Antibody binding specifically to CD40 and use thereof
11905331 ยท 2024-02-20
Assignee
Inventors
- Gil Yong JI (Seoul, KR)
- Kwon Pyo Hong (Cheongju-si, KR)
- Eui Sup LEE (Seoul, KR)
- Yoo Ri MOON (Seoul, KR)
- Sangsoon Yoon (Seoul, KR)
Cpc classification
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/14
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
C07K16/2878
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention relates to an anti-CD40 antibody binding specifically to CD40 and use thereof and, particularly, provides an anti-CD40 antibody or an antigen-binding fragment thereof, and a pharmaceutical composition comprising the same antibody or fragment as an effective ingredient for prevention and/or treatment of cancer, cancer metastasis, infection, and/or immune deficiency diseases.
Claims
1. An anti-CD40 antibody or an antigen-binding fragment thereof, comprising the following complementarity determining region (CDRs): CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 33, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 35, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 36, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22.
2. The anti-CD40 antibody or the antigen-binding fragment thereof according to claim 1, comprising: a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 1, 23, 24, 25, 26, 27, or 28; and a light-chain variable region comprising the amino acid sequence of SEQ ID NO: 3, 29, or 30.
3. The anti-CD40 antibody or the antigen-binding fragment thereof according to claim 1, wherein the anti-CD40 antibody is an animal antibody, a chimeric antibody, or a humanized antibody.
4. The anti-CD40 antibody or the antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is scFv, (scFv)2, Fab, Fab, or F(ab)2 of the anti-CD40 antibody.
5. The anti-CD40 antibody or the antigen-binding fragment thereof according to claim 1, having an agonist activity for CD40.
6. A pharmaceutical composition comprising the anti-CD40 antibody or the antigen-binding fragment thereof according to claim 1.
7. A nucleic acid encoding the anti-CD40 antibody or the antigen-binding fragment thereof according to claim 1.
8. The nucleic acid according to claim 7, encoding either or both of: an amino acid sequence comprising SEQ ID NO: 17 or SEQ ID NO: 33, and SEQ ID NO: 18 or SEQ ID NO: 34, and SEQ ID NO: 19; and an amino acid sequence comprising SEQ ID NO: 20 or SEQ ID NO: 35, and SEQ ID NO: 21 or SEQ ID NO: 36, and SEQ ID NO: 22.
9. The nucleic acid of claim 7, encoding either or both of: the amino acid sequence of SEQ ID NO: 1, 23, 24, 25, 26, 27, or 28; and the amino acid sequence of SEQ ID NO: 3, 29, or 30.
10. A recombinant vector comprising the nucleic acid of claim 8.
11. A recombinant cell comprising the recombinant vector of claim 10.
12. An anti-CD40 antibody or an antigen-binding fragment thereof, which comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of an anti-CD40 antibody produced by the hybridoma of accession number KCLRF-BP-00381.
13. The anti-CD40 antibody or an antigen-binding fragment thereof according to claim 12, which comprise a heavy chain variable region and a light chain variable region of an anti-CD40 antibody produced by the hybridoma of accession number KCLRF-BP-00381.
14. The anti-CD40 antibody or an antigen-binding fragment thereof according to claim 12, wherein the anti-CD40 antibody is produced by the hybridoma of accession number KCLRF-BP-00381.
15. A hybridoma, deposited with accession number KCLRF-BP-00381, for producing an anti-CD40 antibody.
16. A method of treating a disease, the method comprising administering the anti-CD40 antibody or the antigen-binding fragment thereof according to claim 1 to a subject in need of treating the disease, wherein the disease is selected from the group consisting of cancer and cancer metastasis.
17. The method of claim 16, wherein the anti-CD40 antibody or the antigen-binding fragment thereof comprises: a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 1, 23, 24, 25, 26, 27, or 28; and a light-chain variable region comprising the amino acid sequence of SEQ ID NO: 3, 29, or 30.
18. A method of treating a disease, the method comprising administering an anti-CD40 antibody or an antigen-binding fragment thereof to a subject in need of treating the disease, wherein the disease is selected from the group consisting of cancer and cancer metastasis, wherein the anti-CD40 antibody is selected from the group consisting of: an anti-CD40 antibody produced by hybridoma of accession number KCLRF-BP-00381, or an antigen-binding fragment thereof; an anti-CD40 antibody comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the anti-CD40 antibody produced by the hybridoma of accession number KCLRF-BP-00381, or an antigen-binding fragment thereof; and an anti-CD40 antibody comprising the heavy-chain and light-chain variable regions of the anti-CD40 antibody produced by the hybridoma of accession number KCLRF-BP-00381, or an antigen-binding fragment thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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MODE FOR CARRYING OUT THE INVENTION
(21) Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.
Example 1: Production of Anti-CD40 Antibody
(22) 1-1. Mouse Antibody Preparation
(23) 1-1-1. Preparation of Monoclonal Antibody-Producing Cell
(24) Splenocytes from a Balb/c mouse injected with a human recombinant protein CD40 antigen were fused to the myeloma cell line X63-Ag8.653 (ATCC, PTA-8431) from an 8-azaguanine-resistant mouse. To this end, Balb/c mice were each injected with 100 g of the human CD40 recombinant antigen every two weeks for six weeks to induce an immune reaction. On day 3 after the final inoculation, splenocytes were isolated and suspended. According to Koeler and Milstein's method (Koeler & Milstein 1975), 10.sup.8splenocytes and 10.sup.7 myeloma cells were fused to each other using 50% polyethylene glycol 400 in DMEM (Dulbeco's modified Eagle's medium). The fused cells were washed and then resuspended in a DMEM medium supplemented with 20% fetal bovine serum, 100 M hypoxanthine, 0.44 M aminopterin, and 16 M thymidine (HAT culture medium). The cells ere plated into 96-well plates and cultured at 37 C. in an incubator supplied with 5% CO.sub.2.
(25) When colonies were observed two weeks later, the supernatant was taken and analyzed using ELISA (Enzyme-Linked ImmunoSorbent Assay) to examine whether to bind to CD40.
(26) For a positive group, selection was made of wells in which 10.sup.5 or more cells per well were formed in a single moiety. After being taken from the wells where colonies were formed with the supernatant measured to have a high antibody titer, cells were subcloned according to limiting dilution assay to obtain monoclonal cells having a high antibody titer. A supernatant was taken from the monoclonal cell culture and stored until the subsequent experiment.
(27) 1-1-2. Selection of Monoclonal Cell Producing Antibody to Human CD40
(28) Human recombinant CD40 (R&D systems, Cat. No.: P25942) was plated in an amount of 100.0 ng per well into a MaxiSorp ELISA plate and reacted at 37 C. for one hour for antibody coating, followed by blocking by incubation with 200 L of 1 blocking solution (Sigma) per well at 37 C. for one hour.
(29) The monoclonal cell culture was added at a density of 100 L/well, incubated at 37 C. for one hour, and washed three times with phosphate buffered saline before treatment with the secondary antibody goat anti-mouse IgG-HRP. Incubation at 37 C. for 30 minutes was followed by three rounds of washing with phosphate buffered saline. Then, TMB Single Solution (Life Technologies, Cat. No: 002023) was added in an amount of 100 l per well and incubated at room temperature for 5-10 min in a dark place. Color development was stopped with 100 L of 1.0 N sulfuric acid and absorbance at 450 nm was measured.
(30) As a result, the monoclonal cell line 5C2 that produced an antibody reacting specifically with the recombinant antigen human CD40 could be selected. The cell line thus obtained was deposited Nov. 2, 2016, with the Korean Cell Line Research Foundation (KCLRF), located in Yongon-Dong, Chongno-Gu, Seoul, under accession number KCLRF-BP-00381.
(31) 1-1-3. Production of Monoclonal Antibody from Selected Monoclonal Cell Line
(32) The established cell line 5C2 was cultured in a 10% fetal bovine serum-supplemented RPMI medium at 37 C. for two weeks under a 5% CO.sub.2 condition.
(33) After being sterilized by filtration, the cell line culture was loaded into HiTrap Protein G HP column (GE Healthcare, Cat. No.: 17-0405-03) equilibrated with Protein G equilibration buffer (20 mM phosphate, pH7.4), washed by flowing the equilibration buffer through the column, and recovered with an elution buffer (20 mM Citric acid pH3.0). The recovered antibody was named 5C2 (mouse antibody) and dialyzed against phosphate buffered saline to exchange the buffer for use in subsequent tests.
(34) 1-1-4. Assay for Specificity of Monoclonal Anti-CD40 Antibody
(35) For assay for the specificity of the anti-CD40 5C2 antibody (mouse antibody), reactivity against a recombinant antibody human CD40 (rCD40; R&D systems, Cat. No.: P25942), a recombinant antigen human CD40L (Recombinant Human sCD40 Ligand; Peprotech, Cat. No.: 310-02; amino acid sequence: MQKGDQNPQI AAHVISEASS KTTSVLQWAE KGYYTMSNNL VTLENGKQLT VKRQGLYYIY AQVTFCSNRE ASSQAPFIAS LWLKSPGRFE RILLRAANTH SSAKPCGQQS IHLGGVFELQ PGASVFVNVT DPSQVSHGTG FTSFGLLKL (SEQ ID NO: 31)), and a human IgG (Dinona) was analyzed using ELISA. The three antigens were each diluted at a density of 1.0 g/mL in phosphate buffered saline and the dilution was plated at 100 L/well into microplates and incubated at 37 C. for one hour to coat the plates therewith. Then, a 1 blocking solution (Sigma) was added in an amount of 200 L per well and incubated at 37 C. for one hour to block the antigens.
(36) Anti-CD40 antibody 5C2 and anti-CD40L 5C8 (ATCC, ATCC HB-10916; positive control) were each diluted to a density of 1.0 g/mL in phosphate buffered saline, and the dilutions were added in an volume of 100 L per well and incubated at 37 C. for one hour, washed three times with phosphate buffered saline, and treated with the secondary antibody goat anti-mouse IgG-HRP. After 30 min of incubation at 37 C., the wells were washed three times with phosphate buffered saline, added with TMB Single Solution (Life Technologies, Cat. No: 002023) in an amount 100 L per well, and incubated at room temperature for 5-10 min in a dark place. The color development was stopped with 100 L of 1.0 N sulfuric acid and absorbance at 450 nm was measured.
(37) The results obtained are depicted in
(38) 1-2. Preparation of Chimeric Antibody
(39) On the basis of the amino acid sequence of the produced anti-CD40 mouse antibody 5C2, an anti-CD40 chimeric antibody was prepared.
(40) 1-2-1. Plasmid Construction
(41) For use in expressing an anti-CD40 chimeric antibody, a heavy-chain expressing plasmid and a light-chain expressing plasmid were constructed, separately. Both the heavy-chain and light-chain expressing plasmids were based on the pcDNA3.4 vector (Invitrogen).
(42) Heavy- and light-chain variable region-encoding cDNA for antibody expression were cloned using Ig-Primer sets (Novagen), inserted into pCR2.1 vector (Invitrogen, Cat. No.: K200001), and identified by sequencing. A mouse antibody gene was identified with the aid of the IMGT site (www.imgt.org).
(43) The amino acid sequences of heavy- and light-chain variable regions in the mouse 5C2 monoclonal antibody and the nucleotide sequence of the coding gene therefor are as follows.
(44) TABLE-US-00001 TABLE1 AminoacidsequenceofVariableRegioninAnti-CD40Mouse5C2 MonoclonalAntibody Aminoacidsequence Codinggene(DNA)sequence Heavy- QVQMLQSGTELVRPGT CAGGTACAGATGCTGCAGAGCGGAACTGAACTG chain SVKVSCKASGYGFTNY GTTAGACCTGGTACTAGCGTTAAGGTCAGCTGTA variable LIEWVKQRPGQGLEWIG AGGCTAGCGGATACGGTTTCACCAACTACCTGAT region VINPGYGGVNYNEKFK CGAATGGGTCAAGCAGAGGCCAGGACAAGGTTT sequence GKAILTADKSSSTAYMH GGAGTGGATTGGAGTGATTAACCCCGGGTATGG (VH) LTSLTSDDSAVYFCARG GGGCGTGAATTACAATGAGAAGTTTAAAGGCAA GSGFAFWGOGTLVTVS AGCCATACTGACCGCAGACAAATCAAGTAGTAC T(SEQIDNO:1) CGCCTATATGCACCTGACATCTTTGACATCTGAC GATTCTGCCGTGTATTTTTGCGCCCGGGGCGGGA GTGGCTTTGCTTTTTGGGGCCAGGGCACACTTGT GACTGTGTCTACA(SEQIDNO:2) Light- DIQMTQTTSSLSASLGQ GACATCCAAATGACCCAAACCACCTCCTCACTTT chain RVTISCRASQDISNHLN CCGCATCTCTTGGACAAAGAGTCACCATCTCCTG variable WYQQKPNGTVRLLISST FAGGGCAAGTCAAGACATCTCCAACCACCTCAAC region SRLHSGVPSRFSGSGSG FGGTACCAGCAGAAGCCAAACGGAACTGTTAGG sequence TDYSLTISNLEQEDIATY FTGTTGATCTCCAGCACCTCACGTTTGCACTCAG (VL) FCQQGNTLPWTFGGGT GAGTACCATCACGATTCAGCGGTAGTGGTTCTGG KLEIK(SEQIDNO:3) FACAGATTACAGCTTGACCATTAGCAACCTGGAG CAGGAGGATATTGCTACCTACTTCTGCCAGCAGG GCAATACCCTGCCTTGGACATTTGGGGGGGGCAC AAAACTGGAAATTAAG(SEQIDNO:4)
(45) (CDR1, CDR2, and CDR3 (according to the IMGT definition) are sequentially underlined)
(46) In order to allow the variable region-encoding cDNAs and constant region-encoding cDNA to be expresses as sequential amino acid sequences in antibodies, respective gene fragments in which the cloned variable region-encoding nucleotide sequences were linked to nucleotide sequences coding for the constant region (heavy chain) of known human IgG1, human IgG2, and human IgG4 (S228P, serine at position 228 was substituted with proline) and the kappa constant region (light chain) were synthesized (Bioneer). As such, the synthesized heavy- and light-chain expressing genes were digested with restriction enzymes Xho I and EcoR1. The heavy chain and light chain gene fragments thus obtained were ligated to respective pcDNA3.4 vectors to construct antibody-expressing vectors for three heavy chains and one light chain. The amino acid sequences of the heavy and light chains in the three chimeric antibodies thus constructed and nucleotide sequences coding therefor are summarized in Table 2, below:
(47) TABLE-US-00002 TABLE2 Aminoacidsequencesofheavyandlightchainsinthreechimeric antibodiesandnucleotidesequencecodingtherefor (boldsrepresentvariableregions) Aminoacidsequence Nucleotidesequence(cDNA) Heavy QVQMLQSGTELVRP CAGGTACAGATGCTGCAGAGCGGAACTGAACTG chainof GTSVKVSCKASGYGF GTTAGACCTGGTACTAGCGTTAAGGTCAGCTGTA chimeric TNYLIEWVKQRPGQ AGGCTAGCGGATACGGTTTCACCAACTACCTGAT IgG1Ab GLEWIGVINPGYGGV CGAATGGGTCAAGCAGAGGCCAGGACAAGGTTT (comprising NYNEKFKGKAILTAD GGAGTGGATTGGAGTGATTAACCCCGGGTATGG human KSSSTAYMHLTSLTS GGGCGTGAATTACAATGAGAAGTTTAAAGGCAA IgG1 DDSAVYFCARGGSGF AGCCATACTGACCGCAGACAAATCAAGTAGTAC Constant AFWGQGTLVTVSTA CGCCTATATGCACCTGACATCTTTGACATCTGAC region) STKGPSVFPLAPSSKST GATTCTGCCGTGTATTTTTGCGCCCGGGGCGGG SGGTAALGCLVKDYF AGTGGCTTTGCTTTTTGGGGCCAGGGCACACTT PEPVTVSWNSGALTSG GTGACTGTGTCTACAGCTTCAACTAAGGGACCAA VHTFPAVLQSSGLYSL GCGTATTCCCACTTGCTCCATCTAGCAAGAGCACTA SSVVTVPSSSLGTQTYI GCGGAGGAACAGCTGCTTTGGGGTGTTTGGTAAAG CNVNHKPSNTKVDKK GATTACTTTCCCGAACCTGTTACCGTGAGCTGGAAC VEPKSCDKTHTCPPCP AGCGGGGCTTTGACAAGTGGCGTTCATACATTTCCT APELLGGPSVFLFPPKP GCCGTTTTGCAAAGCAGCGGCTTGTATAGCTTGAGC KDTLMISRTPEVTCVV TCTGTTGTTACCGTTCCAAGCTCATCTCTGGGCACA VDVSHEDPEVKFNWY CAAACATACATCTGCAACGTGAACCACAAGCCCTC VDGVEVHNAKTKPRE AAACACCAAGGTGGACAAGAAGGTGGAGCCAAAG EQYNSTYRVVSVLTV TCTTGCGACAAGACCCACACCTGTCCACCTTGTCCA LHQDWLNGKEYKCK GCCCCTGAACTCCTGGGGGGCCCTTCAGTTTTTCTC VSNKALPAPIEKTISKA TTTCCTCCTAAACCTAAAGATACACTCATGATCAGT KGQPREPQVYTLPPSR CGGACCCCTGAAGTTACCTGTGTGGTGGTCGATGTG DELTKNQVSLTCLVK TCTCATGAAGATCCTGAAGTCAAGTTTAACTGGTAT GFYPSDIAVEWESNGQ GTGGACGGCGTGGAGGTGCATAATGCCAAGACCAA PENNYKTTPPVLDSDG GCCTCGGGAGGAGCAATATAATTCTACCTATCGCGT SFFLYSKLTVDKSRWQ CGTCTCTGTCCTCACCGTCCTGCATCAGGACTGGCT QGNVFSCSVMHEALH GAATGGCAAAGAGTATAAGTGCAAAGTCAGTAACA NHYTQKSLSLSPGK AAGCCCTCCCCGCCCCCATAGAGAAAACCATTAGT (SEQIDNO:5) AAAGCCAAAGGGCAGCCCCGCGAGCCCCAGGTCTA TACACTGCCCCCCAGTAGAGACGAGCTGACAAAGA ATCAGGTGTCTCTGACATGCCTGGTGAAAGGCTTTT ATCCCTCTGACATTGCCGTCGAGTGGGAGTCTAATG GGCAGCCCGAGAATAATTATAAGACAACACCCCCC GTGCTGGACAGTGACGGCTCATTTTTCCTGTATTCA AAACTGACAGTGGACAAAAGTCGGTGGCAGCAGGG GAATGTGTTTTCATGCAGTGTCATGCACGAGGCCCT CCACAATCACTATACCCAGAAATCTCTGAGTCTCTC TCCTGGGAAATGA(SEQIDNO:6) Heavy QVQMLQSGTELVRP CAGGTACAGATGCTGCAGAGCGGAACTGAACTG chainof GTSVKVSCKASGYGF GTTAGACCTGGTACTAGCGTTAAGGTCAGCTGTA IgG2 TNYLIEWVKQRPGQ AGGCTAGCGGATACGGTTTCACCAACTACCTGAT chimeric GLEWIGVINPGYGGV CGAATGGGTCAAGCAGAGGCCAGGACAAGGTTT Ab NYNEKFKGKAILTAD GGAGTGGATTGGAGTGATTAACCCCGGGTATGG (comprising KSSSTAYMHLTSLTS GGGCGTGAATTACAATGAGAAGTTTAAAGGCAA human DDSAVYFCARGGSGF AGCCATACTGACCGCAGACAAATCAAGTAGTAC IgG2 AFWGQGTLVTVSTA CGCCTATATGCACCTGACATCTTTGACATCTGAC constant STKGPSVFPLAPCSRST GATTCTGCCGTGTATTTTTGCGCCCGGGGCGGG region) SESTAALGCLVKDYFP AGTGGCTTTGCTTTTTGGGGCCAGGGCACACTT EPVTVSWNSGALTSG GTGACTGTGTCTACAGCTTCCACCAAGGGCCCATC VHTFPAVLQSSGLYSL CGTGTTCCCTCTGGCCCCATGTTCTAGGTCTACATCT SSVVTVPSSNFGTQTY GAGAGCACCGCCGCCCTCGGCTGTCTGGTGAAGGA TCNVDHKPSNTKVDK TTATTTCCCCGAGCCCGTGACCGTGTCTTGGAACAG TVERKCCVECPPCPAP CGGAGCCCTGACTAGCGGAGTGCACACCTTCCCAG PVAGPSVFLFPPKPKD CTGTGCTGCAGAGCTCCGGCCTGTACAGCCTCTCTT TLMISRTPEVTCVVVD CTGTGGTGACCGTGCCCTCTAGCAACTTCGGAACAC VSHEDPEVQFNWYVD AGACCTACACATGTAACGTGGATCACAAGCCTTCC GVEVHNAKTKPREEQ AACACCAAGGTGGATAAGACCGTGGAGAGAAAGTG FNSTFRVVSVLTVVHQ CTGTGTGGAGTGCCCTCCATGTCCTGCCCCACCTGT DWLNGKEYKCKVSN GGCTGGACCTTCTGTGTTTCTGTTCCCTCCAAAGCC KGLPAPIEKTISKTKGQ AAAGGATACCCTGATGATCAGCAGAACTCCTGAGG PREPQVYTLPPSREEM TGACCTGTGTGGTGGTGGACGTGAGCCACGAGGAT TKNQVSLTCLVKGFYP CCTGAGGTGCAGTTTAACTGGTACGTGGATGGCGTG SDIAVEWESNGQPENN GAGGTGCATAACGCTAAGACAAAGCCTAGGGAGGA YKTTPPMLDSDGSFFL GCAGTTTAACAGCACCTTCAGAGTGGTGAGCGTGCT YSKLTVDKSRWQQGN GACCGTGGTGCACCAGGATTGGCTGAACGGCAAGG VFSCSVMHEALHNHY AGTATAAGTGTAAGGTGTCTAACAAGGGCCTGCCA TQKSLSLSPGK(SEQ GCCCCTATTGAGAAGACCATCAGTAAGACCAAGGG IDNO:7) ACAGCCTAGGGAGCCTCAGGTGTACACCCTGCCTCC TTCCAGAGAGGAGATGACAAAGAACCAGGTGAGCC TGACCTGTCTGGTGAAGGGCTTCTACCCTAGCGATA TCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAG AACAACTACAAGACCACCCCACCTATGCTGGACAG CGATGGCTCTTTCTTCCTGTACTCTAAGCTGACCGT GGACAAGAGCAGATGGCAGCAGGGCAACGTGTTTT CTTGTTCTGTGATGCACGAGGCCCTGCACAACCACT ACACCCAGAAGTCTCTGTCTCTGTCTCCAGGCAAGT GA(SEQIDNO:8) Heavy QVQMLQSGTELVRP CAGGTACAGATGCTGCAGAGCGGAACTGAACTG chainof GTSVKVSCKASGYGF GTTAGACCTGGTACTAGCGTTAAGGTCAGCTGTA IgG4 TNYLIEWVKQRPGQ AGGCTAGCGGATACGGTTTCACCAACTACCTGAT chimeric GLEWIGVINPGYGGV CGAATGGGTCAAGCAGAGGCCAGGACAAGGTTT Ab NYNEKFKGKAILTAD GGAGTGGATTGGAGTGATTAACCCCGGGTATGG (comprising KSSSTAYMHLTSLTS GGGCGTGAATTACAATGAGAAGTTTAAAGGCAA human DDSAVYFCARGGSGF AGCCATACTGACCGCAGACAAATCAAGTAGTAC IgG4 AFWGQGTLVTVSTA CGCCTATATGCACCTGACATCTTTGACATCTGAC constant STKGPSVFPLAPCSRST GATTCTGCCGTGTATTTTTGCGCCCGGGGCGGG region) SESTAALGCLVKDYFP AGTGGCTTTGCTTTTTGGGGCCAGGGCACACTT EPVTVSWNSGALTSG GTGACTGTGTCTACAGCTTCCACCAAGGGCCCCTC VHTFPAVLQSSGLYSL CGTGTTCCCTCTCGCCCCTTGCTCCAGATCCACCTCC SSVVTVPSSSLGTKTY GAGTCTACCGCCGCTCTGGGCTGCCTGGTCAAGGAC TCNVDHKPSNTKVDK TACTTCCCCGAGCCTGTGACCGTGTCTTGGAACTCT RVESKYGPPCPPCPAP GGCGCACTGACCAGCGGCGTGCACACCTTCCCTGCC EFLGGPSVFLFPPKPK GTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCC DTLMISRTPEVTCVVV GTCGTGACCGTGCCCTCCTCCAGCCTGGGCACCAAG DVSQEDPEVQFNWYV ACCTACACCTGTAACGTGGACCACAAGCCCTCCAA DGVEVHNAKTKPREE CACCAAGGTGGACAAGCGGGTGGAATCTAAGTACG QFNSTYRVVSVLTVLH GCCCTCCCTGCCCCCCCTGCCCTGCCCCTGAATTTCT QDWLNGKEYKCKVS GGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCC NKGLPSSIEKTISKAKG CAAGGACACCCTGATGATCTCCCGGACCCCCGAAG QPREPQVYTLPPSQEE TGACCTGCGTGGTGGTGGACGTGTCCCAGGAAGAT MTKNQVSLTCLVKGF CCCGAGGTCCAGTTCAATTGGTACGTGGACGGCGT YPSDIAVEWESNGQPE GGAAGTGCACAACGCCAAGACCAAGCCCAGAGAG NNYKTTPPVLDSDGSF GAACAGTTCAACTCCACCTACCGGGTGGTGTCCGTG FLYSRLTVDKSRWQE CTGACCGTCCTGCACCAGGACTGGCTGAACGGCAA GNVFSCSVMHEALHN AGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGC HYTQKSLSLSLGK CCTCCAGCATCGAAAAGACCATCTCCAAGGCCAAG (SEQIDNO:9) GGCCAGCCCCGCGAGCCTCAGGTGTACACCCTGCC CCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGT CCCTGACCTGTCTCGTCAAAGGCTTCTACCCCTCCG ATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCC GAGAACAACTACAAGACCACCCCCCCTGTGCTGGA CTCCGACGGCTCCTTCTTTCTGTACTCTCGGCTGACC GTGGATAAGAGCCGGTGGCAGGAAGGCAACGTCTT CTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCA CTATACCCAGAAGTCCCTGTCCCTGAGCCTGGGCAA ATGA(SEQIDNO:10) Light DIQMTQTTSSLSASL GACATCCAAATGACCCAAACCACCTCCTCACTTT chain GQRVTISCRASQDISN CCGCATCTCTTGGACAAAGAGTCACCATCTCCTG HLNWYQQKPNGTVR TAGGGCAAGTCAAGACATCTCCAACCACCTCAAC LLISSTSRLHSGVPSR TGGTACCAGCAGAAGCCAAACGGAACTGTTAGG FSGSGSGTDYSLTISN TTGTTGATCTCCAGCACCTCACGTTTGCACTCAG LEQEDIATYFCQQGN GAGTACCATCACGATTCAGCGGTAGTGGTTCTG TLPWTFGGGTKLEIK GTACAGATTACAGCTTGACCATTAGCAACCTGGA RTVAAPSVFIFPPSDEQ GCAGGAGGATATTGCTACCTACTTCTGCCAGCA LKSGTASVVCLLNNFY GGGCAATACCCTGCCTTGGACATTTGGGGGGGG PREAKVQWKVDNAL CACAAAACTGGAAATTAAGCGGACTGTTGCTGCTC QSGNSQESVTEQDSKD CATCTGTTTTTATATTTCCTCCCAGCGACGAGCAGC STYSLSSTLTLSKADY TGAAAAGCGGCACTGCCTCTGTGGTGTGTCTGCTGA EKHKVYACEVTHQGL ATAATTTTTACCCCCGGGAAGCCAAAGTCCAGTGG SSPVTKSFNRGEC AAGGTGGATAATGCCCTCCAGTCTGGGAACAGTCA (SEQIDNO:11) GGAAAGTGTGACAGAACAGGATAGTAAGGACTCTA CTTATAGCCTCTCTTCTACACTGACTCTGTCAAAGG CCGACTATGAGAAGCATAAAGTGTATGCCTGCGAG GTGACACATCAGGGCCTGAGTTCACCCGTGACAAA ATCTTTTAACCGCGGCGAGTGCTGA(SEQIDNO:12)
(48) For use as a positive control antibody, the anti-CD40 human antibody CP870,893 (U.S. Pat. No. 7,338,660 B2; 21.4.1 antibody) was also synthesized. A pcDNA3.4 expression vector was constructed in the same manner. For use as a control, an antibody was constructed using the sequence information of CP870,893 antibody (U.S. Pat. No. 7,338,660 B2; 21.4.1 antibody) and named CP870,893 antibody analogue. The amino acid sequences and coding nucleotide sequences used to construct the CP870,893 antibody analogue are summarized in Table 3, below:
(49) TABLE-US-00003 TABLE3 Aminoacidandnucleotidesequencesofanti-CD40humanantibody CP870,893(21.4.1antibody)(signalsequencesunderlinedand variableregionsinbold) Aminoacidsequence Nucleotidesequence(cDNA) Heavy MDWTWRILFLVAAATGA ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAG chain HSQVQLVQSGAEVKKP CAGCCACAGGAGCCCACTCCCAGGTGCAGCTGGTGC GASVKVSCKASGYTFTG AGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC YYMHWVRQAPGQGLE AGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCT WMGWINPDSGGTNYAQ TCACCGGCTACTATATGCACTGGGTGCGACAGGCC KFQGRVTMTRDTSISTA CCTGGACAAGGGCTTGAGTGGATGGGATGGATCA YMELNRLRSDDTAVYY ACCCTGACAGTGGTGGCACAAACTATGCACAGAAG CARDQPLGYCTNGVCS TTTCAGGGCAGGGTCACCATGACCAGGGACACGTC YFDYWGQGTLVTVSSAS CATCAGCACAGCCTACATGGAGCTGAACAGGCTGA TKGPSVFPLAPCSRSTSES GATCTGACGACACGGCCGTGTATTACTGTGCGAGA TAALGCLVKDYFPEPVTV GATCAGCCCCTAGGATATTGTACTAATGGTGTATG SWNSGALTSGVHTFPAVL CTCCTACTTTGACTACTGGGGCCAGGGAACCCTGG QSSGLYSLSSVVTVPSSNF TCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGG GTQTYTCNVDHKPSNTK FCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGA VDKTVERKCCVECPPCPA GAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTAC PPVAGPSVFLFPPKPKDTL FTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCG MISRTPEVTCVVVDVSHE CTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCT DPEVQFNWYVDGVEVHN ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG AKTKPREEQFNSTFRVVS ACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACA VLTVVHQDWLNGKEYKC CCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGT KVSNKGLPAPIEKTISKTK GGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGC GQPREPQVYTLPPSREEM CCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAG FK FCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG NQVSLTCLVKGFYPSDIA ATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGG VEWESNGQPENNYKTTPP ACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTG MLDSDGSFFLYSKLTVDK GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC SRWQQGNVFSCSVMHEA AAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGT LHNHYTQKSLSLSPGK GTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGC (SEQIDNO:13) FGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACA AAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAA AACCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC CCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACACCTCCCATGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCG FGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQIDNO:14) Light MRLPAOLLGLLLLWFPGS ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCT chain RCDIQMTQSPSSVSASVG CTGGTTCCCAGGTTCCAGATGCGACATCCAGATGAC DRVTITCRASQGIYSWL CCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAG AWYQQKPGKAPNLLIY ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGT TASTLQSGVPSRFSGSGS ATTTACAGCTGGTTAGCCTGGTATCAGCAGAAACC GTDFTLTISSLQPEDFAT AGGGAAAGCCCCTAACCTCCTGATCTATACTGCAT YYCQQANIFPLTFGGGT CCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGC KVEIKRTVAAPSVFIFPPS GGCAGTGGATCTGGGACAGATTTCACTCTCACCAT DEQLKSGTASVVCLLNNF CAGCAGCCTGCAACCTGAAGATTTTGCAACTTACT YPREAKVQWKVDNALQS ATTGTCAACAGGCTAACATTTTCCCGCTCACTTTCG GNSQESVTEQDSKDSTYS GCGGAGGGACCAAGGTGGAGATCAAACGAACTGTG LSSTLTLSKADYEKHKVY GCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA ACEVTHQGLSSPVTKSFN GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGC RGEC(SEQIDNO:15) FGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTG GAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACC TACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAG ACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCAC CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTC AACAGGGGAGAGTGTTAG(SEQIDNO:16)
(50) 1-2-2. Chimeric Anti-CD40 Antibody Production
(51) For chimeric antibody production, expression vectors carrying three heavy chains (respectively comprising constant regions of human IgG1, human IgG2, and human IgG4) and one light chain (inclusive of the kappa constant regions) were introduced into the EXPICHO Expression system (Thrmofisher, Cat. No.: A29133) in a transient transformation manner to produce IgG1 chimeric antibody (inclusive of the constant region of human IgG1), IgG2 chimeric antibody (inclusive of the constant region of human IgG2), and IgG4 chimeric antibody (inclusive of the constant region of human IgG4).
(52) The ExpiCHO-S cell line included within the EXPICHO Expression system kit (Termofisher, Cat. No.: A29133) was thawed and added to the Expression medium and incubated at 37 C. in a 8% CO.sub.2 atmosphere with shaking at 120 rpm to secure a necessary number of cells. After being harvested, the ExpiCHO-S cells were seeded at a density of 610.sup.6 cells/mL in the Expression medium to a final volume of 150 mL. As indicated by the manual, 100 g of the light chain vector and 50 g of the heavy chain vector were mixed with OPTIPRO SFM and EXPIFECTAMINE CHO reagent to give a mixture in which the vectors were at a concentration of 1.0 g/mL with the light chain and the heavy chain maintained at a ratio of 2:1. The vector mixture prepared for transformation was added to the ExpiCHO-S cell line and incubated at 37 C. for 24 hours in 8% CO.sub.2 atmosphere with shaking at 120 rpm. At 24 hours of incubation, the EXPICHO Enhancer and Feed were further added. On day five after incubation, the incubation condition was changed to 32 C., 5% CO.sub.2, and 120 rpm. In the new condition, the cells were incubated with the EXPICHO Feed for an additional 12 days.
(53) The culture medium thus obtained was sterilized by filtration and loaded into HITRAP Protein A HP column (GE Healthcare, Cat. No.: 11-0034-93) equilibrated with Protein A equilibration buffer (20 mM phosphate, pH7.4) and washed by flowing the equilibration buffer through the column, followed by antibody recovery with an elution buffer (20 mM Citric acid pH3.0). The recovered antibody was dialyzed against phosphate buffered saline to exchange the buffer for use in subsequent tests.
(54) 1-2-3. Assay for Antigen Binding Force of Chimeric Anti-CD40 Antibody
(55) The three anti-CD40 chimeric antibodies IgG1, IgG2, and IgG4 constructed in Examples 1-2-2 were assayed for antigen reactivity by ELISA. A dilution of the recombinant antigen human CD40 (Sino Biological Inc., Cat. No.: 10774-H02H) at a concentration of 1.0 g/mL in phosphate buffered saline was plated at 100 L/well into microplates and incubated at 37 C. for one hour to coat the plates therewith. Then, a 1 blocking solution (Sigma) was added in an amount of 200 L per well and incubated at 37 C. for one hour to block the antigen.
(56) Each of the three anti-CD40 chimeric antibodies and the control antibody anti-CD40 human antibody CP870,893 analogue (21.4.1 antibody in Table 2) was serially diluted from a concentration of 10.0 g/mL. The dilutions were added in an amount of 100 L/well, incubated at 37 C. for one hour and washed three times with phosphate buffered saline. The antibodies were treated with the secondary antibody goat anti-human IgG F(ab)2-HRP (Jackson Immunoresearch, Cat. No.: 109-035-006) at 37 C. for 30 min and washed three times with phosphate buffered saline. After reaction with 100 L of TMB Single Solution (Life Technologies, Cat. No: 002023) per well at room temperature for 4-10 min in a dark place, the color development was stopped with 100 L of 1.0 N sulfuric acid and then absorbance at 450 nm was measured.
(57) Absorbance at 450 nm of the chimeric antibodies thus obtained are depicted in
(58) 1-3. Preparation of Humanized Antibody
(59) On the basis of the amino acid sequence of the anti-CD40 mouse antibody 5C2, an anti-CD40 humanized antibody was constructed.
(60) 1-3-1. Selection of Recombinant Antibody Sequence by in Silico Humanization
(61) Humanized antibody sequences that retained CDR region sequences of each of the heavy and light chains of mouse CD40 antibody 5C2 and in which on the basis of a germline sequence of a human antibody gene, sequences for the framework region were recombined were selected in an in-silico manner.
(62) To begin with, heavy-chain CDRs (complementarity determining regions) and light-chain CDRs were determined on the basis of the heavy- and light-chain variable regions of the anti-CD40 mouse antibody 5C2 introduced in Table 1 (referring to IMGT/V-QUEST and are summarized in Table 4, below:
(63) TABLE-US-00004 TABLE4 CDR1 CDR2 CDR3 DeterminedaccordingtoIMGT/V-QUEST (http://www.imgt.org/IMGT_vquest/share/textes/) Heavy GYGFTNYL(SEQ INPGYGGV(SEQIDNO: GGSGFAF(SEQ chain IDNO:17) 18) IDNO:19) Light QDISNH(SEQID STS(SEQIDNO:21) QQGNTLP(SEQ chain NO:20) IDNO:22) DeterminedaccordingtoCabatdefinition (http://www.bioinf.org.uk/abs/) Heavy NYLIE(SEQIDNO: VINPGYGGVNYNEKFKG GGSGFAF(SEQ chain 33) (SEQIDNO:34) IDNO:19) Light RASQDISNHLN STSRLHS(SEQIDNO: QQGNTLP(SEQ chain (SEQIDNO:35) 36) IDNO:22)
(64) In Table 5 are summarized human antibody germline genes that were employed as the backbones of humanized recombinant antibody sequences because of the highest similarity in sequence to each of the heavy and light chains of mouse CD40 antibody 5C2:
(65) TABLE-US-00005 TABLE 5 Human Antibody Germline Gene Used for Humanization of Mouse 5C2 Antibody Human Ab Germline Heavy chain Light chain IGHV1-2*02 IGKV1-NL1*01 IGHV1-69*06 IGKV3-15*01 IGHV5-51*01
(66) Six heavy-chain variable regions and two light-chain variable regions for humanized 5C2 antibodies were selected in an in-silico method using the human antibody germline gene sequences and are summarized in Table 6, below:
(67) TABLE-US-00006 TABLE6 5C2humanizedantibodyvariableregionsequenceselectedinsilico Class Sequence SEQIDNO: Heavy VH1 QVQLVQSGAEVKKPGASVKVSCKASGYGFTNYLIEWVRQAP 23 chain GOGLEWIGVINPGYGGVNYNEKFKGRATLTADKSISTAYMEL variable SRLRSDDTAVYFCARGGSGFAFWGQGTLVTVSS region VH2 QVQLVQSGAEVKKPGASVKVSCKASGYGFTNYLIEWVRQAP 24 GQGLEWIGVINPGYGGVNYNEKFKGRVTLTADKSISTAYMEL SRLRSDDTAVYFCARGGSGFAFWGOGTLVTVSS VH3 QVQLVQSGAEVKKPGSSVKVSCKASGYGFTNYLIEWVRQAPG 25 QGLEWIGVINPGYGGVNYNEKFKGRATLTADKSTSTAYMELS SLRSEDTAVYFCARGGSGFAFWGQGTLVTVSS VH4 QVQLVQSGAEVKKPGSSVKVSCKASGYGFTNYLIEWVRQAPG 26 QGLEWIGVINPGYGGVNYNEKFKGRVTITADKSTSTAYMELSS LRSEDTAVYFCARGGSGFAFWGQGTLVTVSS VH5 EVQLVQSGAEVKKPGESVKISCKASGYGFTNYLIEWVROMPG 27 KGLEWIGVINPGYGGVNYNEKFKGQATLSADKSISTAYLQLSS LKASDTAVYFCARGGSGFAFWGQGTLVTVSS VH6 EVQLVQSGAEVKKPGESLKISCKASGYGFTNYLIEWVRQMPG 28 KGLEWIGVINPGYGGVNYNEKFKGQVTISADKSISTAYLOLSS LKASDTAMYFCARGGSGFAFWGQGTLVTVSS Light VL1 DIQMTQSPSSLSASVGDRVTITCRASQDISNHLNWYQQKPGKA 29 variable VKLLISSTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFC region QQGNTLPWTFGQGTKVEIK Chain VL2 EIVMTQSPATLSVSPGERATLSCRASQDISNHLNWYQQKPGOA 30 VRLLISSTSRLHSGIPARFSGSGSGTEYTLTISSLQSEDFAVYFCQ QGNTLPWTFGQGTKVEIK
(68) (CDR1, CDR2, are CDR3 (according to the IMGT definition) are sequentially underlined)
(69) 1-3-2. Expression and Analysis of Humanized Recombinant Antibody
(70) The heavy-chain variable region sequences in-silico selected were linked to the human IgG2 constant region to complete heavy-chain sequences while the light-chain variable region sequences were linked to the kappa light-chain constant region to complete light-chain sequences. The selected amino acid sequences were converted into genetic sequences coding therefor and gene fragments having the sequences are synthesized (Cosmogenetech).
(71) The coding gene fragments of heavy and light chains prepared above were transferred into respective pCDNA3.4 vectors. Four heavy chains (VH1, VH2, VH3, VH5) and two light chains (L1, L2) were combined to make eight humanized antibodies (VH1VL1: a combination of VH1 and VL1; VH2VL1: a combination of VH2 and VL1; VH3VL1: a combination of VH3 and VL1; VH5VL1: a combination of VH5 and VL1; VH6VL1: a combination of VH6 and VL1; VH1VL2: a combination of VH1 and VL2; VH2VL2: a combination of VH2 and VL2; VH3VL2: a combination of VH3 and VL2; VH5VL2: a combination of VH5 and VL2; VH6AVL2: a combination of VH6 and VL2). The antibodies were introduced to be expressed in CHO cells (Sigma, Cat. #: 85050302) with the aid of VIAFECT Transfection Reagent (Promega, Cat. No.: D4981) and incubated for an additional two day before taking the supernatants.
(72) Each of the humanized antibody cultures was assayed for reactivity for a recombinant antigen human CD40 (rCD40; R&D systems, Cat. No.: P25942) by ELISA. For this assay, a dilution of rCD40 at a concentration of 1.0 g/mL in phosphate buffered saline was plated at a density of 100 L/well into microplates and incubated at 37 C. for one hour to coat the microplates with the antigen which was then blocked by adding a 1 blocking solution (Sigma) in an amount of 200 l per well and incubating at 37 C. for one hour.
(73) Following one hour of incubation with 100 L of each of the humanized anti-CD40 antibody cultures at 37 C., the microplates were washed three times with phosphate buffered saline. Treatment with the secondary antibody goat anti-human IgG F(ab)2-HRP (Jackson Immunoresearch, Cat. No.: 109-035-006) at 37 C. for 30 min was followed by three rounds of washing with phosphate buffered saline. Then, TMB Single Solution (Life Technologies, Cat. No: 002023) was added in an amount of 100 l per well and incubated at room temperature for 5-10 min in a dark place. Color development was stopped with 100 L of 1.0 N sulfuric acid and absorbance at 450 nm was measured.
(74) Absorbances at 450 nm of the eight humanized antibodies are depicted in
Example 2: Effect of Anti-CD40 Antibody 5C2 on CD40/CD40L Binding Affinity
(75) A blocking test was performed so as to examine whether the 5C2 antibody (mouse antibody) has an influence on interaction between CD40 and CD40L in the human body.
(76) For buffer change, the 5C2 antibody and the isotype control antibody (Dinona, Cat. No.: 88020R) were each dialyzed against 0.1M sodium bicarbonate buffer and 1.0 mg of each antibody solution was prepared. A solution of FITC Isomer I (Invitrogen, Cat. No.: F1906) at a concentration of 2.6 mg/mL in DMSO (Sigma-Aldrich, Cat. No.: 276855) was added in an amount of 20.0 L to each of the antibody solutions. After FITC conjugation by stirring at room temperature for 2 hours, the resulting antibody solutions were dialyzed against phosphate buffered saline to obtain FITC-labeled antibodies.
(77) Then, 510.sup.6 cells of Ramos cell line (ATCC, ATCC CRL-1596), which expresses human CD40, was incubated at room temperature for 30 min with 100 L of a dilution of the recombinant antigen CD40L (Peprotech, Cat. No.: 310-02) at a concentration of 15.0 g/mL. After washing with phosphate buffered saline, the cells were reacted with 1.0 g/mL of each of the prepared FITC-labeled 5C2 and FITC-labeled isotype antibodies at room temperature for 15 min. The cells were washed again with phosphate buffered saline and then subjected to fluorescent immunoassay using flow cytometry (Stratedigm, S1000EXi).
(78) The flow cytometry analysis results thus obtained are depicted in
Example 3: Assay for Activation of Dendrocyte
(79) 3-1. Preparation of Monocyte-Derived Dendrocyte
(80) In order to examine whether the anti-CD40 antibody activates dendrocytes, monocyte-derived dendrocytes were prepared by differentiation and used for testing the efficacy of the anti-CD40 antibody.
(81) Whole blood sampled from a healthy adult volunteer was transferred to an EDTA (ethylenediaminetetraacetic acid)-treated tube and mixed with one volume of phosphate buffered saline. The whole blood mixture was loaded on FICOLL PAQUE PLUS (GE Healthcare, Cat. No.: 17-1440-03) and centrifuged at 700g at room temperature for 30 min. After centrifugation, only the PBMC (peripheral blood mononuclear cell) layer was transferred to a new tube and added with about two volumes of phosphate buffered saline. Centrifugation at 4 C. at 700 g for 5 min formed a PBMC pellet. A suspension of the PBMC pellet in a 10% fetal bovine serum-supplemented RPMI medium was cultured at 37 C. for 4 hours in a cell culture dish (VOM plastic crop., Cat. No.: V100D) with 5% CO.sub.2.
(82) After removal of non-adherent cells from the cell culture dish, a fresh 10% fetal bovine serum-supplemented RPMI was added to the dish. For differentiation to dendrocytes, the adherent cells were incubated with 510.sup.3 unit/mL of each of rhGM-CSF (Recombinant Human Granulocyte Macrophage Colony Stimulating Factor; JW creagene) and rhIL-4 (JW creagene). On days 3 and 6 after incubation, the 10% fetal bovine serum-supplemented RPMI medium was changed with a fresh one, with each of rhGM-CSF and rhIL-4 added at a concentration of 510.sup.3 unit/mL.
(83) On day 7 of differentiation, the dendrocytes were detached with trypsin-EDTA buffer (Thermo, Cat. No.: R001100), washed with a 10% fetal bovine serum-supplemented RPMI medium, and aliquoted into 24- or 12-well cell culture dishes. The cells were treated with the anti-CD40 antibody and various reagents according to test purposes and cultured for an additional 2 to 5 days before analysis of changes in the dendrocytes
(84) 3-2. Activation of Dendrocyte by Mouse Monoclonal 5C2
(85) 510.sup.5 cells of the monocyte-derived dendrocytes prepared in Example 3-1 were treated with 10 g/mL of the mouse monoclonal antibody 5C2, together with the secondary antibody goat anti-mouse IgG (H+L) (Dionona) for crosslinking so as to amplify the efficacy.
(86) After incubation with the antibody at 37 C. for 2 days in a 5% CO.sub.2 atmosphere, the dendrocytes were detached with trypsin-EDTA (Thermo, Cat. No.: R001100). Dendrocytes were identified by immunostaining with an anti-CD11c antibody-FITC (eBiosceince, Cat. No.: 11-0116-42). Degrees of activation of the dendrocytes were accounted for by expression levels of the co-stimulatory factors CD80, CD83, and CD86 which were measured as fluorescence intensities detected after staining the cells with each of anti-CD80 antibody-PECy5 (eBiosceince, Cat. No.: 15-0809-42), anti-CD86 antibody-PE (eBiosceince, Cat. No.: 12-0869-42), and anti-CD83 antibody-APC (eBiosceince, Cat. No.: 17-0839-42).
(87) The fluorescence intensities obtained above are depicted as relative values to a control (not treated with the antibody, 100%) in
(88) 3-3. Effect of Toll-like Receptor Ligand Used in Combination on Activation of Dendrocyte
(89) Toll-like receptors (TLRs) are usually expressed on sentinel cells, such as macrophages and dendrocytes, which account for innate immunity. The proteins are responsible for the function of recognizing molecules derived from microbes and activating innate immunity. Lipopolysaccharide (LPS), known as a TLR ligand recognized by TLR-4, was used in combination with the 5C2 antibody in order to evaluate the activation of dendrocytes.
(90) For this, 510.sup.5 cells of the monocyte-derived dendrocytes prepared in Example 3-1 were incubated for two days with 1.0 ng/mL of LPS (Sigma-Aldrich, Cat. No.: L3024) in combination of 10 g/mL of 5C2 and 20 g/mL of a secondary antibody goat anti-Mouse IgG(H+L) (Dionona), followed by detaching the dendrocytes with trypsin-EDTA. The dendrocytes were stained with anti-CD11c-FITC (eBiosceince, Cat. No.: 11-0116-42).
(91) Degrees of activation of the dendrocytes were accounted for by expression levels of the co-stimulatory factors CD80 and CD83 which were measured as fluorescence intensities detected after staining the cells with each of anti-CD80 antibody-PECy5 (eBiosceince, Cat. No.: 15-0809-42) and anti-CD86 antibody-PE (eBiosceince, Cat. No.: 12-0869-42).
(92) The fluorescence intensities obtained above are depicted as relative values to a control (not treated with the antibody, 100%) in
(93) 3-4. Activation of Dendrocyte by Chimeric 5C2
(94) Dendrocytes which were differentiated from 510.sup.5 monocytes as described in Example 3-1 were treated with 10 g/mL of each of the two chimeric 5C2 antibodies (chimeric IgG2 and chimeric IgG4) and the CP870,893 analogue (U.S. Pat. No. 7,338,660 B2; 21.4.1 antibody; refer to Table 3) in the absence of a second antibody. After incubation at 37 C. for 5 days in a 5% CO.sub.2 atmosphere, the dendrocytes were detached with trypsin-EDTA(Thermo, Cat. No.: R001100).
(95) Dendrocytes were identified by immunostaining with an anti-CD11c antibody-FITC (eBiosceince, Cat. No.: 11-0116-42). Degrees of activation of the dendrocytes were accounted for by expression levels of the co-stimulatory factors CD80, CD83, and CD86 which were measured as fluorescence intensities detected after staining the cells with each of anti-CD80 antibody-PECy5 (eBiosceince, Cat. No.: 15-0809-42), anti-CD86 antibody-PE (eBiosceince, Cat. No.: 12-0869-42), and anti-CD83 antibody-APC (eBiosceince, Cat. No.: 17-0839-42).
(96) The fluorescence intensities obtained above are depicted as relative values to a control (not treated with the antibody, 100%) in
(97) The three 5C2-derived chimeric antibodies were observed to be not much different in reactivity for human CD40 because of the identical variable region thereof (
(98) 3-5. Synergistic Effect of IFN-Gamma on Activation of Dendrocytes
(99) Dendrocytes indispensably requires interferon gamma for inducing T cell activation to effectively attack foreign matter or cancer cells, and interferon gamma is known to be directly involved in dendritic differentiation.
(100) In order to examine the effect of the 5C2 chimeric antibody on dendrocyte activation and cytokine change in the presence of interferon gamma (IFN), interferon gamma (Pepprotech, Cat. No.: 300-02) was added at a concentration of 1000 unit/mL to the 510.sup.5 monocyte-derived dendrocyte prepared in Example 3-1.
(101) To the resulting cells, each of the two chimeric 5C2 antibodies (chimeric IgG2 and chimeric IgG4) and the CP870,893 analogue (U.S. Pat. No. 7,338,660 B2; 21.4.1 antibody; refer to Table 3) was added at a concentration of 10.0 g/mL.
(102) The dendrocytes were incubated with the antibodies at 37 C. for 2 days in a 5% CO.sub.2 atmosphere and then detached with trypsin-EDTA (Thermo, Cat. No.: R001100). Dendrocytes were identified by immunostaining with an anti-CD11c antibody-FITC (eBiosceince, Cat. No.: 11-0116-42). Degrees of activation of the dendrocytes were accounted for by expression levels of the co-stimulatory factors CD86 and CD83 which were measured as fluorescence intensities detected after staining the cells with each of anti-CD86 antibody-PE (eBiosceince, Cat. No.: 12-0869-42) and anti-CD83 antibody-APC (eBiosceince, Cat. No.: 17-0839-42).
(103) The fluorescence intensities obtained above are depicted as relative values to a control (not treated with the antibody, 100%) in
(104) As shown in
Example 4: Activation Effect of Dendrocyte on CD8 T Cell
(105) After recognizing an external antigen, dendrocytes present a MHC/foreign antigen to T cells. Then, T cells recognize the MHC/foreign antigen and are activated to effectively remove the foreign antigen. In order to examine whether the anti-CD40 antibody provided in the present disclosure induces T cell activation, the Burkitt's lymphoma cell line Ramos was used as a foreign antigen in a T cell activation assay.
(106) After being cultured in a 10% fetal bovine serum RPMI medium, the Ramos cell line (ATCC, ATCC CRL-1596) were repetitively frozen and thawed twice and centrifuged. Absorbance of the supernatant (Ramos lysate) thus obtained was measured at 280 nm and arbitrarily set in the concentration unit mg/mL.
(107) Dendrocytes which were differentiated from 510.sup.5 monocytes as described in Example 3-1 were seeded at a density of 510.sup.5 cells/well into 12-well culture dishes to which the prepared Ramos lysate was then added at a concentration of 20 g/mL. For comparison of effects of the anti-CD40 antibodies, the cells were incubated with 10 g/mL of each of the three chimeric 5C2 antibodies (chimeric IgG1, chimeric IgG2, and chimeric IgG4) and the CP870,893 analogue (U.S. Pat. No. 7,338,660 B2; 21.4.1 antibody; refer to Table 3). On the next day, PBMC (peripheral blood mononuclear cell) was separated from the whole blood from the donor of the dendrocytes and then co-cultured with the prepared dendrocytes.
(108) Dendrocytes were identified by immunostaining with an anti-CD11c antibody-FITC (eBiosceince, Cat. No.: 11-0116-42). Degrees of activation of the dendrocytes were accounted for by expression levels of the co-stimulatory factors CD86 and CD83 which were measured as fluorescence intensities detected after staining the cells with each of anti-CD86 antibody-PE (eBiosceince, Cat. No.: 12-0869-42) and anti-CD83 antibody-APC (eBiosceince, Cat. No.: 17-0839-42).
(109) The fluorescence intensities obtained above are depicted as relative values to a control (not treated with the antibody, 100%) in
(110) For T cell activation assay, PBMC co-cultured with dendrocytes were treated with the antibodies and immunostained with anti-CD8-FITC (Dinona, Cat. No.: 10143B) and anti-CD69-PE (eBioscience, Cat. No.: 12-0699-42) together. CD8-expressing cells (CD8-FITC positive cells) and CD69-expressing cells (CD69 positive cells) were selected with reference to the fluorescence intensities detected. Proportions (% based on numbers of cells) of CD69-positive cells in the selected CD8-FITC positive T cell population are depicted in
Example 5: Assay for Apoptotic Efficacy
(111) 5-1. Assay for Apoptotic Effect of Chimeric 5C2 Antibody In order to identify the direct cytotoxicity of the 5C2 antibody against tumor cell lines, an apoptosis test was conducted. The Ramos cell line (ATCC, ATCC CRL-1596) was washed with phosphate buffered saline, resuspended in a 5.0% fetal bovine serum-supplemented RPMI, and plated at a density of 210.sup.5 cells/well into microplates. The cells were incubated with 1.0 g/mL of each of the three anti-CD40 chimeric antibodies (IgG1, IgG2, and IgG4), the CP870,893 analogue, and Rituxan at 37 C. for 2 days in a 5% CO.sub.2 atmosphere. Cytotoxicity effects were determined by positive cell proportions (%) after staining FITC Annexin-V (BD Pharmingen, Cat. No.: 51-65874X) and 7-AAD (BD Pharmingen, Cat. No.: 51-68981E) according to the manufacturer's instruction. The test results are depicted in
(112) For use in examining the apoptotic efficacy of humanized antibody 5C2, the Raji Burkitt's lymphoma cell line (ATCC, ATCC CCL-86) was washed with phosphate buffered saline, resuspended in a 3.0% fetal bovine serum-supplemented RPMI medium, and plated at a density of 210.sup.5 cells/well into microplates. The cells were incubated with 10.0 g/mL of each of seven humanized antibodies 5C2 IgG2 (VH1VL2, VH2VL1, VH2VL2, VH3VL1, VH3VL2, VH5VL1, and VH5VL2), the CP870,893 analogue, and the human IgG at 37 C. for two days in a 5% CO.sub.2 atmosphere. Subsequently, as described in Example 5-1, the cells were stained with FITC Annexin-V and 7-AAD to determine the apoptotic effect. The result is depicted in
Example 6: T Cell Activation by 5C2 Antibody in Presence of Super-Antigen Staphylococcal Enterotoxin B (SEB)
(113) Because T cell activation is made in an antigen-specific manner, one antigen cannot induce the activation unless T cells have the same TCR. A super-antigen, such as SEB, which can activate T cells in a non-specific manner, should be employed in order to generally activate T cells in in vitro tests using PBMC.
(114) In this test, SEB was applied to PBMC to establish a condition for T cell activation before examining whether treatment with 5C2 antibodies further enhanced an immune reaction.
(115) After being isolated from the whole blood, from a healthy volunteer, PBMC was seeded at a density of 110.sup.6 cells/well into microplates. Then, the cells were treated with 30 ng/mL of SEB, together with 10 g/mL of each of five humanized antibodies 5C2 (VH2VL1, VH2VL2, VH3VL1, and VH3VL2), the CP870,893 analogue, and the human IgG at 37 C. for three days in a 5% CO.sub.2 atmosphere. The medium supernatant was taken and used to measure an amount of human IFN-gamma (Affymetrix, Cat. NO.: 88-7316-22) according to the manufacturer's instruction. The result thus obtained is depicted in
Example 7: Assay for Anti-Tumor Effect of 5C2 in Animal Model
(116) Being able to introduce CD8 T cell activation via dendrocytes, the 5C2 antibodies are expected to have an anti-tumor effector. In addition, the apoptotic effect of the 5C2 antibodies on CD40-expressing tumor cell lines allows the anticipation of an anti-tumor effector accompanied.
(117) The 5C2 antibodies have no cross reactivity to mouse CD40. Thus, effects of the 5C2 antibodies were tested in two models: injected with a tumor cell line alone and in combination with human dendrocytes and peripheral blood mononuclear cell (PBMC).
(118) The leukemia cell line Ramos (ATCC, ATCC CRL-1596) was subcutaneously injected alone or human dendrocytes and PBMC to NSG (NOD.Cg-Prkdcscidll2rgtm1Wjl/SzJ) mice (the Jackson Laboratory, 25 grams, 6-8 weeks old).
(119) In this test, dendrocytes were prepared by taking whole blood from a healthy adult volunteer, separating PBMC from the whole blood with the aid of FICOLL PAQUE PLUS (GE Healthcare, Cat. No.: 17-1440-03), and differentiating the adherent cells in the presence of rhGM-CSF (Recombinant Human Granulocyte Macrophage Colony Stimulating Factor; JW creagene) and rhlL-4 (JW creagene), as in Example 3-1. On day 7 of differentiation, dendrocytes were detached, washed with phosphate buffered saline, and subcutaneously injected at a density of 510.sup.5 cells/mouse to the mouse abdomen.
(120) After being prepared by separation from whole blood taken from the same donor of dendrocytes, with the aid of FICOLL PAQUE PLUS (GE Healthcare, Cat. No.: 17-1440-03), PBMC was washed with phosphate buffered saline and subcutaneously injected at a density of 210.sup.6 cells/mouse to the mouse abdomen.
(121) Subsequent to the cell injection, the chimeric 5C2 IgG2 antibody or phosphate buffered saline was intraperitoneally injected. At the time of one week later, the antibody or phosphate buffered saline was further injected once. The assay procedure is summarized in Table 7, below:
(122) TABLE-US-00007 TABLE 7 Assay for Anti-Tumor Effector of 5C2 in Animal Drug Group Tumor Effector Dose # of Group N Tumor Number Route Type Number Route Type (mg/Kg) Dose Route Group 1 5 Ramos 1 10.sup.7 S.C. None NA S.C. PBS NA 2 I.P. Group 2 5 None NA 5C2 1.0 Group 3 5 DC/PBMC 5 10.sup.5/2 10.sup.6 PBS NA Group 4 5 DC/PBMC 5 10.sup.5/2 10.sup.6 5C2 1.0
(123) After antibody administration, tumor volumes (mm.sup.3) were monitored.
(124) Average values of the tumor volumes (mm.sup.3) that has changed with time are given in Table 8 and depicted in
(125) TABLE-US-00008 TABLE 8 Average Tumor Volume (mm.sup.3) Time after Ab administ. (Day) 0 10 13 17 20 24 27 31 34 38 Group 1 0 24.1 105.6 1090.0 2287.2 5912.3 11481.9 Group 2 0 44.5 111.0 141.2 591.5 1347.6 2816.7 Group 3 0 110.9 308.4 1109.0 2786.2 6073.3 12537.8 Group 4 0 2.8 5.2 23.2 38.4 0 0 0 0 96.2
(126) As shown in Table 8 and