TPBG ANTIBODY AND PREPARATION METHOD THEREFOR, CONJUGATE AND USE THEREOF
20210198377 · 2021-07-01
Assignee
Inventors
- Xiaolan Sun (Shanghai, CN)
- Ying Zhang (Shanghai, CN)
- Yu Zhang (Shanghai, CN)
- Feifei HU (Shanghai, CN)
- Shiyong GONG (Shanghai, CN)
- Lile Liu (Shanghai, CN)
Cpc classification
A61K47/6877
HUMAN NECESSITIES
A61K47/6803
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
A61K47/6851
HUMAN NECESSITIES
C07K2317/33
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K47/68
HUMAN NECESSITIES
Abstract
The present invention provides a TPBG antibody, a preparation method thereof, conjugates and use thereof. The TPBG antibody comprises one or more of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 of the heavy chain variable region of TPBG antibody, and/or one or more of the light chain CDR1, the light chain CDR2 and the light chain CDR3 of the light chain variable region of TPBG antibody, and the amino acid sequences of which are described in the present invention. The TPBG antibody is humanized antibody with high affinity, and the conjugates prepared by coupling the TPBG antibody with the small-molecule drug toxin MMAF can have a cytotoxicity effects on TPBG-positive cells, therefore the TPBG antibody can be used in the preparation of drugs for treating tumors and the like.
Claims
1. An isolated protein comprising one or more of heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of TPBG antibody and/or one or more of light chain CDR1, light chain CDR2 and light chain CDR3 of the TPBG antibody, wherein the amino acid sequence of the heavy chain CDR1 is set forth as SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:66 or SEQ ID NO:74; the amino acid sequence of the heavy chain CDR2 is set forth as SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:51, SEQ ID NO:59, SEQ ID NO:67 or SEQ ID NO:75; the amino acid sequence of the heavy chain CDR3 is set forth as SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:68 or SEQ ID NO:76; the amino acid sequence of the light chain CDR1 is set forth as SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:46, SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:70 or SEQ ID NO:78; the amino acid sequence of the light chain CDR2 is set forth as SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71 or SEQ ID NO:79; the amino acid sequence of the light chain CDR3 is set forth as SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72 or SEQ ID NO:80; or the amino acid sequence of the heavy chain CDR1 has at least 80% sequence identity to the sequence set forth as SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:66 or SEQ ID NO:74; the amino acid sequence of the heavy chain CDR2 has at least 80% sequence identity to the sequence set forth as SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:51, SEQ ID NO:59, SEQ ID NO:67 or SEQ ID NO:75; the amino acid sequence of the heavy chain CDR3 has at least 80% sequence identity to the sequence set forth as SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:68 or SEQ ID NO:76; the amino acid sequence of the light chain CDR1 has at least 80% sequence identity to the sequence set forth as SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:46, SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:70 or SEQ ID NO:78; the amino acid sequence of the light chain CDR2 has at least 80% sequence identity to the sequence set forth as SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71 or SEQ ID NO:79; the amino acid sequence of the light chain CDR3 has at least 80% sequence identity to the sequence set forth as SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72 or SEQ ID NO:80.
2. The protein according to claim 1, wherein the amino acid sequence of the heavy chain CDR1 is set forth as SEQ ID NO:2, the amino acid sequence of the heavy chain CDR2 is set forth as SEQ ID NO:3, and the amino acid sequence of the heavy chain CDR3 is set forth as SEQ ID NO:4; the amino acid sequence of the heavy chain CDR1 is set forth as SEQ ID NO:10, the amino acid sequence of the heavy chain CDR2 is set forth as SEQ ID NO:11, and the amino acid sequence of the heavy chain CDR3 is set forth as SEQ ID NO:12; the amino acid sequence of the heavy chain CDR1 is set forth as SEQ ID NO:18, the amino acid sequence of the heavy chain CDR2 is set forth as SEQ ID NO:19, and the amino acid sequence of the heavy chain CDR3 is set forth as SEQ ID NO:20; the amino acid sequence of the heavy chain CDR1 is set forth as SEQ ID NO:26, the amino acid sequence of the heavy chain CDR2 is set forth as SEQ ID NO:27, and the amino acid sequence of the heavy chain CDR3 is set forth as SEQ ID NO:28; the amino acid sequence of the heavy chain CDR1 is set forth as SEQ ID NO:34, the amino acid sequence of the heavy chain CDR2 is set forth as SEQ ID NO:35, and the amino acid sequence of the heavy chain CDR3 is set forth as SEQ ID NO:36; the amino acid sequence of the heavy chain CDR1 is set forth as SEQ ID NO:42, the amino acid sequence of the heavy chain CDR2 is set forth as SEQ ID NO:43, and the amino acid sequence of the heavy chain CDR3 is set forth as SEQ ID NO:44; the amino acid sequence of the heavy chain CDR1 is set forth as SEQ ID NO:50, the amino acid sequence of the heavy chain CDR2 is set forth as SEQ ID NO:51, and the amino acid sequence of the heavy chain CDR3 is set forth as SEQ ID NO:52; the amino acid sequence of the heavy chain CDR1 is set forth as SEQ ID NO:58, the amino acid sequence of the heavy chain CDR2 is set forth as SEQ ID NO:59, and the amino acid sequence of the heavy chain CDR3 is set forth as SEQ ID NO:60; the amino acid sequence of the heavy chain CDR1 is set forth as SEQ ID NO:66, the amino acid sequence of the heavy chain CDR2 is set forth as SEQ ID NO:67, and the amino acid sequence of the heavy chain CDR3 is set forth as SEQ ID NO:68; the amino acid sequence of the heavy chain CDR1 is set forth as SEQ ID NO:74, the amino acid sequence of the heavy chain CDR2 is set forth as SEQ ID NO:75, and the amino acid sequence of the heavy chain CDR3 is set forth as SEQ ID NO:76; the amino acid sequence of the light chain CDR1 is set forth as SEQ ID NO: 6, the amino acid sequence of the light chain CDR2 is set forth as SEQ ID NO:7, and the amino acid sequence of the light chain CDR3 is set forth as SEQ ID NO:8; the amino acid sequence of the light chain CDR1 is set forth as SEQ ID NO:14, the amino acid sequence of the light chain CDR2 is set forth as SEQ ID NO:15, and the amino acid sequence of the light chain CDR3 is set forth as SEQ ID NO:16; or the amino acid sequence of the light chain CDR1 is set forth as SEQ ID NO: 22, the amino acid sequence of the light chain CDR2 is set forth as SEQ ID NO:23, and the amino acid sequence of the light chain CDR3 is set forth as SEQ ID NO:24; the amino acid sequence of the light chain CDR1 is set forth as SEQ ID NO: 30, the amino acid sequence of the light chain CDR2 is set forth as SEQ ID NO:31, and the amino acid sequence of the light chain CDR3 is set forth as SEQ ID NO:32; the amino acid sequence of the light chain CDR1 is set forth as SEQ ID NO: 38, the amino acid sequence of the light chain CDR2 is set forth as SEQ ID NO:39, and the amino acid sequence of the light chain CDR3 is set forth as SEQ ID NO:40; the amino acid sequence of the light chain CDR1 is set forth as SEQ ID NO: 46, the amino acid sequence of the light chain CDR2 is set forth as SEQ ID NO:47, and the amino acid sequence of the light chain CDR3 is set forth as SEQ ID NO:48; the amino acid sequence of the light chain CDR1 is set forth as SEQ ID NO: 54, the amino acid sequence of the light chain CDR2 is set forth as SEQ ID NO:55, and the amino acid sequence of the light chain CDR3 is set forth as SEQ ID NO:56; the amino acid sequence of the light chain CDR1 is set forth as SEQ ID NO: 62, the amino acid sequence of the light chain CDR2 is set forth as SEQ ID NO:63, and the amino acid sequence of the light chain CDR3 is set forth as SEQ ID NO:64; the amino acid sequence of the light chain CDR1 is set forth as SEQ ID NO: 70, the amino acid sequence of the light chain CDR2 is set forth as SEQ ID NO:71, and the amino acid sequence of the light chain CDR3 is set forth as SEQ ID NO:72; or the amino acid sequence of the light chain CDR1 is set forth as SEQ ID NO: 78, the amino acid sequence of the light chain CDR2 is set forth as SEQ ID NO:79, and the amino acid sequence of the light chain CDR3 is set forth as SEQ ID NO:80.
3. An isolated protein comprising the heavy chain variable region of TPBG antibody and/or the light chain variable region of TPBG antibody; the amino acid sequence of the heavy chain variable region is set forth as SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:65 or SEQ ID NO:73; the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:45, SEQ ID NO:53, SEQ ID NO:61, SEQ ID NO:69 or SEQ ID NO:77.
4. The protein according to claim 3, wherein the amino acid sequence of the heavy chain variable region is set forth as SEQ ID NO:1, the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:5; the amino acid sequence of the heavy chain variable region is set forth as SEQ ID NO:9, the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:13; the amino acid sequence of the heavy chain variable region is set forth as SEQ ID NO:17, the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:21; the amino acid sequence of the heavy chain variable region is set forth as SEQ ID NO:25, the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:29; the amino acid sequence of the heavy chain variable region is set forth as SEQ ID NO:33, the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:37; the amino acid sequence of the heavy chain variable region is set forth as SEQ ID NO:41, the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:45; the amino acid sequence of the heavy chain variable region is set forth as SEQ ID NO:49, the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:53; the amino acid sequence of the heavy chain variable region is set forth as SEQ ID NO:57, the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:61; the amino acid sequence of the heavy chain variable region is set forth as SEQ ID NO:65, the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:69; or the amino acid sequence of the heavy chain variable region is set forth as SEQ ID NO:73, the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:77.
5. The protein according to claim 1 further comprising a heavy chain constant region of the antibody and/or a light chain constant region of the antibody.
6. The protein according to claim 5 wherein the heavy chain constant region of the antibody is a human or mouse heavy chain constant region of the antibody; and the light chain constant region is a human or mouse light chain constant region of the antibody.
7. The protein according to claim 6, wherein the heavy chain constant region of the antibody is a human heavy chain constant region of the antibody; and the light chain constant region is a human light chain constant region of the antibody.
8. The protein according to claim 1, wherein the protein is one or more of a TPBG antibody, monoclonal antibody, an antibody full-length protein, a protein fragment of an antigen-antibody binding domain, a dual-specificity antibody, a multiple-specificity antibody, a single chain antibody, a single domain antibody (sdAb) and a single-domain antibody.
9. A nucleic acid encoding at least one of the proteins according to claim 1.
10. The nucleic acid according to claim 9, wherein the nucleotide sequence encoding the heavy chain variable region is set forth as SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97 or SEQ ID NO:99; and/or the nucleotide sequence encoding the light chain variable region is set forth as SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98 or SEQ ID NO:100.
11. The nucleic acid according to claim 10, wherein the nucleotide sequence encoding the heavy chain variable region is set forth as SEQ ID NO:81, and the nucleotide sequence encoding the light chain variable region is set forth as SEQ ID NO:82; the nucleotide sequence encoding the heavy chain variable region is set forth as SEQ ID NO:83, and the nucleotide sequence encoding the light chain variable region is set forth as SEQ ID NO:84; the nucleotide sequence encoding the heavy chain variable region is set forth as SEQ ID NO:85, and the nucleotide sequence encoding the light chain variable region is set forth as SEQ ID NO:86; the nucleotide sequence encoding the heavy chain variable region is set forth as SEQ ID NO:87, and the nucleotide sequence encoding the light chain variable region is set forth as SEQ ID NO:88; the nucleotide sequence encoding the heavy chain variable region is set forth as SEQ ID NO:89, and the nucleotide sequence encoding the light chain variable region is set forth as SEQ ID NO:90; the nucleotide sequence encoding the heavy chain variable region is set forth as SEQ ID NO:91, and the nucleotide sequence encoding the light chain variable region is set forth as SEQ ID NO:92; the nucleotide sequence encoding the heavy chain variable region is set forth as SEQ ID NO:93, and the nucleotide sequence encoding the light chain variable region is set forth as SEQ ID NO:94; the nucleotide sequence encoding the heavy chain variable region is set forth as SEQ ID NO:95, and the nucleotide sequence encoding the light chain variable region is set forth as SEQ ID NO:96; the nucleotide sequence encoding the heavy chain variable region is set forth as SEQ ID NO:97, and the nucleotide sequence encoding the light chain variable region is set forth as SEQ ID NO:98; or the nucleotide sequence encoding the heavy chain variable region is set forth as SEQ ID NO:99, and the nucleotide sequence encoding the light chain variable region is set forth as SEQ ID NO:100.
12. A recombinant expression vector comprising the nucleic acid according to claim 9.
13. A recombinant expression transformant comprising the recombinant expression vector according to claim 12.
14. A preparation method of a TPBG antibody comprising culturing the recombinant expression transformant according to claim 13 and obtaining the TPBG antibody from culture.
15. An immunoconjugate comprising the protein according to claim 1 which is covalently linked to a cytotoxic agent.
16. The immunoconjugate according to claim 15, wherein 1 equivalent of the protein is linked to y equivalents of cytotoxic agent through x equivalents of linker, which has the structure shown in formula 1,
Ab-(L)x-(D)y Formula 1 wherein Ab is the protein, L is a linker; D is a cytotoxic agent; x is a natural number; y is 0 or a natural number.
17. The immunoconjugate according to claim 16, wherein the linker comprises active esters, carbonates, carbamates, imine phosphate esters, oximes, hydrazones, acetals, orthoesters, aminos, small peptides or nucleotide fragments; and/or the D is selected from a cytotoxin, a chemotherapeutic agent, a radioisotope, a therapeutic nucleic acid, an immunomodulator, an anti-angiogenic agent, an anti-proliferative and pro-apoptotic agent or a cytolytic enzyme.
18. The immunoconjugate according to claim 17, wherein x=y=n in formula 1, and the structure of the immunoconjugate is shown in formula 3, 4 or 5, ##STR00007## in formula 3, m is 1-10; ##STR00008## in formula 4, L is 4-(N-maleimide methyl) cyclohexane-1-carboxylic succinimide; D is N2′-deacetyl-N2′-(3-mercapto-1-oxypropyl) methesteine (DM1); ##STR00009## in formula 5, L is maleimidocaproyl-L-valine-L-citrulline p-amino benzyl alcohol, D is methylauristatin E (MMAE); wherein n is a natural number.
19. A pharmaceutical composition comprising the immunoconjugate according to claim 15 and a pharmaceutically acceptable vehicle.
20. The pharmaceutical composition comprising 0.01-99.99% of the protein according to claim 1 and 0.01-99.99% of pharmaceutical vehicle, wherein the percentage is the mass percentage of the pharmaceutical composition.
21. (canceled)
22. (canceled)
23. (canceled)
24. A method for detecting cells overexpressing a TPBG protein, which comprises the following steps: contacting the protein according claim 1 with the sample to be tested in vitro, and detecting the binding of the protein according to claim 1 with the sample to be tested.
25. A process for treating a patient in need of a medicament for tumor, comprising administering an effective amount of the protein according to claim 1 to the patient.
26. A process for treating a patient in need of a medicament for tumor, comprising administering an effective amount of the immunoconjugate according to claim 15 to the patient.
27. A process for treating a patient in need of a medicament for tumor, comprising administering an effective amount of the pharmaceutical composition according to claim 19 to the subject.
Description
DESCRIPTION OF THE DRAWINGS
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EMBODIMENTS
[0182] The present invention will be further illustrated by the following examples, but is not therefore limited to the scope of the described embodiments. The experimental methods that do not specify the specific conditions in the following embodiments are conventional methods and conditions, or selected according to the instruction of the products.
[0183] The room temperatures described in the examples are conventional room temperatures known in the art, which is generally 10-30° C.
[0184] Unless otherwise specified, PBS described in the examples is PBS phosphate buffer, pH7.2.
Embodiment 1
Preparation of TPBG Antibody
[0185] (1) Preparation of Immunogen A
[0186] The nucleotide sequence containing amino acid sequence 32-355 (Ser32-Ser355) encoding the extracellular domain of human TPBG protein (wherein the accession number of the nucleotide sequence encoding human TPBG protein is Genbank ID: AAH37161.1) was cloned into the pCpC vector including human IgG Fc fragment (purchased from Invitrogen, V044-50) and the plasmid was prepared according to established standard molecular biology protocol. For specific methods, please refer to Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, N.Y.: Cold Spring Harbor Laboratory Press). HEK293 cells (purchased from Invitrogen) were transiently transfected (polyether imide, PEI, purchased from Polysciences) and expanded using FreeStyle™ 293 (purchased from Invitrogen) at 37° C. After 4 days, cell culture was harvested, cell pellets were removed by centrifugation and culture supernatant containing the extracellular domain of TPBG protein was obtained. The culture supernatant was loaded onto protein A affinity chromatography column (Mabselect Sure, purchased from GE Healthcare), and the change in ultraviolet absorption was monitored using a UV detector. Once the sample was loaded, the protein A affinity chromatography column was equilibrated with PBS buffer (pH 7.2) until the ultraviolet absorption value returned to baseline, and then elution was performed using 0.1M glycine hydrochloride acid (pH 2.5), and hFc tagged TPBG protein (i.e. human TPBG-hFc) eluting from the protein A affinity chromatography column was harvested and dialyzed against PBS (pH 7.2) at 4° C. overnight in refrigerator. The dialyzed protein was aseptically filtered through 0.22 μm and stored at −80° C. in aliquots, and purified immunogen A was then obtained.
[0187] Prior to being used, Immunogen A was subjected to a series of quality control tests, e.g. protein concentration, purity, molecular weight, biological activity, etc., and it was found that the immunogen A was qualified and can be used as the antigen for the subsequent preparation of TPBG antibodies.
[0188] (2) Preparation of Immunogen B
[0189] The nucleotide sequence encoding the full-length amino acid sequence of human TPBG (wherein the Genbank accession number of the nucleotide sequence encoding human TPBG protein is Genbank ID: AAH37161.1) was cloned into the pIRES vector (purchased from Clontech) and the plasmid was prepared. Recombinant plasmid (PEI, purchased from Polysciences) was transfected to HEK293 cell line (purchased from Invitrogen), and the transfected cells were selectively cultured in 0.5 μg/ml of DMEM culture medium containing 10% (w/w) fetal bovine serum for two weeks. Subcloning was performed using limiting dilution assay in 96-well plates and the plates were cultured at 37° C., 5% (v/v) CO.sub.2, and after two weeks a portion of the wells containing monoclones were selected and expanded into 6-well plates. The expanded clones were screened by FACS analysis using commercial available TPBG antibody (purchased from Sigma, Lot NO: SAB1404485). Robust monoclonal cell lines with high fluorescence intensity were selected and further expanded and cryopreserved in liquid nitrogen to obtain immunogen B. The specific selection results were shown in Table 3 and
TABLE-US-00003 TABLE 3 FACS screening results of HEK293 cells transfected with human TPBG Cellular MFI Clone number of IgG isotype TPBG NO. transfected cell control antibody 1 293F-hTPBG 4E1 5.2 145.0 2 293F-hTPBG 4A8 3.1 33.4 3 293F-hTPBG 4A9 6.3 203.9 5 293F-hTPBG 4B9 6.3 126.1 6 293F-hTPBG 4C3 3.2 27.2 7 293F-hTPBG 4C5 5.6 171.5 8 293F-hTPBG 4E1 4.8 91.8 9 293F-hTPBG 4F9 3.9 47.7 10 293F-hTPBG 4G1 4.0 131.2 11 293F-hTPBG 4G6 3.1 31.8 12 293F-hTPBG 4H12 4.0 9.5 13 293F-hTPBG 5E6 2.6 13.2 15 293F-hTPBG 5A11 4.6 166.5 16 293F-hTPBG 5A9 5.0 53.2 17 293F-hTPBG 5C12 3.7 75.4 18 293F-hTPBG 5D4 3.0 70.6 19 293F-hTPBG 5E5 5.6 280.5 20 293F-hTPBG 5G11 4.1 11.3 21 293F-hTPBG 5G8 6.0 167.8 22 293F-hTPBG 5H6 3.0 36.4
[0190] (3) Preparation of Hybridoma Cells and Screening of Antibody
[0191] A. Immunization with Immunogen A
[0192] BALB/cAnNCrl mice or SJL/JorllcoCrl mice (both purchased from SLAC Co. Ltd, Shanghai) aged 6-8 weeks were used and raised in SPF condition. In the primary immunization, 0.25 ml of immunogen A being emulsified with Freund's complete adjuvant, i.e. 50 mg of immunogen A per mouse was intraperitoneally injected. In booster immunization, 0.25 ml of immunogen A being emulsified with Freund's incomplete adjuvant, i.e. 50 mg of immunogen A per mouse was intraperitoneally injected. The interval between the primary immunization and first booster is two weeks and the interval between each booster is three weeks. Blood was collected 1 week after each booster, and the antibody titer and specificity of the immunogen in serum were tested by ELISA and FACS. The results were shown in
TABLE-US-00004 TABLE 4 Detection of antibody titers in the serum of Balb/c mice immunized with TPBG protein using ELISA OD.sub.450 nm Serum Dilution Rate Batch 1:100 1:10.sup.3 1:10.sup.4 1:10.sup.5 1:10.sup.6 1:10.sup.7 Blank 731(TB2) 2.8722 2.8084 2.8186 1.5772 0.3892 0.1201 0.0935 732(TB2) 2.8715 2.8171 2.857 1.2767 0.2601 0.1353 0.0985 733(TB2) 2.8411 2.8841 2.9258 1.7943 0.3336 0.1178 0.1418 734(TB2) 2.8735 2.8503 2.861 1.3150 0.3052 0.1129 0.1365 735(TB2) 2.9460 2.9859 2.9761 1.9749 0.4203 0.1463 0.1531
[0193] B. Immunization with Immunogen B
[0194] BALB/cAnNCrl mice or SJL/JorllcoCrl mice (both purchased from SLAC Co. Ltd, Shanghai) at 6-8 weeks of age were used and feed under SPF condition. The pIRES plasmid containing the nucleotide sequence encoding full-length human TPBG (please refer to step 2, Example 1) was transfected into HEK293 cell line and the recombinant HEK293 cell line containing human TPBG was obtained (transfection was performed using X-treme GENE HP DNA Transfection Reagent, purchased from Roche Co. Ltd, and operated according to the manual). Culture was expanded in T-75 flasks to a confluency of 90%, the culture medium was removed via pipetting, and cells were washed twice with DMEM basic medium (purchased from Invitrogen). Then cells were treated using enzyme-free cell dissociation buffer (purchased from Invitrogen) at 37° C. until the cells can be peeled off from the dish wall, and then harvested. The cells were washed twice with DMEM basic medium and diluted to a concentration of 2×10.sup.7 cell/mL using phosphate buffer after cell counting. Every time, 0.5 ml of cell suspension was injected intraperitoneally into each mouse, and there was a two-week interval between the primary and booster immunizations. After that booster immunization was performed every three weeks. Blood sample was taken one week after every immunization except for the primary immunization, and the titers and specificity of the antibody in the serum was detected using FACS. After the second booster immunization, the antibody titer in the serum reached to a value higher than 1:1000.
[0195] Prior to the completion of step A and B, each selected mice was injected intraperitoneally with 100 μg of purified immunogen A (for mice immunized with immunogen A) or recombinant HEK293 cell line containing human TPBG (for mice immunized with immunogen B) for the last immunization, and sacrificed 5 days later. Their spleen cells were harvested, and NH.sub.4OH was added to a final concentration of 1% (w/w) to lyze the red blood cells in the spleen cells, follow by preparing a splenocyte suspension. Cells were washed for three times by centrifugation at 1000 rpm using DMEM basic medium, and the viable cells were mixed with mouse myeloma cell SP2/0 (purchased from ATCC) at a ratio of 5:1. Cell fusion was performed using a high efficient electrofusion method (please refer to METHODS IN ENZYMOLOGY, VOL. 220). Fused cells were diluted in DMEM medium containing 20% (w/w) FBS and 1×HAT, and then plated onto 96-well cell culture plates at the density of 1×10.sup.5 cells/200 ml per well and cultured in 5% (v/v) CO.sub.2 incubator at 37□. After 14 days, the supernatants from the cell fusion plates were screened using ELISA and Acumen (microplate cytometry), and positive clones with an ELISA OD.sub.450nm>1.0 and Acumen MFI value>100 were subjected to expansion culture in DMEM culture medium containing 10% (w/w) HT FBS in 24-wells plate at 37□ in 5% (v/v) CO.sub.2. The culture medium from the expansion culture in 24-wells plate was centrifuged after 3 days of culture and the supernatant was collected and subjected to antibody subclass analysis. Its binding activity to TPBG protein and TPBG-positive cells (for the method of measuring binding activity, refers to the related information in Example 3A and 3B) and mouse TPBG antibody-MMAF indirect cytotoxicity assay (for the detection method for indirect cytotoxic killing assay, refers to the related information in Example 4) were determined using ELISA and FACS.
[0196] Based on the screening results of 24-wells plate, hybridoma cells having ELISA OD.sub.450nm>1.0, FACS MFI>50 and the hybridoma supernatant killing rate on TPBG-positive cells reaching up to 50% in indirect cytotoxicity assay were selected as eligible positive clones. Selected eligible hybridoma cells were subjected to subcloning by limiting dilution assay in 96-well plates and cultured in DMEM medium (purchased from Invitrogen) containing 10% (w/w) HT FBS at 37□ in 5% (v/v) CO.sub.2. 10 days after the subcloning, initial screening was performed using ELISA and Acumen, and single positive monclones were selected and subjected to expansion culture in 24-wells plate. After 3 days, positive antigen binding was confirmed by FACS and mouse antibody-MMAF indirect cytotoxicity assay was performed to evaluate biologic activity, while the criteria for the evaluation is that OD.sub.450nm>1.0 for ELISA, MFI>50 for FACS and the hybridoma supernatant killing rate on TPBG-positive cells shall reach up to 50% in indirect cytotoxicity assay.
[0197] According to the detection results of 24-wells plate, the optimal clones were picked and expanded in DMEM medium (purchased from Invitrogen) containing 10% (w/w) HT FBS at 37□ in 5% (v/v) CO.sub.2. The optimal clones were obtained and stored in liquid nitrogen, and then used for subsequently production and purification of antibody.
Embodiment 2
Production and Purification of Lead Antibody
[0198] The antibody concentration generated by hybridoma cells was relatively low, about only 1-10 mg/mL with a broad-range variation, and the various proteins derived from the culture medium and the contents of FBS contained in the culture medium were interfered by many biologic activity assays to different degrees. Therefore a small-scale (1-5 mg) production and purification of antibody is necessary.
[0199] The hybridoma cells obtained by Embodiment 1 were seeded into T-75 cell culture flask and passaged for three times. Until reaching a robust growing state, cells were seeded into the cell culture spinner flasks. 200 mL of production culture medium were added to each 2L spinner flask with a 1.0×10.sup.5/mL of seeding density. The flask lid was tightened and the spinner flask was placed on the rotary machine in a 37° C. incubator with a 3 rpm of rotation speed. The cell culture was collected after continuous spinning culture for 14 days and cells were removed by filtration through a 0.45 μm filter until the culture supernatant was clear and can be subjected to immediate purification or stored at −30° C.
[0200] The TPBG antibody in the obtained culture supernatant (200 mL) was purified using 2 mL of protein A column (purchased from GE Healthcare). The protein G column was first equilibrated with equilibration buffer (PBS buffer, pH 7.4), and the culture supernatant was then applied to the protein A column at a controlled flow rate of 3 mL/min. The equilibration buffer was used to wash the protein G column once sample loading was complete, and the volume of the equilibration buffer used herein was four times as much as the bed volume of protein A column. The TPBG antibody bound to protein A column was eluted using elution buffer (0.1M sodium citrate buffer, pH 3.5), and the elution was monitored by UV detector (A280nm absorption). The antibody eluate was collected and pH was neutralized by adding with 10% (v/v) 1.0M Tris-HCl buffer, then the eluate was immediately subjected to dialysis against PBS buffer overnight, and the buffer was changed once the next day and the dialysis was continued for 3 h. The dialyzed TPBG antibody was harvested, filtered by 0.22 μm sterile filter, and then stored in sterile condition, therefore a purified TPBG antibody was obtained.
[0201] The purified TPBG antibody was tested for its protein concentration, purity, endotoxin (Lonza kit) and the like. Results shown in table 5 indicates that the concentration of endotoxin in the final antibody product is below 1.0 EU/mg.
TABLE-US-00005 TABLE 5 Analysis of purified TPBG antibody Protein Antibody Concentration Endotoxin Clone Number Purity (mg/mL) (EU/mg) 12B12C7C3 >90% 0.82 <0.12 5G4H10G5 >90% 0.68 <0.12 37H9C5G2 >90% 0.65 <0.12 39A11G5F2 >90% 0.85 <0.12 52C9E9F6 >90% 1.2 <0.12 28D4E6A9 >90% 1.02 <0.12 36A10D8B12 >90% 0.28 <0.12 99E12C7H1 >90% 1.12 <0.12 103E2E9C2 >90% 0.73 <0.12 106D5G3D10 >90% 0.54 <0.12
Embodiment 3
Identification of Lead Antibody
[0202] A. Detection of TPBG Antibody Binding to TPBG Protein By Enzyme-Linked Immunosorbent Assay (ELISA)
[0203] The purified TPBG antibody obtained from Embodiment 2 reacted with human TPBG-hFc.
[0204] The purified immunogen A (as for its method of preparation please refer to step 1 in Example 1) diluting with PBS to a final concentration of 1.0 μg/mL was aliquoted into a 96-well ELISA plate at 100 μL per well, and sealed with a parafilm and incubated at 4° C. overnight. The plate was washed twice with plate washing buffer (PBS containing 0.01% (v/v) Tween 20), and blocked with blocking buffer (PBS containing 0.01% (v/v) Tween 20 and 1% (w/w) BSA) at room temperature for 2 h. The blocking buffer was discarded, and 100 μL per well of the purified antibody obtained from Embodiment 2 was added. After incubating at 37° C. for 2 h, the plate was washed for three times with plate washing buffer (PBS containing 0.01% (v/v) Tween 20). HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Sigma) was added and incubated at 37° C. for 2 h, and the plate was washed for three times using plate washing buffer (PBS containing 0.01% (v/v) Tween 20). 100 μL of TMB substrate was aliquoted into each well, incubated at room temperature for 30 min, and 100 μL of stop buffer (1.0N HCl) per well was subsequently added. A.sub.450nm values were read using the ELISA plate reader (SpectraMax 384plus purchased from Molecular Device), and the results were shown in
TABLE-US-00006 TABLE 6 ELISA detection of the binding reaction between the TPBG antibody and human TPBG-hFc protein OD.sub.450 nm Antibody Concentration(nM) Clone Number 200 20 2 0.2 0.02 0.002 0.0002 Blank 12B12C7C3 2.82 2.83 2.84 1.67 0.29 0.13 0.10 0.11 5G4H10G5 2.72 2.80 2.68 1.76 0.34 0.10 0.08 0.07 37H9C5G2 2.61 2.63 2.52 1.55 0.35 0.11 0.09 0.09 39A11G5F2 2.55 2.50 2.43 1.36 0.30 0.11 0.09 0.09 52C9E9F6 2.65 2.69 2.68 1.39 0.27 0.12 0.09 0.08 28D4E6A9 2.60 2.59 2.64 1.97 0.46 0.14 0.10 0.09 36A10D8B12 2.51 3.07 3.06 2.15 0.51 0.18 0.16 0.19 99E12C7H1 2.93 2.88 2.93 2.62 0.78 0.21 0.11 0.08 103E2E9C2 2.60 2.62 2.64 1.89 0.48 0.14 0.10 0.13 106D5G3D10 2.88 2.82 2.81 2.36 0.56 0.16 0.11 0.10 IgG control 0.24 0.11 0.10 0.11 0.10 0.10 0.11 0.11
[0205] B. The Binding of TPBG Antibody to TPBG-expressing Cells was Detected By Fluorescence Activated Cell Sorting (FACS)
[0206] pIRES plasmid containing the nucleotide sequence encoding human TPBG full-length amino acid sequence in step 2 of Embodiment 1 was transfected into CHO-K1 cell line, and a recombinant CHO-K1 cell line containing human TPBG (herein referred to as CHO-K1-hTPBG stable cell line) was obtained. Similarly, pIRES plasmid containing cynomolgus monkey full-length TPBG gene (the preparation method of which is the same as that of the pCpC vector containing human IgG Fc fragment in the “Preparation of Immunogen A” part of step 1 in Embodiment 1, the full-length amino acid sequence of cynomolgus monkey with Genbank ID: BAE00432.1 was transfected into CHO-K1 cell line, and CHO-K1 stable cell line containing cynomolgus monkey TPBG (herein referred to as CHO-k1-cTPBG stable cell line) was obtained. pIRES plasmid containing full-length mouse TPBG gene (the method of preparation of which is the same as that of the pCpC vector containing human IgG Fc fragment in the “Preparation of Immunogen A” part of step 1 in Example 1, the Genbank ID of mouse TPBG full-length gene sequence can be accessed by: CAA09931.1) was transfected into CHO-K1 cell line, and a recombinant CHO-K1 cell line containing mouse TPBG (herein referred to as CHO-K1-mTPBG stable cell line) was prepared.
[0207] The titers and specificity of the TPBG antibody in serum were measured by FACS. For the detection method, please refer to the method identifying the stable cell line HEK293-hTPBG in the “Preparation of Immunogen B”, step 2 in Embodiment 1. Results were shown in Table 7 and
TABLE-US-00007 TABLE 7 Results of FACS screening and detection of human/monkey/mouse TPBG-transfected CHO-K1 cells Cellular MFI Transfected Cell IgG Anti-TPBG Clone Number Control antibody CHO-k1-hTPBG 3A2F1 3.24 798.37 CHO-k1-cTPBG 3F13G4 2.66 198.35 CHO-k1-mTPBG 3A3 2.38 135.25
[0208] The stable cell lines CHO-k1-hTPBG, CHO-k1-cTPBG, CHO-k1-mTPBG (i.e. the CHO-k1-hTPBG 3A2F1, CHO-k1-cTPBG 3F13G4 and CHO-k1-mTPBG 3A3 shown in table 7) and CHO-K1 cells were expanded to a 90% of confluency in T-75 cell culture flasks, the culture medium were removed, and cells were then rinsed twice with HBSS buffer (Hanks Balanced Salt Solution, purchased from Invitrogen) and lysed with enzyme-free cell dissociation buffer (Versene solution purchased from Life technology) and harvested. Harvested cells were rinsed with HBSS buffer twice and counted before diluting with HBSS buffer to 2×10.sup.6 cells /mL, and 10% goat serum blocking buffer was then added, wherein the percentage is the mass percentage. Cells were incubated on ice for 30 min, and then washed twice with HBSS buffer by centrifugation. The harvested cells were resuspended with FACS buffer (HBSS+1% BSA, wherein the percentage is mass percentage) to a concentration of 2×10.sup.6 cells /mL. 100 μL per well of the cells was aliquoted to 96-well FACS reaction plates, and 100 μL per well of test sample—the purified TPBG antibody obtaining from Embodiment 2 was added before incubating on ice for 2 h. The plate was washed with the FACS buffer twice by centrifugation, and 100 μL per well of fluorescence (Alexa 488) labeled secondary antibody (purchased from Invitrogen) was then added and incubated on ice for 1 h. The plate was washed with the FACS buffer for three times by centrifugation, follow by resuspending in 100 μL of fixation buffer (4% (v/v) paraformaldehyde). Cells were washed with FACS buffer twice after 10 min, follow by resuspending with 100 μL of FACS buffer. The results were measured and analyzed by FACS (FACS Calibur, purchased from BD). Data analysis was performed by software (CellQuest) to obtain mean fluorescence intensity (MFI) of the cells, follow by calculating EC50 value by software (GraphPad Prism) analysis and data fitting. The analysis results were shown in Table 8 and
TABLE-US-00008 TABLE 8 FACS Analysis of Binding Activity of TPBG Antibody to Human/Monkey/Mouse TPBG-expressing Cell Lines EC50(nM) CHO-K1- CHO-K1- CHO-K1- Clone Number hTPBG cTPBG mTPBG CHO-K1 12B12C7C3 0.65 0.59 Negative Negative 5G4H10G5 1.75 0.84 9.07 Negative 37H9C5G2 0.94 0.62 11.05 Negative 39A11G5F2 2.34 1.35 1.88 Negative 52C9E9F6 3.27 1.54 Negative Negative 28D4E6A9 0.63 0.25 Negative Negative 36A10D8B12 0.94 0.60 Negative Negative 99E12C7H1 3.54 2.29 Negative Negative 103E2E9C2 0.85 0.83 Negative Negative 106D5G3D10 1.72 1.26 Negative Negative
Embodiment 4
Cell Cytotoxicity Activity Assay of TPBG Antibody-Drug Conjugate
[0209] After dialyzing the purified TPBG antibody obtained from Embodiment 2 in sodium borate buffer, pH 6.5-8.5, Tris (2-carboxyethyl) phosphine (TCEP) was then added, wherein the molar ratio of TCEP to the purified TPBG antibody was 2. Reaction solution A was obtained after 1 h of reductive reaction at room temperature. The excess TCEP in reaction solution A was removed by G25 desalting column (purchased from GE), and reaction solution B was obtained. MC-MMAF (purchased from Levena Biopharma, Nanjing) was added to reaction solution B, wherein the molar ratio of MC-MMAF to purified TPBG antibody was 5 and the reaction was carried out at RT for 4 h. Cysteine was further added to neutralize excess MC-MMAF and excess small molecules were removed by G25 desalting column to obtain purified TPBG antibody-drug conjugate (for the method of conjugation, please refer to Doronina, 2006, Bioconjugate Chem. 17, 114-124). Parameters of the drug, such as drug antibody ratio, purity etc. were determined by HIC prior to analyzing cytotoxicity effect of cells. The drug antibody ratio (DAR) of all antibody-drug conjugate is 8, wherein DAR (drug antibody ratio) refers to the average number of small-molecule drugs carried by a single antibody molecule after antibody conjugation.
[0210] The obtained purified TPBG antibody-drug conjugates were serially diluted with complete medium, respectively. Each 100 μL of TPBG-positive NCI-H1568 cell suspension (purchased from ATCC, Catalog NO: #CRL5876) containing 2000 cells was added to 96-wells cell culture plate and cultured overnight before adding with each 10 μL of purified TPBG antibody-drug conjugate at different concentrations. After further culturing the cells for 5 days, the cell viability was measured using the CellTiter-Glo kit (purchased from Promega and used according to instructions). Meanwhile, TPBG-negative tumor cell line NCI-H1770 (purchased from ATCC, Catalog NO: #CRL5893) was selected to perform cytotoxicity test as described above. Results were shown in Table 9 and
[0211] In addition, the cytotoxicity effects of the purified TPBG antibody obtained from Embodiment 2 alone were further measured, and the method of which is the same as that in Embodiment 4 for detecting cell cytotoxicity of TPBG antibody-drug conjugate. Results were shown in Table 9 and
TABLE-US-00009 TABLE 9 Detection of Specific Cytotoxicity of TPBG Antibody-Drug Conjugate on TPBG-Positive Cells via Cellular Killing Assay EC50(nM) Clone Number DAR NCI-1568(+) NCI-1770(−) 12B12C7C3-MMAF 8 0.042 Negative 5G4H10G5-MMAF 8 0.626 Negative 37H9C5G2-MMAF 8 0.120 Negative 39A11G5F2-MMAF 8 0.285 Negative 52C9E9F6-MMAF 8 1.485 Negative 28D4E6A9-MMAF 8 0.038 Negative 36A10D8B12-MMAF 8 0.066 Negative 99E12C7H1-MMAF 8 0.844 Negative 103E2E9C2-MMAF 8 0.135 Negative 106D5G3D10-MMAF 8 0.208 Negative 12B12C7C3 0 Negative Negative 5G4H10G5 0 Negative Negative 39A11G5F2 0 Negative Negative 28D4E6A9 0 Negative Negative 36A10D8B12 0 Negative Negative
Embodiment 5
Analysis of Epitope Distribution of TPBG Antibody and Antibody via Competitive ELISA Assay
[0212] The TPBG antibodies were grouped by competitive ELISA in order to identify the binding sites of the antibodies to antigens.
[0213] The purified antibodies to be tested were diluted to 1 μg/mL using PBS, and coated with high-adsorption of 96-well microplates at 50 μg per well. After overnight coating at 4□, 250 μL of blocking buffer (PBS containing 0.01% (v/v) Tween20 and 1% (w/w) BSA) was added to block coating at room temperature for 1 h, and 0.05 μg/mL of biotinylated recombinant TPBG protein was added to each well. Meanwhile, 5 μg/mL final concentration of competitive antibodies, i.e. the purified antibodies obtained from Embodiment 2 with the clone number of 12B12C7C3, 5G4H10G5, 37H9C5G2, 39A11G5F2, 52C9E9F6, 28D4E6A9, 36A10D8B12, 99E12C7H1, 103E2E9C2 and 106D5G3D10 were added and incubated at 25-37° C. for 1-2 h. The plates were washed three times with plate-washing buffer (PBS containing 0.01% (v/v) Tween20) and HRP (horseradish peroxidase)-labeled streptavidin (purchased from Sigma) was added. After incubating at 37° C. for 0.5 h, the plates were washed three times with plate-washing buffer (PBS containing 0.01% (v/v) Tween20), and 100 μL of TMB substrate was added to each well and the plates were further incubated at room temperature for 30 min prior to adding 100 μL of stop buffer (1.0N HCl) to each well. A.sub.450nm values were read using an ELISA microplate reader (SpectraMax 384plus purchased from Molecular Device) and results were shown in
TABLE-US-00010 TABLE 10 Competition Rates Between TPBG Antibodies A B 12B12C7C3 106D5G3D10 5G4H10G5 99E12C7H1 37H9C5G2 12B12C7C3 97% 96% 47% 25% 1% 106D5G3D10 95% 94% 40% 11% 3% 5G4H10G5 9% 3% 97% 95% 5% 99E12C7H1 11% 12% 95% 94% 5% 37H9C5G2 6% 4% 12% 7% 96% 36A10D8B12 21% 14% 17% 25% 96% 39A11G5F2 9% 4% 19% 16% 0% 52C9E9F6 27% 7% 32% 18% 4% 28D4E6A9 24% 5% 11% 11% 0% 103E2E9C2 0% 0% 7% 3% 23% A B 36A10D8B12 39A11G5F2 52C9E9F6 28D4E6A9 103E2E9C2 12B12C7C3 3% 0% 4% 74% 0% 106D5G3D10 1% 1% 0% 1% 0% 5G4H10G5 1% 10% 19% −1% 2% 99E12C7H1 8% 1% 0% 38% 1% 37H9C5G2 94% 3% 5% 58% 38% 36A10D8B12 85% 4% 23% 86% 40% 39A11G5F2 0% 96% 95% 0% 0% 52C9E9F6 19% 97% 97% 17% 0% 28D4E6A9 75% 0% 0% 96% 88% 103E2E9C2 31% 0% 0% 95% 88%
[0214] Among them, the meaning of A in Table 10 is that the concentration of coated antibody in each column is 1 μg/mL; The meaning of B is that the concentration of competitive antibody in each row is 5 μg/mL.
[0215] The results indicate that 12B12C7C3 and 106D5G3D10 can compete with each other and share similar epitopes; 5G4H10G5 and 99E12C7H1 can compete with each other and share similar epitopes; 37H9C5G2 and 36A10D8B12 can compete with each other and share similar epitopes; 39A11G5F2 and 52C9E9F6 can compete with each other and share similar epitopes; 28D4E6A9 and 103E2E9C2 can compete with each other and share similar epitopes.
Embodiment 6
Amino Acid Sequence Determination of the Light Chain and Heavy Chain Variable Regions
[0216] Total RNA Extraction: 5×10.sup.7 hybridoma cells preparing in Embodiment 1 were collected by centrifugation, and 1 mL of Trizol was added, mixed and then the mixture was transferred to a 1.5 mL Eppendorf tube and allowed to stand at room temperature for 5 min. Next, 0.2 mL of chloroform was added, vortexed for 15 s and the tube was centrifugated at 12,000 g, 4° C. for 5 min and allowing to stand for 10 min. The supernatant was transferred to a new 1.5 mL Eppendorf tube, and 0.5 mL of isopropanol was added, and the contents in the tube were mixed gently and allowed to stand for 10 min at room temperature prior to centrifuging at 12,000 g, 4° C. for 15 min. The supernatant was discarded, 1 mL of 75% (v/v) ethanol was added and the pellets were rinsed gently and centrifugated at 12,000 g for 5 min. The supernatant was removed, the pellets were air-dried before dissolving with DEPC-treated water (water bath at 55° C. for 10 min facilitating dissolving), and finally the total RNA was extracted.
[0217] Reverse transcription and PCR: 1 μg of total RNA was took and the reverse transcriptase was added, a 20 μL system was set up and reacted at 42□ for 60 minutes, the reaction was terminated at 85□ for 10 minutes. A 50 μL PCR system was set with 1 μL of cDNA, 25 pmol of each primer, 1 μL of DNA polymerase, 250 μmol of dNTPs and a compatible buffer system. PCR program consisted of pre-denaturing at 95□ for 3 minutes, 35 cycles of denaturing at 95□ for 30 seconds, annealing at 55□ for 30 seconds and extending at 72□ for 35 seconds, followed by extension at 72□ for 5 minutes to obtain a PCR product. The kit used for reverse transcription was PrimeScript RT Master Mix purchased from Takara, with Cat. No. RR036; the kit used for PCR including the Q5 high-fidelity enzyme was purchased from NEB, with Cat. No. M0492.
[0218] Cloning and sequencing: 5 μL of PCR product was used to test by agarose gel electrophoresis, and positive samples were purified by column recovery kit NucleoSpin® Gel & PCR Clean-up, purchased from MACHEREY-NAGEL with a Cat. No. of 740609. Ligation was carried out by using 50 ng of sample, 50 ng of T vector, 0.5 μL of recombinase Exnaseligase and 1 μL of buffer to a final reaction system volume of 10 μL, and reacted at 16□ for half an hour to obtain the ligated product, wherein the ligation kit is T4 DNA ligase purchased from NEB with a Cat. No. of M0402; 5 μL of the ligation product was pipetted into 100 μL of competent cells (Ecos 101 competent cells, purchased from Yeastern, Cat. No. FYE607) and ice-cooled for 5 minutes. Subsequently the competent cells were heat shocked at 42□ for 1 minute in water bath and placed on ice for 1 minutes, then 650 μL of antibiotic-free SOC medium was added and the competent cells were resuscitated at 200 RPM on a shaker at 37□ for 30 minutes, 200 μL of cells suspension was pipetted and spread on LB solid medium containing antibiotic and cultured overnight in 37□ incubator. On the next day, colony PCR was performed in a 30 μL PCR system using primers M13F and M13R specifically deisigned for T vector, bacterial colonies were picked by a tip and pipetted into the PCR system and mixed, and 0.5 μL of suspension was picked onto another LB solid plate containing 100 μg/mL ampicillin to preserve the bacterial strain; 5 μL of product was subjected to the detection of agarose gel electrophoresis when the PCR reaction is completed, and the positive samples were sequenced and analyzed [see Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991)]. The sequencing results were shown in Tables 11-12.
TABLE-US-00011 TABLE 11 Protein Sequences Number of TPBG Antibodies Heavy Chain Protein Light Chain Protein Clone Variable Variable Number Region CDR1 CDR2 CDR3 Region CDR1 CDR2 CDR3 12B12C7C3 1 2 3 4 5 6 7 8 5G4H10G5 9 10 11 12 13 14 15 16 37H9C5G2 17 18 19 20 21 22 23 24 39A11G5F2 25 26 27 28 29 30 31 32 52C9E9F6 33 34 35 36 37 38 39 40 28D4E6A9 41 42 43 44 45 46 47 48 36A10D8B12 49 50 51 52 53 54 55 56 99E12C7H1 57 58 59 60 61 62 63 64 103E2E9C2 65 66 67 68 69 70 71 72 106D5G3D10 73 74 75 76 77 78 79 80
[0219] In Table 11, the numbers are the sequence numbers in sequence listing, e.g. the amino acid sequence of the variable region in the heavy chain protein of 12B12C7C3 is set forth as SEQ ID NO:1, while the CDR1 amino acid sequence in the heavy chain protein variable region of 12B12C7C3 is set forth as SEQ ID NO:2.
TABLE-US-00012 TABLE 12 The gene sequence numbers of TPBG antibody Heavy Chain Protein Light Chain Protein Clone Number Variable Region Variable Region 12B12C7C3 81 82 5G4H10G5 83 84 37H9C5G2 85 86 39A11G5F2 87 88 52C9E9F6 89 90 28D4E6A9 91 92 36A10D8B12 93 94 99E12C7H1 95 96 103E2E9C2 97 98 106D5G3D10 99 100
[0220] The numbers in Table 12 are the sequence numbers in sequence listing, e.g. the nucleotide sequence encoding the variable region in the heavy chain protein of 12B12C7C3 is the nucleotide sequence shown in SEQ ID NO:81;
[0221] wherein the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 12B12C7C3 is the nucleotide sequence from 91 to 105 shown in SEQ ID NO:81;
[0222] the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 12B12C7C3 is the nucleotide sequence from 148 to 198 in SEQ ID NO:81;
[0223] the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 12B12C7C3 is the nucleotide sequence from 295 to 327 in SEQ ID NO:81;
[0224] the nucleotide sequence encoding the CDR1 in the light chain variable region of 12B12C7C3 is the nucleotide sequence from 70 to 114 in SEQ ID NO:82;
[0225] the nucleotide sequence encoding the CDR2 in the light chain variable region of 12B12C7C3 is the nucleotide sequence from 160 to 180 in SEQ ID NO:82;
[0226] the nucleotide sequence encoding the CDR3 in the light chain variable region of 12B12C7C3 is the nucleotide sequence from 277 to 303 in SEQ ID NO:82;
[0227] the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 5G4H10G5 is the nucleotide sequence from 91 to 105 in SEQ ID NO:83;
[0228] the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 5G4H10G5 is the nucleotide sequence from 148 to 198 in SEQ ID NO:83;
[0229] the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 5G4H10G5 is the nucleotide sequence from 295 to 330 in SEQ ID NO:83;
[0230] the nucleotide sequence encoding the CDR1 in the light chain variable region of 5G4H10G5 is the nucleotide sequence from 70 to 102 in SEQ ID NO:84;
[0231] the nucleotide sequence encoding the CDR2 in the light chain variable region of 5G4H10G5 is the nucleotide sequence from 148 to 168 in SEQ ID NO:84;
[0232] the nucleotide sequence encoding the CDR3 in the light chain variable region of 5G4H10G5 is the nucleotide sequence from 265 to 288 in SEQ ID NO:84;
[0233] the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 37H9C5G2 is the nucleotide sequence from 91 to 105 in SEQ ID NO:85;
[0234] the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 37H9C5G2 is the nucleotide sequence from 148 to 198 in SEQ ID NO:85;
[0235] the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 37H9C5G2 is the nucleotide sequence from 295 to 327 in SEQ ID NO:85;
[0236] the nucleotide sequence encoding the CDR1 in the light chain variable region of 37H9C5G2 is the nucleotide sequence from 70 to 102 in SEQ ID NO:86;
[0237] the nucleotide sequence encoding the CDR2 in the light chain variable region of 37H9C5G2 is the nucleotide sequence from 148 to 168 in SEQ ID NO:86;
[0238] the nucleotide sequence encoding the CDR3 in the light chain variable region of 37H9C5G2 is the nucleotide sequence from 265 to 291 in SEQ ID NO:86;
[0239] the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 39A11G5F2 is the nucleotide sequence from 91 to 105 in SEQ ID NO:87;
[0240] the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 39A11G5F2 is the nucleotide sequence from 148 to 198 in SEQ ID NO:87;
[0241] the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 39A11G5F2 is the nucleotide sequence from 295 to 315 in SEQ ID NO:87;
[0242] the nucleotide sequence encoding the CDR1 in the light chain variable region of 39A11G5F2 is the nucleotide sequence from 70 to 102 in SEQ ID NO:88;
[0243] the nucleotide sequence encoding the CDR2 in the light chain variable region of 39A11G5F2 is the nucleotide sequence from 148 to 168 in SEQ ID NO:88;
[0244] the nucleotide sequence encoding the CDR3 in the light chain variable region of 39A11G5F2 is the nucleotide sequence from 265 to 291 in SEQ ID NO:88;
[0245] the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 52C9E9F6 is the nucleotide sequence from 91 to 105 in SEQ ID NO:89;
[0246] the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 52C9E9F6 is the nucleotide sequence from 148 to 198 in SEQ ID NO:89;
[0247] the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 52C9E9F6 is the nucleotide sequence from 295 to 315 in SEQ ID NO:89;
[0248] the nucleotide sequence encoding the CDR1 in the light chain variable region of 52C9E9F6 is the nucleotide sequence from 70 to 102 in SEQ ID NO:90;
[0249] the nucleotide sequence encoding the CDR2 in the light chain variable region of 52C9E9F6 is the nucleotide sequence from 148 to 168 in SEQ ID NO:90;
[0250] the nucleotide sequence encoding the CDR3 in the light chain variable region of 52C9E9F6 is the nucleotide sequence from 265 to 291 in SEQ ID NO:90;
[0251] the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 28D4E6A9 is the nucleotide sequence from 91 to 105 in SEQ ID NO:91;
[0252] the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 28D4E6A9 is the nucleotide sequence from 148 to 198 in SEQ ID NO:91;
[0253] the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 28D4E6A9 is the nucleotide sequence from 295 to 327 in SEQ ID NO:91;
[0254] the nucleotide sequence encoding the CDR1 in the light chain variable region of 28D4E6A9 is the nucleotide sequence from 70 to 102 in SEQ ID NO:92;
[0255] the nucleotide sequence encoding the CDR2 in the light chain variable region of 28D4E6A9 is the nucleotide sequence from 148 to 168 in SEQ ID NO:92;
[0256] the nucleotide sequence encoding the CDR3 in the light chain variable region of 28D4E6A9 is the nucleotide sequence from 265 to 291 in SEQ ID NO:92;
[0257] the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 36A10D8B12 is the nucleotide sequence from 91 to 108 in SEQ ID NO:93;
[0258] the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 36A10D8B12 is the nucleotide sequence from 151 to 198 in SEQ ID NO:93;
[0259] the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 36A10D8B12 is the nucleotide sequence from 295 to 324 in SEQ ID NO:93;
[0260] the nucleotide sequence encoding the CDR1 in the light chain variable region of 36A10D8B12 is the nucleotide sequence from 70 to 102 in SEQ ID NO:94;
[0261] the nucleotide sequence encoding the CDR2 in the light chain variable region of 36A10D8B12 is the nucleotide sequence from 148 to 168 in SEQ ID NO:94;
[0262] the nucleotide sequence encoding the CDR3 in the light chain variable region of 36A10D8B12 is the nucleotide sequence from 265 to 291 in SEQ ID NO:94;
[0263] the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 99E12C7H1 is the nucleotide sequence from 91 to 105 in SEQ ID NO:95;
[0264] the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 99E12C7H1 is the nucleotide sequence from 148 to 198 in SEQ ID NO:95;
[0265] the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 99E12C7H1 is the nucleotide sequence from 295 to 318 in SEQ ID NO:95;
[0266] the nucleotide sequence encoding the CDR1 in the light chain variable region of 99E12C7H1 is the nucleotide sequence from 70 to 102 in SEQ ID NO:96;
[0267] the nucleotide sequence encoding the CDR2 in the light chain variable region of 99E12C7H1 is the nucleotide sequence from 148 to 168 in SEQ ID NO:96;
[0268] the nucleotide sequence encoding the CDR3 in the light chain variable region of 99E12C7H1 is the nucleotide sequence from 265 to 291 in SEQ ID NO:96;
[0269] the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 103E2E9C2 is the nucleotide sequence from 91 to 105 in SEQ ID NO:97;
[0270] the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 103E2E9C2 is the nucleotide sequence from 148 to 198 in SEQ ID NO:97;
[0271] the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 103E2E9C2 is the nucleotide sequence from 295 to 324 in SEQ ID NO:97;
[0272] the nucleotide sequence encoding the CDR1 in the light chain variable region of 103E2E9C2 is the nucleotide sequence from 70 to 105 in SEQ ID NO:98;
[0273] the nucleotide sequence encoding the CDR2 in the light chain variable region of 103E2E9C2 is the nucleotide sequence from 151 to 171 in SEQ ID NO:98;
[0274] the nucleotide sequence encoding the CDR3 in the light chain variable region of 103E2E9C2 is the nucleotide sequence from 268 to 294 in SEQ ID NO:98;
[0275] the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 106D5G3D10 is the nucleotide sequence from 91 to 105 in SEQ ID NO:99;
[0276] the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 106D5G3D10 is the nucleotide sequence from 148 to 198 in SEQ ID NO:99;
[0277] the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 106D5G3D10 is the nucleotide sequence from 295 to 318 in SEQ ID NO:99;
[0278] the nucleotide sequence encoding the CDR1 in the light chain variable region of 106D5G3D10 is the nucleotide sequence from 70 to 99 in SEQ ID NO:100;
[0279] the nucleotide sequence encoding the CDR2 in the light chain variable region of 106D5G3D10 is the nucleotide sequence from 145 to 165 in SEQ ID NO:100;
[0280] the nucleotide sequence encoding the CDR3 in the light chain variable region of 106D5G3D10 is the nucleotide sequence from 262 to 294 in SEQ ID NO:100.
Embodiment 7
Construction, Production and Purification of Mouse-Human Chimeric Antibody
[0281] 1. Plasmid Construction and Preparation: The sequences of heavy chain variable region and light chain variable region of TPBG antibodies were confirmed by the sequencing results from Embodiment 6. The sequences of the heavy chain variable regions in the lead antibodies obtained from Embodiment 2 and Embodiment 3 were recombined into an expression vector containing a signal peptide and a constant region of human heavy chain antibody IgG1 (the expression vector was purchased from Invitrogen, and the recombination process was completed by ChemPartner, Shanghai), and the sequences of the light chain variable regions in the TPBG antibodies were recombined into an expression plasmid containing a signal peptide and a constant region of human light chain kappa, and the recombined plasmids were confirmed by sequencing (the sequencing method of which is the same as that in Embodiment 6). High-purity of recombinant plasmid was extracted by an alkaline lysis kit (purchased from MECHEREY-NAGEL), and more than 500 μg of plasmid was collected and filtered through a 0.22 μm membrane filter (purchased from Millipore) for following transfection.
[0282] 2. Cell Transfection: 293E cells (purchased from Invitrogen) were cultured in Freestyle 293 expression medium (purchased from Invitrogen). The shaker was set at 37° C., 130 rpm and 8% CO.sub.2 (v/v). During transfection, 10% (v/v) of F68 (purchased from Invitrogen) was added to Freestyle 293 expression medium to a final F68 concentration of 0.1% (v/v), and the Freestyle 293 culture medium containing 0.1% (v/v) F68, i.e. culture medium A was prepared. 5 mL of culture medium A was mixed with 200 μg/mL PEI (purchased from Sigma), obtaining culture medium B. 5 mL of culture medium A was mixed with 100 μg/mL of recombinant plasmid obtained in step 1 to prepare culture medium C. After 5 min, culture medium B and C were pooled and mixed, and allowed to stand for 15 min to prepare mixture D. 10 mL of mixture D was slowly added into 100 mL of Freestyle 293 expression medium containing 293E cells, with simultaneously oscillation to avoid over-aggregation of PEI, until the density of the 293E cells reach 1.5×10.sup.6/mL and the cells were cultured in the shaker. Peptone was added the next day until its concentration reached 0.5% (w/v). From day 5 to day 7, the titer of antibodies in the culture were tested. From day 6 to day 7, the supernatant was collected by centrifugation (3500 rpm, 30 min) and filtered through a 0.22 μm of membrane filter to prepare a filtered cell supernatant for purification.
[0283] 3. Antibody Purification: Endotoxin-free chromatography columns and Protein A filler in continuous production state were treated with 0.1M NaOH for 30 min or washed with 5 column bed volumes of 0.5M NaOH. Fillers and chromatography columns without being used for a long time were soaked in 1M NaOH for at least 1 h, follow by neutralizing with endotoxin-free water and rinsing with 10 column bed volumes of 1% Triton X100. The column was equilibrated with 5 column bed volumes of PBS, and the filtered cells supernatant were loaded on the column and the flow-through portion of the sample was collected if necessary. After finishing sample loading, 5 column bed volumes of PBS was used to wash the column. 5 column bed volumes of 0.1M pH 3.0 Glycine-HCl was used to elute, follow by collecting eluate and neutralizing column with 1/10 volume of 1M Tris-HCl, pH 8.5. The antibody was harvested and then dialyzed in 1×PBS overnight to prevent endotoxin contamination. Then, the concentration was determined by spectrophotometer or kit, the purity of the antibody was determined by HPLC-SEC, and the content of endotoxin was determined by endotoxin detection kit (purchased from Lonza).
[0284] In the following embodiments, the first segment of characters in the names of the chimeric antibodies were selected from the first 3-5 characters in their corresponding lead antibodies, e.g. the clone number of the lead antibody corresponding to the chimeric antibody 12B12-MMAF is 12B12C7C3, and the clone number of the lead antibody corresponding to the chimeric antibody 5G4-MMAF is 5G4H10G5, etc.
Embodiment 8
In Vitro Pharmacodynamic Studies of Chimeric Antibodies
[0285] The purified TPBG chimeric antibodies obtained from Embodiment 7 were coupled with MC-MMAF using the method described in Embodiment 4. After dialysis with sodium borate buffer pH 6.5-8.5, Tris (2-carboxyethyl) phosphine (TCEP) was added, wherein the molar ratio of TCEP to the purified TPBG antibodies was 2. Reaction solution A was obtained by reductive reaction at room temperature for 1 h. The reaction solution A was desalted on G25 (purchased from GE) to remove excess TCEP and hence reaction buffer B was obtained. MC-MMAF was added to reaction buffer B, wherein the molar ratio of MC-MMAF to the purified TPBG antibody was 5, and the reaction was carried out at room temperature for 4 h. Cysteine was added to neutralize excess MC-MMAF, excess small molecules were removed by G25 desalting, follow by obtaining purified TPBG antibody-drug conjugate (for the method of conjugation, refers to Doronina, 2006, Bioconjugate Chem. 17, 114-124). Drug-antibody ratio of the drugs were analyzed by HIC, the purity of the antibody-drug conjugates was analyzed by SEC, and the cytotoxicity was analyzed subsequently. The drug-antibody ratio (DAR) of all antibody-drug conjugates was 3.0-5.0, wherein DAR (drug-antibody ratio) refers to the average number of small-molecule drugs carried on a single antibody molecule after antibody conjugation.
[0286] The obtained purified TPBG antibody-drug conjugates were serially diluted with complete medium, respectively. 100 μL of TPBG-positive NCI-H1299 cell suspension was added to a 96-well plate at 2000 cells per well. After incubating the cell suspension overnight, 10 μL of purified TPBG chimeric antibody-drug conjugate diluents at different concentrations were added to each well. After 5 days further culturing, the cell viability was measured using the CellTiter-Glo kit (purchased from Promega and used according to instructions). Results were shown in Table 13 and
TABLE-US-00013 TABLE 13 Detection of Specific Cytotoxicity of Purified TPBG Chimeric Antibody-Drug Conjugates on TPBG-Positive NCI-H1299 Cells via Cellular Killing Assay IC50 (nM) Sample Name NCI-H1299(+) Chimeric Antibody 12B12-MMAF 0.13 Chimeric Antibody 5G4-MMAF 87.27 Chimeric Antibody 39A11-MMAF 4.30 Chimeric Antibody 28D4-MMAF 0.21 Chimeric Antibody 36A10-MMAF 0.70 Control hIgG-MMAF >100
Embodiment 9
In Vivo Pharmacodynamic Studies of Chimeric Antibodies
[0287] 200 μL of NCI-H1299 (non-small cell lung cancer cell line, ATCC, CRL-5803) (5×10.sup.6 cells) were inoculated subcutaneously in the right rib of Balb/c nude mice and allowed to grow for 7-10 days until the tumor volumes reach 200 mm.sup.3. After removing the mice with too large or too small body weight and tumor size, the mice were randomly divided into several groups, i.e. 7 mice for each group, according to the tumor size. Antibody was injected intravenously to the tail at D0, and the injection was performed once every four days with a total of four injections. Tumor volume and body weight of mice were measured every two weeks, and the data were recorded. Formulation for calculating tumor volume is: V=½×a x b2; wherein a and b represents length and width, respectively. Grouping was shown in Table 14.
TABLE-US-00014 TABLE 14 In Vivo Pharmacodynamic Studies of TPBG Chimeric Antibodies and Its Antibody-Drug Conjugates Injected Number of Treating Dose Amount Route of Administration Group Animals Group (mg/kg) (μl/g) Administation Plan 1 7 Solvent — 10 Tail Administer Control intravenous once every 4 injection i.v. days × 4 times 2 7 Chimeric 1 10 Tail Administer Antibody intravenous once every 4 12B12-MMAF injection i.v. days × 4 times 3 7 Chimeric 10 10 Tail Administer Antibody intravenous once every 4 12B12-MMAF injection i.v. days × 4 times 4 7 Chimeric 1 10 Tail Administer Antibody intravenous once every 4 5G4-MMAF injection i.v. days × 4 times 5 7 Chimeric 10 10 Tail Administer Antibody intravenous once every 4 5G4-MMAF injection i.v. days × 4 times 6 7 Chimeric 1 10 Tail Administer Antibody intravenous once every 4 39A11-MMAF injection i.v. days × 4 times 7 7 Chimeric 10 10 Tail Administer Antibody intravenous once every 4 39A11-MMAF injection i.v. days × 4 times 8 7 Chimeric 1 10 Tail Administer Antibody intravenous once every 4 28D4-MMAF injection i.v. days × 4 times 9 7 Chimeric 10 10 Tail Administer Antibody intravenous once every 4 28D4-MMAF injection i.v. days × 4 times 10 7 Chimeric 1 10 Tail Administer Antibody intravenous once every 4 36A10-MMAF injection i.v. days × 4 times 11 7 Chimeric 10 10 Tail Administer Antibody intravenous once every 4 36A10-MMAF injection i.v. days × 4 times 12 7 Chimeric 10 10 Tail Administer Antibody intravenous once every 4 12B12 injection i.v. days × 4 times 13 7 Chimeric 10 10 Tail Administer Antibody intravenous once every 4 5G4 injection i.v. days × 4 times 14 7 Chimeric 10 10 Tail Administer Antibody intravenous once every 4 39A11 injection i.v. days × 4 times 15 7 Chimeric 10 10 Tail Administer Antibody intravenous once every 4 28D4 injection i.v. days × 4 times 16 7 Chimeric 10 10 Tail Administer Antibody intravenous once every 4 36A10 injection i.v. days × 4 times 17 7 Control 10 10 Tail Administer hIgG-MMAF intravenous once every 4 injection i.v. days × 4 times
[0288] Results were shown in
Embodiment 10
In Vitro Pharmacodynamic Studies of Antibody-drug Conjugates Coupled with Different Linker-Toxins
[0289] Purified TPBG chimeric antibodies 12B12 and 28D4 obtained from Embodiment 7 were coupled with MC-MMAF and MC-VC-PAB-MMAE, respectively, using the method described in Embodiment 8. After dialysis with sodium borate buffer with pH 6.5-8.5, tris(2-carboxyethyl) phosphine (TCEP) was added, wherein the molar ratio of TCEP to the purified TPBG antibodies was 2. Reductive reaction was carried out at room temperature for 1 h, and reaction solution A was obtained. Excess TCEP was removed by desalting reaction solution A with G25 (purchased from GE), and then reaction solution B was obtained. MC-MMAF or MC-VC-PAB-MMAE (purchased from Levena Biomart, Nanjing) was added to reaction solution B, wherein the molar ratio of MC-MMAF or MC-VC-PAB-MMAE to purified TPBG antibody was 5. The reaction was carried out at room temperature for 4 h. Cysteine was further added to neutralize excess MC-MMAF or MC-VC-PAB-MMAE, and excess small molecules were removed by G25 desalting, follow by obtaining purified TPBG antibody-drug conjugate—i.e. the chimeric antibodies 12B12-MMAF, 12B12-MMAE, 28D4-MMAF, 28D4-MMAE listed in the table (as for the method of conjugation please refer to Doronina, 2006, Bioconjugate Chem. 17, 114-124). Drug-antibody ratio of the drugs were analyzed by HIC, the purity of the antibody-drug conjugates was analyzed by SEC, follow by determining the cytotoxicity of conjugates.
[0290] The purified chimeric antibodies 12B12 and 28D4 obtained from Embodiment 7 were coupled with SMCC, respectively. After dialyzing the purified antibodies obtained in Embodiment 7 with phosphate buffer pH 6.5-7.4, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) was added in the presence of 30% (v/v) dimethylacetamide (DMA), wherein the molar ratio of SMCC to the purified TPBG chimeric antibodies was 8. Reaction solution A was obtained after the reaction was carried out at room temperature for 1 h. Excess small molecules were removed by desalting reaction solution A with G25 column (purchased from GE) to obtain reaction solution B. DMA was added to reaction solution B until a final volume percent of 10% was reached, and DM1 (N2′-deacetyl-N2′-(3-mercapto-1-oxypropyl)-maytansine) was then added, wherein the molar ratio of DM1 to purified TPBG antibodies was 9. Reaction solution C was obtained once the reaction was carried out at room temperature for 3.5 h. Reaction solution C was subjected to desalting by G25 (purchased from GE) to remove excess small molecules, and the purified TPBG chimeric antibody-drug conjugates—i.e. the chimeric antibodies 12B12-DM1 and 28D4-DM1 listed in the table (for the coupling method please refer to U.S. Pat. No. 5,208,020) were obtained. After the drug-antibody ratio was determined by LC-MS and the purity of the antibody-drug conjugates is analyzed by SEC, the cytotoxicity effects of conjugates were determined as well. The drug-antibody ratios (DAR) of all chimeric antibody-drug conjugates were about 3.0-5.0, wherein DAR (drug antibody ratio) refers to the average number of small-molecule drugs carried by a single antibody molecule after antibody conjugation.
[0291] The obtained purified TPBG antibody-drug conjugates were serially diluted with complete medium. 100 μL of TPBG-positive NCI-H1299 (purchased from ATCC, Catalog NO: #CRL5803) or NCI-H1568 (purchased from ATCC, Catalog NO: #CRL5876) cell suspension was added to the 96-well plate at 2000 cells per cell, cultured overnight, and 104 of purified TPBG chimeric antibody-drug conjugate diluents of different concentrations were added to each well. After further culturing the cells for 5 days, cell viability was measured using the CellTiter-Glo kit (purchased from Promega and used according to instructions). Results were shown in Table 15 and
TABLE-US-00015 TABLE 15 Detection of Specific Cytotoxicity of Purified TPBG Chimeric Antibody-Drug Conjugates on TPBG-Positive HCI-H1299 and HCI-H1568 Cells via Cellular Killing Assay IC.sub.50 (nM) Sample Name NCI-H1299 NCI-H1568 Chimeric Antibody 12B12 None None Chimeric Antibody 12B12-MMAF 0.04 0.025 Chimeric Antibody 12B12-MMAE >100 >100 Chimeric Antibody 12B12-DM1 10.42 1.271 Chimeric Antibody 28D4 None None Chimeric Antibody 28D4-MMAF 0.132 0.066 Chimeric Antibody 28D4-MMAE 368.6 >100 Chimeric Antibody 28D4-DM1 6.084 2.507
[0292] The obtained purified TPBG antibody-drug conjugates were serially diluted with complete medium. 100 μL of TPBG-expressing 293-hTPBG recombinant cell line (the method of preparation please refer to Example 1: Preparation of Immnogen B) or 293 cell line not expressing TPBG (purchased from ATCC, Catalog NO: #CRL-1573) was added to a the 96-well plate at 2000 cells per cell, cultured overnight, and 10 μL of purified TPBG chimeric antibody-drug conjugate diluents of different concentrations were added to each well. After further culturing the cells for 5 days, cell viability was measured by the CellTiter-Glo kit (purchased from Promega and used according to instructions). Results were shown in Table 16 and
TABLE-US-00016 TABLE 16 Detection of Specific Cytotoxicity of Purified TPBG Chimeric Antibody-Drug Conjugates on TPBG-Positive 293-hTPBG and TPBG-Negative 293Cells via Cellular Killing Assay IC50 (nM) Sample Name 293-hTPBG 293 Chimeric Antibody 12B12 13.58 None Chimeric Antibody 12B12-MMAF 0.001 >100 Chimeric Antibody 12B12-MMAE 0.033 >100 Chimeric Antibody 12B12-DM1 0.154 8.885 Chimeric Antibody 28D4 1.137 None Chimeric Antibody 28D4-MMAF 0.024 >100 Chimeric Antibody 28D4-MMAE 0.08 >100 Chimeric Antibody 28D4-DM1 0.406 7.658
[0293] It should be understood that after reading the above contents of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.