Humanized anti-TPBG antibody, preparation method therefor, conjugate thereof, and applications
11608384 · 2023-03-21
Assignee
Inventors
- Ying Zhang (Shanghai, CN)
- Shiyong GONG (Shanghai, CN)
- Xiaolan Sun (Shanghai, CN)
- Tengjiao Xu (Shanghai, CN)
- Fei Peng (Shanghai, CN)
- Yuzhu Chen (Shanghai, CN)
- Lile Liu (Shanghai, CN)
Cpc classification
A61K39/395
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K47/6851
HUMAN NECESSITIES
C12N15/63
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
C07K16/00
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
A61K39/3955
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
A61K39/3955
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
C12N15/00
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
International classification
A61K47/68
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
C12N15/63
CHEMISTRY; METALLURGY
Abstract
The present invention provides a humanized anti-TPBG antibody, a preparation method therefor, a conjugate thereof, and applications. The humanized anti-TPBG antibody comprises: (a), a framework region comprising a residue of a human antibody framework region; and (b) one or more CDRs of a light-chain variable region as shown in the SEQ ID NO:4 or 8 or one or more CDRs of a heavy-chain variable region as shown in the SEQ ID NO:2 or 6.
Claims
1. A humanized anti-TPBG antibody comprising: (a) a framework region containing residues of a human antibody framework region; and (b) one or more CDRs of the mouse-derived antibody light chain variable region set forth in SEQ ID NO: 4 or 8, or one or more CDRs of the mouse-derived antibody heavy chain variable region set forth in SEQ ID NO: 2 or 6; wherein the humanized anti-TPBG antibody comprises at least one heavy chain variable region and one light chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 18 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 26 or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 20 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 26; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 24 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 26; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 16 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 28; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 18 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 28; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 20 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 28; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 22 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 28; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 24 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 28; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 18 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 30; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 20 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 30; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 24 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 30; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 16 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 32; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 18 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 32; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 20 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 32; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 22 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 32; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 24 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 32; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 34 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 42; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 36 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 42; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 38 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 42; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 40 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 42; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 34 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 44; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 36 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 44; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 38 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 44; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 40 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 44; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 34 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 36 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 38 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 40 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46.
2. The humanized anti-TPBG antibody according to claim 1, wherein the humanized anti-TPBG antibody further comprises a heavy chain constant region and a light chain constant region of human antibody.
3. The humanized anti-TPBG antibody according to claim 1, wherein the humanized anti-TPBG antibody is a full-length protein of antibody, a protein fragment of antigen-antibody binding domain, a dual-specificity antibody, a multiple-specificity antibody, a single chain antibody, a single domain antibody or a single-region antibody; or, the humanized anti-TPBG antibody is a monoclonal antibody or a polyclonal antibody; or, the humanized anti-TPBG antibody is a super-humanized antibody or a double antibody.
4. A nucleic acid encoding the humanized anti-TPBG antibodies according to claim 1.
5. The nucleic acid according to claim 4, wherein the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 18, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 26; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 20, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 26; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 24, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 26; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 16, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 28; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 18, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 28; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 20, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 28; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 22, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 28; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 24, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 28; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 18, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 30; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 20, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 30; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 24, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 30; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 16, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 32; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 18, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 32; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 20, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 32; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 22, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 32; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 24, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 32; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 34, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 42; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 36, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 42; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 38, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 42; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 40, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 42; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 34, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 44; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 36, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 44; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 38, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 44; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 40, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 44; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 34, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 46; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 36, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 46; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 38, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 46; or, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO: 40, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO: 46.
6. A recombinant expression vector comprising the nucleic acid according to claim 4.
7. A recombinant expression transformant comprising the recombinant expression vector according to claim 6.
8. A preparation method of humanized anti-TPBG antibody according claim 1, which comprises following steps: culturing the recombinant expression vector according to claim 7, obtaining humanized anti-TPBG antibody from culture.
9. An immunoconjugate comprising the humanized anti-TPBG antibody according to claim 1 which is covalently linked to a cytotoxic agent.
10. The immunoconjugate according to claim 9, wherein the humanized anti-TPBG antibody according to claim 1 is linked to y equivalent of cytotoxic agent through x equivalent of linker, which has the structure shown in formula 1,
Ab-(L).sub.x-(D).sub.y Formula 1 wherein Ab is the humanized anti-TPBG antibody according to claim 1, L is linker; D is cytotoxic agent; x is natural number.
11. The immunoconjugate according to claim 10, wherein the linker L is active esters, carbonates, carbamates, imine phosphate esters, oximes, hydrazones, acetals, orthoesters, aminos, small peptides or nucleotide fragments.
12. The immunoconjugate according to claim 11, wherein the linker L is selected from maleimidocaproyl (MC), maleimidocaproyl-L-valine-L-citrulline p-amino benzyl alcohol (MC-VC-PAB), 4-(N-maleimide methyl) cyclohexane-1-carboxylic succinimide (SMCC); and/or, the D is selected from cytotoxin, chemotherapeutic agent, radioisotope, therapeutic nucleic acid, immunomodulator, anti-angiogenic agents, anti-proliferative and pro-apoptotic agent or cytolytic enzyme.
13. The immunoconjugate according to claim 9, 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; D is methylauristatin F; ##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.
14. A pharmaceutical composition comprising the immunoconjugate according to claim 9 and pharmaceutically acceptable vehicle.
15. A package of medical kits, comprising a component A and a component B, the component A is the humanized anti-TPBG antibody according to claim 1, or the immunoconjugate according to claim 9, or the pharmaceutical composition according to claim 14, wherein the component B is the other anti-tumor antibodies or pharmaceutical composition comprising other anti-tumor antibodies.
16. A method for treating a tumor in a subject in need thereof, comprising: administering a humanized anti-TPBG antibody according to claim 1, or an immunoconjugate according to claim 9, or a pharmaceutical composition according to claim 14, or a package of medical kits to the subject.
17. A method for detecting cells overexpressing TPBG protein, which comprises the following steps: contacting the humanized anti-TPBG antibody according to claim 1 with the sample to be tested in vitro, and detecting the binding of the humanized anti-TPBG antibody according to claim 1 with the sample to be tested.
18. The humanized anti-TPBG antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 34 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 42; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 36 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 42; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 38 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 42; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 40 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 42; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 34 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 44; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 36 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 44; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 38 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 44; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 40 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 44; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 34 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 36 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 38 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 40 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46.
19. The humanized anti-TPBG antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 38 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 42; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 40 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 42; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 38 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 44; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 40 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 44; or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 40 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46.
20. The humanized anti-TPBG antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 38 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 42.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
(39) The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure. The experimental methods in the following examples which do not specify the specific conditions are selected according to conventional methods and conditions, or according to the product specifications.
(40) The room temperature described in the examples is a conventional room temperature in the art, generally 10 to 30° C.
(41) Unless otherwise specified, the PBS described in the examples is PBS phosphate buffer, pH 7.2.
Embodiment 1 Preparation of Chimeric Antibodies 12B12 and 28D4 and Humanized TPBG Antibodies
(42) 1. Preparation of Mouse Antibodies 12B12 and 28D4
(43) (1) Preparation of Immunogen a (Human TPBG-hFc Protein)
(44) A nucleotide sequence containing the nucleic acid encoding amino acid sequence 32-355 (Ser32-Ser355) of 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 a pCpC vector with 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 at 37° C. using FreeStyle™293 (purchased from Invitrogen). 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 (A280 nm) was monitored using a UV detector. Once the sample was loaded, the protein A affinity chromatography column was equilibrated with PBS buffer (pH7.2) until the ultraviolet absorption value returned to baseline. Subsequently, elution was performed using 0.1M glycine hydrochloride acid (pH2.5), 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 human TPBG-hFc protein was then obtained.
(45) Prior to being used, human TPBG-hFc protein 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 human TPBG-hFc protein was qualified and can be used as the antigen for the subsequent preparation of TPBG antibodies.
(46) (2) Preparation of Immunogen B
(47) 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 of which was prepared. Recombinant plasmid (PEI, purchased from Polysciences) was transfected to HEK293 cell line (purchased from ATCC), and the transfected cells were selectively cultured in 0.5 g/ml DMEM culture medium containing 10% (w/w) fetal bovine serum for two weeks. Subcloning was performed using limiting dilution assay in 96-wells plate and the plate 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-wells plate. The expanded clones were screened by FACS analysis using commercially 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, i.e., immunogen B was obtained. The specific selection results were shown in Table 2. The IgG isotype control herein was mouse IgG. Table 2 indicates that a series of TPBG expression-positive HEK 293 cell lines have been prepared. Table 2 demonstrates that 293F-hTPBG 5E5 is a cell strain with high level of TPBG expression, wherein the MFI of the cells labeled with anti-TPBG antibody is 280.5, and the migration rate of the cells is 98.5%.
(48) TABLE-US-00003 TABLE 2 Results of FACS screening of HEK293 cells transfected with human TPBG protein Clone number of Cellular MFI NO transfected cell IgG isotype control TPBG 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
(49) (3) Preparation of Hybridoma Cells and Screening of Antibody
(50) A. Immunization with Immunogen A
(51) BALB/cAnNCrl mice or SJL/JorllcoCrl mice (both purchased from SLAC Co. Ltd, Shanghai) aged 6-8 weeks were raised in SPF condition. For the primary immunization, 0.25 ml of immunogen A prepared in step 1 being emulsified with Freund's complete adjuvant, i.e. 50 mg of immunogen A per mouse was intraperitoneally injected. For 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 3. Table 3 indicates that the serums of mouse immunized with immunogen A have different degrees of binding to immunogen A and show antigen-antibody reaction, wherein the highest dilution rate is around one million. The blank control is 1% (w/w) BSA, and batch refers to the type of mouse serum at day 7 after the second booster immunization. The data in the table refers to OD450 nm values.
(52) 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
(53) B. Immunization with Immunogen B
(54) 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. According above-described step (2), the pIRES plasmid containing the nucleotide sequence encoding full-length human TPBG was transfected into HEK293 cell line and the recombinant HEK293 stable cell line containing human TPBG was obtained (293F-hTPBG 5E5) (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 was over 1:1000.
(55) Prior to the completion of steps 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 stable 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 lyse the erythrocyte that is mixed with splenocyte, 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 highly 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-wells cell culture plates at the density of 1×10.sup.5 cells/200 μl per well and cultured in 5% (v/v) CO.sub.2 incubator at 37° C. After 14 days, the supernatants from the cell fusion plates were screened using ELISA and Acumen (microplate cytometry), and positive clones with an ELISA OD450 nm>1.0 and Acumen MFI value >100 were subjected to expansion culture in 24-wells plate. Positive clones were cultured in DMEM culture medium containing 10% (w/w) HT FBS at 37° C. in 5% (v/v) CO.sub.2. The culture medium from the expansion culture in 24-wells plate was centrifuged after 3 days of culturing and the supernatant was collected and subjected to antibody subclass analysis. Its binding activity to TPBG protein and TPBG-positive cells were determined using ELISA and FACS.
(56) Based on the screening results of 24-wells plate, hybridoma cells having ELISA OD450 nm>1.0, FACS MFI>50 and the hybridoma supernatant killing rate on TPBG-positive cells reaching 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-wells plate and cultured in DMEM medium (purchased from Invitrogen) containing 10% (w/w) HT FBS at 37° C. in 5% (v/v) CO.sub.2. 10 days after the subcloning, initial screening was performed using ELISA and Acumen, and single positive monoclones were selected and subjected to expansion culture in 24-wells plate. After 3 days, positive antigen binding was confirmed by FACS, while the criteria for the evaluation is that OD450 nm>1.0 for ELISA, and MFI>50 for FACS.
(57) According to the detection results of 24-wells plate, the optimal clone was picked and expanded in DMEM medium (purchased from Invitrogen) containing 10% (w/w) HT FBS at 37° C. in 5% (v/v) CO.sub.2. The optimal clone was obtained and stored in liquid nitrogen, and then used for subsequently production and purification of lead antibody.
(58) (4) Production and Purification of Lead Antibody
(59) The antibody concentration generated by hybridoma cells was relatively low, i.e. about only 1-10 mg/mL with a broad-range variation, and the various proteins produced by cell culturing and the contents of FBS contained in the culture medium have interfered many biologic activity assays to different degrees. Therefore, a small-scale (1-5 mg) production and purification of antibody is necessary.
(60) The hybridoma cells obtained by section (3) were seeded into T-75 cell culture flask and passaged for three times. Until reaching a robust growing state, cells were seeded into the spinner flasks for cell culturing. 200 mL of production culture medium were added to each 2 L spinner flask at a seeding density of 1.0×10.sup.5/mL. 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 which is ready for immediate purification or stored at −30° C.
(61) The TPBG antibody containing 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, pH7.4), and then loaded by the culture supernatant at a controlled flow rate of 3 mL/min. Four bed volumes of equilibration buffer was used to wash the protein G column once sample loading was complete. The TPBG antibody bound to protein A column was eluted using elution buffer (0.1M sodium citrate buffer, pH3.5), and the elution was monitored by UV detector (A280 nm absorption). The antibody eluate was collected and pH was neutralized by adding with 10% (v/v) of 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, and filtered by 0.22 m sterile filter, thereby a purified TPBG antibody was obtained and stored in sterile condition.
(62) The purified TPBG antibody was tested for its protein concentration (A.sub.280 nm/1.4), purity, endotoxin (Lonza kit) and the like. Results shown in Table 4 indicates that the concentration of endotoxin in the final antibody product is below 1.0 EU/mg.
(63) TABLE-US-00005 TABLE 4 Analysis of purified TPBG antibody Protein Antibody Concentration Endotoxin Clone Number Purity (mg/mL) (EU/mg) 12B12C7C3 (12B12 for short) >90% 0.82 <0.12 28D4E6A9 (28D4 for shot) >90% 1.02 <0.12
(64) (5) Identification of Lead Antibody
(65) A. Detection of TPBG Antibody Binding to TPBG Protein by Enzyme-Linked Immunosorbent Assay (ELISA)
(66) The purified TPBG antibody reacted with human TPBG-hFc.
(67) The purified human TPBG-hFc obtained from step (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 t L per well of the purified antibody TPBG 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.450 nm values were read using the ELISA plate reader (SpectraMax 384plus purchased from Molecular Device), and the results were shown in Table 5. Table 5 indicates that purified TPBG antibody can combine with recombinant TPBG protein at the level of ELISA. In Table 5 the IgG control herein is a mice IgG control, the data in the Table are OD.sub.450 nm values, and Blank represents the OD.sub.450 nm value of PBS buffer in the plate.
(68) TABLE-US-00006 TABLE 5 ELISA detection of the binding reaction between the TPBG antibody and human TPBG-hFc protein OD.sub.450 nm Clone Antibody Concentration(nM) 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 28D4E6A9 2.60 2.59 2.64 1.97 0.46 0.14 0.10 0.09 IgG control 0.24 0.11 0.10 0.11 0.10 0.10 0.11 0.11
(69) B. The Binding of TPBG Antibody to TPBG-Expressing Cells was Detected by Fluorescence Activated Cell Sorting (FACS)
(70) The nucleotide sequence encoding the full-length amino acid sequence of human TPBG (wherein the accession numbers of amino acid sequence and nucleotide sequence encoding human TPBG protein in Genbank are Genbank ID AAH37161.1 and Gene ID: 7162, respectively) was cloned into the pIRES vector (purchased from Clontech) and a plasmid was prepared. pIRES plasmid containing the nucleotide sequence encoding human TPBG full-length amino acid sequence (PEI, purchased from Polysciences) was transfected into CHO-k1 cell line (purchased from ATCC), and a recombinant CHO-k1 cell line containing human TPBG (herein referred to as CHOk1-hTPBG stable cell line) was obtained. Similarly, the nucleotide sequence encoding the full-length amino acid sequence of cynomolgus TPBG (wherein the accession numbers of amino acid sequence and nucleotide sequence encoding cynomolgus TPBG protein in Genbank are Genbank ID BAE00432.1 and Gene ID: 102132149, respectively) was cloned into the pIRES vector (purchased from Clontech) and a plasmid was prepared. pIRES plasmid containing the nucleotide sequence encoding cynomolgus TPBG full-length amino acid sequence (PEI, purchased from Polysciences) was transfected into CHO-k1 cell line (purchased from ATCC), and a recombinant CHO-k1 cell line containing cynomolgus TPBG (herein referred to as CHOk1-cTPBG stable cell line) was obtained. Similarly, the nucleotide sequence encoding the full-length amino acid sequence of mouse TPBG (wherein the accession numbers of amino acid sequence and nucleotide sequence encoding mouse TPBG protein in Genbank are Genbank ID: CAA09931.1 and Gene ID: 21983, respectively) was cloned into the pIRES vector (purchased from Clontech) and a plasmid was prepared. pIRES plasmid containing the nucleotide sequence encoding mouse TPBG full-length amino acid sequence (PEI, purchased from Polysciences) was transfected into CHO-k1 cell line (purchased from ATCC), and a recombinant CHO-k1 cell line containing mouse TPBG (herein referred to as CHOk1-mTPBG stable cell line) was obtained.
(71) The titers and specificity of the TPBG antibody in serum were measured by FACS. For the detection method, please refer to the method for identifying the stable cell line HEK293-hTPBG in the “Preparation of Immunogen B”, step (2) as described above. Results were shown in Table 6. Results in Table 6 indicate that human, cynomolgus or mouse TPBG protein are overexpressed on the cell membrane of the stable cell lines CHOk1-hTPBG, CHOk1-cTPBG and CHOk1-mTPBG, respectively, which can be used for the screening of TPBG antibody.
(72) TABLE-US-00007 TABLE 6 Results of FACS screening and detection of human/cynomolgus/mouse TPBG-transfected CHOK1 cells Transfected Cell Cellular MFI Clone Number Control IgG Anti-TPBG antibody CHOk1-hTPBG 3A2F1 3.24 798.37 CHOk1-cTPBG 3F13G4 2.66 198.35 CHOk1-mTPBG 3A3 2.38 135.25
(73) The stable cell lines CHOk1-hTPBG, CHOk1-cTPBG, CHOk1-mTPBG (i.e. the CHOk1-hTPBG 3A2F1, CHOk1-cTPBG 3F13G4 and CHOk1-mTPBG 3A3 shown in Table 6) and CHO-k1 cells were expanded to a 90% of confluency in T-75 cell culture flasks, and the culture medium were removed. 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 was 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 step (4) as described above 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). The data was analyzed by software (CellQuest) to obtain the 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 7. Data in Table 7 is the EC50 values converted from the MFI, and Table 7 indicates that TPBG antibody is able to bind cell-surface TPBG proteins.
(74) TABLE-US-00008 TABLE 7 FACS Analysis of Binding Activity of TPBG Antibody to Human/Cynomolgus/Mouse TPBG-expressing Cell Lines EC50 (nM) CHOk1- CHOk1- CHOk1- Clone Number hTPBG cTPBG mTPBG CHO-k1 12B12C7C3 0.65 0.59 Negative Negative 28D4E6A9 0.63 0.25 Negative Negative
(75) (6) Analysis of Epitope Distribution of TPBG Antibody and Antibody Via Competitive ELISA Assay
(76) The aforementioned TPBG antibodies were grouped by competitive ELISA in order to identify the binding sites of the antibodies to antigens.
(77) The purified antibodies to be tested were diluted to 1 g/mL using PBS, and coated to high-adsorption of 96-wells microplate at 50 μL per well. After overnight coating at 4° C., 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 g/mL of biotinylated recombinant TPBG protein was added to each well. Meanwhile, 5 g/mL final concentration of competitive antibodies, i.e. the purified antibodies with the clone number of 12B12C7C3 and 28D4E6A9 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.450 nm values were read using an ELISA microplate reader (SpectraMax 384plus purchased from Molecular Device). The competition rates between the antibodies were calculated according to the A.sub.450 nm values, and results were shown in Table 8. The higher the values of the competition rate, the closer the antigen surface of the two antibodies are.
(78) TABLE-US-00009 TABLE 8 Competition Rates Between antibody 12B12C7C3 and antibody 28D4E6A9 A B 12B12C7C3 28D4E6A9 12B12C7C3 97% 74% 28D4E6A9 24% 96%
(79) The results indicate that epitopes of 12B12C7C3 and 28D4E6A9 are different.
(80) (7) Determination of Amino Acid Sequences of the Light Chain and Heavy Chain Variable Regions
(81) Total RNA Extraction: 5×10.sup.7 hybridoma cells prepared in previous step (3) 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 allowed to stand for 10 min and then centrifugated at 12,000 g, 4° C. for 5 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 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 (dissolution was promoted in a water bath at 55° C. for 10 min), and finally the total RNA was extracted.
(82) 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° C. for 60 minutes prior to terminating reaction at 85° C. for 10 minutes. A 50 μL PCR system was set containing 1 μL of cDNA, 25 pmol of each primer, 250 μmol of dNTPs, 1 μL of DNA polymerase and a compatible buffer system. PCR program consisted of pre-denaturing at 95° C. for 3 minutes, 35 cycles of denaturing at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds and extending at 72° C. for 35 seconds, followed by another extension at 72° C. for 5 minutes and then PCR products were harvested. The kit used for reverse transcription was PrimeScript RT Master Mix purchased from Takara, with Cat. No. RR036; and the kit used for PCR including the Q5 high-fidelity enzyme was purchased from NEB, with Cat. No. M0492.
(83) Cloning and sequencing: 5 μL of PCR products were determined 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 at 16° C. for half an hour in 10 μL of final reaction system comprising 50 ng of sample, 50 ng of T vector, 0.5 μL of ligase and 1 μL of buffer and the ligated products were harvested, wherein the ligation kit is T4 DNA ligase purchased from NEB with a Cat. No. of M0402. Later on, 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° C. 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 recovered at 200 RPM on a shaker at 37° C. for 30 minutes, followed by pipetting and spreading 200 μL of cells suspension on LB solid medium containing antibiotic and culturing overnight in 37° C. incubator. On the next day, colony PCR was performed in a 30 μL PCR system using primers M13F and M13R specifically designed 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 plated onto another LB solid plate containing 100 μg/mL ampicillin to preserve the bacterial strain. 5 μL of product was subjected to the detection by 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)].
(84) The amino acid sequence of heavy chain variable region is shown in SEQ ID NO: 2, and the light chain variable region sequence is shown in SEQ ID NO: 4 for the antibody 12B12C7C3.
(85) wherein the amino acid sequence of CDR1 in the heavy chain variable region corresponds to positions 31 to 35 of SEQ ID NO: 2, the amino acid sequence of CDR2 in the heavy chain variable region corresponds to positions 50 to 66 of SEQ ID NO: 2, the amino acid sequence of CDR3 in the heavy chain variable region corresponds to positions 99 to 109 of SEQ ID NO: 2;
(86) The amino acid sequence of CDR1 in the light chain variable region of the mouse antibody corresponds to positions 24 to 38 of SEQ ID NO: 4, the amino acid sequence of CDR2 corresponds to positions 54 to 60 of SEQ ID NO: 4, the amino acid sequence of CDR3 corresponds to positions 93 to 101 of SEQ ID NO: 4.
(87) The amino acid sequence of heavy chain variable region of the antibody 28D4E6A9 is shown in SEQ ID NO: 6, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.
(88) wherein the amino acid sequence of CDR1 in the heavy chain variable region corresponds to positions 31 to 35 of SEQ ID NO: 6, the amino acid sequence of CDR2 in the heavy chain variable region corresponds to positions 50 to 66 of SEQ ID NO: 6, the amino acid sequence of CDR3 in the heavy chain variable region corresponds to positions 99 to 109 of SEQ ID NO: 6;
(89) The amino acid sequence of CDR1 in the light chain variable region of the mouse antibody corresponds to positions 24 to 34 of SEQ ID NO: 8, the amino acid sequence of CDR2 corresponds to positions 50 to 56 of SEQ ID NO: 8, the amino acid sequence of CDR3 corresponds to positions 89 to 97 of SEQ ID NO: 8.
(90) Nucleotide sequencing results are as follows:
(91) The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 3 for antibody 12B12C7C3;
(92) the nucleotide sequence of heavy chain variable region of the is shown in SEQ ID NO: 5, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 7 for antibody 28D4E6A9;
(93) wherein the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 12B12C7C3 corresponds to positions from 91 to 105 of SEQ ID NO: 1;
(94) the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 12B12C7C3 corresponds to positions from 148 to 198 of SEQ ID NO: 1;
(95) the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 12B12C7C3 corresponds to positions from 295 to 327 of SEQ ID NO: 1;
(96) the nucleotide sequence encoding the CDR1 in the light chain variable region of 12B12C7C3 corresponds to positions from 70 to 114 of SEQ ID NO: 3;
(97) the nucleotide sequence encoding the CDR2 in the light chain variable region of 12B12C7C3 corresponds to positions from 160 to 180 of SEQ ID NO: 3;
(98) the nucleotide sequence encoding the CDR3 in the light chain variable region of 12B12C7C3 corresponds to positions from 277 to 303 of SEQ ID NO: 3;
(99) the nucleotide sequence encoding the CDR1 in the heavy chain variable region of 28D4E6A9 corresponds to positions from 91 to 105 of SEQ ID NO: 5;
(100) the nucleotide sequence encoding the CDR2 in the heavy chain variable region of 28D4E6A9 corresponds to positions from 148 to 198 of SEQ ID NO: 5;
(101) the nucleotide sequence encoding the CDR3 in the heavy chain variable region of 28D4E6A9 corresponds to positions from 295 to 327 of SEQ ID NO: 5;
(102) the nucleotide sequence encoding the CDR1 in the light chain variable region of 28D4E6A9 corresponds to positions from 70 to 102 of SEQ ID NO: 7;
(103) the nucleotide sequence encoding the CDR2 in the light chain variable region of 28D4E6A9 corresponds to positions from 148 to 168 of SEQ ID NO: 7;
(104) the nucleotide sequence encoding the CDR3 in the light chain variable region of 28D4E6A9 corresponds to positions from 265 to 291 of SEQ ID NO: 7;
(105) 2. Preparation of Mouse-Human Chimeric Antibody 12B12 and 28D4
(106) The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TPBG mouse monoclonal antibody 12B12 were exemplified in SEQ ID NO: 2,4, respectively. The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TPBG mouse monoclonal antibody 28D4 were exemplified in SEQ ID NO: 6,8, respectively.
(107) (1) Plasmid Construction and Preparation: The sequences of the heavy chain variable regions in the mouse lead antibodies were incorporated 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 incorporated into an expression plasmid containing a signal peptide and a constant region of human light chain kappa (the expression vector was purchased from Invitrogen, and the recombination process was completed by ChemPartner, Shanghai), and the recombined plasmids were confirmed by sequencing (The experimental principle and steps of the above plasmid recombination can be found in the ‘Molecular Cloning: A Laboratory Manual’ (Third Edition), (US) J. Sambrook et al). High-purity of recombinant plasmid was extracted by an alkaline lysis kit (purchased from MECHEREY-NAGEL), and more than 500 g of plasmid was harvested and filtered through a 0.2 m membrane filter (purchased from Millipore) for following transfection.
(108) (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 supplied with 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), thus obtaining culture medium B. 5 mL of culture medium A was mixed with 100 μg/mL of recombinant plasmid (here is a mixed recombinant plasmid in which the above-mentioned heavy chain recombinant plasmid and light chain recombinant plasmid were mixed in a conventional equal ratio) 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 day5 to day7, the titer of antibodies in the culture were tested. From day6 to day7, the supernatant was collected by centrifugation (3500 rpm, 30 min) and filtered through a 0.22 m of membrane filter to prepare a filtered cell supernatant for further purification.
(109) (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, the column was washed by 5 column bed volumes of PBS. Eluent was eluted by 5 column bed volumes of 0.1M pH3.0 Glycine-HCl and collected, and the column was then neutralized with 1/10 volume of 1M Tris-HCl, pH8.5. The antibody was harvested and then dialyzed in 1×PBS overnight to prevent endotoxin contamination. Then, the concentration of the antibody 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).
(110) Purified TPBG chimeric antibodies 12B12 and 28D4 were harvested respectively
(111) 3. Preparation of Humanized TPBG Antibody
(112) Human germline templates of antibody heavy chain variable region and light chain variable region that match the non-CDR regions of chimeric antibody 12B12 or 28D4 described above optimally were selected in the Germline database. The human ‘acceptor’ sequence of the humanized TPBG antibody is selected from the human germline exon V.sub.H, J.sub.H, V.sub.k and J.sub.k sequences. The template for the heavy chain variable region of the 12B12 antibody comprises VH3-48 and VH3-30 of the V.sub.H exon of the human germline antibody heavy chain V.sub.H exon, and JH-6 of the J.sub.H exon, and the template of the light chain variable region comprises B3 and A2 of the human germline antibody light chain V.sub.K exon, and /J.sub.K-2 of the J.sub.K exon. The template for the heavy chain variable region of the 28D4 antibody comprises VH3-11 of the human germline antibody heavy chain V.sub.H exon, and J.sub.H-6 of the J.sub.H exon, and the template of the light chain variable region comprises O18 and A2 of human germline antibody light chain V.sub.K Exons, and JK exon of J.sub.K-5.
(113) The heavy and light chain CDRs of chimeric antibody 12B12 or 28D4, amino acid residues of which determined according to the Kabat definition, were each grafted into a selected human germline template, and the CDR regions of the human germline template were replaced to obtain a humanized antibody. Based on the three-dimensional structure of the mouse antibody, reverse mutation on the embedding residues, residues that directly interact with CDRs region and ones that have an important impact on conformation of VL and VH were conducted, and the humanized antibody was obtained. Briefly, synthetic overlapping oligonucleotides spanning human VH or VL domain were generated and each domain was assembled using PCR overlap extension. The VH domain was directionally cloned into an expression vector containing signal peptide and constant region of humanized antibody heavy chain IgG1, and the V.sub.L domain was directionally cloned into an expression vector containing signal peptide and human antibody light chain kappa using restriction site for incorporation of PCR product. The obtained recombinant plasmids were confirmed by sequencing, and high-purity of recombinant plasmids were extracted by an alkaline lysis kit (purchased from MACHEREY-NAGEL). More than 500 g of plasmid was collected and subjected to a 0.22 μm membrane filter (purchased from Millipore) for following transfection.
(114) Sequence alignment of the heavy and light chain variable regions among humanized anti-TPBG antibody variants, the human germline and chimeric antibodies are shown in
(115) Human germline heavy chain variable region template VH3-48/JH6 (SEQ ID NO: 9) is as follows
(116) TABLE-US-00010 VH3-48: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSY ISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR; JH6: WGQGTTVTVSS.
(117) Human germline heavy chain variable region template VH3-30/JH6 (SEQ ID NO: 10) is as follows
(118) TABLE-US-00011 VH3-30: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV ISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR; JH6: WGQGTTVTVSS.
(119) Human germline light chain variable region template B3/Jk2 (SEQ ID NO: 11) is as follows
(120) TABLE-US-00012 B3: DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC; JK2: FGQGTKLEIK.
(121) Human germline light chain variable region template A2/Jk2 (SEQ ID NO: 12) is as follows
(122) TABLE-US-00013 A2: DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQPPQ LLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC; JK2: FGQGTKLEIK.
(123) Human germline heavy chain variable region template VH3-11/JH6 (SEQ ID NO: 13) is as follows
(124) TABLE-US-00014 VH3-11: QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSY ISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR; JH6: WGQGTTVTVSS.
(125) Human germline light chain variable region template O18/Jk5 (SEQ ID NO: 14) is as follows
(126) TABLE-US-00015 O18: DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC; JK5: FGQGTRLEIK.
(127) Several framework positions were selected to reintroduce mouse donor residues. Humanized 12B12 and 28D4 variants can be produced by incorporating different combinations of mouse framework donor residues in VH domain or human CDR residues in VL domain. These variants are summarized in Tables 9 and 10 below. These two tables show the variable regions of these variants, without including the constant regions. In Tables 9 and 10, a c initial means chimeric antibody and a h initial means humanized antibody; wherein, in the columns of donor framework residues and reverse mutation, for example, ‘A93S’ in each humanized anti-TPBG antibody 12B12 heavy chain variable region h12B12.VH1 and its variants indicates that the 93rd amino acid shown in
(128) TABLE-US-00016 TABLE 9 Light Heavy chain chain variable variable region region template Donor template Donor VH of the framework Vk of the framework germ line of residues and SEQ germ line residues and SEQ Heavy human reverse ID Light of human reverse ID Antibody chain antibody mutation NO: chain antibody mutation NO: c12B12 VH None CDR 2 Vk None CDR 4 h12B12-1 VH.1a VH3- CDR-grafted, 18 Vk.1 B3/Jk2 CDR-grafted 26 48/JH6 A93S h12B12-2 VH.1b VH3- CDR-grafted, 20 Vk.1 B3/Jk2 CDR-grafted 26 48/JH6 S49A, A93S h12B12-3 VH.2a VH3- CDR-grafted, 24 Vk.1 B3/Jk2 CDR-grafted 26 30/JH6 Q1E, A93S h12B12-4 VH.1 VH3- CDR-grafted 16 Vk.1a B3/Jk2 CDR-grafted, 28 48/JH6 M4L, Y49K h12B12-5 VH.1a VH3- CDR-grafted, 18 Vk.1a B3/Jk2 CDR-grafted, 28 48/JH6 A93S M4L, Y49K h12B12-6 VH.1b VH3- CDR-grafted, 20 Vk.1a B3/Jk2 CDR-grafted, 28 48/JH6 S49A, A93S M4L, Y49K h12B12-7 VH.2 VH3- CDR-grafted, 22 Vk.1a B3/Jk2 CDR-grafted, 28 30/JH6 Q1E M4L, Y49K h12B12-8 VH.2a VH3- CDR-grafted, 24 Vk.1a B3/Jk2 CDR-grafted, 28 30/JH6 Q1E, A93S M4L, Y49K h12B12-9 VH.1a VH3- CDR-grafted, 18 Vk.2 A2/Jk2 CDR-grafted 30 48/JH6 A93S h12B12- VH.1b VH3- CDR-grafted, 20 Vk.2 A2/Jk2 CDR-grafted 30 10 48/JH6 S49A, A93S h12B12- VH.2a VH3- CDR-grafted, 24 Vk.2 A2/Jk2 CDR-grafted 30 11 30/JH6 Q1E, A93S h12B12- VH.1 VH3- CDR-grafted 16 Vk.2a A2/Jk2 CDR-grafted, 32 12 48/JH6 M4L, Y49K h12B12- VH.1a VH3- CDR-grafted, 18 Vk.2a A2/Jk2 CDR-grafted, 32 13 48/JH6 A93S M4L, Y49K h12B12- VH.1b VH3- CDR-grafted, 20 Vk.2a A2/Jk2 CDR-grafted, 32 14 48/JH6 S49A, A93S M4L, Y49K h12B12- VH.2 VH3- CDR-grafted, 22 Vk.2a A2/Jk2 CDR-grafted, 32 15 30/JH6 Q1E M4L, Y49K h12B12- VH.2a VH3- CDR-grafted, 24 Vk.2a A2/Jk2 CDR-grafted, 32 16 30/JH6 Q1E, A93S M4L, Y49K
(129) TABLE-US-00017 10 Heavy chain Light chain variable variable region region template VH Donor template Vk Donor of the germ framework of the germ framework line of residues and SEQ line of residues and SEQ Heavy human reverse ID Light human reverse ID Antibody chain antibody mutation NO: chain antibody mutation NO: c28D4 VH None CDR 6 Vk None CDR 8 h28D4-1 VH.1 VH3-11/JH6 CDR-grafted 34 Vk.1 O18/Jk5 CDR-grafted 42 h28D4-2 VH.1a VH3-11/JH6 CDR-grafted, 36 Vk.1 O18/Jk5 CDR-grafted 42 Q1E, A93I, R94M h28D4-3 VH.1b VH3-11/JH6 CDR-grafted, 38 Vk.1 O18/Jk5 CDR-grafted 42 Q1E, A93I, R94M, S49A, N76K h28D4-4 VH.1c VH3-11/JH6 CDR-grafted, 40 Vk.1 O18/Jk5 CDR-grafted 42 Q1E, A93I, R94M, S49A, N76K, I37A, G44R h28D4-5 VH.1 VH3-11/JH6 CDR-grafted 34 Vk.1a O18/Jk5 CDR- 44 grafted, F71Y h28D4-6 VH.1a VH3-11/JH6 CDR-grafted, 36 Vk.1a O18/Jk5 CDR- 44 Q1E, grafted, A93I, R94M F71Y h28D4-7 VH.1b VH3-11/JH6 CDR-grafted, 38 Vk.1a O18/Jk5 CDR- 44 Q1E, grafted, A93I, R94M, F71Y S49A, N76K h28D4-8 VH.1c VH3-11/JH6 CDR-grafted, 40 Vk.1a O18/Jk5 CDR- 44 Q1E, grafted, A93I, R94M, F71Y S49A, N76K, I37A, G44R h28D4-9 VH.1 VH3-11/JH6 CDR-grafted 34 Vk.1b O18/Jk5 CDR- 46 grafted, F71Y, A43T, P44V, Y87F h28D4- VH.1a VH3-11/JH6 CDR-grafted, 36 Vk.1b O18/Jk5 CDR- 46 10 Q1E, grafted, A93I, R94M F71Y, A43T, P44V, Y87F h28D4- VH.1b VH3-11/JH6 CDR-grafted, 38 Vk.1b O18/Jk5 CDR- 46 11 Q1E, A93I, grafted, R94M, F71Y, S49A, N76K A43T, P44V, Y87F h28D4- VH.1c VH3-11/JH6 CDR-grafted, 40 Vk.1b O18/Jk5 CDR- 46 12 Q1E, grafted, A93I, R94M, F71Y, 49A, A43T, N76K, I37A, P44V, Y87F G44R
(130) Note: The “/” in the table means “and” and the contents to the left and right of ‘/’ are in parallel.
(131) cDNAs were synthesized based on the amino acid sequences of the light chain variable region and heavy chain variable region of each humanized antibody (i.e., the sequences shown in SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45 of the sequence listing, respectively). Heavy chain cDNAs were digested by FspAI and AfeI, and light chain cDNAs were treated by FspAI and BsiwI. Those cDNA fragments were inserted into expression vector containing a signal peptide and a constant region of human heavy chain antibody IgG1 and expression plasmid containing a signal peptide and a constant region of human light chain kappa (the expression vector was purchased from Invitrogen, and the recombination process was completed by ChemPartner, Shanghai) by FspAI/AfeI or FspAI/BsiwI restriction sites, respectively. The recombined plasmids were confirmed by sequencing. High-purity of recombinant plasmid was extracted by an alkaline lysis kit (purchased from MECHEREY-NAGEL), and more than 500 g of plasmid was collected and subjected to a 0.22 μm membrane filter (purchased from Millipore) for following transfection.
(132) Before transfection, 293E cells (purchased from Invitrogen) were cultured in Freestyle 293 expression medium (purchased from Invitrogen). 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 g/mL PEI (purchased from Sigma), obtaining culture medium B. 5 mL of culture medium A was mixed with 100 μg of heavy and light chain recombinant plasmids (mass ratio of heavy chain recombinant plasmid to light chain recombinant plasmid is 1:1 to 1:3) 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. The cells were then cultured in the shaker, and the shaker was set at 37° C., 130 rpm and 8% CO.sub.2 (v/v). Peptone was added the next day until its concentration reached 0.5% (w/v). From day5 to day7, the titer of antibodies in the culture were tested. From day6 to day7, the supernatant was collected by centrifugation (3500 rpm, 30 min) and filtered through a 0.22 m of membrane filter to prepare a filtered cell supernatant for purification.
(133) In antibody purification, continuous endotoxin-free chromatography columns and protein A filler (purchased from GE) were washed with 5 column bed volumes of 0.5M NaOH. The column was equilibrated with 5 column bed volumes of PBS (PBS buffer, pH 7.4), 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 pH3.0 Glycine-HCl was used to elute, immediately follow by collecting eluate and neutralizing TPBG antibody with 1/10 volume of 1M Tris-HCl, pH8.5. All of the solutions used above need to be freshly prepared. The antibody was harvested and then dialyzed in 1×PBS for 4 hours, meanwhile endotoxin contamination shall be prevented. 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). Obtained TPBG antibody was characterized (the procedure is as described in Embodiment 2, Embodiment 3, and Embodiment 4 below).
Embodiment 2 Detection of Binding of Humanized TPBG Antibody to TPBG Protein by Enzyme-Linked Immunosorbent Assay (ELISA)
(134) The purified humanized TPBG antibody obtained from Embodiment 1 was reacted with human TPBG-hFc, for the method, see (5) Identification of lead antibody in Embodiment 1. The results are shown in
(135) TABLE-US-00018 TABLE 11 ELISA detection of the binding reaction between humanized h12B12 antibody variants and human TPBG-hFc protein Antibody EC50 (nM) Chimeric antibody 12B12 (control) 0.137 Humanized antibody 12B12-1 0.248 Humanized antibody 12B12-2 0.274 Humanized antibody 12B12-3 0.333 Humanized antibody 12B12-4 0.227 Humanized antibody 12B12-5 0.253 Humanized antibody 12B12-6 0.362 Humanized antibody 12B12-7 0.367 Humanized antibody 12B12-8 0.287 Humanized antibody 12B12-9 0.223 Humanized antibody 12B12-10 0.25 Humanized antibody 12B12-11 0.239 Humanized antibody 12B12-12 0.206 Humanized antibody 12B12-13 0.289 Humanized antibody 12B12-14 0.257 Humanized antibody 12B12-15 0.193 Humanized antibody 12B12-16 0.2
(136) TABLE-US-00019 TABLE 12 ELISA detection of the binding reaction between humanized h28D4 antibody and human TPBG-hFc protein Antibody EC50 (nM) Chimeric antibody 28D4 (control) 0.165 Humanized antibody 28D4-1 0.364 Humanized antibody 28D4-2 0.181 Humanized antibody 28D4-3 0.16 Humanized antibody 28D4-4 0.147 Humanized antibody 28D4-5 0.782 Humanized antibody 28D4-6 0.205 Humanized antibody 28D4-7 0.161 Humanized antibody 28D4-8 0.162 Humanized antibody 28D4-9 0.218 Humanized antibody 28D4-10 0.224 Humanized antibody 28D4-11 0.191 Humanized antibody 28D4-12 0.167
Embodiment 3 Characterization and Analysis of Humanized TPBG Antibodies (Biacore)
(137) To assess the binding specificity and affinity of the humanized anti-TPBG antibody, Biacore analysis was performed using human TPBG antigen immobilized on a CM5 chip. Biacore technique utilizes the change in the refractive index of surface layer after antibody binding to the TPBG antigen immobilized on the surface layer. The binding was determined by surface plasmon resonance (SPR) through detecting the laser refracted from the surface. Non-specific and specific interactions were distinguished by analysis of signal kinetics binding rate and dissociation rate. Chimeric antibodies 12B12 and 28D4 were used as controls.
(138) TABLE-US-00020 TABLE 13 Results of Biacore Test Antibody ka (1/Ms) kd (1/s) KD (M) Chimeric Antibody 12B12 1.61E+06 1.64E−05 1.02E−11 Humanized Antibody 12B12-4 1.58E+06 3.46E−05 2.20E−11 Humanized Antibody 12B12-5 1.17E+06 2.10E−05 1.79E−11 Humanized Antibody 12B12-12 1.23E+06 2.29E−05 1.86E−11 Humanized Antibody 12B12-15 1.77E+06 <1e−5 <5.66E−12 Humanized Antibody 12B12-16 1.59E+06 3.65E−05 2.29E−11 Chimeric Antibody 28D4 2.50E+06 3.24E−05 1.30E−11 Humanized Antibody 28D4-3 1.12E+06 4.57E−05 4.06E−11 Humanized Antibody 28D4-4 1.55E+06 3.58E−05 2.31E−11 Humanized Antibody 28D4-7 3.68E+06 9.42E−05 2.56E−11 Humanized Antibody 28D4-8 1.02E+06 3.01E−05 2.96E−11 Humanized Antibody 28D4-12 1.03E+06 3.94E−05 3.81E−11
(139) Biacore results show that KD values of humanized anti-TPBG antibodies 12B12 and 28D4 variants are very similar to that of chimeric antibodies 12B12 and 28D4, demonstrating that humanized anti-TPBG antibodies did not significantly reduce antigen binding activity compared to chimeric antibodies.
Embodiment 4 the Binding of TPBG Antibody to TPBG-Expressing Cells was Detected by Fluorescence Activated Cell Sorting (FACS)
(140) For the preparation method, FACS detection method and interpretation of the results of CHO-k1 stable cell line of human TPBG (CHOk1-hTPBG stable cell line), CHO-k1 stable cell line of cynomolgus TPBG (CHOk1-cTPBG stable cell line), and CHO-k1 stable cell line of mouse TPBG (CHOk1-mTPBG stable cell line), see (5) “Identification of lead antibody” in Embodiment 1.
(141) The analysis results are shown in Tables 14, 15 and
(142) TABLE-US-00021 TABLE 14 FACS analyzing the binding activity of humanized antibody 12B12 variants and TPBG expressing cell line EC50 (nM) CHOK1- CHOK1- CHOK1- Clone Number hTPBG cTPBG mTPBG CHOK1 Chimeric Antibody 0.33 0.67 Negative Negative 12B12 Humanized Antibody 0.33 10.31 Negative Negative 12B12-12 Humanized Antibody 0.37 0.87 Negative Negative 12B12-15
(143) TABLE-US-00022 TABLE 15 FACS analyzing the binding activity of humanized antibody 28D4 variants and TPBG expressing cell line EC50 (nM) CHOK1- CHOK1- CHOK1- Clone Number hTPBG cTPBG mTPBG CHO-k1 Chimeric Antibody 0.33 0.94 Negative Negative 28D4 Humanized Antibody 0.26 0.62 Negative Negative 28D4-3 Humanized Antibody 0.38 0.72 Negative Negative 28D4-4 Humanized Antibody 0.37 0.27 Negative Negative 28D4-7
Embodiment 5 In Vitro Pharmacodynamic Studies of Humanized TPBG Antibody-Drug Conjugate
(144) The purified humanized TPBG antibodies obtained from Embodiment 1 were conjugated with MC-MMAF. 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 3. 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 humanized TPBG antibody was 10, and the reaction was carried out at room temperature for 4 h. Cysteine was added to neutralize excess MC-MMAF, and excess small molecules were removed by G25 desalting, thereby obtaining purified humanized TPBG antibody-drug conjugate (for the method of conjugation, refers to Doronina, 2006, Bioconjugate Chem. 17, 114-124). Drug-antibody ratio and purity of the drugs were analyzed by HPLC-HIC and HPLC-SEC, and the cytotoxicity was analyzed subsequently. The drug-antibody ratio (DAR) of all humanized TPBG 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-drug conjugation.
(145) Humanized TPBG antibody-drug conjugates were serially diluted with complete medium, respectively. 100 μL of cell suspension of TPBG-positive non-small cell lung carcinoma cell line NCI-H1568 (Purchased from ATCC, item #CRL-5876) 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).
(146) Results were shown in Tables 16, 17 and
(147) TABLE-US-00023 TABLE 16 Detection of Cytotoxicity of Humanized 12B12 Variants and its Antibody-drug Conjugates on TPBG-Positive Cells via Cellular Killing Assay Clone Number IC50(nM) Negative antibody control Negative Chimeric Antibody 12B12 Negative Humanized Antibody 12B12-12 Negative Humanized Antibody 12B12-15 Negative Negative antibody control -MMAF Negative Chimeric Antibody 12B12-MMAF 0.063 Humanized Antibody 12B12-12-MMAF 0.066 Humanized Antibody 12B12-15-MMAF 0.058
(148) TABLE-US-00024 TABLE 17 Detection of Cytotoxicity of Humanized 28D4 Variants and its Antibody-drug Conjugates on TPBG-Positive Cells via Cellular Killing Assay Clone Number IC50(nM) Chimeric Antibody 28D4 Negative Humanized Antibody 28D4-3 Negative Humanized Antibody 28D4-4 Negative Humanized Antibody 28D4-7 Negative Chimeric Antibody 28D4-MMAF 0.091 Humanized Antibody 28D4-3-MMAF 0.061 Humanized Antibody 28D4-4-MMAF 0.074 Humanized Antibody 28D4-7-MMAF 0.066
Embodiment 6 Antibody-Drug Conjugates Coupled with Different Linker-Toxins
(149) Purified humanized antibodies 12B12 and 28D4 obtained from Embodiment 1 were coupled with MC-MMAF and MC-VC-PAB-MMAE, respectively. 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 3. 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 humanized TPBG antibody was 10. 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, thereby obtaining purified humanized TPBG antibody-drug conjugate (as for the method of conjugation please refer to Doronina, 2006, Bioconjugate Chem. 17, 114-124). Drug-antibody ratio and the purity of the drugs were analyzed by HPLC-HIC and HPLC-SEC, follow by determining the cytotoxicity of conjugates in vitro and pharmacodynamic studies in vivo. The drug-antibody ratios (DAR) of all humanized 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-drug conjugation.
(150) The purified humanized antibodies 12B12-12 and 28D4-3 obtained from Embodiment 1 were coupled with succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), respectively. After dialyzing the purified antibodies with phosphate buffer (pH 6.5-7.4), 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. N, N-dimethylacetamide (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 humanized TPBG chimeric antibody-drug conjugates (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 was analyzed by SEC, the cytotoxicity of conjugates in vitro and pharmacodynamic studies of conjugates in vivo were determined. The drug-antibody ratios (DAR) of all humanized 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-drug conjugation.
Embodiment 7 In Vitro Pharmacodynamic Studies of Humanized TPBG Antibody-Drug Conjugate
(151) The purified humanized TPBG antibody-drug conjugates obtained in Embodiment 6 were serially diluted with complete medium, respectively. Each 100 μL of cell suspension of TPBG-positive non-small cell lung carcinoma cell line NCI-H1568 (purchased from ATCC, Catalog NO: #CRL-5876) 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, the non-small cell lung carcinoma cell line NCI-H1975 (purchased from ATCC, Catalog NO: #CRL-5908) and the breast cancer cell line MDA-MB-468 (purchased from ATCC, Catalog NO: #HTB-132) with weak expression of TPBG were selected to perform cytotoxicity test as described above.
(152) Results were shown in Table 18 and
(153) TABLE-US-00025 TABLE 18 Detection of Cytotoxicity of Humanized TPBG Antibody-Drug Conjugates on TPBG-Positive Cells via Cellular Killing Assay IC50 (nM) Sample Name NCI-1568 NCI-1975 MDA-MB-468 Humanized Antibody 0.43 1.20 0.27 12B12-12-MMAE Humanized Antibody 0.05 0.13 0.06 12B12-12-MMAF Humanized Antibody 0.38 5.93 0.44 12B12-12-DM1 Humanized Antibody Negative Negative Negative 12B12-12 Human IgG Negative Negative Negative
Embodiment 8 Mouse Xenograft Model of Non-Small Cell Lung Carcinoma Cell Line NCI-H1975
(154) 200 μL of NCI-H1299 cells (non-small cell lung carcinoma cell line, ATCC, CRL-5908) (2×10.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 excluding 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. Grouping was shown in Table 19, therapeutic groupings of humanized 28D4-3-MMAF antibody-drug conjugate prepared in Embodiment 6 were prepared in different doses, as well as the same dose of humanized 12B12-12-MMAF and humanized 28D4-3-MMAF antibody-drugs conjugate treatment groups. Grouping was shown in Table 20, therapeutic grouping of humanized 28D4-3-MMAF, 28D4-3-MMAE and 28D4-3-DM1 antibody-drug conjugates coupled with different linker-toxins were prepared in the same dose. Antibody was injected intravenously to the tail at DO, and the injection was performed once every four days with a total of four injections. Tumor volume and body weight of mice were measured twice every week, and the data were recorded. Formula for calculating tumor volume is: V=½×a×b.sup.2; wherein a and b represent length and width, respectively.
(155) TABLE-US-00026 TABLE 19 In Vivo Pharmacodynamic Studies of Different Doses of Humanized TPBG Antibody-drug Conjugates in NCI-H1975 Mouse Xenograft Model Number of Dose Injected Route of Administration Group Animals Treating Group (mg/kg) Amount Administration Plan 1 7 Vehicle control — 10 μl/g Tail Administer intravenous once every 4 injection i.v. days × 4 times 2 7 Humanized 1 10 μl/g Tail Administer Antibody 28D4- intravenous once every 4 3-MMAF injection i.v. days × 4 times 3 7 Humanized 3 10 μl/g Tail Administer Antibody 28D4- intravenous once every 4 3-MMAF injection i.v. days × 4 times 4 7 Humanized 10 10 μl/g Tail Administer Antibody 28D4- intravenous once every 4 3-MMAF injection i.v. days × 4 times 5 7 Humanized 10 10 μl/g Tail Administer Antibody 12B12- intravenous once every 4 12-MMAF injection i.v. days × 4 times
(156) TABLE-US-00027 TABLE 20 In Vivo Pharmacodynamic Studies of humanized TPBG Antibody-drug Conjugates Coupled with Different Linker-Toxins in NCI-H1975 Mouse Xenograft Model Number of Dose Injected Route of Administration Group Animals Treating Group (mg/kg) Amount Administration Plan 1 7 Vehicle control — 10 μl/g Tail Administer intravenous once every 4 i.v. days × 4 times 2 7 Humanized 10 10 μl/g Tail Administer Antibody 28D4- intravenous once every 4 3-MMAF i.v. days × 4 times 3 7 Humanized 10 10 μl/g Tail Administer Antibody 28D4- intravenous once every 4 3-MMAE i.v. days × 4 times 4 7 Humanized 10 10 μl/g Tail Administer Antibody 28D4- intravenous once every 4 3-DM1 i.v. days × 4 times
(157)
(158)
Embodiment 9 Mouse Xenograft Model of Non-Small Cell Lung Carcinoma NCI-H1568
(159) 200 μL of NCI-H1568 cells (non-small cell lung carcinoma cell line, ATCC, CRL-5876) (1×10.sup.7 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 excluding the mice with too large or too small body weight and tumor size, the mice were randomly divided into several groups, i.e. 6 mice for each group, according to the tumor size. Grouping was shown in Table 21, therapeutic groupings of humanized 28D4-3-MMAF, 28D4-3-MMAE and 28D4-3-DM1 antibody-drug conjugates coupled with different linker-toxins were prepared in different doses. Antibody was injected intravenously to the tail at DO, and the injection was performed once every four days with a total of four injections. Tumor volume and body weight of mice were measured twice every week, and the data were recorded. Formula for calculating tumor volume is: V=½×a×b.sup.2; wherein a and b represent length and width, respectively.
(160) TABLE-US-00028 TABLE 21 In Vivo Pharmacodynamic Studies of Humanized TPBG Antibody-drug Conjugates in NCI-H1568 Mouse Xenograft Model Number of Treating Dose Injected Route of Administration Group Animals Group (mg/kg) Amount Administration Plan 1 6 Vehicle — 10 μl/g Tail Administrate control intravenous once every 4 injection i.v. days × 4 times 2 6 Humanized 2 10 μl/g Tail Administrate Antibody intravenous once every 4 12B12-12- injection i.v. days × 4 times MMAF 3 6 Humanized 10 10 μl/g Tail Administrate Antibody intravenous once every 4 12B12-12- injection i.v days × 4 times MMAF 4 6 Humanized 2 10 μl/g Tail Administrate Antibody intravenous once every 4 12B12-12- injection i.v. days × 4 times MMAE 5 6 Humanized 10 10 μl/g Tail Administrate Antibody intravenous once every 4 12B12-12- injection i.v days × 4 times MMAE 6 6 Humanized 10 10 μl/g Tail Administrate Antibody intravenous once every 4 12B12-12- injection i.v days × 4 times DM1
(161)
Embodiment 10 Mouse Xenograft Model of Breast Cancer MDA-MB-468
(162) 200 μL of MDA-MB-468 cells (purchased from ATCC, HTB-132) (1×10.sup.7 cells) which were suspended in L-15 basal medium adding with 50% Matrigel were inoculated subcutaneously in the right rib of CB17 SCID mice and allowed to grow for 7-10 days until the tumor volumes reach 200 mm.sup.3. After excluding the mice with too large or too small body weight and tumor size, the mice were randomly divided into several groups, i.e. 6 mice for each group, according to the tumor size. Grouping was shown in Table 22, therapeutic groupings of humanized 12B12-12-MMAF, 12B12-12-MMAE and 12B12-12-DM1 antibody-drug conjugates coupled with different linker-toxins were prepared in different doses. Antibody was injected intravenously to the tail at DO, and the injection was performed once every four days with a total of four injections. Tumor volume and body weight of mice were measured twice a week, and the data were recorded. Formula for calculating tumor volume is: V=½×a×b.sup.2; wherein a and b represent length and width, respectively.
(163) TABLE-US-00029 TABLE 22 In Vivo Pharmacodynamic Studies of Humanized TPBG Antibody-drug Conjugates in MDA-MB-468 Mouse Xenograft Model Number of Dose Injected Route of Administration Group Animals Treating Group (mg/kg) Amount Administration Plan 1 6 Vehicle control — 10 μl/g Tail Administrate intravenous once every 4 injection i.v. days × 4 times 2 6 Humanized 0.5 10 μL/g Tail Administrate Antibody intravenous once every 4 12B12-12- injection i.v. days × 4 times MMAF 3 6 Humanized 2 10 μL/g Tail Administrate Antibody intravenous once every 4 12B12-12- injection i.v. days × 4 times MMAF 4 6 Humanized 0.5 10 μL/g Tail Administrate Antibody intravenous once every 4 12B12-12- injection i.v. days × 4 times MMAE 5 6 Humanized 2 10 μL/g Tail Administrate Antibody intravenous once every 4 12B12-12- injection i.v. days × 4 times MMAE 6 6 Humanized 2 10 μL/g Tail Administrate Antibody intravenous once every 4 12B12-12- injection i.v. days × 4 times DM1
(164)
Embodiment 11 Stability of Humanized Anti-TPBG Antibody-Drug Conjugates in Rat Serum
(165) 3 mg/kg of humanized 28D4-MMAE conjugate was injected into Sprague-Dawley rats (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) by a single intravenous injection of tail vein. 200 μl of whole blood was collected respectively before injection, 10 minutes after injection, 1 hour, 4 hours, 8 hours, and 1, 2, 4, 7, 14, 21 and 28 days, and serum was collected after centrifugation at 14,000 rpm for 5 minutes. The concentration of total anti-TPBG antibody (the total anti-TPBG antibody comprises naked antibody and antibody-drug conjugates) and the antibody drug conjugates coupled to at least one cytotoxic drug in serum were determined by ELISA. The concentration of total humanized anti-TPBG antibody in the serum was detected by ELISA, and captured using anti-human Fc antibody and detected using horseradish peroxidase (HRP) conjugated anti-mouse Fc antibody. The concentration of the antibody drug conjugate carrying at least one cytotoxic drug in the serum was detected by ELISA, and captured using an anti-MMAE antibody and detected using a horseradish peroxidase (HRP)-conjugated anti-mouse Fc antibody, wherein, anti-MMAE antibody (trade name anti-MMAF-mIgG1, purchased from Shanghai Ruizhi Chemical Research Co., Ltd.) was prepared by using hybridoma technology. Anti-human Fc antibody and anti-MMAE antibody were separately diluted with PBS to a certain concentration (2-5 μg/ml), coated on a 96-well ELISA plate at 4° C. overnight. The liquid in the well was discarded and spin dried, and the blocking solution (PBS, 1% BSA, 0.05% Tween-20) was added and blocked for 1 hour at room temperature, and then washed 3 times with PBST (PBS, 0.05% Tween-20) for later use. Diluted the standard sample to a certain concentration with PBST, and prepare serum sample as appropriate. Added 200 μl/well of the diluted serum sample or standard sample to the coated 96-well plate and incubated for 1-3 hours at room temperature. The supernatant was discarded and the plate was washed 3 times with PBST; then horseradish peroxidase (HRP)-conjugated anti-mouse Fc antibody was diluted with PBS to an appropriate concentration, and 100 μl of which was added to each well of a 96-well plate and incubated for 30 minutes at 37° C. After the supernatant was discarded, the plate was washed 3 times with PBST; 100 μl of TMB coloring solution was added to each well, and incubated at room temperature for 5-10 minutes. 100 μl of 2N sulfuric acid stop solution was added to each well to terminate the reaction, and the ratio of OD 450 nm/630 nm was measured by a microplate reader. The data of concentration of total antibody and conjugate thereof in serum at different sampling time points from each animal were analyzed using a two compartment model with IV bolus input, first order elimination and macro rate constant (Model 8, WinNonlinear Pro v. 5.0.1, Pharsight Corporation, Mountain View, Calif.) to analyze the in vivo stability of the humanized anti-TPBG antibody drug conjugate.
(166) The results of the 28-day pharmacokinetic analysis performed in rats were shown in
(167) TABLE-US-00030 TABLE 23 Drug Metabolism of Anti-TPBG Antibody MMAE conjugate 28D4-3-MMAE in Rats PK Parameter CL Vss V1 Alpha t.sub.1/2 Beta t.sub.1/2 AUC Unit mL/day/kg mL/kg mL/kg day day day * μg/mL Total 17.2 ± 1.8 225 ± 16.1 60.7 ± 0.61 0.39 ± 0.016 10.4 ± 1.5 176 ± 17.8 Antibody Conjugated 51.3 ± 2.7 246 ± 23.4 70.8 ± 0.73 0.52 ± 0.052 6.18 ± 1.0 58.5 ± 3.0 Antibody
(168) From the results, conjugated antibody and total antibody had similar pharmacokinetic characteristics, such as long half-life, nonlinear distribution and elimination. The difference is that conjugated antibody shows a higher clearance rate (CL) and a lower elimination half-life (Beta t.sub.1/2) and exposure (AUC) compared to total antibody, which is consistent with the classical ADC drug metabolism. The reason may be that the ADC drug is a mixture of various molecules with high heterogeneity, and some of the conjugates degrade in the blood circulation or induce immune response, so that the body generates antibodies against the ADC drug, resulting in the clearance of the ADC drug.
Embodiment 12 Pharmacodynamic Experiment of Humanized Anti-TPBG Antibody-Drug Conjugate in Human Tumor Patient-Derived Xenograft Model (PDX)
(169) The PDX model is a non-small cell lung carcinoma (NSCLC) human tumor model (a xenograft model in which a patient's tumor tissue is inoculated into a mouse and grow relying on the environment provided by the mouse; the PDX model of the present invention is purchased from Shanghai Ruizhi Chemical Research Co., Ltd.). The expression of TPBG in the NSCLC PDX model was assessed by immunohistochemistry. Briefly, fresh tumor tissue of PDX model was quick-frozen using liquid nitrogen and stored in −80° C. refrigerator. Fresh tumor tissue of PDX model was sliced with a microtome, fixed with 4% paraformaldehyde for 10 minutes at room temperature; incubated with 3% hydrogen peroxide at room temperature for 5 minutes at room temperature to block endogenous peroxidase; and incubated with 5% fetal bovine serum at room temperature to block non-specific protein binding sites for 15 minutes. Tissue slices were incubated with humanized anti-TPBG antibody 12B12-12 or isotype control antibody hIgG for 1 hour at room temperature; incubated with Goat anti-human IgG (H+L) Secondary Antibody, HRP conjugate (purchased from Thermo) at room temperature for 30 minutes, and DAB chromogenic for 5 minutes. The slice was then placed in a tank containing tap water to terminate the reaction; counterstained with hematoxylin for 10 seconds, and rinsed with tap water for 3 times; followed by differentiating by hydrochloric acid alcohol for 1 second, and rinsing with tap water for 3 minutes; subsequently, the slice was dehydrated with 75% alcohol for 2 minutes, 95% alcohol for 2 minutes, 100% alcohol for 3 times, and xylene for 3 times for 2 minutes each. Then, the slice was sealed with neutral gum, and the staining results were observed under a microscope and photographed. The expression level of TPBG in the PDX model was shown in
(170) The cryopreserved P1 generation (the first generation of tumor tissue samples of the above-described PDX model) tumor samples (FP1) were taken out from the liquid nitrogen storage tank, and then thawed and subcutaneously inoculated into SCID mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), and named FP1+1. The health status of the mice was monitored daily and tumor growth was initially monitored twice a week by visual inspection. Mouse body weight was recorded 2 to 3 times a week. The tumor diameter was measured with vernier caliper 2 to 3 times a week. The tumor volume is calculated as: V=½a×b.sup.2, and a and b represent the long and short diameters of the tumor, respectively. When the tumor grows to 1000 mm.sup.3, the mice are euthanized, and fresh tumor tissue of mice is taken out, cut into small pieces and subcutaneously inoculated into female nude mice. After being passaged three times or more by this method, such mice were tested for pharmacodynamic studies (named FP1+3).
(171) Female Nu/Nu mice with 6-8 weeks age and 16-19 g weight were purchased (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). Experiment began 7 days after the animals arrived and fed in IVC (Individual Ventilated Cages) in the SPF animal room. Since individual differences in mice may result in difference in the growth rate of tumor tissue, therefore, it is necessary to prepare at least extra 50% of the number of animals to allow the minimum tumor volume variance at randomization. Female Nu/Nu mice were inoculated via the above-mentioned resuscitation tumor tissue, and the health status of the mice was monitored daily and tumor growth was initially monitored twice a week by visual inspection, followed by recording the body weight of the mice 2 to 3 times per week. When the tumor can be touched, the tumor diameter was measured with vernier caliper 2 to 3 times a week and the tumor volume was calculated. After the tumor volume was as big as 100 mm.sup.3, the mice with too large or too small body weight and tumor size were excluded, and remaining mice were randomly grouped according to the tumor volume, with 10 mice in each group (groupings was shown in Table 24). Therapeutic groupings of humanized 12B12-12-MMAF, 12B12-12-MMAE and 12B12-12-DM1 antibody-drug conjugates coupled with different linker-toxins were prepared in different doses. Mice were injected with the antibody into the tail vein on DO, and administrated once every 4 days×4 times. The tumor volume was measured twice a week, the rats were weighed, and the data were recorded. The tumor volume (V) is calculated as: ½a×b.sup.2; wherein a and b represent length and width, respectively.
(172) TABLE-US-00031 TABLE 24 Pharmacodynamic Studies of Humanized Anti-TPBG Antibody-drug Conjugate in Human Tumor Patient-derived Xenograft Model (PDX) Number of Treating Dose Injected Route of Administration Group Animals Group (mg/kg) Amount Administration Plan 1 10 Vehicle — 10 μl/g Tail Administrate control intravenous once every 4 injection i.v. days × 4 times 2 10 Humanized 1 10 μL/g Tail Administrate Antibody intravenous once every 4 28D4-3- injection i.v. days × 4 times MMAE 3 10 Humanized 3 10 μL/g Tail Administrate Antibody intravenous once every 4 28D4-3- injection i.v. days × 4 times MMAE 4 10 Humanized 10 10 μL/g Tail Administrate Antibody intravenous once every 4 28D4-3- injection i.v. days × 4 times MMAE
(173)
Embodiment 13 Inhibition of Tumor Growth and Formation of Immunological Memory in Mice Under Treatment of Humanized Anti-TPBG Antibody-Drug Conjugate Combined with Anti-PD-1 Antibody
(174) Preparation method and flow cytometry detection method of CT26 stable cell line expressing human TPBG (CT26-hTPBG stable cell line) (purchased from Shanghai Ruizhi Chemical Research Co., Ltd.), are the same with Embodiment 1 (b) “Preparation of Immunogen B”, in which mouse colon cancer cell CT26 was purchased from ATCC.
(175) 50 μL of CT26-TPBG cells (1×10.sup.6 cells) were inoculated subcutaneously in the right rib of Balb/c nude mice and allowed to grow for 7-10 days. 32 animals with tumor volumes of 50-100 mm.sup.3 were selected. Mice were randomly divided into 4 groups according to tumor volume, 8 in each group. Those groups comprise vehicle group, treated group of humanized 28D4-3-MMAE antibody-drug conjugate alone, treated group of anti-PD-1 alone, and combination treated group comprising humanized 28D4-3-MMAE antibody-drug conjugate and anti-PD-1. The grouping was shown in Table 25.
(176) Humanized 28D4-3-MMAE antibody-drug conjugate was administered once i.v. on the day of grouping (defined as DO). Anti-PD-1 antibody was administered i.p. from DO twice a week for 8 times in total. The body weight of the mice was recorded twice a week, and the length and diameter of the tumor were measured with vernier caliper. The tumor volume V was calculated by the following formula: V=½a×b.sup.2; wherein a and b represent the long diameter and short diameter of the tumor, respectively. Animals with a tumor volume of 2000 mm.sup.3, which were deemed to have reached the end of the experiment, were euthanized and the survival of the animals were analyzed.
(177) TABLE-US-00032 TABLE 25 In vivo Pharmacodynamic Studies of Humanized TPBG Antibody Drug Conjugates or/with Anti-PD-1 Antibodies in Tumor Models Number of Dose Injected Route of Schedule of Group Animals Treating Group (mg/kg) Amount Administration Administration 1 8 Vehicle control — 10 μl/g Tail Administrate intravenous twice a week × i.v. 4 times 2 8 Humanized 1 10 μl/g Tail Administrate 28D4-3-MMAE intravenous on D0 Antibody-drug i.v. Conjugate 3 8 Anti-PD-1 5 10 μl/g Intraperitoneal Administrate Antibody i.p. twice a week × 4 times 4 8 humanized ADC: 1; 10 μl/g Tail Administrate 28D4-3-MMAE Antibody: 5; intravenous on D0; Antibody-drug i.v. Administer Conjugate + Intraperitoneal twice a week × Anti-PD-1 i.p. 4 times Antibody Conjugates
(178) Tumor growth curves of each group showed that tumor growth was significantly inhibited in each drug-administered group at D14 days after administration compared with vehicle group, and the tumor suppression effect of the combination treatment group was significantly better than that of the monotherapy group (
(179) In conclusion, the humanized 28D4-3-MMAE antibody-drug conjugate significantly enhanced the anti-tumor activity of anti-PD-1 antibody against CT26-hTPBG mouse model. The complete remission rate of combination therapy of the two drugs reached 37.5%. The combination therapy of humanized 28D4-3-MMAE antibody-drug conjugate and anti-PD-1 antibody could significantly prolong the survival of the mice compared to humanized 28D4-3-MMAE antibody-drug conjugate or anti-PD-1 monotherapy, and the mice with complete tumor remission after combination treatment will form immunological memory.
(180) The specific embodiments of the present invention described above are merely illustrative, and 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 without departing from the principles and spirits of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.