NOVEL LINKER, PREPARATION METHOD, AND APPLICATION THEREOF

Abstract

Provided in the present invention is a linker and a preparation method thereof, wherein one end of the linker may covalently link a small molecule compound and the like and the other end may specifically and covalently link a targeting substance site under the action of Sortase enzyme. The linker of the present invention can be used to prepare a targeting drug conjugate.

Claims

1.-60. (canceled)

61. A coupling intermediate having the structure of formula (Ill) or (IV):
PCA1−(LA).sub.a−CCA1−Payload.sub.h   (Ill),
or
Payload.sub.h−CCA2−(LA).sub.a−PCA2   (IV), wherein: Payload is a nucleic acid; h is an integer from 1 to 1000; when h>1, Payload is same or different; PCA1 is a receptor substrate recognition sequence of Sortase; PCA2 is a donor substrate recognition sequence of Sortase; each of CCA1 and CCA2 is chemical conjugate region for connecting a payload to be connected; LA is a connecting region, to connect PCA and CCA, wherein a is 0 or 1; PCA1−(LA).sub.a−CCA1 (I) is selected from linkers 1-25 below; and CCA2−(LA).sub.a−PCA2 (II) is selected from linkers 26-35 below: ##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005## ##STR00006## ##STR00007## wherein n is an integer of 1-100, m is 0 or an integer 1-1000, and X is −OH or −NH.sub.2.

62. The coupling intermediate according to claim 61, wherein CCA2−(LA).sub.a−PCA2 (II) is linker 26: ##STR00008## linker 26 and wherein n is an integer of 1-100, m is 0 or an integer 1-1000, and X is —OH or —NH.sub.2.

63. The coupling intermediate according to claim 61, wherein X is —OH.

64. The coupling intermediate according to claim 61, wherein the nucleic acid is a siRNA.

65. The coupling intermediate according to claim 64, wherein the sequence of the siRNA is: TABLE-US-00002 5′-GUAUGACAACAGCCUCAAGdTdT-3′ 3′-dTdTCAUACUGUUGUCGGAGUUC-5′.

66. The coupling intermediate according to claim 61, wherein in preparation of the nucleic acid, a modification group of thiol, hydroxyl, carboxyl, amino, alkoxy-amino, alkynyl, azide or tetrazine is introduced at a preferred position, in order to covalently link with PCA1−(LA).sub.a−CCA1 (I) or CCA2−(LA).sub.a−PCA2 (II).

67. The coupling intermediate according to claim 66, the modification group is thiol.

68. The coupling intermediate according to claim 61, wherein the coupling intermediate has the structure of the following formula 47: ##STR00009##

69. A targeting drug conjugate, wherein the said conjugate having a structure represented by the formula (V) or (VI):
T−PCA1−(LA).sub.a−CCA1−Payload.sub.h   (V) or
Payload.sub.h−CCA2−(LA).sub.a−PCA2−T   (VI) wherein: Payload is a nucleic acid; T is a targeting moiety; h is an integer from 1 to 1000, when h>1, Payload is same or different; and h is an integer from 1 to 1000; when h>1, Payload is same or different; PCA1 is a receptor substrate recognition sequence of Sortase; PCA2 is a donor substrate recognition sequence of Sortase; each of CCA1 and CCA2 is chemical conjugate region for connecting a payload to be connected; LA is a connecting region, to connect PCA and CCA, wherein a is 0 or 1; PCA1−(LA).sub.a−CCA1 (I) is selected from linkers 1-25 below; and CCA2−(LA).sub.a−PCA2 (II) is selected from linkers 26-35 below: ##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016## wherein n is an integer of 1-100, m is 0 or an integer 1-1000, and X is —OH or —NH.sub.2, and wherein the PCA1−(LA).sub.a−CCA1 (I) or CCA2−(LA).sub.a−PCA2 (II) is site-specifically coupled to the targeting moiety.

70. The coupling intermediate according to claim 69, wherein CCA2−(LA).sub.a−PCA2 (II) is linker 26: ##STR00017## wherein X is —OH or —NH.sub.2, and wherein the linker 26 is site-specifically coupled to the targeting moiety.

71. The targeting drug conjugate according to claim 69, wherein the nucleic acid is a siRNA.

72. The targeting drug conjugate according to claim 71, wherein the sequence of the siRNA is: TABLE-US-00003 5′-GUAUGACAACAGCCUCAAGdTdT-3′ 3′-dTdTCAUACUGUUGUCGGAGUUC-5′.

73. The targeting drug conjugate according to claim 69, wherein in preparation of the nucleic acid, a modification group of thiol, hydroxyl, carboxyl, amino, alkoxy-amino, alkynyl, azide or tetrazine is introduced at a preferred position, in order to covalently link with PCA1-(LA).sub.a−CCA1 (I) or CCA2−(LA).sub.a−PCA2 (II).

74. The targeting drug conjugate according to claim 73, wherein the modification group is thiol.

75. The targeting drug conjugate according to claim 69, wherein the targeting moiety is an antibody, a single chain antibody, a nano-antibody, a single domain antibody, an antibody fragment, an antibody mimetics, or a cell specific binding protein/peptide.

76. The targeting drug conjugate according to claim 69, wherein the targeting moiety is capable of binding to a target cell of: a tumor cell, a commonly used genetic engineering transfected cell, a virus-infected cell, a microorganism infected cell or a primary cultured cell.

77. A pharmaceutical composition, wherein the said composition comprises the targeting drug conjugate according to claim 69 and a pharmaceutically acceptable carrier or excipient.

78. A method for treatment of a disease of a subject comprising administration of the pharmaceutical composition according to claim 77 in an effective amount to the subject.

79. The method for treatment of a disease of a subject according to claim 78, wherein the said disease is selected from cancers, autoimmune diseases, inflammatory diseases, cardiovascular diseases and neurodegenerative diseases.

80. A method for preparing the targeting drug conjugate according to claim 69, which comprises connecting the coupling intermediate according to claim 61, with a targeting moiety (T) in a site specific way with Sortase.

Description

DESCRIPTION OF THE DRAWINGS

[0073] FIG. 1 A general structure of linker 1 (n=1-100, X is OH or NH2)

[0074] FIG. 2 A general structure of linker 2 (n=1-100, X is OH or NH2)

[0075] FIG. 3 A general structure of linker 3 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0076] FIG. 4 A general structure of linker 4 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0077] FIG. 5 A general structure of linker 5 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0078] FIG. 6 A general structure of linker 6 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0079] FIG. 7 A general structure of linker 7 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0080] FIG. 8 A general structure of linker 8 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0081] FIG. 9 A general structure of linker 9 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0082] FIG. 10 A general structure of linker 10 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0083] FIG. 11 A general structure of linker 11 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0084] FIG. 12 A general structure of linker 12 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0085] FIG. 13 A general structure of linker 13 (n=1-100, X is OH or NH2)

[0086] FIG. 14 A general structure of linker 14 (n=1-100, X is OH or NH2)

[0087] FIG. 15 A general structure of linker 15 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0088] FIG. 16 A general structure of linker 16 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0089] FIG. 17 A general structure of linker 17 (n=1-100, X is OH or NH2)

[0090] FIG. 18 A general structure of linker 18 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0091] FIG. 19 A general structure of linker 19 (n=1-100, X is OH or NH2)

[0092] FIG. 20 A general structure of linker 20 (n=1-100, X is OH or NH2)

[0093] FIG. 21 A general structure of linker 21 (n=1-100, X is OH or NH2)

[0094] FIG. 22 A general structure of linker 22 (n=1-100, X is OH or NH2)

[0095] FIG. 23 A general structure of linker 23 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0096] FIG. 24 A general structure of linker 24 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0097] FIG. 25 A general structure of linker 25 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0098] FIG. 26 A general structure of linker 26 (X is OH or NH2)

[0099] FIG. 27 A general structure of linker 27 (m=0, 1-1000, X is OH or NH2)

[0100] FIG. 28 A general structure of linker 28 (X is OH or NH2)

[0101] FIG. 29 A general structure of linker 29 (X is OH or NH2)

[0102] FIG. 30 A general structure of linker 30 (X is OH or NH2)

[0103] FIG. 31 A general structure of linker 31 (X is OH or NH2)

[0104] FIG. 32 A general structure of linker 32 (X is OH or NH2)

[0105] FIG. 33 A general structure of linker 33 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0106] FIG. 34 A general structure of linker 34 (X is OH or NH2)

[0107] FIG. 35 A general structure of linker 35 (m=0, 1-1000, X is OH or NH2)

[0108] FIG. 36 The preparation process of antibody-dugs and antibody-siRNA conjugates

[0109] FIG. 37 The chemical structure of linker 1

[0110] FIG. 38 The UPLC profile of linker 1

[0111] FIG. 39 The ES I-MS profile of linker 1

[0112] FIG. 40 The UPLC profile of maysteine derivative DM1

[0113] FIG. 41 The ES I-MS profile of maysteine derivative DM1

[0114] FIG. 42 The structure of a coupling intermediate made of maysteine derivative DM1

[0115] FIG. 43 The UPLC-MS profile of an coupling intermediate made of maysteine derivative DM1

[0116] FIG. 44 The chemical structure of linker 26

[0117] FIG. 45 The HPLC profile of linker 26

[0118] FIG. 46 The ES I-MS of linker 26

[0119] FIG. 47 The structure of a coupling intermediate of GAPDH siRNA-linker 26

[0120] FIG. 48 The coupling efficiency of GAPDH siRNA with linker 26, checked by SDS PAGE 1: GAPDH siRNA; 2: the coupling intermediate GAPDH siRNA-linker 26

[0121] FIG. 49 The structure of coupling product: GAPDH siRNA-linker 26-GFP

[0122] FIG. 50 The coupling efficiency of GAPDH siRNA-linker 26 with GFP checked by native PAGE 1: GAPDH siRNA-linker 26; 2: 0 min, 3: 60 min, 4 120 min, *: final product siRNA-GFP; **: the coupling intermediate GAPDH siRNA-linker 26

[0123] FIG. 51 The structure of linker 2

[0124] FIG. 52 The HPLC profile of linker 2

[0125] FIG. 53 The ESI-MS of linker 2

[0126] FIG. 54 The structure of linker 3

[0127] FIG. 55 The HPLC profile of linker 3

[0128] FIG. 56 The ES I-MS of linker 3

[0129] FIG. 57 The structure of linker 9

[0130] FIG. 58 The HPLC profile of linker 9

[0131] FIG. 59 The ES I-MS of linker 9

DETAILED DESCRIPTION

[0132] The present disclosure is further illustrated with the following specific examples, which, however, are not limitations to the present disclosure.

[0133] 1. The preparation of linker 1

[0134] When n=5, X is —OH, the general formula of linker 1 shown in FIG. 1 is shown in

[0135] FIG. 37. The linker was prepared via solid phase peptide synthesis protocol on Wang resin using Fmoc Chemistry. The c-amino group of lysine was deprotected, and N-Succinim idyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC) was chemically coupled to it in DMF. The linker was then cleaved from the resin and all protection groups were removed. The crude linker 1 was purified by HPLC, and characterized by ESI-MS. As shown in FIG. 38, the purity of the linker was 95.49%, and the found MS was 708.5 (M+1) shown in FIG. 39 (expected MW 707). This linker thus obtained will be coupled with small molecules, nucleic acids or tracer molecules.

[0136] 2. The preparation of a coupling intermediate made of linker 1 and DM1

[0137] Maytansine derivative DM1 was purchased from Jiangyin Concortis Bio-Technology Co., Ltd. UPLC analysis showed a purity of 91.43% and ESI-MS showed a molecular weight of 738.5(expected 738). The results were shown in FIGS. 40 and 41.

[0138] The synthetic linker 1 obtained above and the maytansine derivative DM1 were dissolved in a suitable solvent in equimolar ratio, the mixture was incubated at room temperature. The structure of the coupling intermediate is shown in FIG. 42. It was subjected to UPLC-MS analysis, and the results shown in FIG. 43. The coupling efficiency was 100%, expected molecular weight is 1447.9, ESI-MS found 1447(M-1).

[0139] The product obtained from the above procedure was site-specifically connected to a tumor-specific antibody or antibody analogue. The antibody-drug conjugate thus obtained was highly homogeneous, i.e., the number of drugs and the sites of coupling are highly specific. This highly homogenous ADC drugs can be used in a variety of tumor targeted therapies, including but not limited to breast cancer, stomach cancer, lung cancer, ovarian cancer and leukemia. In comparison with the ADCs already on the market, the highly homogenous new drugs prepared by the current invention, offer many advantages including but not limited to stability, reliability, efficacy and safety.

[0140] 3. The preparation of linker 26

[0141] When X is —OH, the general linker structure shown in 26 becomes the structure shown in FIG. 44.

[0142] A similar method as used for the preparation of linker 1 was used. The crude product was purified by HPLC, characterized by ESI-MS analysis. As shown in FIG. 45, the purity of linker 26 was more than 99%; the expected molecular weight of 765, ESI-MS found 764 (M-1), as shown in FIG. 46.

[0143] The linker 26 and those alike may be used to react with small molecules, nucleic acids or tracer molecules.

[0144] 4. The preparation of a conjugate intermediate with siRNA as the payload

[0145] A 5′-terminal thiol modified mice GAPDH siRNA was purchased from Genepharm Shanghai Ltd. The sequence of the said siRNA is:

TABLE-US-00001 5′-GUAUGACAACAGCCUCAAGdTdT-3′ 3′-dTdTCAUACUGUUGUCGGAGUUC-5′.

[0146] The modified siRNA and an excess of linker 26 were incubated in 1 x PBS buffer (pH7.4) at room temperature for 1-24 h. The extra linker 26 was removed by ultrafiltration to give a GAPDH siRNA-linker intermidiate as shown in FIG. 47. SDS PAGE indicated that the coupling efficency was >90% as shown in FIG. 48.

[0147] 5. Enzyme catalysed site specific coupling of siRNA and Green Florecein Proten(GFP)

[0148] Recombinant GFP was purified by nickel affinity purification, treated with TEV enzyme to release the polyglycine sequence as the substrate for Sortase, and the resulted GGG-GFP protein was collected.

[0149] Excess amount of GAPDH siRNA linker intermediate 26 and GGG-GFP was site-specifically coupled by a genetically engineered Sortase A in 1× PBS buffer (containing Tris pH8.0, NaCI, CaCl2) at 37° C. for 2h. Samples were taken at different time intervals. The structure of the final product is shown in FIG. 49. 15% non-denaturing SDS PAGE showed that the coupling efficebcy was 80% in 2 h (FIG. 50).

[0150] This result clearly indicated that siRNA was site-specific coupling to a protein. An important application of this method is the site specific coupling of a tumor targeting antibody or antibody analogue with siRNA of therapeutic value, creating a new generation of targeting siRNA drugs. Another important application of this method is the coupling of tumor targeting antibody or antibody analogue with a tracer molecule which offers a new generation of tumor tracing agents.

[0151] 6. The preparation of linkers 2, 3 and 9

[0152] When n=3, X is —NH2, the structure in formulus 2 become linker 2 (FIG. 51). A similar method as described for linker 1 was used to prepare linker 2. After purification, it was analized by ESI-MS. As shown in FIGS. 52, the purity of linker 2 is 97.3492%. The expected MS of linker 2 is 535 and found 536 (M+1) (FIG. 53).

[0153] When n=5, m=4, X is —OH, the chemical structure of linker 3 was specified and shown in FIG. 54. Similar protocol as descrobed for linker 1 was applied with modification. The crude product was purified by HPLC. After purification, it was analized by ESI-MS. As shown in FIGS. 55, the purity of linker 3 is 99.3650%. The expected MS of linker 3 is 954 and found 953 (M +−1) (FIG. 56).

[0154] When n=5, m=4, X is —OH, the chemical structure of linker 9 was specified and shown in FIG. 57. Similar protocol as descrobed for linker 1 was applied with modification. The crude product was purified by HPLC. After purification, it was analized by ESI-MS. As shown in FIGS. 58, the purity of linker 3 is 99.3650%. The expected MS of linker 9 is 1249 and found 1248 (M-1) (FIG. 59).

[0155] Linkers 2, 3, 9 thus obtained can be used to couple with small molecules, nucleic acids, or tracer molecules. Linker 9 has two reactive functional groups which can react with two small molecules, nucleic acids or tracer molecules.