ANTIBODY CONJUGATE AND APPLICATION OF PHARMACEUTICAL COMPOSITION THEREOF

Abstract

An application of an antibody conjugate in the preparation of a drug for treating CD30 positive tumors, which is characterized in that: the antibody conjugate is F0002-ADC; the general formula of the structural thereof is Ab-L.sub.m-Y.sub.n; and the CD30-positive tumors are CD30-positive tumors that express a multidrug resistance gene 1. In addition, a pharmaceutical combination and a pharmaceutical composition containing the antibody conjugate, which may be applied to the preparation of a drug used for treating CD30-positive tumors.

Claims

1. A method for the treatment of CD30-positive tumors in a subject in need thereof; comprising: administering an effective amount of an antibody conjugate to the subject, wherein, the antibody conjugate is F0002-ADC with a structural general formula of Ab-L.sub.m-Y.sub.n: the CD30-positive tumor is a CD30-positive tumor expressing multidrug resistance gene 1; wherein, Ab is an anti-human CD30 antibody cAC10, an active fragment thereof, or a variant thereof; the Ab is only connected with the L; Y is Mertansine as shown in formula DM1; ##STR00009## the Y is only connected with the L; m is 3.3-10; n is 3.3-3.9; and m≥n; when both ends of L are respectively connected with the Ab and the Y, the L is ##STR00010## its left end forms an amide bond with the amino in lysine of the Ab, and its right end forms a thioether bond with S in the DM1; when the L is only connected with the Ab, the L is ##STR00011## and its left end forms an amide bond with the amino in lysine of the Ab.

2. The method according to claim 1, wherein, the n of the antibody conjugate is 3.6; or, the similarity between the variant of the anti-human CD30 antibody cAC10 and the amino acid sequence of cAC10 is not less than 90%, and the mutations related to lysine is not more than 80%; or, the m is equal to the n, the general structural formula is Ab-(L-Y)n, and the structure is as follows: ##STR00012## or, the CD30-positive tumor expressing the multidrug resistance gene 1 is Hodgkin lymphoma expressing the multidrug resistance gene 1.

3. The method according to claim 2, wherein, in the F0002-ADC, the m is equal to the n, the general structural formula is Ab-(L-Y)n, and the structure is as follows: is the following structure: ##STR00013## the distribution of different DAR values is as follows: TABLE-US-00022 D0 D1 D2 D3 D4 D5 D6 D7 3% 10% 17% 20% 18% 16% 9% 7% n=3.6; or, the cells of the Hodgkin lymphoma expressing multidrug resistance gene 1 are CD30-positive Hodgkin lymphoma cells L428 expressing multidrug resistance gene 1 or CD30-positive Hodgkin lymphoma cells L540 expressing multidrug resistance gene 1.

4. A method for the treatment of CD30-positive tumors in a subject in need thereof; comprising: administering an effective amount of an antibody conjugate to the subject, wherein, the antibody conjugate is the F0002-ADC as defined in claim 1; the CD30-positive tumor is a CD30-positive tumor resistant to Adcetris.

5. The method according to claim 4, wherein, the CD30-positive tumor resistant to Adcetris is CD30-positive Hodgkin lymphoma resistant to Adcetris.

6. A method for the treatment of CD30-positive tumors in a subject in need thereof; comprising: administering an effective amount of an antibody conjugate to the subject, wherein, the antibody conjugate is the F0002-ADC as defined in claim 1; the CD30-positive tumor is CD30-positive Hodgkin lymphoma.

7. The method according to claim 6, wherein, the cells of CD30-positive Hodgkin lymphoma are CD30-positive Hodgkin lymphoma cells L428 or CD30-positive Hodgkin lymphoma cells L540.

8. (canceled)

9. A pharmaceutical combination, wherein, the pharmaceutical combination comprises an antibody conjugate X and a substance Y; the antibody conjugate X is the F0002-ADC as defined in claim 1; the substance Y is one or more of substances Y1, Y2, Y3, Y4, Y5, Y6, Y7 and Y8; the substance Y1 is Y1-1, Y1-2 or Y1-3; Y1-1 is Doxorubicin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof, Y1-2 is Epirubicin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof, Y1-3 is Daunorubicin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y2 is Y2-1, Y2-2, Y2-3, Y2-4 or Y2-5; Y2-1 is Bleomycin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof, Y2-2 is Boanmycin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y2-3 is Boningmycin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y2-4 is Pingyangmycin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y2-5 is Peplomycin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y3 is Y3-1, Y3-2, Y3-3 or Y3-4; Y3-1 is Vinblastine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof, Y3-2 is Vincristine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y3-3 is Vinorelbine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof, Y3-4 is Vindesine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y4 is Y4-1 or Y4-2; Y4-1 is Dacarbazine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof, Y4-2 is Temozolomide, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y5 is Y5-1 or Y5-2; Y5-1 is Etoposide, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y5-2 is Teniposide, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y6 is Y6-1 or Y6-2; Y6-1 is Cyclophosphamide, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof, Y6-2 is Ifosfamide, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y7 is Procarbazine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y8 is Y8-1 or Y8-2; Y8-1 is Prednisone, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y8-2 is Prednisone, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof.

10. The pharmaceutical combination according to claim 9, wherein, the substance Y is scheme 1, scheme 2, scheme 3, scheme 4 or scheme 5; scheme 1 is substances Y1, Y3 and Y4; or, scheme 2 is substances Y1, Y2, Y3 and Y4; or, scheme 3 is substances Y2, Y5, Y1, Y6, Y3, Y7 and Y8; or, scheme 4 is substances Y6, Y1, Y3 and Y8; or, scheme 5 is substances Y6, Y3 and Y8; or, or, the antibody conjugate X and “all or part of the substance Y” are administered simultaneously or separately; or, the pharmaceutical combination is in the form of a mixture of all components, or in the form that each component is independent, or in the form that each component is divided into several groups.

11. A pharmaceutical composition A, wherein, the pharmaceutical composition A comprises the F0002-ADC as defined in claim 1 and a pharmaceutical excipient.

12. A pharmaceutical composition B, wherein, the pharmaceutical composition B comprises the pharmaceutical combination as defined in claim 9 and a pharmaceutical excipient.

13. A method for the treatment of CD30-positive tumors in a subject in need thereof; comprising: administering an effective amount of an antibody conjugate to the subject, wherein, in the method, the antibody conjugate is used in combination with the substance Y; the antibody conjugate is the F0002-ADC as defined in claim 1; the substance Y is one or more of substances Y1, Y2, Y3, Y4, Y5, Y6, Y7 and Y8; the substance Y1 is Y1-1, Y1-2 or Y1-3:Y1-1 is Doxorubicin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y1-2 is Epirubicin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y1-3 is Daunorubicin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y2 is Y2-1, Y2-2, Y2-3, Y2-4 or Y2-5:Y2-1 is Bleomycin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y2-2 is Boanmycin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y2-3 is Boningmycin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y2-4 is Pingyangmycin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y2-5 is Peplomycin, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y3 is Y3-1, Y3-2, Y3-3 or Y3-4:Y3-1 is Vinblastine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y3-2 is Vincristine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y3-3 is Vinorelbine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y3-4 is Vindesine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y4 is Y4-1 or Y4-2:Y4-1 is Dacarbazine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y4-2 is Temozolomide, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y5 is Y5-1 or Y5-2:Y5-1 is Etoposide, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y5-2 is Teniposide, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y6 is Y6-1 or Y6-2:Y6-1 is Cyclophosphamide, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y6-2 is Ifosfamide, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y7 is Procarbazine, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; the substance Y8 is Y8-1 or Y8-2:Y8-1 is Prednisone, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof; Y8-2 is Prednisone, a pharmaceutically acceptable salt thereof, a solvate thereof, or, a solvate of the pharmaceutically acceptable salt thereof.

14. The method according to claim 13, wherein, the CD30-positive tumor is a CD30-positive lymphoma; or, the CD30-positive tumor is a CD30-positive tumor expressing multidrug resistance gene 1; or, the CD30-positive tumor is CD30-positive tumor resistant to Adcetris.

15. A method for the treatment of CD30-positive tumors by administering an effective dose of the pharmaceutical composition as defined in claim 11 to a patient.

16. The method according to claim 15, wherein, the CD30-positive tumor is a CD30-positive lymphoma: or, the CD30-positive tumor is a CD30-positive tumor expressing multidrug resistance gene 1; or, the CD30-positive tumor is CD30-positive tumor resistant to Adcetris.

17. The method according to claim 2, wherein, the distribution of different DAR values is as follows: TABLE-US-00023 D0 D1 D2 D3 D4 D5 D6 D7 3% 10% 17% 20% 18% 16% 9% 7%

18. The method according to claim 5, wherein, the cells of the CD30-positive tumor resistant to Adcetris are CD30-positive Hodgkin lymphoma cells L428 resistant to Adcetris or CD30-positive Hodgkin lymphoma cells L540 resistant to Adcetris.

19. The pharmaceutical combination according to claim 10, wherein, scheme 1 is substances Y1-1, Y3-2 and Y4-1; or, scheme 2 is substances Y1-1, Y2-1, Y3-2 and Y4-1; or, scheme 3 is substances Y2-1, Y5-1, Y1-1, Y6-1, Y3-2, Y7-1 and Y8-1; or, scheme 4 is substances Y6-1, Y1-1, Y3-2 and Y8-1; or, scheme 5 is substances Y6-1, Y3-1 and Y8-1.

20. The pharmaceutical combination according to claim 19, wherein, scheme 1 is substances Y1-1, Y3-2 and Y4-1; the molar ratio of the antibody conjugates X:Y1-1:Y3-2:Y4-1 is 1:(400-800):(11-400):(550000-3000000); or, scheme 2 is substances Y1-1, Y2-1, Y3-2 and Y4-1; the molar ratio of the antibody conjugates X:Y1-1:Y2-1:Y3-2:Y4-1 is 1:(400-800):(30000-45000):(11-400):(550000-3000000); or, scheme 3 is substances Y2-1, Y5-1, Y1-1, Y6-1, Y3-2, Y7-1 and Y8-1; the molar ratio of the antibody conjugates X:Bleomycin:Etoposide:Doxorubicin:Cyclophosphamide:Vincristine:Procarbazine:Prednisone is 1:30000:700000:400:8000000:400:6500000:1500000; or, scheme 4 is substances Y6-1, Y1-1, Y3-2 and Y8-1; more preferably the molar ratio of the antibody conjugates X:Y6-1:Y1-1:Y3-2:Y8-1 is 1:(1700000-12000000):(800-1200):(11-600):(550000-1400000); or, scheme 5 is substances Y6-1, Y3-1 and Y8-1; the molar ratio of the antibody conjugates X:Y6-1:Y3-1:Y8-1 is 1:(8000000-12000000):(400-600):(1500000-5500000).

21. The method according to claim 14, wherein, the CD30-positive lymphoma is CD30-positive Hodgkin lymphoma, CD30-positive anaplastic large cell lymphoma, CD30-positive diffuse histiocytic lymphoma or CD30-positive cutaneous T cell lymphoma; or, the CD30-positive tumor is a CD30-positive Hodgkin lymphoma expressing multidrug resistance gene 1; or, the CD30-positive tumor is CD30-positive Hodgkin lymphoma resistant to Adcetris.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0126] The present disclosure will be further explained by way of embodiments below, but the present disclosure is not limited to the scope of the described embodiments. Experimental methods for which specific conditions are not indicated in the following embodiments are selected according to conventional methods and conditions, or according to the commercial specification.

Embodiment 1 Construction and Expression of Anti-Human CD30 Antibody cAC10

[0127] The fully synthesized DNA fragments encoding the heavy and light chains of cAC10 monoclonal antibody were (see U.S. Pat. No. 7,090,843, B1RECOMBINANT ANTI-CD30 ANTIBODIES AND USES THEREOF, Seattle Genetics, Inc., 2006, SEQ ID NO:1 and SEQ ID No:9; and International Nonproprietary Names for Pharmaceutical Substances (INN). WHO Drug Information Vol. 24, No. 2, 2010) cloned into the pEE12.4 eukaryotic expression vector of Lonza Company, respectively, and the enzymatic cleavage and ligation were performed according to the instructions of the commercially available kit (DNA Ligation kit Ver2.0, TAKARA).

[0128] The constructed heavy chain and light chain expression vectors were transformed into E. coli DH5a respectively, and the positive clones were selected and inoculated in 500 mL LB medium for amplification. DNA was extracted and purified using Qiagen's Ultrapure Plasmid DNA Purification Kit according to the manufacturer's instructions. The above plasmid DNA containing heavy and light chain coding sequences was co-transfected into CHO-K1 (Chinese hamster ovary cells, purchased from ATCC) in a certain ratio using Invitrogen's liposome method kit, and the procedure was performed according to the manufacturer's instructions.

[0129] The DMEM/F12 (1:1) cell medium (Invitrogen) was replaced with GMEM screening medium (Sigma) containing the screening drug 24-48 hours after transfection, and the screening medium was changed every 3-4 days until cell clone formation. When the diameter of the cell clone reached 1 mm-2 mm, the monoclonal was picked from the plate by clone ring and transferred to a 24-well plate containing 1 mL of screening medium. When single cell clones grew to 50%-70% full layer in 24-well plates, the culture supernatant of each clone was taken for ELISA detection, and the cell clones with high expression were selected for drug-pressurized amplification screening. When the concentration of the screened drugs rose to the highest level, the expression level of each cloned single cell was detected, and cells with high expression levels and good cell growth status were selected for amplification culture. Recombinant cell culture supernatant was collected and purified by protein A affinity chromatography for functional evaluation.

[0130] Recombinant human CD30 antigen (coating antigen, R&D Systems) was diluted to a certain concentration (0.5 μg/mL) with a coating solution (pH 9.6 CBS) and coated on 96-well plates, 100 μL/well, and placed at 2° C.-8° C. overnight. The liquid in the hole was discarded, then the mixture was washed with PBST for 3 times, dried by spin, and then added with 400 μL/hole sealing solution (1% BSA PBST) at room temperature for 2 hours, then washed with PBST for 3 times and spin-dried. The standard was diluted with diluent to a certain concentration, and the expression supernatant was diluted appropriately according to the situation, the sample was added to a 96-well plate with 100 μL/well replicate, incubated at 37° C. for 1 hour, the liquid was discarded, the plate was washed 3 times, and spin-dried. Goat anti-human IgG(Fc)-HRP (enzyme-linked antibody, PIERCE company) was diluted with diluent at a ratio of 1:20,000, added to a 96-well plate at 100 μL/well, the reaction was carried out at 37° C. for 1 hour, the liquid was discarded, the plate was washed 3-6 times, and spin-dried. The substrate mixture was prepared, added into a 96-well plate at 100 μL/well, and incubated at 37° C. for 20 min. 100 μL/well of stopping solution was added to stop the reaction. The wavelength of 655 nm was used as the reference wavelength, and the absorbance was measured at 450 nm; the content of the samples was calculated according to the standard curve, and the clones with high expression were selected for amplification and culture. Recombinant cell culture supernatant was collected and purified by protein A affinity chromatography for subsequent ligation experiments.

Embodiment 2 Detection Method of DAR

[0131] DAR detection was based on the UV absorption of Ab and DM1, and the coupling degree was calculated by measuring the absorbance at 252 nm and 280 nm, the average number of DM1 connected to each antibody molecule was determined. Ultraviolet/visible spectrophotometry (UV/Vis) was a simple and convenient method, which can be used to determine the protein concentration and the average number of drugs conjugated by the antibody in the antibody-drug conjugate (ADC). DAR can be determined by using ADC absorbance measurements and extinction coefficients of corresponding antibodies and drugs.

[0132] When the absorption coefficient of a pure substance under certain conditions was known, the tested sample can be prepared into a solution under the same conditions, and its absorbance was measured, and the content of the substance can be calculated by the following formula: A=E×c×1, wherein A is absorbance, E is absorption coefficient, c is protein content, and 1 is liquid layer thickness (cm). This formula is also applicable to multi-component systems; if these components have different absorption spectra and there is no interaction between them, the light absorption of these components in the sample solution can be added. At this time, the absorbance Aλ=(E1λ×C1+E2λ×C2+ . . . +Enλ×cn)×1, n is the number of different absorption components, and Enλ is the extinction coefficient of the nth component; cn is the concentration of the nth component.

[0133] The F0002-ADC sample is known to have chromophores in the ultraviolet region, and the cAC10 monoclonal antibody (labeled as mab in the following formula) has an obvious maximum absorption value at 280 nm±3 nm, while the drug (DM1, labeled as a drug in the following formula) has a maximum absorption value at 252 nm±3 nm, and the presence of a drug does not affect the light absorption properties of the antibody. Therefore, by applying the above formula:

A.sub.280=(E.sub.drug.sup.280×C.sub.drug+E.sub.mab.sup.280×C.sub.mab)×l

A.sub.252=(E.sub.drug.sup.252×C.sub.drug+E.sub.mab.sup.252×C.sub.mab)×l

[0134] to reduce the systematic error, the value of the reference wavelength A.sub.320 was subtracted from the measured values of both A.sub.280 and A.sub.252, and then A.sub.280 and A.sub.252 were substituted into the above equation and combined with the two equations above to obtain the concentrations of antibody and drug.

[00001]$C_{\mathrm{mab}}=\frac{A_{280\times }{E}_{\mathrm{drug}}^{252}-A_{252\times }{E}_{\mathrm{drug}}^{280}}{\left(E_{\mathrm{mab}}^{280}{E}_{\mathrm{drug}}^{252}-E_{\mathrm{mab}}^{252}{E}_{\mathrm{drug}}^{280}\right)\times l}$$C_{\mathrm{durg}}=\frac{A_{280\times }{E}_{\mathrm{mab}}^{252}-A_{252\times }{E}_{\mathrm{mab}}^{280}}{\left(E_{\mathrm{drug}}^{280}{E}_{\mathrm{mab}}^{252}-E_{\mathrm{drug}}^{252}{E}_{\mathrm{mab}}^{280}\right)\times l}$

[0135] Then the average drug antibody coupling ratio (DAR) was calculated as follows:

[00002]$\mathrm{DAR}=\frac{C_{\mathrm{drug}}}{C_{\mathrm{mab}}}$

[0136] E.sub.drug.sup.280=5700 L/mol.Math.cm; E.sub.drug.sup.252=26790 L/mol.Math.cm;

[0137] E.sub.mab.sup.280=235454 L/mol.Math.cm; E.sub.mab.sup.252=83816 L/mol.Math.cm.

Embodiment 3 Preparation of Antibody Conjugate

[0138] The present disclosure ensured the stability of the average coupling ratio (DAR) of the toxic antibody by controlling the feeding ratio, pH, temperature and stirring of the coupling reaction.

[0139] A. Linker SMCC Modified Antibody:

[0140] Configuration of buffer: 50 mM dipotassium hydrogen phosphate-potassium dihydrogen phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5. The medium of the antibody was changed with buffer by 10 times volume ultrafiltration, and the final concentration of the antibody was 10 mg/mL, argon gas was added until it was full. 1.5 mL of 20 mM concentration of SMCC (dissolved in DMA) was added to 20 mL of cAC10 monoclonal antibody (Brentuximab, i.e., brentuximab) solution and the reaction was carried out for 4 hours at room temperature. The reaction mixture was filtered by a Sephadex G25 gel column, and the column was first equilibrated with buffer at 5 times the column volume. The characteristic peaks of the antibodies were collected at OD280 to obtain the antibodies modified by SMCC.

[0141] B. Coupling of Modified Antibodies with Mertansine (DM1):

[0142] The antibody modified by SMCC was diluted to a final concentration of 3 mg/mL with buffer for a total of 62 mL. Then, 1.7 mL of DM1 solution dissolved in DMA (concentration was 4.0 mM) was added to the antibody diluent. The reaction was carried out at room temperature (20° C.-30° C.) for 16 hours under the protection of argon. The reaction solution was chromatographed by Superdex 200, and the characteristic peak of the antibody was collected under OD280 to obtain the target product.

[0143] The structural formula of the obtained F0002-ADC antibody conjugate was (wherein mAb was cAC10 monoclonal antibody):

##STR00008##

[0144] According to the detection method in embodiment 2, the DAR of the antibody conjugate obtained in this embodiment was 3.6. The distribution of their different DAR values by LC-MS analysis was as follows:

TABLE-US-00003 DAR D0 D1 D2 D3 D4 D5 D6 D7 3% 10% 17% 20% 18% 16% 9% 7% 3.6

[0145] The proportions of different DAR distributions were obtained by LC-MS and then multiplied by the corresponding DAR values, and finally summed to obtain an average DAR value of 3.6.

Effect Embodiment 1 In Vitro Killing

[0146] Human degenerative large cell lymphoma cells Karpas299 (Nanjing Cobioer) were seeded at 5×10.sup.4 cells/mL, and serially diluted samples of the antibody conjugate prepared above, cAC10 monoclonal antibody and commercially available Adcetris were added. After the sample was added, it was cultured in 5% CO.sub.2 at 37° C. for 77±2 hours, stained with AlamarBlue fluorescent dye, cultured for 19±2 hours, and read at 530 nm (excitation)/590 nm (emission). The IC.sub.50 value of the semi-inhibitory concentration of each sample was calculated by fitting with the computer's four-parameter equation software. The assay results of Karpas299 showed that, with the increase of the concentration of F0002-ADC, the survival rate of tumor cells decreased significantly, showing a strong cell killing effect. However, cAC10 monoclonal antibody had basically no inhibitory effect on cell growth. As a comparative embodiment, the killing activity of Adcetris was equivalent to that of F0002-ADC.

TABLE-US-00004 TABLE 1 Summary table of cell survival rate of Karpas299 cells from different samples F0002 monoclonal F0002-ADC antibody Adcetris Cell survival Cell survival Cell survival Concentration rate Concentration rate Concentration rate (ng/mL) (%) (ng/mL) (%) (ng/mL) (%) 50 27 50 92 50 24 25 28 25 93 25 28 12.5 34 12.5 97 12.5 39 6.25 51 6.25 96 6.25 70 4.17 76 4.17 96 4.17 82 2.78 87 2.78 98 2.78 91 1.85 90 1.85 99 1.85 93 0.74 96 0.74 100 0.74 97 0.30 96 0.30 100 0.30 98

[0147] Furthermore, compared to Adcetris, the killing activity of F0002-ADC in different CD30-positive tumor cells, such as Hodgkin lymphoma cells L428, cutaneous somatic lymphoma HH cells, Hodgkin lymphoma cells L540 and human degenerative large cell lymphoma cells SU-DHL-1, was measured and characterized by IC.sub.50 value, the results were shown in table 2.

TABLE-US-00005 TABLE 2 Comparison of killing activity in vitro against different CD30-positive tumor cells IC.sub.50 (ng/mL) Karpas299 L428 HH L540 SU-DHL-1 F0002-ADC 10.6 5.7 3.2 8.6 8.1 adcetris 14.8 54314 3.2 5.9 12.5

[0148] As shown in table 2, wherein, Adcetris showed significant resistance to Hodgkin lymphoma cells L428 cells with a IC.sub.50 value up to 54,314 ng/mL, which may be related to the clinical insensitivity of Adcetris in some patients. In contrast, F0002-ADC showed significant killing activity against L428 cells.

[0149] It is known in the art that small molecule sensitivity does not mean that the ADC drug coupled to the antibody still has sensitive activity. For example, the small molecule MMAE in adcetris is sensitive to L428, but adcetris is not sensitive to L428. In the present disclosure, the antibody of F0002-ADC is the same as that of adcetris, but the difference lies in the linker and cytotoxic agent; however, during the project development, we unexpectedly found that F0002-ADC is sensitive to L428 and has significant killing activity through the above experiments; the unexpected results were obtained.

Effect Embodiment 2 Identification of MDR1 Expression

[0150] The reason for drug resistance of Hodgkin lymphoma cells L428 is that there is no accumulation of MMAE in cells, which leads to not enough active small molecules to kill tumor cells, this may be related to the expression of drug resistance pump in this cell; the expression level of multidrug resistance pump on the surface of different CD30 tumor cells was analyzed by flow cytometry. The sample was taken for 1×10.sup.6 cells/tube, centrifuged at 100 g for 5 min, washed once with 1 mL PBS, added with 100 μL PBS to resuspend the cells, then added with 5 μL/tube of CD243 (ABC B1) monoclonal antibody (UIC 2) and PE antibody (ThermoFisher), then 5 μL/tube of PBS was added to the negative control, and the cells were incubated on ice for 30 min, washed twice with cold PBS. The expression level of MDR1 was evaluated by flow cytometry after resuspension of cells in 0.2 mL PBS/tube to detect the fluorescence signal. The results showed that L428 cells specifically expressed MDR1 on the surface, while other CD30 cells did not express MDR 1; with the addition of 12.5 μg/mL Verapamil, the killing effect of Adcetris on L428 can reach the activity level equivalent to that of F0002-ADC. It was demonstrated that the main reason for Adcetris resistance in L428 cells was attributed to the high expression of MDR1.

TABLE-US-00006 TABLE 3 expression level of MDR1 in different cell lines Cells MDR1 binding ratio HH 2.6 Karpas 299 1.2 L540 1.2 L428 16.1

TABLE-US-00007 TABLE 4 effect of MDR1 inhibitor on the killing effect of Adcetris on L428 Adcetris + F0002-ADC Verapamil (12.5 μg/mL) Adcetris Concentration Cell survival Concentration Cell survival Concentration Cell survival (ng/mL) rate (%) (ng/mL) rate (%) (ng/mL) rate (%) 500 36 500 16 500000 30 20 38 20 36 50000 32 8 40 8 89 5000 41 3.20 46 3.20 94 500 90 1.28 48 1.28 96 50 100 0.51 67 0.51 100 5 102 0.20 86 0.20 100 0.50 100 0.08 100 0.08 99 0.05 100 0.03 102 0.03 100 0.01 100

Effect Embodiment 3 Adcetris Induced Drug-Resistant Cell Lines

[0151] A concentration gradient increasing, pulsating method was employed, i.e., the cell density was 2.5×10.sup.4/mL, the killing effect time was chosen to be 4 days, the recovery culture time was 3 days, and the concentration gradient of Adcetris was chosen to be 1×IC.sub.50, 2×IC.sub.50, 5×IC.sub.50, 10×IC.sub.50, with IC.sub.50 referring to the previous cytotoxicity results. The Adcetris resistant cell lines Karpas299, L428, HH, L540 and SU-DHL-1 were screened, and the drug resistant cell lines Karpas299-R, L428-R, HH-R, L540-R and SU-DHL-1-R were screened out respectively.

Effect Embodiment 4 Killing Evaluation of Drug Resistant Cells

[0152] The killing activity of F0002-ADC compared to Adcetris was evaluated separately for the above drug-resistant cell lines treated with Adcetris induction and characterized by IC.sub.50 values. As shown in Table 6 below, HH, Karpas299, L540 and SU-DHL-1 drug-resistant cells induced by Adcetris had significant drug-resistant activity to Adcetris. Further analysis showed that the drug resistance of HH, Karpas299 and SU-DHL-1 cells was mainly caused by the down-regulation of CD30 antigen abundance, and they also had drug resistance activity to F0002-ADC. The drug resistance of L540 cells to Adcetris was mainly caused by the high expression of MDR1, and its mRNA was increased by 6.3 times compared with before drug resistance. The results showed that F0002-ADC still had sensitive killing activity against L540 cells after drug resistance, indicating that F0002-ADC had specific and sensitive killing activity against the occurrence of MDR1 high expression after treatment with Adcetris.

TABLE-US-00008 TABLE 5 sensitivity of cells to Adcetris before and after L540 drug resistance L540 cells L540-R cells F0002-ADC Adcetris F0002-ADC Adcetris Cell Cell Cell Cell Concentration survival Concentration survival Concentration survival Concentration survival (ng/mL) rate (%) (ng/mL) rate (%) (ng/mL) rate (%) (ng/mL) rate (%) 50 22 50 30 500000 15 500000 17 25 34 25 34 250000 13 250000 18 12.5 42 12.5 50 125000 14 125000 19 8.33 65 8.33 65 62500 15 62500 26 5.56 73 5.56 78 31250 16 31250 34 3.70 78 3.70 88 15625 17 15625 39 1.48 78 1.48 92 7812.5 19 7812 46 0.59 78 0.59 94 3906.2 20 3906 47 0.24 76 0.24 95 1953.1 19 1953 47 / 976.6 20 976.6 51 488.3 20 488.3 50 244.1 20 244.1 49 122.1 21 122.1 53 61.04 20 61.04 51 30.52 20 30.52 55 15.26 23 15.26 58 7.63 46 7.63 65 3.81 72 3.81 72

TABLE-US-00009 TABLE 6 comparison of antigen abundance, MDR1 level and killing activity of different tumor cells before and after drug resistance CD30 binding IC.sub.50 (ng/mL) MDR1 Cells ratio F0002-ADC Adcetris mRNA HH 254 1.5 1.5 Not detected HH-R 48 3921 36714 Not detected Karpas 299 112 3.5 4.4 Not detected Karpas 299-R 16 249 63.7 Not detected SU-DHL-1 68 9.8 7.0 Not detected SU-DHL-1-R 13 891 63.7 Not detected L540 104 10.9 8.8 1 L540-R 42 8.2 3053 6.3 L428 48 2.9 27157 8.2 Note: CD30 binding ratio indicates the ratio of CD30 flow signal with negative cell romas.

Effect Embodiment 5 Verification of In Vivo System Model

[0153] The above embodiments confirmed that F0002-ADC maintains killing sensitive activity on tumor cells with high MDR1 expression, and has good tumor killing inhibitory activity on cells with high MDR1 expression induced by Adcetris treatment, indicating that F0002-ADC can specifically overcome MDR1 drug resistance; the purpose of this embodiment is to further verify the anti-tumor efficacy of F0002-ADC and Adcetris on the in vivo system model of L428 cells expressing MDR1.

[0154] To establish the L428 in vivo systemic model, 11 to 12 week-old female NPG mice were injected with 1×10.sup.7 human lymphoma cells (L428) dissolved in 100 μL of PBS solution via tail vein. On the 8th day after cell inoculation, the mice were randomly divided into three treatment groups, with 6 mice in each group, namely blank control group/F0002-ADC group (3 mg/kg)/Adcetris group (3 mg/kg). The first dosing was started on the day of grouping (D8), F0002-ADC and Adcetris were prepared into 2.5 mL target solutions with a concentration of 0.6 mg/mL, the dosing method was tail vein administration, the dosing cycle was Q3W*2 (D8 and D29), the body weight of the experimental animals was measured twice a week and the status of the experimental animals was observed, after the end of the second dosing, the status of the experimental animals was closely monitored; and mice with deteriorating physical condition, near death or unable to feed and drink normally were euthanized, until all animals in the treated group died due to disease progression; after the experiment, SPSS was used for statistical analysis, and Kaplan-Meier method was used to draw the survival curve of each group. Compared with the blank control group, both F0002-ADC and Adcetris significantly prolonged the survival period of the lymphoma model mice, the blank group showed animal death from D53 after cell inoculation, and all the mice in the group died on D79; and the F0002-ADC group showed animal death from D66 after cell inoculation, and all the mice in the group died on D114; the mice in the Adcetris group showed animal death from D58 after cell inoculation, and all the mice in the group died on D90. The F0002-ADC group (3 mg/kg) had a good effect on prolonging the survival of human lymphoma model mice compared with the blank control group, P<0.001 was statistically different and highly significant; the Adcetris group (3 mg/kg) had a certain effect on prolonging the survival of model mice compared with the blank control group, but P>0.05 was not statistically different. There was a statistical difference between the F0002-ADC group and the Adcetris group (p<0.05).

TABLE-US-00010 TABLE 7 comparison of survival time Days/survival rate Blank control (%) F0002-ADC (%) Adcetris (%) 0 100 100 100 53 83 100 100 57 83 100 100 58 83 100 83 60 83 100 67 64 50 100 67 66 33 83 67 68 33 83 50 73 33 83 33 75 17 83 33 79 0 83 33 85 0 67 33 87 0 50 33 90 0 50 0 94 0 33 0 114 0 0 0

Effect Embodiment 6 Combined ABVD/AVD Scheme

[0155] Cell proliferation method was used to investigate the killing effect of drugs on cells. L428 cells were inoculated in 96-well cell culture plates at 5×10.sup.4 cell/mL, i.e., 5000 cells per well. Doxorubicin was diluted to 259.5 ng/mL, 129.8 ng/mL, 64.88 ng/mL, 32.44 ng/mL, 21.63 ng/mL, 14.42 ng/mL, 9.611 ng/mL, 3.844 ng/mL, 1.538 ng/mL using cell culture medium, for a total of 9 concentrations. Serial dilutions of Doxorubicin were added to the culture plates of the seeded plates and incubated for 77±2 hours at 37° C. in a 5% CO.sub.2 incubator. The samples were stained with AlamarBlue fluorescent dye and incubated at 37° C. in a 5% CO.sub.2 incubator for 19±2 hours. Readings were performed at wavelength of 50 nm (excitation)/590 nm (emission). The IC.sub.50 value of Doxorubicin was calculated by fitting it with four-parameter equation.

[0156] The killing curves of four single drugs of ABVD and F0002-ADC against L428 and L540 and IC.sub.50 values of single drug were obtained by the same method described above, wherein the dose-effect concentration range of the drugs could be obtained by pre-experiments.

[0157] According to Hodgkin lymphoma drug combination scheme, the classic combination mode of ABVD was selected first, and the combination effect of F0002-ADC with ABVD or AVD scheme was studied. Single drug killing was first performed: single drug experiments were performed on F0002-ADC, Doxorubicin, Bleomycin, Vincristine and Dacarbazine against L428 and L540 cells, respectively, to obtain the IC.sub.50 values of inhibitory killing at different dose concentrations, as shown in Table 8 below.

TABLE-US-00011 TABLE 8 summary table of IC.sub.50 value of each cell of single drug Drug name L428 (IC.sub.50, nM) L540 (IC.sub.50, nM) F0002-ADC 0.075 0.070 Doxorubicin 27.79 58.49 Bleomycin 2043 3198 Vincristine 28.08 0.772 Dacarbazine 178140 31346

[0158] The combination method was chosen as a constant combination ratio (i.e., the ratio of IC.sub.50), and F0002-ADC was combined with ABVD or AVD for killing experiments against L428 and L540 cells. Calcusyn, the software for calculating the combination index, was chosen to calculate the CI of the combination index at different killing levels,

[00003]$\mathrm{CI}=\underset{i=1}{\overset{n}{.Math.}}\frac{\left(D\right)i}{\left(D_x\right)j},$

wherein D is the single dose and Dx is the combination dose. CI value less than 1 was synergistic, equal to 1 was additive, and greater than 1 was antagonistic.

[0159] (1) In the combination scheme of F0002-ADC+ABVD (Doxorubicin, Bleomycin, Vincristine and Dacarbazine), the dosing molar ratio of F0002-ADC:Doxorubicin:Bleomycin:Vincristine:Dacarbazine was 1:400:30000:400:3000000; L428 cells were treated with multiple concentrations for 96 hours, in order to investigate the killing results of ABVD scheme combined with F0002-ADC, and compared with F0002-ADC; the combined index CI value was less than 1 when the cell killing rate was 500%, 7500 and 900%; the results showed that F0002-ADC had synergistic effect after combined with AB VD, and could obtain a better killing effect.

TABLE-US-00012 TABLE 9 F0002-ADC combined with ABVD in L428 cells F0002-ADC + ABVD A B V D F0002-ADC F0002-ADC (Doxorubicin) (Bleomycin) (Vincristine) (Dacarbazine) Concentration Cell survival Concentration Concentration Concentration Concentration Concentration Cell survival (nM) rate (%) (nM) (nM) (μM) (nM) (μM) rate (%) 0.33 29 0.066 26.40 1.98 26.40 198 17 0.17 33 0.034 13.60 1.02 13.60 102 19 0.08 35 0.016 6.40 0.48 6.40 48 20 0.04 46 0.008 3.20 0.24 3.20 24 28 0.03 65 0.006 2.40 0.18 2.40 18 44 0.02 79 0.004 1.60 0.12 1.60 12 73 0.01 87 0.002 0.80 0.06 0.80 6 82 0.003 88 0.001 0.24 0.02 0.24 1.80 89

[0160] (2) Also in L540 cells, in the combination scheme of F0002-ADC+ABVD, the molar ratio of F0002-ADC:Doxorubicin:Bleomycin:Vincristine:Dacarbazine was 1:800:45000:11:550000; the cells were treated with multiple concentrations for 96 hours, and the combined index CI value obtained by calculation was less than 1 when the cell killing rate was 5000, 7500 and 900%; the results showed that F0002-ADC had synergistic effect after combined with ABVD, and could obtain better killing effect.

TABLE-US-00013 TABLE 10 F0002-ADC combined with ABVD in L540 cells F0002-ADC + ABVD A B V D F0002-ADC F0002-ADC (Doxorubicin) (Bleomycin) (Vincristine) (Dacarbazine) Concentration Cell survival Concentration Concentration Concentration Concentration Concentration Cell survival (nM) rate (%) (nM) (nM) (μM) (nM) (μM) rate (%) 0.33 27 0.066 52.80 2.97 0.73 36.30 14 0.17 43 0.034 27.20 1.53 0.37 18.70 27 0.08 58 0.016 12.80 0.72 0.18 8.80 33 0.04 66 0.008 6.40 0.36 0.09 4.40 44 0.03 69 0.006 4.80 0.27 0.07 3.30 58 0.02 77 0.004 3.20 0.18 0.04 2.20 73 0.01 84 0.002 1.60 0.09 0.02 1.10 90 0.003 97 0.001 0.48 0.03 0.01 0.33 93

[0161] (3) In the combination scheme of F0002-ADC+AVD (Doxorubicin, Vincristine and Dacarbazine), the dosing molar ratio of F0002-ADC:Doxorubicin:Vincristine:Dacarbazine was 1:400:400:3000000; L428 cells were treated with multiple concentrations to examine the killing results of the AVD scheme combined with F0002-ADC. The combined index CI value obtained by calculation was less than 1 when the cell killing rate was 500%, 7500 and 900%; the results showed that F0002-ADC had synergistic effect after combined with AVD, and could obtain better killing effect.

TABLE-US-00014 TABLE 11 F0002-ADC combined with AVD in L428 cells F0002-ADC + AVD A V D F0002-ADC F0002-ADC (Doxorubicin) (Vincristine) (Dacarbazine) Concentration Cell survival Concentration Concentration Concentration Concentration Cell survival (nM) rate (%) (nM) (nM) (nM) (μM) rate (%) 0.33 29 0.086 33 33 247.5 17 0.17 33 0.042 17 17 127.5 19 0.08 35 0.020 8 8 60 20 0.04 46 0.010 4 4 30 37 0.03 65 0.008 3 3 22.50 54 0.02 79 0.005 2 2 15 68 0.01 87 0.002 1 1 7.50 68 0.003 88 0.001 0.30 0.30 2.25 69

[0162] (4) In the combination scheme of F0002-ADC+AVD, the dosing molar ratio of F0002-ADC:Doxorubicin:Vincristine:Dacarbazine was 1:800:11:550000; cells were treated with multiple concentrations in order to investigate the killing results of AVD scheme combined with F0002-ADC, and the combined index CI value obtained by calculation was less than 1 at 500%, 7500 and 900% of cell killing; the results indicated that F0002-ADC had a synergistic effect after combining with AVD, and could obtain a better killing effect.

TABLE-US-00015 TABLE 12 F0002-ADC combined with AVD in L540 cells F0002-ADC + AVD A V D F0002-ADC F0002-ADC (Doxorubicin) (Vincristine) (Dacarbazine) Concentration Cell survival Concentration Concentration Concentration Concentration Cell survival (nM) rate (%) (nM) (nM) (nM) (μM) rate (%) 0.33 27 0.082 66 0.91 45.38 16 0.17 43 0.042 34 0.47 23.38 23 0.08 58 0.020 16 0.22 11 33 0.04 66 0.010 8 0.11 5.50 43 0.03 69 0.008 6 0.08 4.12 60 0.02 77 0.005 4 0.06 2.75 79 0.01 84 0.002 2 0.03 1.39 80 0.003 97 0.001 0.60 0.01 0.41 88

Effect Embodiment 7 Other Combination Schemes

[0163] A single drug scheme was used to study the inhibitory effects of Bleomycin, Etoposide, Doxorubicin, Cyclophosphamide, Vincristine, Procarbazine and Prednisone, etc., on L428, L540 cells and Karpas299 cells; the IC.sub.50 values were shown in the table below, and then the drugs were combined to kill, and constant combination ratio was used in the combination method, i.e., IC.sub.50 ratio. See table 13 below.

TABLE-US-00016 TABLE 13 summary table of IC.sub.50 value of each cell of single drug L428 cells L540 cells Karpas299 cells Drug name (IC.sub.50, nM) (IC.sub.50, nM) (IC.sub.50, nM) F0002-ADC 0.075 0.070 0.033 Bleomycin 2043 3198 1680 Etoposide 54160 30089 64111 Doxorubicin 27.79 58.49 40.51 Cyclophosphamide 590444 120550 385446 Vincristine 28.08 0.772 18.54 Procarbazine 500438 24689 26899 Prednisone 112854 98544 186715

[0164] (1) In the combination scheme of F0002-ADC+BEACOPP (Bleomycin, Etoposide, Doxorubicin, Cyclophosphamide, Vincristine, Procarbazine and Prednisone), the dosing molar ratio of F0002-ADC:Bleomycin:Etoposide:Doxorubicin:Cyclophosphamide:Vincristine:Procarbazine:Prednisone was 1:30000:700000:400:8000000:400:6500000:1500000; L428 cells were treated with multiple concentrations for 96 hours, and the killing effect of the combination on L428 was investigated and compared with F0002-ADC; the results showed that the combined BEACOPP scheme had a synergistic effect and achieved better killing effect.

TABLE-US-00017 TABLE 14 F0002-ADC combined with BEACOPP in L428 cells F0002-ADC + BEACOPP B E A F0002-ADC F0002-ADC (Bleomycin) (Etoposide) (Doxorubicin) Concentration Cell survival Concentration Concentration Concentration Concentration (nM) rate (%) (nM) (μM) (μM) (nM) 0.33 29 0.041 1.238 28.88 16.50 0.17 33 0.021 0.638 14.88 8.50 0.08 35 0.010 0.300 7.00 4.00 0.04 46 0.005 0.150 3.50 2.00 0.03 65 0.004 0.112 2.62 1.50 0.02 79 0.002 0.075 1.75 1.00 0.01 87 0.001 0.038 0.88 0.50 0.003 88 0.0004 0.011 0.26 0.15 F0002-ADC combined with BEACOPP in L428 cells F0002-ADC + BEACOPP C O P P (Cyclophosphamide) (Vincristine) (Procarbazine) (Prednisone) Concentration Concentration Concentration Concentration Cell survival (μM) (nM) (μM) (μM) rate (%) 330 16.50 268.12 61.88 13 170 8.50 138.12 31.88 22 80 4.00 65 15 22 40 2.00 32.5 7.5 33 30 1.50 24.38 5.62 50 20 1.00 16.25 3.75 84 10 0.50 8.125 1.88 90 3 0.15 2.44 0.56 93

[0165] (2) In the combination scheme of F0002-ADC+CHOP (Cyclophosphamide, Doxorubicin, Vincristine, Prednisone), the dosing molar ratio of F0002-ADC:Cyclophosphamide:Doxorubicin:Vincristine:Prednisone was 1:1700000:800:11:1400000; L540 cells were treated with multiple concentrations for 96 hours, and the killing effect of the combination on L540 was investigated and compared with F0002-ADC; the results showed that the combined CHOP scheme had a synergistic effect and achieved better killing effect.

TABLE-US-00018 TABLE 15 F0002-ADC combined with CHOP in L540 cells F0002-ADC + CHOP C H O P F0002-ADC F0002-ADC (Cyclophosphamide) (Doxorubicin) (Vincristine ) (Prednisone) Concentration Cell survival Concentration Concentration Concentration Concentration Concentration Cell survival (nM) rate (%) (nM) (μM) (nM) (nM) (μM) rate (%) 0.33 27 0.066 1122 52.8 0.73 92.40 14 0.17 43 0.034 578 27.2 0.37 47.60 19 0.08 58 0.016 272 12.8 0.18 22.40 27 0.04 66 0.008 136 6.4 0.09 11.20 45 0.03 69 0.006 102 4.8 0.07 8.40 64 0.02 77 0.004 68 3.2 0.04 5.60 82 0.01 84 0.002 34 1.6 0.02 2.80 83 0.003 97 0.001 10.2 0.48 0.01 0.84 86

[0166] In the combination scheme of F0002-ADC+CHOP (Cyclophosphamide, Doxorubicin, Vincristine, Prednisone), the dosing molar ratio of F0002-ADC:Cyclophosphamide:Doxorubicin:Vincristine:Prednisone was 1:12000000:1200:600:5500000; Karpas 299 cells were treated with multiple concentrations for 96 hours, and the killing effect of the combination on Karpas 299 was investigated and compared with F0002-ADC; the results showed that the combined CHOP scheme had a synergistic effect and achieved better killing effect.

TABLE-US-00019 TABLE 16 F0002-ADC combined with CHOP in Karpas 299 cells F0002-ADC + CHOP C H O P F0002-ADC F0002-ADC (Cyclophosphamide) (Doxorubicin) (Vincristine) (Prednisone) Concentration Cell survival Concentration Concentration Concentration Concentration Concentration Cell survival (nM) rate (%) (nM) (μM) (nM) (nM) (μM) rate (%) 0.33 24 0.066 792 79.20 39.60 363 11 0.17 27 0.034 408 40.80 20.40 187 17 0.08 34 0.016 192 19.20 9.60 88 16 0.04 44 0.008 96 9.60 4.80 44 30 0.03 53 0.006 72 7.20 3.60 33 43 0.02 66 0.004 48 4.80 2.40 22 56 0.01 89 0.002 24 2.40 1.20 11 92 0.003 97 0.0006 7.20 0.72 0.36 3.30 97 0.001 94 0.0002 2.40 0.24 0.12 1.10 98

[0167] (3) In the combined scheme of F0002-ADC+CVP (Cyclophosphamide, Vinblastine and Prednisone), the molar ratio of F0002-ADC:Cyclophosphamide:Vinblastine:Prednisone was 1:800000:400:1500000; L428 cells were treated with multiple concentrations for 96 hours, the killing results of the combination combined on L428 were examined and compared with F0002-ADC; and the results showed that the combined CVP scheme had better killing effect on L428.

TABLE-US-00020 TABLE 17 F0002-ADC combined with CVP in L428 cells F0002-ADC + CVP C V P F0002-ADC F0002-ADC (Cyclophosphamide) (Vincristine) (Prednisone) Concentration Cell survival Concentration Concentration Concentration Concentration Cell survival (nM) rate (%) (nM) (μM) (nM) (μM) rate (%) 0.33 29 0.082 660 33 124 14 0.17 33 0.042 340 17 64 16 0.08 35 0.020 160 8 30 20 0.04 46 0.010 80 4 15 37 0.03 65 0.008 60 3 11 50 0.02 79 0.005 40 2 8 73 0.01 87 0.002 20 1 4 77 0.003 88 0.0008 6 0.30 1 87

[0168] In the combined scheme of F0002-ADC+CVP (Cyclophosphamide, Vinblastine and Prednisone), the molar ratio of F0002-ADC:Cyclophosphamide:Vinblastine:Prednisone was 1:12000000:600:5500000; Karpas 299 cells were treated with multiple concentrations for 96 hours, the killing results of the combination on Karpas 299 were examined and compared with F0002-ADC; and the results showed that the combined CVP scheme had a better killing effect on Karpas 299.

TABLE-US-00021 TABLE 18 F0002-ADC combined with CVP in Karpas 299 cells F0002-ADC + CVP C V P F0002-ADC F0002-ADC (Cyclophosphamide) (Vincristine) (Prednisone) Concentration Cell survival Concentration Concentration Concentration Concentration Cell survival (nM) rate (%) (nM) (μM) (nM) (μM) rate (%) 0.33 24 0.082 990 49.50 453.8 14 0.17 27 0.042 510 25.50 233.8 19 0.08 34 0.020 240 12 110 24 0.04 44 0.010 120 6 55 28 0.03 53 0.008 90 1.50 41.25 40 0.02 66 0.005 60 3 27.50 51 0.01 89 0.002 30 1.50 13.75 92 0.003 97 0.0008 9 0.45 4.13 97 0.001 98 0.0002 3 0.15 1.38 98

Effect Embodiment 8 Acute Toxicity Study

[0169] The tolerance difference between F0002-ADC and ADCERIS was compared in the acute toxicity test of monkeys: F0002-ADC 30 mg/kg and ADCERIS 6 mg/kg groups were set, 4 cynomolgus monkeys in each group, half male and half female, were administered intravenously once, and were dissected 4 weeks after recovery. The detection indexes included clinical observation, body weight, food intake, clinical pathology (hematology, serum biochemistry and blood coagulation) and gross anatomy.

[0170] The mortality of 6 mg/kg ADCETRIS group was 2/4 on D14 after single administration to cynomolgus monkeys. Histopathological examination showed that the death was caused by drug toxicity, which was related to liver, thymus (decreased immune function) and secondary lung lesions, besides skin and mucous membrane lesions were also observed. No animal died in the F0002-ADC 30 mg/kg group. Clinical observation showed skin erythema after administration in all animals of the two groups, and skin papules (3/4) were also observed on D13 in the Adcetris 6 mg/kg group. The body weight of F0002-ADC 30 mg/kg group decreased significantly (3/4), which was still lower than that before administration after 4 weeks of drug withdrawal, which was consistent with the loss of appetite; the appetite and body weight of ADCETRIS 6 mg/kg group also decreased obviously, but the appetite and body weight of the surviving animals (2/4) returned to normal from D18. The incidence of hematological changes in F0002-ADC 30 mg/kg group was low, mainly a decrease in #NEUT (1 case each on D8, D21 and D28) and a decrease in PLT (1 case each on D5 and D8), which recovered after 7 days. Both of them had reversible changes in skin, hematology and liver function. All lesions can be recovered after 4 weeks after the withdrawal of drug. Cynomolgus monkeys were given a single dose of F0002-ADC 30 mg/kg and ADCETRIS 6 mg/kg; the tolerance of animals to F0002-ADC 30 mg/kg was higher than that of ADCETRIS 6 mg/kg; the tolerable dose of F0002-ADC was 30 mg/kg, and the lethal dose of Adcetris was 6 mg/kg. Compared with ADCETRIS, F0002-ADC has obvious safety advantages, which supports the safety improvement of this drug.

Effect Embodiment 9 Study on Long Toxicity in Rats

[0171] Repeated administration toxicity tests for rats were performed in F0002-ADC blank formulation group, F0002-ADC 5, 10 and 20 mg/kg groups (calculated by DM1, they were 510, 1020 and 2040 μg DM1/m2 respectively), and DM1 0.1 mg/kg group (600 μg DM1/m2); 30 SD rats, half male and half female in each group. There were 4 males and 4 females in each group. The drug was administered by tail vein injection, once on D1, D8, D15 and D22, for 4 times in total. Anatomy was performed at the end of administration and recovery period on D26 and D54 respectively; the evaluation indexes included clinical observation, body weight, food intake, ophthalmic examination, hematology, serum biochemistry, serum electrolyte, blood coagulation and urine analysis, gross anatomy, organ weight, histopathological examination and bone marrow smear examination. F0002-ADC was administered intravenously to SD rats in 4 repeated cycles at a maximum tolerated dose (MTD) of 20 mg/kg; in this test, the toxicity of F0002-ADC was mainly manifested as toxic reactions related to immune hematopoietic organs, liver, kidney and reproductive system. 4 weeks after the withdrawal of drug, the changes of male reproductive system were recovered in the 5 mg/kg group.

Effect Embodiment 10 Study on Long Toxicity in Monkey

[0172] Repeated administration toxicity tests for cynomolgus monkeys were performed with F0002-ADC blank formulation, F0002-ADC 3, 10 and 20 mg/kg, and F0002 monoclonal antibody 20 mg/kg. Fifty cynomolgus monkeys were divided into 5 groups, with 10 in each group, half male and half female. Intravenous infusion once every 21 days was set as one cycle, 4 cycles of continuous administration was performed, the administration rate was 1.5 mL/min, and the recovery period was 6 weeks. The detection indexes include clinical observation, body weight, body temperature, eye examination, food intake, clinical pathology, electrocardiogram, immunogenicity (anti-drug antibody and neutralizing antibody), toxicokinetics, safe pharmacology, local stimulation, lymphocyte typing, circulating immune complex, gross anatomy, histopathological examination and bone marrow smear examination. Cynomolgus monkeys were given F0002-ADC for 4 consecutive cycles, no apparent toxic reaction dose (NOAEL) was 3 mg/kg, the highest non severely toxic dose (HNSTD) was 10 mg/kg; and the minimum lethal dose (MLD) was 20 mg/kg. The main toxic reactions observed with F0002-ADC were skin changes, decreased body weight and food intake, hematological changes (decreased WBC, #NEUT, red lineage and platelets), serum biochemical changes (increased AST, ALP, CK and GLB, decreased ALB and A/G), hemagglutination changes (prolonged APTT and TT, increased FIB), and toxic target organs were sciatic nerve, spinal cord, liver, spleen, kidney, thymus, adrenal gland, breast, sternum (including bone marrow) and seminal vesicles. No significant toxic reactions were observed in the F0002 monoclonal antibody 20 mg/kg group.

[0173] The MTD of F0002-ADC was 30 mg/kg in the acute monkey toxicity test, and the unrestricted toxicity dose (HNSTD) was 10 mg/kg in monkey long-term toxicity test. An enzymatically non-degradable linker was used in F0002-ADC; and F0002-ADC is stable in vivo, has low levels of toxic small molecule shedding, and has a low non-specific cytotoxic activity of the active metabolite Lys-MCC-DM1, thus showing a good safety in non-clinical animal studies, and is expected to be a safer and more effective option for targeting CD30 for the treatment of HL, ALCL and CTCL.

[0174] Although the above describes specific embodiments of the present disclosure, it should be understood by those skilled in the art that these are merely illustrative embodiments and that a variety of changes or modifications can be made to these embodiments without departing from the principles and substance of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.