Glycosylated Region of Humanized Antibody
(293) It was found that there was the amino acid sequence having the possibility of glycosylation in the light chain variable region (SEQ ID NO: 105) of the humanized anti-CD43 antibody obtained from the example 18 (257-10; comprising SEQ ID NO: 93 (heavy chain variable region)+SEQ ID NO: 105 (light chain variable region)), and the mutant antibody was prepared to remove it.
(294) Since the glycosylation of light chain epitope (light chain variable region) is highly likely to cause negative effects on the antigen binding capacity and physical properties of antibody, and may affect the productivity degradation in the subsequent mass production, the possibility of antibody development as a therapeutic agent was increased by substituting the amino acid having the potential possibility of glycosylation into other amino acids.
(295) The 50th position of amino acid residue asparagine (Asn, N) and the 52th position of amino acid residue serine (Ser, S) were selected as the amino acids having the possibility of glycosylation of light chain variable region (SEQ ID NO: 105) of humanized anti-CD43 antibody (refer to
(296) TABLE-US-00008 [SEQ ID NO 107 (S52A)] DTQMTQSPSSVSASVGDRVTITCRTSQDISNYLNWYQQKPGKAPKLLIYN TARLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNMFPYTFGQ GTKLEIK [SEQ ID NO 108 (N50Q)] DTQMTQSPSSVSASVGDRVTITCRTSQDISNYLNWYQQKPGKAPKLLIYQ TSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNMFPYTFGQ GTKLEIK [SEQ ID NO 109 (N50A)] DTQMTQSPSSVSASVGDRVTITCRTSQDISNYLNWYQQKPGKAPKLLIYA TSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNMFPYTFGQ GTKLEIK
(297) The IgG1 type antibody comprising the heavy chain variable region (SEQ ID NO: 93) and the light chain variable region of the obtained modified antibody 55 was prepared, and western blotting was performed by using Concanavalin A-HRP binding to glycosylated region (Sigma-Aldrich).
(298) The obtained result was shown in
(299) In addition, by measuring the binding capacity of the antibody to the CD43 positive cell, CEM7 cell, the antigen binding capacity was analyzed. The obtained result was shown in
(300) The chimeric antibody (DNP001) and modified humanized antibody removing glycosylation (heavy chain variable region; SEQ ID NO: 93; light chain variable region: SEQ ID NO: 109) were bound to antigen (CD43) positive cell, CEM7 cell, and they were analyzed by flow cytometry. The obtained result was shown in