Stable antibody-drug conjugate, preparation method therefor, and use thereof

Abstract

The present invention provides a conjugate and preparation method thereof, a pharmaceutical composition comprising the conjugate and use of the pharmaceutical composition in the manufacture of a medicament for the treatment or prevention of a disease.

Claims

1. A compound of Formula (I) or (II): ##STR00025## or a pharmaceutically acceptable salt thereof, wherein PCA is a specific substrate recognition sequence of a ligase; LA1 and LA2 are linker moieties; a and p are independently 0 or 1, that is, LA1 and LA2 are independently present or absent; CCA is a chemical conjugation moiety; represents a single or double bond; Y is a radioactive label, a fluorescent label, an affinity purification tag, a tracer molecule, an anticancer drug or a cytotoxic molecule; z is any of the integers between 1 and 1000.

2. The compound according to claim 1, wherein Y is maytansine, DM1, DM4, paclitaxel, Auristatin, monomethyl auristatin E(MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD), epothilone, or a vinca alkaloid compound.

3. A compound wherein the compound is selected from the group consisting of: ##STR00026## ##STR00027## or a pharmaceutically acceptable salt thereof, wherein n represents any of the integers between 1 and 100, m is 0 or any of the integers between 1 and 1000, and x is OH or NH.sub.2 group.

4. The compound according to claim 1, which is prepared by the following steps: A) a solution of Y and a solution of ##STR00028## are mixed and incubated to obtain a solution of the conjugate: ##STR00029## or a solution of Y and a solution of ##STR00030## are mixed and incubated to obtain a solution of the conjugate ##STR00031## in this case, a S-(LA2).sub.p group is connected to Y; B) to a solution of the conjugate ##STR00032## prepared in A) is added a Tris base solution or other solution which facilitates ring-opening.

5. The compound according to claim 4, wherein the preparation further comprises purifying the obtained product by HPLC.

6. The compound according to claim 5, wherein the total molar content of the compounds of Formula (I) or (II) is more than 50%.

7. The compound according to claim 1, wherein the PCA is 3 series-connected structure units which are selected from the group consisting of one or more glycine and alanine.

8. A compound, wherein the compound is: ##STR00033## wherein n is 3 and x is OH or NH.sub.2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. The schematic diagram of ring-open reaction.

(2) FIG. 2, The schematic diagram of enzyme-catalyzed coupling technology.

(3) FIG. 3. The chemical structure of linker 1 (n is an integer from 1-100, x is OH or NH.sub.2 group)

(4) FIG. 4. The chemical structure of linker 2 (n is an integer from 1-100, x is OH or NH.sub.2 group)

(5) FIG. 5. The chemical structure of linker 3 (n is an integer from 1-100, m is 0 or any of the integers from 1-1,000, X is OH or NH.sub.2 group)

(6) FIG. 6. The chemical structure of linker 4 (n is an integer from 1-100, m is 0 or any of the integers from 1-1,000, X is OH or NH.sub.2 group)

(7) FIG. 7. The chemical structure of linker 5 (n is an integer from 1-100, x is OH or NH.sub.2 groups)

(8) FIG. 8. The chemical structure of linker 6 (n is an integer from 1-100, x is OH or NH.sub.2 groups)

(9) FIG. 9. The chemical structure of linker 7 (n is an integer from 1-100, m is 0 or any of the integers from 1-1,000, x is OH or NH.sub.2 group)

(10) FIG. 10. The chemical structure of linker 8 (n is an integer from 1-100, m is 0 or any of the integers from 1-1,000, x is OH or NH.sub.2 groups)

(11) FIG. 11. The molecular schematic diagram of linker 1-DM1 intermediate (n is an integer from 1-100, x is OH or NH.sub.2 group)

(12) FIG. 12. The molecular schematic diagram of linker 2-DM1 intermediate (n is an integer from 1-100, x is OH or NH.sub.2 group)

(13) FIG. 13. The molecular schematic diagram of linker 3-DM1 intermediate (n is an integer from 1-100, m is 0 or any of the integers from 1-1000, x is OH or NH.sub.2 group)

(14) FIG. 14. The molecular schematic diagram of linker 4-DM1 intermediate (n is an integer from 1-100, m is 0 or any of the integers from 1-1000, x is OH or NH.sub.2 group)

(15) FIG. 15A and FIG. 15B. The ring-open molecular schematic diagram of linker 1-DM1 intermediate (n is an integer from 1-100, x is OH or NH.sub.2 group; FIG. 15A and FIG. 15B are isomers)

(16) FIG. 16A and FIG. 16B. The ring-open molecular schematic diagram of linker 2-DM1 intermediate (n is an integer from 1-100, x is OH or NH.sub.2 group; FIG. 16A and FIG. 16B are isomers)

(17) FIG. 17A and FIG. 17B. The ring-open molecular schematic diagram of linker 3-DM1 intermediate (n is an integer from 1-100, m is 0 or any of the integers between 1-1000, x is OH or NH.sub.2 group; FIG. 17A and FIG. 17B are isomers)

(18) FIG. 18A and FIG. 18B. The ring-open molecular schematic diagram of linker 4-DM1 intermediate (n is an integer from 1-100, m is 0 or any of the integers between 1-1000, x is OH or NH.sub.2 group; FIG. 18A and FIG. 18B are isomers)

(19) FIG. 19. The chemical structure of linker 5-MMAE intermediate (n is an integer from 1-100, x is OH or NH.sub.2 groups)

(20) FIG. 20. The chemical structure of linker 6-MMAE intermediate (n is an integer from 1-100, x is OH or NH.sub.2 groups)

(21) FIG. 21. The chemical structure of linker 7-MMAE intermediate (n is an integer from 1-100, m is 0 or any of the integers from 1-1000, x is OH or NH.sub.2 group)

(22) FIG. 22. The chemical structure of linker 8-MMAE intermediate (n is an integer from 1-100, m is 0 or any of the integers from 1-1000, x is OH or NH.sub.2 group)

(23) FIG. 23A and FIG. 23B. The ring-open molecular schematic diagram of linker 5-MMAE intermediate (n is an integer from 1-100, x is OH or NH.sub.2 group; FIG. 23A and FIG. 23B are isomers)

(24) FIG. 24A and FIG. 24B. The ring-open molecular schematic diagram of linker 6-MMAE intermediate (n is an integer from 1-100, x is OH or NH.sub.2 group; FIG. 23A and FIG. 23B are isomers)

(25) FIG. 25A and FIG. 25B. The ring-open molecular schematic diagram of linker 7-MMAE intermediate (n is an integer from 1-100, x is OH or NH.sub.2 group, m is 0 or any of the integers from 1-1000; FIG. 25A and FIG. 25B are isomers)

(26) FIG. 26A and FIG. 26B. The ring-open molecular schematic diagram of linker 8-MMAE intermediate (n is an integer from 1-100, x is OH or NH.sub.2 group, m is 0 or any of the integers from 1-1000; FIG. 26A and FIG. 23B are isomers)

(27) FIG. 27A and FIG. 27B. The molecular schematic diagram of preferred ADC1 molecules (n is an integer from 1-100, d is any of the integers from 1-20, X in ligase recognition sequence LPXT of is glutamic acid (E) or any other natural/unnatural amino acid; Ab is an antibody, LA3 is linker moiety, comprising 1 to 100 series-connected structure units which are selected from the group consisting of one or more glycine and alanine; each b is independently 0 or 1, indicating the presence or absence of LA3; x is OH or NH.sub.2 group; FIG. 27A and FIG. 27B are isomers).

(28) FIG. 28A and FIG. 28B. The molecular schematic diagram of preferred ADC2 molecules (n is an integer from 1-100, d is any of the integers from 1-20, X in ligase recognition sequence LPXT of is glutamic acid (E) or any other natural/unnatural amino acid; Ab is an antibody, LA3 is linker moiety, comprising 1 to 100 series-connected structure units which are selected from the group consisting of one or more glycine and alanine; each b is independently 0 or 1, indicating the presence or absence of LA3; x is OH or NH.sub.2 group; FIG. 28A and FIG. 28B are isomers).

(29) FIG. 29A and FIG. 29B. The molecular schematic diagram of preferred ADC3 molecules (n is an integer from 1-100, d is any of the integers from 1-20, X in ligase recognition sequence LPXT of is glutamic acid (E) or any other natural/unnatural amino acid; m is 0 or any of the integers from 1-1000, Ab is an antibody, LA3 is linker moiety, comprising 1 to 100 series-connected structure units which are selected from the group consisting of one or more glycine and alanine; each b is independently 0 or 1, indicating the presence or absence of LA3; x is OH or NH.sub.2 group; FIG. 29A and FIG. 29B are isomers).

(30) FIG. 30A and FIG. 30B. The molecular schematic diagram of preferred ADC4 molecules (n is an integer from 1-100, d is any of the integers from 1-20, X in ligase recognition sequence LPXT of is glutamic acid (E) or any other natural/unnatural amino acid; m is 0 or any of the integers from 1-1000, Ab is an antibody, LA3 is linker moiety, comprising 1 to 100 series-connected structure units which are selected from the group consisting of one or more glycine and alanine; each b is independently 0 or 1, indicating the presence or absence of LA3; x is OH or NH.sub.2 group; FIG. 30A and FIG. 30B are isomers).

(31) FIG. 31A and FIG. 31B. The molecular schematic diagram of preferred ADCS molecules (n is an integer from 1-100, d is any of the integers from 1-20, X in ligase recognition sequence LPXT of is glutamic acid (E) or any other natural/unnatural amino acid; Ab is an antibody, LA3 is linker moiety, comprising 1 to 100 series-connected structure units which are selected from the group consisting of one or more glycine and alanine; each b is independently 0 or 1, indicating the presence or absence of LA3; x is OH or NH.sub.2 group; FIG. 31A and FIG. 31B are isomers).

(32) FIG. 32A and FIG. 32B. The molecular schematic diagram of preferred ADC6 molecules (n is an integer from 1-100, d is any of the integers from 1-20, X in ligase recognition sequence LPXT of is glutamic acid (E) or any other natural/unnatural amino acid; Ab is an antibody, LA3 is linker moiety, comprising 1 to 100 series-connected structure units which are selected from the group consisting of one or more glycine and alanine; each b is independently 0 or 1, indicating the presence or absence of LA3; x is OH or NH.sub.2 group; FIG. 32A and FIG. 32B are isomers).

(33) FIG. 33. The UPLC results of linker 2-DM1 intermediate (n=3, ring closed).

(34) FIG. 34. The MS results of linker 2-DM1 intermediate (n=3, ring closed).

(35) FIG. 35A, FIG. 35B, FIG. 35C, FIG. 35D, and FIG. 35E. The ring-open process of linker 2-DM1 intermediate (n=3, ring closed), FIG. 35A, FIG. 35B, FIG. 35C, FIG. 35D, and FIG. 35E are the UPLC results of ring-open reaction which was carried out for 20 minutes, 40 minutes, 60 minutes, 2 hours and 4 hours respectively.

(36) FIG. 36. The UPLC results of linker 2-DM1 intermediate (n=3, ring open).

(37) FIG. 37. The MS results of linker 2-DM1 intermediate (n=3, ring open).

(38) FIG. 38. The SDS-PAGE results of the ADC drug GQ1001

(39) FIG. 39A and FIG. 39B. The high accuracy molecular weight mass spectrometry (ESI-MS) results of ADCs GQ1001 light chain. FIG. 39A, light chain spectrum; FIG. 39B, Relative molecular weight of light chain (25281) after deconvolution with software ProMass 2.8.

(40) FIG. 40. The HIC-HPLC results of ADC GQ1001.

(41) FIG. 41. SEC-HPLC results of ADC GQ1001.

(42) FIG. 42. The binding affinity of ADC GQ1001 with BT474 cell surface ErbB2/Her2 receptor.

(43) FIG. 43. The binding affinity of ADC GQ1001 with SK-BR-3 cell surface ErbB2/Her2 receptor.

(44) FIG. 44. The effect of GQ1001, Kadcyla, Herceptin, DM1 on MCF-7 cell proliferation.

(45) FIG. 45. The effect of GQ1001, Kadcyla, Herceptin, DM1 on MDA-MB-468 cell proliferation.

(46) FIG. 46. The effect of GQ1001, Kadcyla, Herceptin, DM1 on BT-474 cell proliferation.

(47) FIG. 47. The effect of GQ1001, Kadcyla, Herceptin, DM1 on SK-BR-3 cell proliferation.

(48) FIG. 48. The effect of GQ1001, Kadcyla, Herceptin, DM1 on HCC1954 cell proliferation.

(49) FIG. 49. The effect of GQ1001, Kadcyla, DM1 on SK-OV-3 cell proliferation.

(50) FIG. 50. The effect of GQ1001, Kadcyla, Herceptin, DM1 on NCI-N87 Cell Proliferation.

(51) FIG. 51. The pharmacokinetic analysis of rats given a single intravenous injection of GQ1001, Kadcyla. SD rats are injected GQ1001 (10 mg/kg) or Kadcyla (10 mg/kg) via the tail vein, ELISA method is used to detect total ADC concentration in rat serum.

(52) FIG. 52. ADCs GQ1001 inhibit the xenograft tumor in HCC1954 nude mice (n=10, Mean?SEM).

(53) FIG. 53. The weight change of rats after a single intravenous injection of GQ1001 and Kadcyla. Healthy adult female rats are administered GQ1001 (6, 60 mg/kg) or Kadcyla (60 mg/kg) by a single injection via the tail vein. Rats in GQ administration group show no significant difference (P>0.05) in weight gain compared to rats in the vehicle control group, rats in Kadcyla administration group are significantly lower (P<0.05 vs Vehicle) in weight,

(54) FIG. 54A and FIG. 54B. The change of ALT (FIG. 54A) and AST (FIG. 54B) level in rats after a single intravenous injection of GQ1001 and Kadcyla. Healthy adult female rats are administered GQ1001 (6, 60 mg/kg) or Kadcyla (60 mg/kg) by a single injection via the tail vein. Rats in GQ1001 administration group show no significant change (P>0.05) in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) compared to rats in the vehicle control group. ALT and AST in rats in Kadcyla administration group are significantly increased (P<0.05 vs Vehicle).

(55) FIG. 55. The HPLC results of a ring-open reaction solution of linker 5-Mc-Val-Cit-Pab-MMAE drug intermediates (n=3).

(56) FIG. 56, The MALDI-TOF mass results of a ring-open reaction solution of linker 5-Mc-Val-Cit-Pab-MMAE drug intermediates (n=3).

BEST MODE FOR CARRYING OUT THE INVENTION

(57) The present invention is further illustrated in combination with specific examples shown below. It should be understood that these examples are merely intend to illustrate the present invention but not to limit the scope of the invention.

(58) Unless otherwise stated, all scientific and technical terms used herein are of the same meaning as those understood by a person skilled in the art. In addition, any methods and materials similar or equivalent to the contents described is applicable in the method of the present invention. The preferred implementation method and the material described herein are exemplary only.

EXAMPLE

Example 1the Production, Purification and Characterization of Anti-Human ErbB2/Her2 Antibody T-LCCT.SUB.L.-HC

(59) 1) The Production of Antibody T-LCCT.sub.L-HC

(60) SEQ ID No. 1 antibody T-LCCTL-HC encoding plasmid construct was transfected into CHO cells and the cell population was established and screened for a highly expressed cell population, which was cultured with reference to the culture process of Trastuzumab in a 5-10 L reactor, and supernatant was collected.

(61) 2) The Purification of Antibody T-LCCT.sub.L-HC

(62) The purification of T-LCCT.sub.L-HC was carried out in a standard process using the combination of Mab Select affinity chromatography and Sepharose S cation exchange chromatography, the purified products were dissolved in the original Trastuzumab drug buffer (5 mM histidine-HCl, 2% Trehalose, 0.009% Polysorbate 20, PH 6.0), and frozen in small aliquots.

(63) 3) The Quality Control of Antibody T-LCCT.sub.L-HC

(64) The purity of the above purified antibody T-LCCT.sub.L-HC is 98.5% by SDS-PAGE; the content of high molecular weight polymer of the sample is less than 0.4% by SEC-HPLC; endotoxin content is less than 0.098 EU/mg.

Example 2The Preparation of Linker 2-DM1 Intermediate (n=3, Ring-Open)

(65) 1) The Preparation and Quality Control of Linker 2-DM1 Intermediate (n=3, Ring-Closed)

(66) Linker 2 (n=3) and DM1 was weighed in a 1:1 molar ratio, mixed and dissolved sufficiently and react at 0-40? C. for 0.5-20 h, to give linker 2-DM1 intermediate (n=3, ring-closed, structure as shown in FIG. 12). The purity and molecular weight of linker 2-DM1 intermediate (n=3, ring-closed) were detected by UPLC-M, and results showed that the apparent purity is 100% (a mixture of isomers, roughly in 1:1 ratio, shown in FIG. 33), the found molecular weight is 1274 (FIG. 34), which is consistent with expectation.

(67) 2) The Ring-Open Reaction and Purification of Linker 2-DM1 Intermediate (n=3, Ring-Closed)

(68) The solution of linker 2-DM1 intermediate (n=3, ring-closed) was mixed with an appropriate amount of Tris Base solution or other solution to promote the ring-open reaction, the reaction was carried out at 0-40? C. for 0.2-20 h, the resulting structure of linker 2-DM1 intermediate (n=3, ring-open) is shown in FIG. 16. UPLC results of ring-open reaction at 20 minutes, 40 minutes, 60 minutes, 2 hours and 4 hours were shown in FIG. 35A-E. As the reaction proceeds, the ratio of linker 2-DM1 intermediate (n=3, ring-open) in the reaction mixture increased (from 10 to 73%). The preparation of a highly pure linker 2-DM1 intermediate (n=3, ring-open) can be achieved by semi-preparative/preparative HPLC, regardless of the succinimide ring-open efficiency in linker 2-DM1 intermediate (n=3, ring-open), thus ensuring the subsequent use in antibody coupling.

(69) 3) Quality Control of Linker 2-DM1 Linker Intermediate (n=3, Ring-Open)

(70) An appropriate amount of linker 2-DM1 intermediate (n=3, ring-open) was weighed and the purity and molecular weight was detected by UPLC-MS, the results are shown in FIG. 36 and FIG. 37. The purity of the HPLC-purified linker 2-DM1 intermediate (n=3, ring-open) is 100%, the found mass is 1291.8, which is consistent with expectation, laying a solid foundation for the subsequent production of ADCs GQ-1001.

Example 3Preparation of ADC GQ1001

(71) The ADC of this invention is prepared by site-specific coupling of linker 2-DM1 intermediate (n=3, the ring-open) with antibody T-LCCT.sub.L-HC under the catalysis of a transpeptidase (FIG. 28), wherein, n is 3, d is 2, the X in the ligase recognition sequence LPXT is a glutamic acid (E).

(72) 1) The Treatment of Antibody T-LCCT.sub.L-HC

(73) The storage buffer of antibody T-LCCT.sub.L-HC was exchange to 1? ligase buffer by ultrafiltration, dialysis or desalination. The main component of 1? ligase buffer was 50 mM Tris-HCl (pH 5-8), 150 mM NaCl, with or without CaCl.sub.2.

(74) 2) Solid-Phase Preparation of ADC GQ1001

(75) The present invention utilizes the coupling reaction of an optimized and engineered transpeptidase catalyzed antibody T-LCCT.sub.L-HC based on Sortase with linker 2-DM1 intermediate (n=3, ring-open), to produce the ADC GQ1001.

(76) In the 1? transpeptidase buffer, the antibody T-LCCT.sub.L-HC and linker 2-DM1 intermediate (n=3, ring-open) were fully mixed at an appropriate mole ratio (1:1 to 1:100), and the mixture was injected into a solid phase coupling column. There are immobilized transpeptidase on the solid-phase matrix, which catalyze the coupling reaction between antibody T-LCCT.sub.L-HC and linker 2-DM1 intermediate (n=3, ring-open). The coupling reaction is carried out at 4-40? C. for 0.5 to 20 hours. After reaction, the reaction mixture was removed from the solid phase coupling column, and treated by ultrafiltration or dialysis to remove unreacted drug intermediates. Purified ADC GQ1001 was stored in the original Kadcyla buffer (10 mM Sodium Succinate, pH5.0; 100 mg/ml Trahelose; 0.1% (w/v) Polysorbate 20; with reference to Kadcyla Formulation), and stored at 4? C. or ?80? C.

Example 4SDS-PAGE Analysis of ADC GQ1001

(77) The coupling efficiency and purity of GQ1001 can be detected by SDS-PAGE after the coupling reaction. As shown in FIG. 38, the coupling took place on the light chains of antibody T-LCCT.sub.L-HC in a site-specific manner, and an obvious molecular weight change was observed for the DM1 coupled light chain of GQ1001, in comparison with the uncoupled T-LCCT.sub.L-HC light chain. There is not any uncoupled light chain in the coupled product, which indicating a coupling efficiency as high as 95%. the purity of the coupled product is in consistent with expectation.

Example 5the High Accuracy Molecular Weight Mass (ESI-MS) Analysis of ADC GQ1001

(78) High accuracy molecular weight mass spectrometry was used to analyze the light chain of ADC GQ1001, and the results showed that the apparent mass is 25281, while the theoretical molecular weight is 25284, which is consistent with expectation, confirming that there is a cytotoxin coupled to the end of each light chain. The results of the high accuracy molecular weight mass spectrum (ESI-MS) are shown in FIGS. 39A and B.

Example 6HIC-HPLC Analysis of the ADC GQ1001

(79) Butyl-HIC column is used to detect the DAR distribution of ADC GQ1001, and the result is shown in FIG. 40. The cytotoxin-free antibody T-LCCT.sub.L-HC is less than 5%; the majority of coupled product is GQ1001 with a DAR of 2, and the overall DAR of ADC GQ1001 is about 1.8.

Example 7SEC-HPLC Analysis of the ADC GQ1001

(80) SEC-HPLC was used to detect the degree of high molecular weight aggregation of ADC GQ1001. The result is shown in FIG. 41, high molecular weight polymer is not found in the ADC GQ1001, which indicated the damage caused by the coupling reaction is almost negligible.

Example 8the Binding Affinity of ADC GQ1001 to the Cell Surface ErbB2/Her2

(81) 1) Human breast cancer BT-474 cells or SK-BR-3 cells were collected and made into single cell suspension, adjusted the cell density to (0.5-5)?10.sup.6/ml. Take 5?10.sup.5 cells/test, and 6.25 nM of Herceptin, T-LCCTL-HC, or GQ1001 was added respectively. The mixture was incubated at 4? C. for 60 min. 1 ml washing solution (PBS+1% BSA) was added, centrifuged at 1000 rpm for 5 min, and the supernatant was removed. The treatment was repeated twice.

(82) 2) 100 ?L FITC-Goat anti-human IgG antibody dilution was added to Herceptin, T-LCCTL-HC and GQ1001 incubated cells respectively, incubated at 4? C. for 30 min in dark. 1 ml washing solution was added, centrifuged at 1000 rpm for 5 min, and the supernatant was removed. The treatment was repeated twice. The cells are resuspended in 500 ?L PBS, pass through a 300 mesh sieve, and stored in an ice box in dark, flow cytometer detection was carried out by the BD C6. The results were shown in FIGS. 42-43, The binding affinity of Herceptin, T-LCCT.sub.L-HC, GQ1001 to the ErbB2/Her2 receptor on the surfaces of BT-474 and SK-BR-3 cells has no significant difference.

Example 9the Effect of ADCs GQ1001 on the Proliferation of Tumor Cells with Different Levels of ErbB2/Her2 Expression

(83) 1) ErbB2/Her2 low expressing human breast cancer cells MCF-7, MDA-MB-468, ErbB2/Her2 high expressing human breast cancer cells BT-474, SK-BR-3, HCC1954, human ovarian cancer cells SK-OV-3, human gastric cancer cell NCI-N87 was seeded into a 96-well plate at 100 ?l/well (containing 1000 to 10000 cells), and incubated in a cell culture incubator overnight (37? C., 5% CO2, 95% air, 100% humidity).

(84) 2) The cells incubated overnight was added GQ1001, Kadcyla, Herceptin, and DM1 at different concentrations (30, 10, 3.333, 1.111, 0.370, 0.123, 0.041, 0.014, 0.005 nM), the control group was added 50 ?M Puromycin, incubated at 37? C. for a further 48?96 h.

(85) 3) The cell plate was removed from the incubator, equilibrated for about 30 minutes to room temperature. Each well was added 100 ?l CellTiter Glo reagent, shocked in an oscillator for 2 min, then left stand for 10 min at room temperature in dark, the relative light units (RLU) is measured with a BioTech Gen5 microplate reader.

(86) 4) The results of effects of different drugs on the inhibition of tumor cell proliferation are shown in Table 1 and FIGS. 44-50. DM1 has a significant inhibition effect on the proliferation of all cells, either with high or low ErbB2/Her2 expression, but Herceptin and GQ1001 only have a significant inhibition effect on the proliferation of cells with high ErbB2/Her2 expression, and no significant inhibition was observed for cells with low ErbB2/Her2 expression. Kadcyla only inhibits the proliferation of cells with low ErbB2/Her2 expression at high concentrations.

(87) TABLE-US-00001 TABLE 1 Inhibition effects of different drugs on tumor cell proliferation (IC.sub.50, nM) Drug Cell line GQ1001 Kadcyla Herceptin DM1 MCF-7 5.708 MDA-MB-468 4.218 BT-474 0.410 0.144 16.270 SK-BR-3 0.140 0.030 1.918 HCC-1954 0.149 0.049 2.897 SK-OV-3 0.144 0.049 1.593 NCI-N87 0.113 0.031 0.229 7.185 NOTE: Not measured

Example 10In Vivo Pharmacokinetic Study in Rats

(88) 1) 160?180 g SPF grade female SD rats were randomly divided into GQ1001 group, Kadcyla group and blank control group, with 4 rats in each group.

(89) 2) 10 mg/kg GQ1001 (batch number: 20141128, purity>98%) or Kadcyla (Lot N1003), were administered via intravenous injection, and an equal volume of PBS (pH 7.4) was administered for the control group.

(90) 3) 100?200 ?L blood samples (with no anticoagulant added) were taken from the intraocular angular vein at 1 h, 1 day, 2 days, 3 days, 4 days, 6 days, 8 days, 13 days, 17 days, 21 days, 28 days after the drug administration. The collected blood samples were placed on ice for 1?2 h, then centrifuged at 4000 rpm for 20 min (4? C.), the supernatant was divided into small aliquots and added to new EP tubes, and stored at ?80? C. for subsequent use.

(91) 4) The total contents of GQ1001 and Kadcyla in serum were detected by ELISA.

(92) 5) The results show that, one day after the administration, the blood concentrations of GQ1001 and Kadcyla decreased rapidly, which were respectively 44.7% and 42.5% of those detected 1 h after administration, no significant difference was observed between the two. This reduction is due to the rapidly systemic distribution of the ADCs after administration. 6 days after administration, GQ1001 and Kadcyla were respectively 20.9% and 20.5% of those detected 1 h after administration; 13 days after administration, GQ1001 and Kadcyla were respectively 9.9% and 12.2% of those detected 1 h after administration. 21 days after administration, GQ1001 and Kadcyla were respectively 5.5% and 7.7% of those detected 1 h after administration. 28 days after the administration, GQ1001 and Kadcyla were respectively 3.5% and 5.2% of those detected 1 h after administration. These results indicate that GQ1001 and Kadcyla showed no significant difference in the attenuation rate in female rats (FIG. 51).

Example 11In Vivo Pharmacodynamics Evaluation of ADC GQ1001

(93) 1) HCC1954 breast cancer cells in the logarithmic growth phase were collected and adjusted with matrigel buffer (PBS: BD Matrigel=1:1) to a cell density of 2.5?10.sup.7/ml. 0.2 ml of this prepared HCC1954 cell suspension was injected subcutaneously to the right scapula of each BALB/c nude mouse (6 to 8 week-old, SPF grade, female).

(94) 2) 7 days after cell inoculation, the diameter of the tumor was measured by caliper and the tumor volume was calculated according to the Formula: V=0.5a?b.sup.2 (a is the longest diameter of the tumor, b is the shortest diameter of the tumor). Animals with tumor volumes of 130?140 mm.sup.3 were randomized into 5 groups: the vehicle control group, the GQ1001 0.5 mg/kg, the GQ1001 5 mg/kg group, the Kacyla 5 mg/kg group and the Herceptin 5 mg/kg group, with 10 animals in each group. Administration was via the tail vein injection, and the control group received an equal volume of the vehicle. The tumor size was measured twice a week within 31 days, then once a week afterwards. The tumor volumes at each time point were calculated and compared between groups. At the same time, the T-C or T/C value was used as the index to evaluate the anti-tumor activity of each drug. T-C value is calculated as the follows: T is the average time (Days) when the average tumor volume of each treatment group reaches a preset size (500 mm.sup.3), C is the average time (Day) when the average tumor volume of the controlled group reaches the preset size (500 mm.sup.3). While T/C (percentage) is the index for tumor inhibition effect, T is the average tumor volume of all the drug treatment groups at a fixed time point, and C is the average tumor volume of the control group at the fixed time point.

(95) 3) The tumor volumes in the GQ1001 5 mg/kg group and Kadcyla 5 mg/kg group were significantly smaller than control group 10 days after administration, and some tumors even became undetectable. No sign of tumor regrowth appeared until the end of the treatment, 38 days after the administration (Table 2, FIG. 52). These results indicate that both GQ1001 and Kadcyla have significant inhibition on ErbB2/Her2 positive breast cancer at the dosage of 5 mg/kg.

(96) TABLE-US-00002 TABLE 2 The growth inhibition of ADC GQ1001 on HCC1954 xenograft in mice T-C (days) Tumor Size (mm.sup.3).sup.a T/C.sup.b at 500 p Treatment at day 31 (%) mm.sup.3 value Vehicle 2301 ? 566 GQ1001 (0.5 mg/kg) 1045 ? 211 45.4 7 0.359 GQ1001 (5 mg/kg) 34 ? 6 1.5 >17 0.026 Kadcyla (5 mg/kg) 29 ? 4 1.3 >17 0.026 Herceptin (5 mg/kg) 1333 ? 155 57.9 3 0.587 .sup.aMean ? SEM. .sup.bTumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

Example 12Toxicity Studies of Single Injection of ADC GQ1001

(97) 1) Healthy adult female SD rats were randomly divided into four groups (n=6/group): the vehicle control group (0 mg/kg), the GQ1001 6 mg/kg group, the GQ1001 60 mg/kg group, and the Kadcyla 60 mg/kg group. The drug was administered by a single injection via tail vein. The administration dose is 10 ml/kg, and the administration rate is about 1 ml/min. During the experiment, the animals were subjected to clinical inspection, and were examined for body weight, food intake, blood count, blood biochemical indices. At the end of the experiment, all animals were euthanized, anatomized and checked systematically. All major organs were weighed, organ coefficient was calculated and any visible lesions were recorded.

(98) 2) The results showed that, during the experiment, no clinical abnormality was observed for any animal in both GQ1001 groups; For the Kadcyla 60 mg/kg group, visible nasal secretions (2/6), ears flushing (ear portions) and swelling (6/6), fluffy coat (6/6), weight loss (1/6), arched (1/6), ears and limbs pale (1/6) were observed 5 days after administration (D5); wherein ear swelling disappeared on D6, nasal secretions disappeared on D7; 4 out of the 6 animals went back to normal on D8, and the rest 2 out of the 6 animals went all back to normal before being euthanized on D15 except visible hair fluffy. Compared with the vehicle control group, no change in body weight associated with administration in both GQ1001 groups was observed; while in the Kadcyla 60 mg/kg group, a significant weight loss was observed after administration (D2?D12). The results are shown in FIG. 53.

(99) Hematology and clinical biochemical analysis results showed that no significant toxic reaction in both GQ1001 groups, while animals in the Kadcyla 60 mg/kg group had reduced erythroid index (RBC, HGB, HCT, Retic), and increased white blood cell and its subgroup count, ALT, AST, TBIL, GGT increased or had a tendency to increase, especially, the ALT and AST increased significantly, suggesting a drug-related liver toxicity of Kadcyla in a single injection at a dose of 60 mg/kg. The results are shown in FIG. 54.

(100) Systematic anatomy and gross observation showed that, no abnormal changes were observed for animals in each GQ1001 dose group, while general changes associated with administration were observed for animals in Kadcyla 60 mg/kg dose group, including the spleen large (6/6), blunt liver edge round (4/6), thymus (1/6).

(101) The above results suggested that, at the equivalent doses of 60 mg/kg, acute toxicity of GQ1001 is significantly lower than that of Kadcyla.

Example 13the Preparation of Stable Linker 5-Mc-Val-Cit-Pab-MMAE Drug Intermediate (n=3, Ring-Open)

(102) 1) The Preparation and Quality Control of Linker 5-Mc-Val-Cit-Pab-MMAE Drug Intermediate (n=3, Ring-Closed)

(103) Linker 5 (n=3) and Mc-Val-Cit-PAB-MMAE were weighed at 1:1 molar ratio, dissolved and fully mixed, and kept at 0-40? C. for 0.5-20 h, to obtain the linker 5-Mc-Val-Cit-PAB-MMAE (n=3, ring-closed), as shown in FIG. 19.

(104) 2) The Ring-Open Reaction of Linker 5-Mc-Val-Cit-PAB-MMAE Drug Intermediate (n=3, Ring-Closed)

(105) Linker 5-Mc-Val-Cit-PAB-MMAE drug intermediate (n=3, ring-closed) was treated with an appropriate amount of Tris Base solution or other solution to promote the ring-open reaction, the reaction was carried out at 0-40? C. for 0.2-20 h, to give the ring-open form of the intermediate as shown in FIG. 23. The purity and molecular weight of the intermediates (ring-open) was analyzed by HPLC, and the results are shown in FIG. 55, the isomers cannot be separated by common HPLC. The MALDI-TOF mass spectrum was used to detect the ring-open reaction mixture and a series of molecule weight was obtained, as shown in FIG. 56, the theoretical mass is 1681, and the found mass is 1702, 1718, corresponding to Na and K salts respectively, fully in consistent with expectation, confirming that an expected ring-opened product was obtained. The preparation of a highly pure ring-open intermediate can be achieved by semi-preparative/preparative HPLC regardless of the succinimide ring-open efficiency, thus ensuring the subsequent use in antibody coupling.