Multispecific bioconjugate linker and synthesis method thereof

Abstract

This application discloses a multispecific bioconjugate linker and a method for synthesizing the same. Specifically, the application discloses a compound or its tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof. The application also discloses a method for preparing the compound and its use in tumor treatment.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a multispecific bioconjugation linker and a method for synthesizing the same. Background Art

[0002] Multispecific drug molecules are a hot topic in cancer treatment today. Compared to traditional drug design, which relies on a one-drug-per-target approach, multispecific drugs can simultaneously target multiple targets, enabling more precise and efficient attack on cancer cells while minimizing toxic side effects caused by off-target effects.

[0003] Although bispecific antibodies and multispecific antibodies have shown good effects in tumor treatment, the development of multispecific antibody drugs still faces many challenges. One of the most important problems is the preparation of multispecific antibodies. Traditional multispecific antibody preparation platforms mostly use antibody fusion expression for production. This method has the problem of antibody mismatch, which makes the downstream purification process extremely difficult, making it difficult to expand production. There are some multifunctional compounds, but these compounds do not have specificity and bioorthogonality, are prone to mutual interference, and cannot truly achieve precise coupling of multiple compounds. Summary of the Invention

[0004] The present application provides a compound, which can be a multispecific bioconjugate linker (T-Linker) or a multispecific conjugate (T-Body). The present application can have one or more of the following effects: (1) The compound of the present application can be a multispecific compound, and the multispecific conjugate can exhibit good anti-tumor activity, IC 50 The value can be lower than 10 nanomolar; (2) In the preparation method of the compound of the present application, the reaction can achieve fixed-point, quantitative, and modular connection, and ultimately a multispecific conjugate with clear structure, uniformity, and controllable quality can be obtained.

[0005] The present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which has a structure as shown in Formula Ia or Formula Ib:

[0006]

[0007] Said Wa is a trivalent group, and Wb is a tetravalent group;

[0008] Said A, B and C are each independently selected from the following groups:

[0009]

[0010] where R 12 , R 13 and R14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0011] Among them, A, B and C, Cannot exist simultaneously;

[0012] Among them, A, B and C, Cannot exist simultaneously;

[0013] Among them, A, B and C, Cannot exist simultaneously;

[0014] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not an alkynyl group;

[0015] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino,

[0016] A, B and C are not the same

[0017] In one embodiment, the compound wherein:

[0018] (1) A is B is C is

[0019] (2) A is C is or

[0020] (3) A is B is C is or

[0021] (4) A is B is C is or

[0022] (5) A is B is C is or

[0023] (6) A is B is C is or

[0024] (7) A is B is C is or

[0025] (8) A is B is C is or

[0026] (9) A is B is C is or

[0027] (10) A is B is C is or

[0028] (11) A is B is C is or

[0029] (12) A is B is C is or

[0030] (13) A is B is C is or

[0031] (14) A is B is C is

[0032] (15) A is B is C is

[0033] where R 12 , R 13 and R 14are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0034] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not an alkynyl group;

[0035] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino.

[0036] In one embodiment, the compound wherein:

[0037] (1) A is B is C is or

[0038] (2) A is B is C is or

[0039] (3) A is B is C is

[0040] where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0041] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino.

[0042] In one embodiment, the compound wherein:

[0043] (1) A is B is C is or

[0044] (2) A is B is C is or

[0045] (3) A is B is C is

[0046] where R 12 is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0047] When A, B and C, When both exist, the R 12 Not amino.

[0048] In one embodiment, the compound wherein:

[0049] (1) A is B is C is or

[0050] (2) A is B is C is or

[0051] (3) A is B is C is

[0052] where R 12 selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the junction site.

[0053] In one embodiment, the compound wherein: A is B is C is

[0054] where R 12 a group selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and chain alkyl, Represents the junction site.

[0055] In one embodiment, the compound wherein:

[0056]

[0057] Said W is a trivalent group or a tetravalent group, and said W is optionally substituted by one or more R,

[0058] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0059] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0060] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0061] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 Each is independently selected from the group consisting of alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0062] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Each independently selected from the following group: 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1-C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0063] R, R 1 , R 1a and R 1b are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0064] wherein m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 are each independently selected from a number greater than or equal to 0.

[0065] In one embodiment, the compound wherein J 1 , J 2 , and J 3 Each independently selected from the group consisting of: chain alkyl, and polyethylene glycol, or any combination thereof;

[0066] wherein m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3.

[0067] In one embodiment, the compound wherein X 1 , X 2 , and X 3 are each independently selected from the group consisting of -NH- and -O-.

[0068] In one embodiment, the compound wherein K 1 , K 2 , and K 3 Each independently selected from the group consisting of: chain alkyl, and polyethylene glycol, or any combination thereof;

[0069] wherein n1, n2, and n3 are each independently selected from the following group: 0, 1, 2, and 3.

[0070] In one embodiment, the compound wherein (K 1 )n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the group consisting of: -alkane-polyethylene glycol-alkane-, alkane and polyethylene glycol, or n1, n2 or n3 is each independently 0.

[0071] In one embodiment, the compound wherein (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 are each independently 0.

[0072] In one embodiment, the compound wherein Y 1 , Y 2 , and Y 3 Each independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-;

[0073] R 1 selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkanyl;

[0074] wherein n1, n2, and n3 are each independently selected from the following group: 0, 1, 2, and 3.

[0075] In one embodiment, the compound, wherein (Y 1 ) p1 ,(Y 2 ) p2 , and (Y 3 ) p3 Each independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkanyl; or p1, p2 or p3 are each independently 0.

[0076] In one embodiment, the compound wherein L 1 , L 2 , and L 3Each is independently selected from the following group: chain alkyl, and aryl, or any combination thereof; wherein q1, q2, and q3 are each independently selected from the following group: 0, 1, 2, and 3.

[0077] In one embodiment, the compound, wherein (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the group consisting of -alkyl-aryl-, alkyl and polyethylene glycol, or q1, q2 and q3 are each independently 0.

[0078] In one embodiment, the compound, wherein (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 are each independently selected from the group consisting of -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 are each independently 0.

[0079] For example, La, Lb and Lc can be each independently selected from the following group: -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)8-NH-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-, - C(=O)-NH-(CH2)2-NH-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0080] For example, La, Lb and Lc can each be independently selected from the following groups: -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0081] In one embodiment, the compound wherein:

[0082] W is selected from the following group:

[0083] trivalent alkanyl, trivalent cycloalkyl, trivalent heterocycloalkyl, trivalent alkenyl, trivalent alkynyl, trivalent aryl, and trivalent heteroaryl.

[0084] In one embodiment, the compound wherein:

[0085] W is selected from the following group:

[0086]

[0087] The X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 and X 18 Each independently selected from the group consisting of C, N, O, S, P, CH, CH2, NH, P(O) and P(S);

[0088] W is selected from the following group:

[0089]

[0090]

[0091] In one embodiment, the compound is selected from the group consisting of:

[0092]

[0093]

[0094]

[0095]

[0096] In one embodiment, the compound is selected from the group consisting of:

[0097]

[0098] A compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, having a structure as shown in Formula IIa or Formula IIb, wherein:

[0099]

[0100] The Wc is a trivalent group, and Wd is a tetravalent group;

[0101] The A 2 , B 2 and C 2 Each independently selected from the following group:

[0102] and covalent bonds;

[0103] where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0104] Represents the junction site,

[0105] The A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect;

[0106] Among them, P 1 , P 2 and P 3 Each is independently selected from the group consisting of lipids, proteins, nucleic acids, small molecules and polysaccharides, or any combination thereof.

[0107] In one embodiment, the compound wherein:

[0108] (1)A 2 for B 2 for C 2 for or covalent bond; or

[0109] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0110] (3)A 2 for B 2 for C 2 for or covalent bond; or

[0111] (4)A 2 for B 2 for C 2 for or covalent bond; or

[0112] (5)A 2 for B 2 for C 2 for or covalent bond; or

[0113] (6)A 2 for B 2 for C 2 for or covalent bond; or

[0114] (7)A 2 for B2 for C 2 for or covalent bond; or

[0115] (8)A 2 for B 2 for C 2 for or covalent bond; or

[0116] (9)A 2 for B 2 for C 2 for or covalent bond; or

[0117] (10)A 2 for or covalent bond, B 2 is a covalent bond, C 2 is a covalent bond; or

[0118] (11)A 2 for B 2 for C 2 for or covalent bond; or

[0119] (12)A 2 for B 2 for C 2 for or covalent bond; or

[0120] (13)A 2 for B 2 for C 2 for or covalent bonds;

[0121] where R 12 , R 13 and R 14are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0122] Represents the junction site,

[0123] The A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

[0124] In one embodiment, the compound wherein:

[0125] (1)A 2 for B 2 for C 2 for or covalent bond; or

[0126] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0127] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0128] where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0129] Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

[0130] In one embodiment, the compound wherein:

[0131] (1)A 2 for B 2 for C 2 for or covalent bond; or

[0132] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0133] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0134] where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0135] Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

[0136] In one embodiment, the compound wherein:

[0137] (1)A 2 for B 2 for C 2 for or covalent bond; or

[0138] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0139] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0140] where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol,

[0141] Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

[0142] In one embodiment, the compound wherein: A 2 for B 2 for C 2 for or covalent bond; where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

[0143] In one embodiment, the compound wherein: A 2 for B 2 for, C 2 for or covalent bond; where R 12 selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

[0144] In one embodiment, the compound wherein:

[0145] for

[0146] for

[0147] Said W is a trivalent group or a tetravalent group, and said W is optionally substituted by one or more R,

[0148] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0149] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0150] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3-(L 3 ) q3 -;

[0151] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 Each is independently selected from the group consisting of alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0152] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Each independently selected from the following group: 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0153] R, R 1 , R 1a and R 1b are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0154] wherein m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 are each independently selected from a number greater than or equal to 0.

[0155] In one embodiment, the compound wherein J 1, J 2 , and J 3 Each independently selected from the group consisting of: chain alkyl, and polyethylene glycol, or any combination thereof;

[0156] wherein m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3.

[0157] In one embodiment, the compound wherein X 1 , X 2 , and X 3 are each independently selected from the group consisting of -NH- and -O-.

[0158] In one embodiment, the compound wherein K 1 , K 2 , and K 3 Each is independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof; wherein n1, n2, and n3 are each independently selected from the following group: 0, 1, 2, and 3.

[0159] In one embodiment, the compound wherein (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the group consisting of: -alkane-polyethylene glycol-alkane-, alkane and polyethylene glycol, or n1, n2 or n3 is each independently 0.

[0160] In one embodiment, the compound wherein (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 are each independently 0.

[0161] In one embodiment, the compound wherein Y 1 , Y 2 , and Y 3 Each independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-; R 1Selected from the group consisting of hydrogen, protium, deuterium, tritium, and chain alkyl; wherein n1, n2, and n3 are each independently selected from the group consisting of 0, 1, 2, and 3.

[0162] In one embodiment, the compound, wherein (Y 1 ) p1 ,(Y 2 ) p2 , and (Y 3 ) p3 Each independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkanyl; or p1, p2 or p3 are each independently 0.

[0163] In one embodiment, the compound wherein L 1 , L 2 , and L 3 Each is independently selected from the following group: chain alkyl, and aryl, or any combination thereof; wherein q1, q2, and q3 are each independently selected from the following group: 0, 1, 2, and 3.

[0164] In one embodiment, the compound, wherein (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the group consisting of -alkyl-aryl-, alkyl and polyethylene glycol, or q1, q2 and q3 are each independently 0.

[0165] In one embodiment, the compound, wherein (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 are each independently selected from the group consisting of -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 are each independently 0.

[0166] In one embodiment, the compound wherein:

[0167] W is selected from the following group:

[0168] trivalent alkanyl, trivalent cycloalkyl, trivalent heterocycloalkyl, trivalent alkenyl, trivalent alkynyl, trivalent aryl, and trivalent heteroaryl.

[0169] In one embodiment, the compound wherein:

[0170] W is selected from the following group:

[0171]

[0172] The X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 and X 18 Each independently selected from the group consisting of C, N, O, S, P, CH, CH2, NH, P(O) and P(S);

[0173] The --- represents a double bond or a single bond.

[0174] In one embodiment, the compound wherein:

[0175] W is selected from the following group:

[0176]

[0177]

[0178]

[0179] In one embodiment, the compound wherein P 1 , P 2 and P 3 Each is independently selected from the following group: a nucleic acid molecule, a dye molecule, a cytokine, an antigen, and an antibody or an antigen-binding fragment thereof, or any combination thereof.

[0180] In one embodiment, the compound, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and a nanobody.

[0181] In one embodiment, the compound, wherein the antibody targets a target selected from the group consisting of 4-1BB, EGFR, CD3, Her2, CD47, and CD20.

[0182] In one embodiment, the compound, wherein the amino acid sequence of the antibody is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0183] In one embodiment, the compound is selected from the group consisting of:

[0184]

[0185]

[0186] Among them, P 1 , P 2 and P 3 Each is independently selected from antibodies targeting a target selected from the group consisting of 4-1BB, EGFR, CD3, Her2, CD47, and CD20.

[0187] In one embodiment, the compound is:

[0188]

[0189] Among them, P 1 , P 2 and P 3 The amino acid sequences can be:

[0190] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0191] SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 3, or

[0192] SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 3, or

[0193] SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 3, or

[0194] SEQ ID NO: 10, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0195] SEQ ID NO: 11, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0196] SEQ ID NO: 12, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0197] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 10, or

[0198] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, or

[0199] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, or

[0200] SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0201] In one embodiment, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which may have a structure as shown in Formula Ia or Formula Ib:

[0202]

[0203] in:

[0204] for

[0205] for

[0206] in:

[0207] (1) A is B is C is or

[0208] (2) A is B is C is or

[0209] (3) A is B is C is

[0210] where R 12 selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, represents the junction site;

[0211] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 )p1 -(L 1 ) q1 -;

[0212] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0213] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0214] Among them, J 1 , J 2 , and J 3 Each is independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof; m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3;

[0215] X 1 , X 2 , and X 3 are each independently selected from the group consisting of: -NH- and -O-;

[0216] (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -alkanyl-polyethylene glycol-alkanyl-, alkanyl and polyethylene glycol, or n1, n2 or n3 are each independently 0;

[0217] Y 1 , Y 2 , and Y 3 Each independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-; R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkanol; n1, n2, and n3 are each independently selected from the group consisting of 0, 1, 2, and 3;

[0218] (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the following group: -alkanyl-aryl-, alkanyl and polyethylene glycol, or q1, q2 and q3 are each independently 0;

[0219] W is selected from the following group:

[0220]

[0221]

[0222] For example, La, Lb and Lc can be each independently selected from the following group: -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)8-NH-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-phenyl-, - C(=O)-NH-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-NH-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, and -C(=O)-O-.

[0223] For example, La, Lb and Lc can each be independently selected from the following groups: -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0224] In one embodiment, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which may have a structure as shown in Formula Ia or Formula Ib:

[0225]

[0226] in:

[0227] for for

[0228] Among them: A is B is C is where R 12 a group selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and chain alkyl, represents the junction site;

[0229] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0230] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0231] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0232] J 1 , J 2 , and J 3 Each is independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof, m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3;

[0233] X 1 , X 2 , and X 3 are each independently selected from the group consisting of: -NH- and -O-;

[0234] (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 are each independently selected from the group consisting of -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 are each independently 0;

[0235] (Y 1 ) p1 ,(Y 2 ) p2 , and (Y 3 ) p3 Each independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2, or p3 are each independently 0;

[0236] (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 are each independently selected from the group consisting of -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2, and q3 are each independently 0;

[0237] W is

[0238]

[0239] For example, La, Lb and Lc can be each independently selected from the following group: -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)8-NH-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-, - C(=O)-NH-(CH2)2-NH-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0240] For example, La, Lb and Lc can each be independently selected from the following groups: -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0241] In one embodiment, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which may have a structure as shown in Formula IIa or Formula IIb:

[0242]

[0243] in,

[0244] for for

[0245] in,

[0246] A 2 for B 2 for C 2 for or covalent bond; where R 12 , R13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect;

[0247] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0248] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0249] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0250] Among them, J 1 , J 2 , and J 3 Each is independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof; m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3;

[0251] X 1 , X 2 , and X 3 are each independently selected from the group consisting of: -NH- and -O-;

[0252] (K 1 )n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -alkanyl-polyethylene glycol-alkanyl-, alkanyl and polyethylene glycol, or n1, n2 or n3 are each independently 0;

[0253] Y 1 , Y 2 , and Y 3 Each independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-; R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkanol; n1, n2, and n3 are each independently selected from the group consisting of 0, 1, 2, and 3;

[0254] (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the following group: -alkanyl-aryl-, alkanyl and polyethylene glycol, or q1, q2 and q3 are each independently 0;

[0255] W is selected from the following group:

[0256]

[0257]

[0258] P 1 , P 2 and P 3 Each is independently selected from the following group: a nucleic acid molecule, a dye molecule, a cytokine, an antigen, and an antibody or an antigen-binding fragment thereof, or any combination thereof.

[0259] For example, La, Lb and Lc can be each independently selected from the following group: -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)8-NH-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-, - C(=O)-NH-(CH2)2-NH-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0260] For example, La, Lb and Lc can each be independently selected from the following groups: -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0261] In one embodiment, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which may have a structure as shown in Formula IIa or Formula IIb:

[0262]

[0263] in,

[0264] for

[0265] for

[0266] in,

[0267] A 2 for B 2 for, C 2 for or covalent bond; where R12 selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect;

[0268] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0269] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0270] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0271] J 1 , J 2 , and J 3 Each is independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof, m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3;

[0272] X 1 , X 2 , and X 3 are each independently selected from the group consisting of: -NH- and -O-;

[0273] (K 1 ) n1 , (K2 ) n2 , and (K 3 ) n3 are each independently selected from the group consisting of -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 are each independently 0;

[0274] (Y 1 ) p1 ,(Y 2 ) p2 , and (Y 3 ) p3 Each independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2, or p3 are each independently 0;

[0275] (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 are each independently selected from the group consisting of -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2, and q3 are each independently 0;

[0276] W is

[0277]

[0278] P 1 , P 2 and P 3 Each is independently selected from the following group: an antibody targeting 4-1BB, an antibody targeting EGFR, an antibody targeting CD3, an antibody targeting Her2, an antibody targeting CD47, an antibody targeting CD20, a branched peptide containing CMV, neo2 protein, IFNα protein, fluorescein isothiocyanate (FITC), and CPG nucleic acid.

[0279] For example, La, Lb and Lc can be each independently selected from the following group: -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)8-NH-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-, - C(=O)-NH-(CH2)2-NH-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0280] For example, La, Lb and Lc can each be independently selected from the following groups: -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0281] In one aspect, the present application provides a method for preparing the compound described herein, comprising:

[0282] reacting the compound represented by formula (Ia-I) with reactant 1, reactant 2, and reactant 3;

[0283] or reacting the compound represented by formula (Ia-I) with reactant 1 and reactant 2;

[0284] or reacting the compound represented by formula (Ia-I) with reactant 1;

[0285]

[0286] The reactant 1 is HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -A,

[0287] The reactant 2 is HX1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -B,

[0288] The reactant 3 is HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -C,

[0289] Said W is a trivalent group, said W is optionally substituted by one or more R,

[0290] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 Each is independently selected from the group consisting of alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0291] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Each independently selected from the following group: 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0292] R, R 1, R 1a and R 1b each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl;

[0293] wherein m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 are each independently selected from a number greater than 0;

[0294] A, B and C are each independently selected from the following group:

[0295]

[0296] where R 12 , R 13 and R 14 each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl;

[0297] Among them, A, B and C, Cannot exist simultaneously;

[0298] Among them, A, B and C, Cannot exist simultaneously;

[0299] Among them, A, B and C, Cannot exist simultaneously;

[0300] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not an alkynyl group;

[0301] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino,

[0302] A, B and C are not the same

[0303] Among them, when A, B or C is When the compound represented by the formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, Replace with After the compound represented by formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, an acid is added to remove Boc.

[0304] In one embodiment of the preparation method, the order of adding reactant 1, reactant 2 and reactant 3 is determined according to the order of introducing A, B and C. The order of introducing A, B and C from first to last is:

[0305] where R 12 , R 13 and R 14 Each is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl.

[0306] In one aspect, the present application provides a method for preparing the compound described herein, comprising:

[0307] reacting the compound of formula Ia described in any one of the present invention with reactants 4, 5, and 6;

[0308] The reactant 4 is A 1 -P 1 ,

[0309] The reactant 5 is B 1 -P 2 ,

[0310] The reactant 6 is C 1 -P 3 ,

[0311] Among them, A of reactant 4 1 React with A of the compound described in formula Ia, B of reactant 5 1 With the compound B of formula Ia, C of reactant 6 1 Reaction with C of the compound described in Formula Ia;

[0312] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one for

[0313] When at least one of A, B or C is When the corresponding A1 , B 1 or C 1 At least one for

[0314] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0315] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0316] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0317] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0318] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0319] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0320] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0321] When at least one of A, B or C is When the corresponding A 1 , B 1 or C1 At least one for

[0322] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0323] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0324] where R 12 , R 13 and R 14 Each is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl.

[0325] In one embodiment, the preparation method

[0326] in, Cu was added to the reaction + As a catalyst.

[0327] In one embodiment, the preparation method,

[0328] in, 1-100% equivalent of SrtA ligase was added to the reaction as a catalyst.

[0329] A pharmaceutical composition comprising the compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0330] A kit comprising the compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or the pharmaceutical composition described herein.

[0331] Use of the pharmaceutical composition described herein and / or the kit described herein containing the compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salts thereof, in the preparation of a medicament for treating and / or preventing tumors.

[0332] In one embodiment of the use, the tumor is selected from tumors associated with the expression of the following group: 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0333] In one embodiment of the use, the tumor is selected from the group consisting of solid tumors and hematological cancers.

[0334] In one embodiment of the use, the tumor is selected from the group consisting of lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0335] A method for treating and / or preventing tumors, comprising administering to a subject in need thereof the compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition described herein, and / or the kit described herein.

[0336] According to the method for treating and / or preventing tumors described in the present application, the tumor may be a tumor associated with the expression of the following group: 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0337] According to the method for treating and / or preventing tumors described in the present application, the tumor is selected from the following group: solid tumors and blood cancers.

[0338] According to the method for treating and / or preventing tumors described in the present application, the tumor can be selected from the following group: lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer and esophageal cancer.

[0339] A compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition described herein and / or the kit described herein, for use in treating and / or preventing tumors.

[0340] A compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition described herein and / or the kit described herein, for treating and / or preventing tumors selected from the following groups: solid tumors and blood cancers.

[0341] A compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition described herein and / or the kit described herein, for treating and / or preventing tumors, wherein the tumor can be selected from tumors associated with the expression of the following groups: 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0342] A compound described in the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition comprising the same, for use in treating and / or preventing tumors, wherein the tumor can be selected from the following group: lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer and esophageal cancer.

[0343] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0344] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0345] Figure 1 Shown is the H NMR spectrum of the multispecific bioconjugate scaffold 1 described in the present application.

[0346] Figure 2 Shown is the mass spectrum of the Scaffold 1 of a multi-specific bioconjugate described in this application

[0347] Figure 3 Shown is the nuclear magnetic resonance 1H spectrum of the multi-specific bioconjugate scaffold 2 described in this application

[0348] Figure 4 Shown is the mass spectrum of Scaffold 2, a multi-specific bioconjugate described in this application.

[0349] Figure 5Shown is the N3-NHS-NHS H NMR spectrum of the intermediate N3-NHS-NHS in the synthesis of the multispecific bioconjugate scaffold 1 (Scaffold 1) or scaffold 2 (Scaffold 2) described in the present application.

[0350] Figure 6 Shown is an SDS-PAGE image of a multispecific conjugate EGFR-CD3-FITC described in the present application.

[0351] Figure 7 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-FITC described in this application.

[0352] Figure 8 Shown is an SDS-PAGE image of a multispecific conjugate EGFR-CD3-CPG described in the present application.

[0353] Figure 9 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-CPG described in the present application.

[0354] Figure 10 Shown is an SDS-PAGE image of a multispecific conjugate EGFR-CD3-CMV described in the present application.

[0355] Figure 11 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-CMV described in this application.

[0356] Figure 12 Shown is an SDS-PAGE image of a multispecific conjugate EGFR-CD3-neo2 described in the present application.

[0357] Figure 13 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-neo2 described in the present application.

[0358] Figure 14 Shown is an SDS-PAGE image of a multispecific conjugate EGFR-CD3-IFNα described in the present application.

[0359] Figure 15 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-IFNα described in the present application.

[0360] Figure 16 Shown is an SDS-PAGE image of a multispecific conjugate EGFR-CD3-4-1BB described in the present application.

[0361] Figure 17 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-4-1BB described in the present application.

[0362] Figure 18 Shown are the tumor cell growth inhibition results of a multispecific conjugate EGFR-CD3-4-1BB described in this application.

[0363] Figure 19 Shown is the reaction scheme of a multispecific conjugate EGFR-CD3-CMV described in this application.

[0364] Figure 20 Shown is the reaction route of a multispecific conjugate EGFR-CD3-4-1BB described in this application.

[0365] Figure 21 Shown are the reaction routes of the control groups EGFR-TZ-4-1BB and EGFR-CD3-Gly described in this application.

[0366] Figure 22 Shown is the H NMR spectrum of the multispecific bioconjugate scaffold 3.

[0367] Figure 23 Shown is the mass spectrum of multispecific bioconjugate Scaffold 3.

[0368] Figure 24 Shown is the H NMR spectrum of the multispecific bioconjugate scaffold 4.

[0369] Figure 25 Shown is the mass spectrum of the multispecific bioconjugate Scaffold 4.

[0370] Figure 26 Shown is the mass spectrum of the intermediate N3-NHS-NHS* in the synthesis of multispecific bioconjugate tether 3 (Scaffold3) or tether 4 (Scaffold4).

[0371] Figure 27 Shown is the SDS-PAGE representation of EGFR-CD3-LCFA.

[0372] Figure 28 Shown is the mass spectrometric representation of EGFR-CD3-LCFA.

[0373] Figure 29 Shown is the SDS-PAGE representation of EGFR-CD3-ASO.

[0374] Figure 30 Shown is the mass spectrometry characterization of EGFR-CD3-ASO.

[0375] Figure 31Shown is the reaction route for the preparation of EGFR-CD3-M1.

[0376] Figure 32 Shown is the SDS-PAGE representation of EGFR-CD3-M1.

[0377] Figure 33 Shown is the mass spectrometry characterization of EGFR-CD3-M1.

[0378] Figure 34 Shown is the reaction route for the preparation of EGFR-CD3-PDL1.

[0379] Figure 35 Shown is the SDS-PAGE representation of EGFR-CD3-PDL1.

[0380] Figure 36 Shown is the mass spectrometry characterization of EGFR-CD3-PDL1.

[0381] Figure 37 Shown is the reaction route for the preparation of HER2-CD3-PDL1.

[0382] Figure 38 Shown is the mass spectrometry characterization of HER2-CD3-PDL1.

[0383] Figure 39 A-39B shows the binding results of EGFR-CD3-PDL1 with EGFR, CD3, and PDL1 target proteins. DETAILED DESCRIPTION

[0384] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0385] Definition of terms

[0386] In this application, the terms "protein" or "polypeptide" are used interchangeably and generally refer to a chain of at least two (2) or more amino acids linked together by peptide or amide bonds, and independent of post-translational modifications (e.g., glycosylation, acylation, phosphorylation, etc.). Antibodies are specifically intended to be included within the scope of this definition. The polypeptides of the present invention may comprise more than one subunit, wherein each subunit is encoded by a separate DNA sequence. Proteins may also comprise more than one polypeptide unit, including dimers, trimers, tetramers, or various higher order structures.

[0387] In this application, the terms "equivalent" or "equivalent percentage" are used interchangeably and generally refer to the number of moles of "X" relative to the number of moles of "Y." For example, 5 molar equivalents of X relative to Y means that if 1 mole of Y is used, 5 moles of X can be used.

[0388] In this application, the term "corresponding" of the X group to the Y group generally means that when the X group is a certain group, the Y group is another specific group. For example, the selection of A1 of the reactant 4 and the corresponding A of the compound described in formula Ia can be when A is When, accordingly, A1 can choose Corresponding to it.

[0389] In this application, the term "sequence of introduction" generally refers to the order in which a plurality of different specific groups are introduced into a starting compound. The order of introducing the starting compounds may, in one embodiment, be by first adding a Reactant 1 is added with Reactant 2, finally add Reactant 3 is first introduced reintroduction Finally introduced For example, introducing Before, you can Replace with Acid was then added to remove Boc.

[0390] In this application, the term "group X can be connected to group Y and group Z in either orientation" generally means that when group X is used to connect group Y and group Z, the two or more connection sites of group X can be connected to group Y or group Z at will. For example, group The linkage to the group W and the group P can be in any orientation and can be the group The C atom connecting group W, the N atom connecting group P, can also be a group The N atom connects the group W, and the C atom connects the group P.

[0391] In this application, the term "enantiomers" generally refers to two stereoisomers of a compound that are non-superimposable mirror images of each other.

[0392] In this application, the term "polysaccharide" generally refers to a molecule composed of a chain of monosaccharide units connected by glycosidic bonds. For example, the term "polysaccharide" can include molecules composed of two or more monosaccharide units, such as molecules in which the longest monosaccharide chain is 3 to 9 monosaccharide units. The term "polysaccharide" can include both linear and branched molecules, isolated molecules and molecules associated with polypeptides, and sialylated and non-sialylated molecules.

[0393] In this application, the term "diastereomer" generally refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, for example, melting points, boiling points, spectral properties and reactivity.

[0394] In this application, the terms "tautomer" or "tautomeric form" are used interchangeably and generally refer to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via reorganization of some of the bonding electrons.

[0395] As used herein, the term "meso" generally refers to a molecule containing asymmetric atoms but possessing symmetry such that the total optical rotation is zero. The term "racemate" or "racemic mixture" refers to a composition composed of equimolar amounts of two enantiomeric species.

[0396] In this application, the term "lipid" generally refers to a straight-chain, branched, saturated or unsaturated aliphatic carboxylic acid, a phospholipid or a sterol. Examples of aliphatic carboxylic acids are lauric acid, palmitic acid, stearic acid, oleic acid and (CH3(CH2) n )2CHCOOH, wherein n is a number of at least 1. The phospholipid may be phosphatidylethanolamine, such as dioleoylphosphatidylethanolamine.

[0397] In this application, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" generally refer to polymers of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) and include naturally occurring (adenine, guanine, cytosine, uracil and thymine), non-naturally occurring and modified nucleic acids. The term is not limited by the length of the polymer (e.g., the number of monomers). Nucleic acids can be single-stranded or double-stranded and generally contain a 5'-3' phosphodiester bond, although in some cases, nucleotide analogs can have other connections. The monomers are generally referred to as nucleotides. The term "non-natural nucleotide" or "modified nucleotide" refers to a nucleotide containing a modified nitrogenous base, sugar or phosphate group, or a nucleotide with a non-natural portion incorporated into its structure. Examples of non-natural nucleotides include dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated and fluorescently labeled nucleotides.

[0398] In this application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound of the present application may be an organic compound, for example, the compound of the present application may be a compound with a molecular weight of less than 500, a compound with a molecular weight of less than 1000, a compound with a molecular weight of more than 1000, or a compound with a molecular weight of more than 1000 or more than 10000. In this application, a compound may also refer to a compound connected by chemical bonds, for example, a compound in which one or more molecules with a molecular weight of less than 1000 are connected to a biomacromolecule by a chemical bond, and the biomacromolecule may be a polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound of the present application may include a compound in which a protein is connected to one or more molecules with a molecular weight of less than 1000, a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000, or a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000.

[0399] In this application, the term "mixture" generally refers to a blend of two or more individual compounds.

[0400] In this application, the term "antibody" or "antibody or its antigen-binding fragment" generally refers to immunological binding agents extending to all antibodies from all species, including dimers, trimers and multimers, bispecific antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, recombinant and reconstructed antibodies, nanobodies and their fragments. The term "antibody or its fragment" can refer to any antibody-like molecule with an antigen-binding region. The term includes antigen-binding active substance fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), linear antibodies, diabodies, nanobodies and the like. The term "antigen-binding fragment" can refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. For example, a fragment of a full-length antibody can be used to perform the antigen-binding function of an antibody. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. In one embodiment, it can refer to one or more of anti-EGFR antibodies, anti-CD3 antibodies, and anti-41-BB antibodies.

[0401] In this application, the term "Nanobody" generally refers to those antibodies as defined in WO 2008 / 020079 or WO 2009 / 138519, and thus in certain aspects generally refers to a VHH, a humanized VHH or a camelized VH (e.g. a camelized human VH) or generally a sequence-optimized VHH (e.g. optimized for chemical stability and / or solubility, maximal overlap with known human framework regions and maximal expression).

[0402] In this application, the term "any combination" generally means that the group can be a group selected from any number of groups in the group in any order. For example, L 1 Selected from the following groups: chain alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof, in one embodiment, may refer to L 1 It can be an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or a polyethylene glycol group. In one embodiment, it can refer to L 1 It may be a combination of an alkyl group and an aryl group or a combination of an alkyl group and polyethylene glycol. In one embodiment, it may refer to -alkyl group-aryl group or -alkyl group-polyethylene glycol-alkyl group.

[0403] In this application, the term "trivalent" group or "trivalent group" generally refers to a group having three bonding positions in the group. For example, trivalent groups include but are not limited to trivalent alkanes, trivalent cycloalkyls, trivalent heterocycloalkyls, trivalent alkenyls, trivalent alkynyls, trivalent aryls, and trivalent heteroaryls. For example, in one embodiment, it may include but is not limited to

[0404] In the present application, the term "quaternary group" generally refers to a group having 4 bonding positions in the group.

[0405] In this application, the term "polyethylene glycol" or "PEG" generally refers to a polyethylene glycol having the general formula -(OCH2CH2) n -denoted by -, wherein n is an integer of 3, 4, 5, 6, 7, 8, 9 or greater. The polyethylene glycol chain comprises a polymer of ethylene glycol having an average total molecular weight selected from the range of about 500 to about 40,000 daltons. The average molecular weight of the PEG chain is indicated by a number, for example, PEG-5,000 refers to a polyethylene glycol chain having an average total molecular weight of about 5,000. The polyethylene glycol can be substituted or unsubstituted.

[0406] In this application, the term "halogen" is generally meant to include fluorine, chlorine, bromine and iodine.

[0407] In this application, the term "urea" generally refers to -(HN-CO-)2N-.

[0408] In the present application, the term "alkanyl" generally refers to a residue derived from an alkanyl group by removing a hydrogen atom. Alkanyl groups can be substituted or non-substituted, substituted or non-substituted. The term "alkanyl" generally refers to a saturated straight or branched aliphatic hydrocarbon group having a residue derived from the same carbon atom or two different carbon atoms of a parent alkane by removing a hydrogen atom, which can be a straight or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, for example, an alkanyl group containing 1 to 6 carbon atoms. Non-limiting examples of alkanyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc. Alkyl groups may be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituents may be substituted at any available point of attachment, and the substituents may be independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo, for example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, or C 1-6 Aliphatic group.

[0409] In this application, the term "cycloalkyl" generally refers to a residue derived from the same carbon atom or multiple different carbon atoms of a carbocyclic ring by removing hydrogen atoms. The term "cycloalkane" generally refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon, the carbocyclic ring containing 3 to 20 carbon atoms, can contain 3 to 12 carbon atoms, can contain 3 to 10 carbon atoms, can contain 3 to 8 carbon atoms. Non-limiting examples of monocyclic carbocycles include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, cyclooctane, etc.; polycyclic carbocycles can include spirocyclic, fused ring and bridged ring carbocycles. Cycloalkyl groups can be substituted or unsubstituted.

[0410] In this application, the term "heterocycloalkyl" generally refers to a stable non-aromatic 3- to 7-membered monocyclic ring structure, a fused 7- to 10-membered bicyclic heterocyclic ring structure, or a bridged 6- to 10-membered bicyclic heterocyclic ring structure. These ring structures may be saturated or partially saturated. In addition to carbon atoms, these ring structures may contain one or more heteroatoms, wherein the heteroatoms may be selected from the following group: oxygen, sulfur, and nitrogen. For example, the heteroatoms may contain 1 to 4 heteroatoms as defined above. When used to refer to atoms in a heterocyclic ring structure, the term "nitrogen" may include nitrogen that has undergone substitution reactions. Heterocycloalkyl groups may be substituted or unsubstituted.

[0411] In this application, the term "alkenyl" generally refers to a straight or branched hydrocarbon group containing one or more double bonds. Illustrative examples of alkenyl groups include allyl, homoallyl, vinyl, crotyl, butenyl, pentenyl, and hexenyl. C 2-6 Illustrative examples of alkenyl groups include butadienyl, pentadienyl, hexadienyl and hexatrienyl and their branched forms. The position of the unsaturated bond (double bond) can be at any position of the carbon chain. The alkenyl group can be substituted or unsubstituted.

[0412] In the present application, the term "alkynyl" generally refers to an unsaturated straight-chain or branched alkynyl group, such as ethynyl, 1-propynyl, propargyl, butynyl, etc. Alkynyl groups can be substituted or unsubstituted.

[0413] In this application, the term "aryl" generally refers to a residue derived from the removal of hydrogen atoms from the same carbon atom or multiple different carbon atoms of an aromatic ring. The term "aromatic ring" can refer to a 6- to 14-membered all-carbon monocyclic ring or a fused polycyclic ring (i.e., a ring sharing adjacent pairs of carbon atoms) with a conjugated π electron system, which can be 6- to 10-membered, such as benzene and naphthalene. The aromatic ring can be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring connected to the parent structure is the aryl ring. The aryl group can be substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups, which are independently selected from the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0414] As used herein, the term "heteroaryl" generally refers to a residue derived from the removal of hydrogen atoms from the same carbon atom or multiple different carbon atoms of a heteroaromatic ring. The term "heteroaromatic ring" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms can be selected from the group consisting of oxygen, sulfur, and nitrogen. A heteroaryl group can be 5 to 10-membered, and can be 5- or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring can be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0415] In this application, the term "alkoxy" generally refers to an alkyl group to which an oxygen group is attached. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0416] In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance can but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group can but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0417] In this application, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example up to 5, for example 1 to 3 hydrogen atoms, which are independently replaced by a corresponding number of substituents. The substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) whether substitution is possible or not without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0418] Unless otherwise indicated, the structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds identical to the structures described herein except for the replacement of a hydrogen atom by deuterium or tritium, or the replacement of a carbon atom by carbon-13 or carbon-14 are within the scope of this application.

[0419] In this application, the term "small molecule" generally refers to any chemical moiety or other moiety having a molecular weight lower than about 5000 Daltons (Da). In some embodiments, the molecular weight of the small molecule is lower than about 2500 Daltons, about 1000 Daltons, or about 500 Daltons. In some embodiments, the small molecule may not be a polymer. In some embodiments, the small molecule may not be a protein. In some embodiments, the small molecule may not be a nucleic acid. In some embodiments, the small molecule may not be a polysaccharide. In some embodiments, the small molecule may have biological activity and / or play an effect that affects a biological process. In some embodiments, the small molecule may be a natural product or first prepared through chemical synthesis. For example, in one embodiment, the small molecule may be a therapeutic drug, a chemical toxin, or a detection substance, such as the dye molecule FITC.

[0420] In this application, the term "antigen" generally refers to peptides, polypeptides and proteins encoded by the gene constructs of the present invention, which serve as targets for inducing an immune response. The target protein can be an immunogenic protein for which immunity is to be induced, and it can have at least one identical epitope with a protein from a pathogen or an undesirable cell type, such as a cancer cell or a cell involved in an autoimmune disease. An immune response directed against a target protein can protect an individual against a specific infection or disease associated with the target protein, or can treat the above-mentioned infection or disease in an individual.

[0421] In this application, the term "cytokine" generally includes, for example, interleukins, interferons, chemokines, hematopoietic growth factors, tumor necrosis factors and transforming growth factors. Generally speaking, they can be low molecular weight proteins that can regulate the maturation, activation, proliferation and differentiation of cells of the immune system.

[0422] In the present application, the term "dye molecule" generally refers to a substance that dyes a target substance. For example, the "dye molecule" can include fluorescent markers, and can also include near-infrared fluorescent markers. Fluorescent labeling is achieved using chemically reactive derivatives of fluorophores. Common reactive groups can include amine-reactive isothiocyanate derivatives such as FITC and TRITC (derivatives of fluorescein and rhodamine), amine-reactive succinimidyl esters such as NHS-fluorescein, and sulfhydryl-reactive maleimide-activated fluorescein (fluor) such as fluorescein-5-maleimide. Any of these reactive dyes can produce a stable covalent bond formed between a fluorophore and a labeled molecule with the reaction of another molecule. Other suitable fluorescent markers encompassed by the present application are Alexa Fluor, DylightFluor, and ATTO dyes. For example, fluorescent proteins encompassed by the present application are such as GFP, YFP, or CFP. For example, activatable dye molecules activated by pH changes or voltage and temperature are encompassed by the present application.

[0423] In the present application, the terms "pharmaceutically acceptable salt", "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" generally refer to salts of the compounds of the present application. Such salts may be safe and / or effective when used in mammals and may have the desired biological activity. The compounds of the present application may form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0424] In this application, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. For example, the carrier can be suitable for parenteral (e.g., intravenous, intramuscular, subcutaneous, intrathecal) administration (e.g., by injection or infusion). Depending on the route of administration, the active compound can be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0425] In this application, the term "Boc" or "t-Butyloxy carbonyl" generally refers to an amino protecting group commonly used in organic synthesis.

[0426] In this application, the term "Cu+" or "monovalent copper" generally refers to a monovalent copper catalyst. For example, Cu+ can be CuCN, CuSCN, CuI or CuBr.

[0427] In this application, the term "SrtA ligase" generally refers to a ligase that mediates a conjugation reaction. The principle of the SrtA ligase-mediated conjugation reaction (Chen, Long, et al. "Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency." Scientific reports 6.1(2016):1-12.) is that SrtA ligase can specifically recognize the LPETG sequence shown in SEQ ID NO: 7 and connect the substrate molecule with a naked glycine at the N-terminus to the target protein.

[0428] In this application, the term "specific" generally refers to the selective recognition of a specific epitope of an antigen by an antibody. For example, natural antibodies are monospecific. As used in this application, the term "multispecific" refers to the selectivity of having two or more antigen binding sites, at least two of which bind to different antigens or different epitopes of the same antigen. For example, it can be multispecific for at least two different antigens (i.e., EGFR as a first antigen and 4-1BB as a second antigen). In one embodiment of the invention, the multispecific antibody according to the present invention can be bispecific. In another embodiment of the invention, the multispecific antibody according to the present invention can be trispecific.

[0429] In this application, the term "coupling" generally refers to the connection of two compounds by a covalent bond or by a strong non-covalent interaction, such as a connection by a covalent bond. For example, this application provides a connection in which an N3 group of one compound can react with an alkynyl group of another compound to form a covalent bond, thereby forming a conjugate of the two compounds.

[0430] As used herein, the term "linker" generally refers to a central compound to which various other compounds can be attached. The central compound can be linked to one or more other compounds via covalent bonds or strong non-covalent interactions to form a larger compound. For example, the linker herein can be linked to one or more compounds comprising lipids, proteins, nucleic acids, small molecules, or polysaccharides, or any combination thereof, to form a conjugate.

[0431] In this application, the term "comprising" generally refers to including the features specified but not excluding other elements. The terms "above" and "below" generally refer to including the number.

[0432] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value. Detailed Description of the Invention

[0434] In one aspect, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, having a structure as shown in Formula Ia or Formula Ib:

[0435]

[0436] Said Wa may be a trivalent group, and Wb may be a tetravalent group;

[0437] Said A, B and C can be independently selected from the following groups:

[0438]

[0439] where R 12 , R 13 and R 14 may be each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0440] Among them, A, B and C, Cannot exist simultaneously;

[0441] Among them, A, B and C, Cannot exist simultaneously;

[0442] Among them, A, B and C, Cannot Exist at the same time; when A, B and C, When both exist, the R 12 , R 13 and R 14 Not an alkynyl group;

[0443] When A, B and C, When both exist, the R 12 , R 13 and R14 Not amino,

[0444] A, B and C are not the same

[0445] In another embodiment, A, B and C can be independently selected from the following groups:

[0446] Among them, R, R 12 each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, and each X is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, and halogen;

[0447] Among them, when R is C5H 11 , X is H, or R is CH2OH, X is H, or R is CH2CH3, X is F, in A, B and C, Cannot exist simultaneously;

[0448] Among them, when R is CH3 and X is F, or R is CH2CH3 and X is F, among A, B and C, Cannot Existing at the same time,

[0449] Among them, A, B and C, Cannot Existing at the same time,

[0450] Among them, A, B and C, Cannot Exist at the same time.

[0451] In one embodiment, the selection of A, B and C groups can be the following group:

[0452] (1) A is B is C is or

[0453] (2) A is B is C is or

[0454] (3) A is B is C is or

[0455] (4) A is B is C is or

[0456] (5) A is B is C is or

[0457] (6) A is B is C is or

[0458] (7) A is B is C is or

[0459] (8) A is B is C is or

[0460] (9) A is B is C is or

[0461] (10) A is B is C is or

[0462] (11) A is B is or

[0463] (12) A is B is C is or

[0464] (13) A is B is C is or

[0465] (14) A is B is C is

[0466] where R 12 , R13 and R 14 may be each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0467] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not an alkynyl group;

[0468] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino.

[0469] In another embodiment, the selection of A, B and C groups can be the following group:

[0470] (1) A is B is C is or

[0471] (2) A is B is C is or

[0472] (3) A is B is C is

[0473] where R 12 , R 13 and R 14 may be each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0474] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino.

[0475] In another embodiment, the selection of A, B and C groups can be the following group:

[0476] (1) A is B is C is or

[0477] (2) A is B is C is or

[0478] (3) A is B is C is

[0479] where R 12 may be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0480] When A, B and C, When both exist, the R 12 Not amino.

[0481] For example, where R 12 can be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the junction site.

[0482] For example, where: A can be B can be C can be where R 12 A group which may be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and chain alkyl, Represents the junction site.

[0483] In the present application, Wa may be a trivalent group, and Wb may be a tetravalent group; for example:

[0484] Can be

[0485] Can be

[0486] Said W may be a trivalent group or a tetravalent group, and said W may be optionally substituted by one or more R,

[0487] The La can be -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 )p1 -(L 1 ) q1 -;

[0488] The Lb can be -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0489] The Lc can be -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0490] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 can be independently selected from the following group: alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0491] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Can be each independently selected from the following group: -NR 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0492] R, R 1 , R 1a and R 1b may be each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0493] Among them, m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 can each be independently selected from a number greater than 0.

[0494] In another embodiment, wherein J 1 , J 2 , and J 3 Each of m1, m2, and m3 can be independently selected from the following group: 0, 1, 2, and 3.

[0495] In another embodiment, wherein X 1 , X 2 , and X 3 may be each independently selected from the group consisting of -NH- and -O-.

[0496] In another embodiment, wherein K 1 , K 2 , and K 3 Each of n1, n2, and n3 can be independently selected from the following group: 0, 1, 2, and 3.

[0497] For example, where (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each may be independently selected from the group consisting of: -alkane-polyethylene glycol-alkane-, alkane and polyethylene glycol, or n1, n2 or n3 may be each independently 0.

[0498] For example, where (K 1 ) n1 , (K 2 ) n2 , and (K 3) n3 can be each independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 can each independently be 0.

[0499] In another embodiment, wherein Y 1 , Y 2 , and Y 3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-; R 1 Selected from the group consisting of hydrogen, protium, deuterium, tritium, and chain alkyl; wherein n1, n2, and n3 can each be independently selected from the group consisting of 0, 1, 2, and 3.

[0500] For example, where (Y 1 ) p1 ,(Y 2 ) p2 , and (Y 3 ) p3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkanyl; or p1, p2 or p3 may each independently be 0.

[0501] In another embodiment, wherein L 1 , L 2 , and L 3 can be independently selected from the following groups: chain alkyl, and aryl, or any combination thereof; wherein, q1, q2, and q3 can be independently selected from the following groups: 0, 1, 2, and 3.

[0502] For example, where (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 may be each independently selected from the group consisting of -alkanyl-aryl-, alkanyl and polyethylene glycol, or q1, q2 and q3 may each independently be 0.

[0503] For example, where (L 1 ) q1 ,(L 2 ) q2 , and (L3 ) q3 may be each independently selected from the group consisting of -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 may each independently be 0.

[0504] In another embodiment, wherein:

[0505] W can be selected from the following group:

[0506] trivalent alkanyl, trivalent cycloalkyl, trivalent heterocycloalkyl, trivalent alkenyl, trivalent alkynyl, trivalent aryl, and trivalent heteroaryl.

[0507] For example, where:

[0508] W can be selected from the following group:

[0509]

[0510] The X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 and X 18 may be each independently selected from the group consisting of C, N, O, S, P, CH, CH2, NH, P(O) and P(S);

[0511] The --- represents a double bond or a single bond.

[0512] For example, where:

[0513] W is selected from the following group:

[0514]

[0515]

[0516] In another embodiment, the compound can be

[0517]

[0518] The La is -(J 1 )m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0519] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0520] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0521] Among them, J 1 , J 2 , and J 3 can be independently selected from the following groups: chain alkyl, and polyethylene glycol, or any combination thereof; wherein m1, m2, and m3 can be independently selected from the following groups: 0, 1, 2, and 3;

[0522] Among them, X 1 , X 2 , and X 3 may each be independently selected from the group consisting of: -NH- and -O-;

[0523] Among them, (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 may be each independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 may each independently be 0;

[0524] (Y 1 ) p1 ,(Y 2) p2 , and (Y 3 ) p3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and chain alkyl; or p1, p2 or p3 may each independently be 0;

[0525] (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 may be independently selected from the following group: -CH2-aryl-, -(CH2)2-, -CH2-, chain alkyl and polyethylene glycol, or q1, q2 and q3 may be independently 0;

[0526] W can be selected from the following group:

[0527]

[0528] A can be B can be C can be where R 12 A group which may be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and chain alkyl, Represents the junction site.

[0529] For example, W can be selected from the following group:

[0530]

[0531] In another embodiment, the La, Lb and Lc can be each independently selected from the following group: a linear hydrocarbon chain, a linear heterohydrocarbon chain containing one or more heteroatoms, a branched hydrocarbon chain containing one or more branches, and a linear heterohydrocarbon chain containing one or more heteroatoms and containing one or more branches.

[0532] In another embodiment, the La, Lb and Lc may be each independently selected from the group consisting of a linear alkyl chain, and a linear heteroalkyl chain containing one or more heteroatoms.

[0533] In another embodiment, the La, Lb and Lc can be each selected from -C nx H 2nx-, where nx is a number greater than 0. For example, nx is a number greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, or greater than 9. For example, nx is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0534] In another embodiment, the La, Lb and Lc can each be independently selected from a polymer.

[0535] In another embodiment, the La, Lb and Lc can be each independently selected from the following group: polysaccharide, polyamino acid, polynucleotide, and polylactic acid.

[0536] In another embodiment, described La, Lb and Lc can be each independently a polysaccharide.Polysaccharide can be a monomer or polymer of a sugar residue, and can be straight or branched.Polysaccharide can include natural sugar residues (for example, glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose etc.) and / or modified sugar (for example, 2'-fluororibose, 2'-deoxyribose, mannose phosphate, 6'-sulfo N-acetylglucosamine etc.).In another embodiment, described La, Lb and Lc can be each independently a homopolymer and heteropolymer of a sugar residue.

[0537] In another embodiment, the La, Lb and Lc can each independently be a polynucleotide. Polynucleotides can include single-stranded and double-stranded nucleotide polymers. The nucleotides that make up the polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. In another embodiment, the La, Lb and Lc can each independently be a base-modified polynucleotide,

[0538] In another embodiment, La, Lb, and Lc can each independently be polyglycine, for example, a polyglycine polymerized from one or more glycine groups. In certain embodiments, the polyglycine polymerized from one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more glycine groups. In certain embodiments, the polyglycine polymerized from one, two, three, four, five, six, seven, eight, nine, or ten glycine groups.

[0539] In another embodiment, each of the groups can be independently selected from the following groups, which may mean that each group is selected from one or more of the following groups, or it may mean that some of the groups are selected from one or more of the following groups, and another part of the groups are selected from one or more of the following groups in any definition of the group in this application.

[0540] In another embodiment, the compound can be selected from the following group:

[0541]

[0542]

[0543]

[0544]

[0545] In another embodiment, the compound can be selected from the following group:

[0546]

[0547] In another aspect, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which has a structure as shown in Formula IIa or Formula IIb, wherein:

[0548]

[0549] The Wc is a trivalent group, and Wd is a tetravalent group;

[0550] The A 2 , B 2 and C 2 Each independently selected from the following group:

[0551] and covalent bonds;

[0552] where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0553] Represents the junction site,

[0554] The A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect;

[0555] Among them, P 1 , P2 and P 3 Each is independently selected from the group consisting of lipids, proteins, nucleic acids, small molecules and polysaccharides, or any combination thereof.

[0556] In one embodiment, wherein A 2 , B 2 and C 2 can be independently selected from the following groups:

[0557] (1)A 2 for B 2 for C 2 for or covalent bond; or

[0558] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0559] (3)A 2 for B 2 for C 2 for or covalent bond; or

[0560] (4)A 2 for B 2 for C 2 for or covalent bond; or

[0561] (5)A 2 for B 2 for C 2 for or covalent bond; or

[0562] (6)A 2 for B 2 for C2 for or covalent bond; or

[0563] (7)A 2 for B 2 for C 2 for or covalent bond; or

[0564] (8)A 2 for B 2 for C 2 for or covalent bond; or

[0565] (9)A 2 for B 2 for C 2 for or covalent bond; or

[0566] (10)A 2 for or covalent bond, B 2 is a covalent bond, C 2 is a covalent bond; or

[0567] (11)A 2 for B 2 for C 2 for or covalent bond; or

[0568] (12)A 2 for B 2 for C 2 for or covalent bond; or

[0569] (13)A 2 for B 2 for C 2 for or covalent bonds;

[0570] where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0571] Represents the junction site,

[0572] The A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

[0573] For example, where A 2 , B 2 and C 2 can be independently selected from the following groups:

[0574] (1)A 2 for B 2 for C 2 for or covalent bond; or

[0575] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0576] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0577] where R 12 , R 13 and R 14are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0578] Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

[0579] For example, where A 2 , B 2 and C 2 can be independently selected from the following groups:

[0580] (1)A 2 for B 2 for C 2 for or covalent bond; or

[0581] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0582] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0583] where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0584] Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

[0585] For example, R 12 , R 13 and R 14 can be each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl,

[0586] Represents the connection site, the A 2 The two linking sites can be arbitrarily connected with Wc and P 1 Connect the B 2 The two linking sites can be arbitrarily connected with Wc and P 2 Connect the C 2 The two linking sites can be arbitrarily connected with Wc and P 3 connect.

[0587] For example, where A 2 , B 2 and C 2 Can be: A 2 for B 2 for C 2 for or covalent bond; where R 12 , R 13 and R 14 can be each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl, Represents the connection site, the A 2 The two linking sites can be arbitrarily connected with Wc and P 1 Connect the B 2 The two linking sites can be arbitrarily connected with Wc and P 2 Connect the C 2 The two linking sites can be arbitrarily connected with Wc and P 3 connect.

[0588] For example, A 2 Can be B 2 Can be, C 2 Can be or covalent bond; where R 12 can be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the connection site, the A 2The two linking sites can be arbitrarily connected with Wc and P 1 Connect the B 2 The two linking sites can be arbitrarily connected with Wc and P 2 Connect the C 2 The two linking sites can be arbitrarily connected with Wc and P 3 connect.

[0589] In another embodiment, Can be Can be

[0590] Said W may be a trivalent group, said W being optionally substituted by one or more R,

[0591] The La can be -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0592] The Lb can be -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0593] The Lc can be -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0594] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3can be independently selected from the following group: alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0595] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Can be each independently selected from the following group: -NR 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0596] R, R 1 , R 1a and R 1b may be each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0597] Among them, m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 can each be independently selected from a number greater than 0.

[0598] In another embodiment, wherein J 1 , J 2 , and J 3 Each of m1, m2, and m3 can be independently selected from the following group: 0, 1, 2, and 3.

[0599] In another embodiment, wherein X 1 , X 2 , and X 3may be each independently selected from the group consisting of -NH- and -O-.

[0600] In another embodiment, wherein K 1 , K 2 , and K 3 Each of n1, n2, and n3 can be independently selected from the following group: 0, 1, 2, and 3.

[0601] For example, where (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each may be independently selected from the group consisting of: -alkane-polyethylene glycol-alkane-, alkane and polyethylene glycol, or n1, n2 or n3 may be each independently 0.

[0602] For example, where (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 can be each independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 can each independently be 0.

[0603] In one embodiment, wherein Y 1 , Y 2 , and Y 3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-; R 1 Selected from the group consisting of hydrogen, protium, deuterium, tritium, and chain alkyl; wherein n1, n2, and n3 can each be independently selected from the group consisting of 0, 1, 2, and 3.

[0604] For example, where (Y 1 ) p1 ,(Y 2 ) p2 , and (Y 3 ) p3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkanyl; or p1, p2 or p3 may each independently be 0.

[0605] In one embodiment, wherein L 1 , L 2 , and L 3 can be independently selected from the following groups: chain alkyl, and aryl, or any combination thereof; wherein, q1, q2, and q3 can be independently selected from the following groups: 0, 1, 2, and 3.

[0606] For example, where (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 may be each independently selected from the group consisting of -alkanyl-aryl-, alkanyl and polyethylene glycol, or q1, q2 and q3 may each independently be 0.

[0607] For example, where (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 may be each independently selected from the group consisting of -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 may each independently be 0.

[0608] In one embodiment, W can be selected from the following group:

[0609] trivalent alkanyl, trivalent cycloalkyl, trivalent heterocycloalkyl, trivalent alkenyl, trivalent alkynyl, trivalent aryl, and trivalent heteroaryl.

[0610] For example, W can be selected from the following group:

[0611]

[0612] The X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X16 , X 17 and X 18 may be each independently selected from the group consisting of C, N, O, S, P, CH, CH2, NH, P(O) and P(S);

[0613] The --- represents a double bond or a single bond.

[0614] For example, W can be selected from the following group:

[0615]

[0616]

[0617] In another embodiment, wherein P 1 , P 2 and P 3 Each is independently selected from the group consisting of lipids, proteins, nucleic acids, small molecules and polysaccharides, or any combination thereof.

[0618] For example, the lipid in the present application can be a fatty acid. For example, the lipid in the present application can be a long-chain fatty acid LCFA.

[0619] In another embodiment, wherein P 1 , P 2 and P 3 Each can be independently selected from the following group: a nucleic acid molecule, a dye molecule, a cytokine, an antigen, a fusion protein, and an antibody or an antigen-binding fragment thereof.

[0620] For example, the nucleic acid molecule may comprise CPG. For example, the nucleic acid molecule of the present application may comprise an antisense oligonucleotide ASO. For example, the nucleic acid molecule of the present application may comprise an ASO targeting STAT3. For example, the nucleic acid molecule of the present application may comprise Me C*T*A *T*T*T*G*G*A*T*G*T* Me C* A*G* Me C , the sequence shown in SEQ ID NO: 13 (underlined represents locked nucleic acid LNA, for example, its nucleoside may include a bicyclic sugar connecting the 4'-position and the 2'-position bridge, MeC represents methyl C, and * represents the internucleoside linkage of phosphorothioate.

[0621] For example, the proteins of the present application may include the following groups: branched peptides containing CMV antigens, neo2 protein (a fusion protein of cytokines IL2 / IL5), IFNα protein, and M1 protein (a short peptide that penetrates the blood-brain barrier).

[0622] For example, the antibody can be selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and a nanobody.

[0623] For example, wherein the antibody targeting can be selected from the group consisting of: 4-1BB, EGFR, CD3, Her2, CD47, and CD20. For example, wherein the antibody targeting can be selected from the group consisting of: 4-1BB, EGFR, CD3, Her2, and PD-L1.

[0624] For example, the amino acid sequence of the antibody can be selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. For example, the amino acid sequence of the antibody can be selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 15.

[0625] In another embodiment, the compound can be

[0626]

[0627] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0628] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0629] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 )q3 -;

[0630] Among them, J 1 , J 2 , and J 3 can be independently selected from the following groups: chain alkyl, and polyethylene glycol, or any combination thereof; wherein m1, m2, and m3 can be independently selected from the following groups: 0, 1, 2, and 3;

[0631] Among them, X 1 , X 2 , and X 3 may each be independently selected from the group consisting of: -NH- and -O-;

[0632] Among them, (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 may be each independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 may each independently be 0;

[0633] (Y 1 ) p1 ,(Y 2 ) p2 , and (Y 3 ) p3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and chain alkyl; or p1, p2 or p3 may each independently be 0;

[0634] (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 may be independently selected from the following group: -CH2-aryl-, -(CH2)2-, -CH2-, chain alkyl and polyethylene glycol, or q1, q2 and q3 may be independently 0;

[0635] W can be selected from the following group:

[0636]

[0637] A2 Can be B 2 Can be, C 2 Can be or covalent bond; where R 12 can be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the junction site,

[0638] Among them, P 1 , P 2 and P 3 Each independently selected from the group consisting of lipids, proteins, nucleic acids, small molecules and polysaccharides, or any combination thereof;

[0639] The A 2 The two linking sites can be arbitrarily connected with Wc and P 1 Connect the B 2 The two linking sites can be arbitrarily connected with Wc and P 2 Connect the C 2 The two linking sites can be arbitrarily connected with Wc and P 3 connect.

[0640] For example, The a-linked site and P 1 Connection, b connection site and Wc (L 1 ) q1 connect, The b-linking site and P 2 Connection, a connection site and Wc (L 2 ) q2 Connect, C 2 The a-linked site and P 3 Connection, a connection site and Wc (L 3 ) q3 connect.

[0641] For example, W can be selected from the following group:

[0642]

[0643] In another embodiment, the one compound may be selected from the group consisting of:

[0644]

[0645] Among them, P 1 , P 2 and P 3 Each may be independently selected from the group consisting of lipids, proteins, nucleic acids, small molecules and polysaccharides, or any combination thereof.

[0646] In one embodiment, P 1 , P 2 and P 3 Can be independently selected from lipids. 1 , P 2 and P 3 Can be independently selected from the following groups: aliphatic carboxylic acids, phospholipids and sterols. In one embodiment, P 1 , P 2 and P 3 Each can be independently selected from linear, branched, saturated or unsaturated aliphatic carboxylic acids.

[0647] In one embodiment, P 1 , P 2 and P 3 Can be independently selected from proteins. In one embodiment, P 1 , P 2 and P 3 Each can be independently selected from a polypeptide. The protein or polypeptide may include post-expression modifications, such as glycosylation, acetylation, phosphorylation, etc. The protein or polypeptide may include one or more amino acid analogs, polypeptides with substituted bonds, and modified polypeptides known in the art, including both naturally occurring and non-naturally occurring modifications.

[0648] In one embodiment, P 1 , P 2 and P 3 Can be each independently selected from nucleic acid. The nucleic acid can refer to a polymer of nucleotides (such as ribonucleotides or deoxyribonucleotides), and includes naturally occurring (adenine, guanine, cytosine, uracil and thymine), non-naturally occurring and modified nucleic acids. The nucleic acid may not be limited by the length (such as the number of monomers) of the polymer. The nucleic acid can be single-stranded or double-stranded, and generally contains a 5'-3' phosphodiester bond, and nucleotide analogs may also have other connections. The monomers of the nucleic acid are generally referred to as nucleotides. The non-natural nucleotides or the modified nucleotides refer to nucleotides containing modified nitrogenous bases, sugars or phosphate groups, or nucleotides incorporating non-natural parts into their structure. The non-natural nucleotides can include dideoxynucleotides, biotinylation, amination, deamination, alkylation, benzylation and fluorescently labeled nucleotides.

[0649] In one embodiment, P 1 , P 2 and P 3Each of the small molecules can be independently selected from small molecules. The small molecules can be mitotic inhibitors, antitumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, anti-hormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors and radiosensitizers.

[0650] In one embodiment, P 1 , P 2 and P 3 Each of the polysaccharides can be independently selected from polysaccharides. The polysaccharide can include molecules composed of two or more monosaccharide units, for example, molecules in which the longest monosaccharide chain is 3 to 9 monosaccharide units. The polysaccharide can include linear and branched molecules. Any combination of the polysaccharide with lipids, proteins, nucleic acids, or small molecules can include isolated molecules and molecules bound to polypeptides, sialylated and non-sialylated molecules.

[0651] Among them, P 1 , P 2 and P 3 Each may be independently selected from antibodies targeting a target selected from the group consisting of 4-1BB, EGFR, CD3, Her2, CD47, and CD20.

[0652] For example, P 1 , P 2 and P 3 The amino acid sequences can be:

[0653] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0654] SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 3, or

[0655] SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 3, or

[0656] SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 3, or

[0657] SEQ ID NO: 10, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0658] SEQ ID NO: 11, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0659] SEQ ID NO: 12, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0660] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 10, or

[0661] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, or

[0662] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, or

[0663] SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0664] On the other hand, P in this application 1 , P 2 and P 3 Can be each independently a protein having a ligase recognizable sequence, and the ligase can connect the substrate molecule to the protein. On the other hand, P in this application 1 , P 2 and P 3 Each of the proteins may independently be a protein having an LPETG sequence, and the LPETG sequence as shown in SEQ ID NO: 7 can be specifically recognized by a ligase, such as SrtA ligase.

[0665] On the other hand, P in this application 1 , P 2 and P 3 Each can be independently selected from VHH nanoantibodies targeting EGFR, VHH nanoantibodies targeting CD3, VHH nanoantibodies targeting 4-1BB, VHH nanoantibodies targeting Her2, VHH nanoantibodies targeting CD47, VHH nanoantibodies targeting CD20, branched peptides containing CMV antigens, fusion proteins of cytokines IL2 / IL5, cytokines IFNα, dye molecules FITC and CPG nucleic acid molecules.

[0666] Preparation method of compound

[0667] In one aspect, the present application provides a method for preparing a compound, comprising reacting a compound represented by formula (Ia-I) with reactant 1, reactant 2, and reactant 3;

[0668] Alternatively, the compound represented by formula (Ia-I) may be reacted with reactant 1 and reactant 2;

[0669] Alternatively, the compound represented by formula (Ia-I) may be reacted with reactant 1;

[0670]

[0671] The reactant 1 can be HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -A,

[0672] The reactant 2 can be HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -B,

[0673] The reactant 3 can be HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -C,

[0674] Said W may be a trivalent group, and said W may be optionally substituted by one or more R,

[0675] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 can be independently selected from the following group: alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0676] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Can be each independently selected from the following group: -NR 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1-C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0677] R, R 1 , R 1a and R 1b may be each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl;

[0678] wherein m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 can each be independently selected from a number greater than 0;

[0679] A, B and C can each be independently selected from the following groups:

[0680]

[0681] where R 12 , R 13 and R 14 may be each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl;

[0682] Among them, A, B and C, Cannot exist simultaneously;

[0683] Among them, A, B and C, Cannot exist simultaneously;

[0684] Among them, A, B and C, Cannot exist simultaneously;

[0685] When A, B and C, When both exist, the R12 , R 13 and R 14 Not an alkynyl group;

[0686] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino,

[0687] A, B and C are not the same

[0688] Among them, when A, B or C is When the compound represented by the formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, Replace with After the compound represented by formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, an acid may be added to remove Boc.

[0689] For example, Boc removal can be performed by adding acid (0%-300% hydrochloric acid or trifluoroacetic acid).

[0690] In another embodiment, the order of adding reactant 1, reactant 2, and reactant 3 can be determined according to the order of introducing A, B, and C. The order of introducing A, B, and C from first to last can be:

[0691] where R 12 , R 13 and R 14 Each is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl.

[0692] For example, when you need to introduce When you can first introduce reintroduction Finally introduced To decide to add N3-Amine first, then TZ-Amine, and finally Boc-Gly-Amine.

[0693] For example, in the above steps, the compound represented by formula (Ia-I) is reacted with reactant 1, reactant 2, and reactant 3 in an equivalent ratio ranging from 20 / 1 to 1 / 20, and 1%-100% molar equivalents of triethylamine, ethylenediamine, dimethylaminopyridine, pyridine, or N,N-diisopropylethylamine are added as a catalyst. The reaction conditions are 4-100° C., and the reaction solvent can be water, other water-based solutions, DMF, DMSO, methanol, acetonitrile, tetrahydrofuran, dichloromethane, or their corresponding mixed solvents. The reaction concentration can be in the range of 1 nanomolar to 10 molar, and the reaction time can be 0-24 hours.

[0694] For example, step 1: the starting material NHS reagent can be reacted with N3-Amine in a ratio of 1 / 1.2. The reaction conditions can be DMF (N,N-dimethylformamide) as a solvent, a catalytic amount of TEA (triethylamine) as a catalyst, and the reaction is carried out at 25° C. for 2 hours. After the reaction is completed, the solvent can be removed by rotary evaporation, and then it can be purified by HPLC (high performance liquid chromatography) to obtain the first intermediate N3-NHS-NHS.

[0695] Step 2: N3-NHS-NHS and TZ-Amine can be reacted in a ratio of 1 / 1.2. The reaction conditions can be DMF (N,N-dimethylformamide) as a solvent, a catalytic amount of TEA (triethylamine) as a catalyst, and the reaction is carried out at 25°C for 2 hours. After the reaction, the solvent can be removed by rotary evaporation, and then purified by HPLC (high performance liquid chromatography) to obtain a second intermediate, which is the multispecific bioconjugated linker 2 (Scaffold 2).

[0696] Step 3: The second intermediate, Scaffold 2, a multispecific bioconjugate, can be reacted with Boc-Gly-Amine in a ratio of 1:1.2. The reaction conditions can be DMF (N,N-dimethylformamide) as the solvent, a catalytic amount of TEA (triethylamine) as the catalyst, and the reaction is carried out at 25°C for 2 hours. After the reaction, the solvent can be removed by rotary evaporation, and then purified by HPLC (high performance liquid chromatography). The purified product can be dissolved in DCM (dichloromethane), and then 20% by volume of TFA (trifluoroacetic acid) can be added. The Boc (tert-butyloxycarbonyl) group can be removed by reacting at room temperature for 5 minutes. The final product, Scaffold 1, a multispecific bioconjugate, can then be purified by HPLC (high performance liquid chromatography).

[0697] On the other hand, the present application also provides a method for preparing a compound, which can react the compound described in any one of Formula Ia in the present application with reactant 4, reactant 5 and reactant 6;

[0698] The reactant 4 is A 1 -P 1 ,

[0699] The reactant 5 is B 1 -P 2 ,

[0700] The reactant 6 is C 1 -P 3 ,

[0701] Among them, A of reactant 4 1 React with A of the compound described in formula Ia, B of reactant 5 1 With the compound B of formula Ia, C of reactant 6 1 Reaction with C of the compound described in Formula Ia;

[0702] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one for

[0703] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0704] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0705] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0706] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0707] When at least one of A, B or C is When the corresponding A 1 , B1 or C 1 At least one for

[0708] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0709] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0710] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0711] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0712] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0713] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0714] where R 12 , R 13 and R 14 Each may be independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl.

[0715] In some embodiments, when at least one of A, B, or C is When the corresponding A 1 , B1 or C 1 At least one for

[0716] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0717] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0718] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0719] When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for

[0720] Among them, R, R 12 each X may be independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, and each X may be independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen.

[0721] For example, the groups in A, B, or C are the same as the corresponding A 1 , B 1 or C 1 The group A in the B is C is When A 1 for B 1 for C 1 for where R 12 It may be hydrogen.

[0722] For example, Cu can be added to the reaction +As a catalyst. For example, adding a catalytic amount of Cu+ to generate Cu+ in other forms acts as a catalyst.

[0723] For example, 1-100% equivalent of SrtA ligase can be added to the reaction as a catalyst.

[0724] For example, the reaction of the compound described in Formula Ia with reactant 4, reactant 5 and reactant 6 can be carried out in a molar ratio range of 20 / 1 to 1 / 20, and the reaction solvent can be water, other water-based solutions, DMF, DMSO, methanol, acetonitrile, tetrahydrofuran, dichloromethane or their corresponding mixed solvents, etc. The reaction concentration can be in the range of 1 nanomolar to 100 molar, and the reaction time can be 0-24 hours.

[0725] Treatment methods and uses

[0726] In one aspect, the present application provides a pharmaceutical composition, which may contain any compound of the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0727] On the one hand, the present application provides a kit, which may contain the compound described in any one of the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and / or the pharmaceutical composition described in any one of the present application.

[0728] On the other hand, the present application provides the use of the compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereoisomer, or mixture thereof, or pharmaceutically acceptable salt thereof, the pharmaceutical composition of the present application, and / or the kit of the present application in the preparation of a medicament for treating and / or preventing tumors. For example, the tumor can be selected from tumors associated with the expression of the following groups: 4-1BB, EGFR, CD3, Her2, CD47, and CD20. For example, the expression correlation can refer to correlation with high expression and / or target positivity of the target therein. For example, the expression correlation can refer to correlation with high expression of the target therein. For example, the expression correlation can refer to correlation with positive expression of the target therein. For example, the tumor can be selected from the following groups: solid tumors and blood cancers. For example, the tumor can be selected from the following groups: lung cancer, renal cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0729] On the other hand, the present application provides a method for treating and / or preventing tumors, comprising administering to a subject in need thereof a compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition of the present application, and / or a kit of the present application. For example, the tumor can be selected from tumors associated with the expression of the following groups: 4-1BB, EGFR, CD3, Her2, CD47, and CD20. For example, the expression correlation can refer to being associated with high expression and / or target positivity of the target therein. For example, the expression correlation can refer to being associated with high expression of the target therein. For example, the expression correlation can refer to being associated with high expression of the target therein. For example, the expression correlation can refer to being associated with being positive for the target therein. For example, the tumor can be selected from the following groups: solid tumors and blood cancers. For example, the tumor can be selected from the following groups: lung cancer, renal cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0730] On the other hand, the present application provides a compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition of the present application, and / or a kit of the present application, for treating and / or preventing tumors. For example, the tumor can be selected from tumors associated with the expression of the following groups: 4-1BB, EGFR, CD3, Her2, CD47, and CD20. For example, the expression correlation can refer to being associated with high expression and / or target positivity of the target therein. For example, the expression correlation can refer to being associated with high expression of the target therein. For example, the expression correlation can refer to being associated with positive expression of the target therein. For example, the tumor can be selected from the following groups: solid tumors and blood cancers. For example, the tumor can be selected from the following groups: lung cancer, renal cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0731] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the present invention.

[0732] Example

[0733] The materials and equipment used in this application include:

[0734] N3-Amine, TZ-Amine, Boc-Gly-Amine, FITC-Amine, DBCO-Gly, and BCN-Gly were purchased from Xi'an Dianhua Biotechnology Co., Ltd. or Click Chemistry Tools. Other compounds and solvents, unless otherwise specified, were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. or Beijing J&K Technology Co., Ltd.

[0735] CPG-Amine nucleic acid was purchased from Suzhou Jinweizhi Biotechnology Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO: 4, specifically: 5'-TCC ATG ACG TTC CTG ACG TT-3', wherein the 5' portion contains a C12 amino modification.

[0736] The branched peptide containing CMV antigen was purchased from Nanjing Source Peptide Biotechnology Co., Ltd., and its structure is shown in the formula CMV:

[0737]

[0738] The amino acids of the three branches are shown in SEQ ID NO: 5, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0739] All VHH nanobodies were expressed in Escherichia coli.

[0740] The amino acid sequence of the VHH nanobody targeting EGFR is shown in SEQ ID NO: 2, specifically:

[0741]

[0742] The amino acid sequence of the VHH nanobody targeting CD3 is shown in SEQ ID NO: 3, specifically:

[0743]

[0744] The amino acid sequence of the VHH nanobody targeting 4-1BB is shown in SEQ ID NO: 1, specifically:

[0745]

[0746] SrtA ligase (e.g., the amino acid sequence can be as shown in SEQ ID NO: 16) is expressed in Escherichia coli. The principle of the SrtA ligase-mediated coupling reaction (Chen, Long, et al. Scientific Reports 6.1 (2016): 1-12) is that SrtA ligase specifically recognizes the LPETG sequence shown in SEQ ID NO: 7 and transfers the substrate molecule with a naked glycine at the N-terminus to the target protein.

[0747] The neo2 protein of the present application is a fusion protein of cytokines IL2 / IL5, as shown in SEQ ID NO: 8, specifically:

[0748]

[0749] The IFNα protein of the present application is shown in SEQ ID NO: 9, specifically:

[0750] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVR KYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKELPETGHHHHHH. The instruments used included: Waters preparative high performance liquid chromatography (HPLC), Waters liquid chromatography-mass spectrometry (LC-MS), using a C18 column; size exclusion gel-filtration system, using a Superdex 75HR 16 / 600 column; Bio-red gel imaging system; and Bruker 400M nuclear magnetic resonance.

[0751] Module preparation method:

[0752] (1) Preparation of EGFR-DBCO

[0753] EGFR-DBCO is prepared using a SrtA ligase-mediated coupling reaction. The EGFR-targeting VHH nanobody contains the LPETG sequence. SrtA ligase can link the substrate molecule DBCO-Gly, which has a naked glycine at the N-terminus, to the EGFR nanobody. Specifically, 1 millimole of DBCO-Gly and 5 micromolar SrtA ligase are added to a 100 micromolar VHH nanobody solution in PBS. After reacting for 2 hours at room temperature, EGFR-DBCO is obtained. After size exclusion purification, the yield is approximately 80%.

[0754] (2) Preparation of CD3-BCN

[0755] CD3-BCN is prepared using a SrtA ligase-mediated coupling reaction. The CD3-targeting VHH nanobody contains the LPETG sequence. SrtA ligase can link the substrate molecule BCN-Gly, which has a naked glycine at the N-terminus, to the CD3 nanobody. To a 100 micromolar solution of the CD3-targeting VHH nanobody in PBS, 1 millimolar BCN-Gly and 5 micromolar SrtA ligase are added. After a 2-hour reaction at room temperature, CD3-BCN is obtained. After size exclusion purification, the yield is approximately 90%.

[0756] Example 1 Preparation of Multispecific Bioconjugate Linker

[0757] The method for preparing a multispecific bioconjugate linker (T-Linker) provided in this application synthesizes linker 1 (Scaffold 1) and linker 2 (Scaffold 2). The specific reaction formula is as follows:

[0758]

[0759] Step 1: The starting material NHS reagent and N3-Amine are reacted in a ratio of 1 / 1.2. The reaction conditions are DMF (N,N-dimethylformamide) as a solvent and a catalytic amount of TEA (triethylamine) as a catalyst. The reaction is carried out at 25°C for 2 hours. After the reaction is completed, the solvent is removed by rotary evaporation and then purified by HPLC (high performance liquid chromatography) to obtain the first intermediate N3-NHS-NHS with a yield of 74%.

[0760] Step 2: N3-NHS-NHS and TZ-Amine were reacted in a ratio of 1 / 1.2 under the following reaction conditions: DMF (N,N-dimethylformamide) as solvent, catalytic amount of TEA (triethylamine) as catalyst, reaction at 25°C for 2 hours, and after completion of the reaction, the solvent was removed by rotary evaporation, and then purified by HPLC (high performance liquid chromatography) to obtain the second intermediate, namely the multispecific bioconjugated linker 2 (Scaffold 2), with a yield of 65%.

[0761] Step 3: The second intermediate, Scaffold 2, a multispecific bioconjugate, was reacted with Boc-Gly-Amine in a ratio of 1 / 1.2. The reaction conditions were DMF (N,N-dimethylformamide) as a solvent and a catalytic amount of TEA (triethylamine) as a catalyst. The reaction was carried out at 25°C for 2 hours. After the reaction, the solvent was removed by rotary evaporation and then purified by HPLC (high performance liquid chromatography). The purified product was dissolved in DCM (dichloromethane), followed by the addition of 20% by volume of TFA (trifluoroacetic acid) and the reaction was carried out at room temperature for 5 minutes to remove the Boc (tert-butyloxycarbonyl) group. The final product, Scaffold 1, a multispecific bioconjugate, was then purified by HPLC (high performance liquid chromatography) with a yield of 48%.

[0762] use 1 The prepared Scaffold 1 and Scaffold 2 were characterized by H-NMR and liquid chromatography-mass spectrometry (ESI-MS). Figure 1-4 The results shown are analyzed as follows:

[0763] Scaffold 1 1 The H-NMR characterization results are: 1 H NMR(CD3OD,400MHz)δ(ppm):10.28(s,1H),8.45-8.43(d,2H),8.38-8.36 (m,4H),7.89-7.87(d,1H),7.62-7.60(d,1H),7.51-7.49(d,2H),7.43-7. 40(d,1H),7.35-7.33(d,1H),5.45(s,1H),3.84(s,4H),3.64(s,2H),3.6 0-3.38(m,18H),3.26-3.22(m,6H),1.89-1.84(m,6H),1.72-1.66(m,6H). ESI-MS characterization results are: C53H82N14O15, 1154.3 (m / z); [M+H] + :1155.5,[M+Na] + :1177.5(m / z).

[0764] Scaffold2 1 The H-NMR characterization results are: 1H NMR (CD3OD, 400 MHz) δ (ppm): 10.43 (s, 1H), 8.69 (d, 1H), 8.66 (m, 2H), 8.47-8.45 (d, 2H), 8.35-8.31 (m, 2H), 7.59-7.57 (d, 2H), 7.53-7.47 (m, 2H), 3.88 (s, 2H), 3.67 (s, 2H), 3.61-3.43 (m, 12H), 3.31-3.26 (m, 4H), 1.91-1.84 (m, 4H), 1.74-1.67 (m, 4H). ESI-MS characterization results are: C45H60N12O14, 992.4 (m / z); [M+H] + :993.4,[M+Na] + :1015.3(m / z).

[0765] use 1 The intermediate N3-NHS-NHS in the synthesis of Scaffold 1 and Scaffold 2 was characterized by H-NMR. Figure 5 The results shown are analyzed as follows: 1 H NMR(CD2Cl2,400MHz)δ(ppm):8.97-8.96(t,1H),8.875-8.871(d,2H),7.79-7.77(t,1H),6.92(s,1H),3.93(s,2H),3.69- 3.60(m,8H),3.56-3.54(m,2H),3.46-3.4(m,4H),3.37-3.32(m,2H),2.17(s,8H),1.96-1.90(dd,2H),1.69-1.75(dd,2H).

[0766] The present application provides methods and steps similar to those described above for preparing Scaffold 3 and Scaffold 4. The specific reaction formula can be shown as follows:

[0767]

[0768] Step 1: The starting material NHS reagent is reacted with N3-Amine* in a ratio of 1:1.2. The reaction conditions are DMF (N,N-dimethylformamide) as a solvent and a catalytic amount of TEA (triethylamine) as a catalyst. The reaction is carried out at 25°C for 2 hours. After the reaction is completed, the solvent is removed by rotary evaporation and then purified by HPLC (high performance liquid chromatography) to obtain the first intermediate N3-NHS-NHS* with a yield of 74%.

[0769] Step 2: N3-NHS-NHS* and TZ-Amine* were reacted in a ratio of 1 / 1.2 in the presence of DMF (N,N-dimethylformamide) as a solvent and a catalytic amount of TEA (triethylamine) as a catalyst at 25°C for 2 hours. After the reaction, the solvent was removed by rotary evaporation and then purified by HPLC (high performance liquid chromatography) to obtain the second intermediate, Scaffold 4, a multispecific bioconjugate, with a yield of 75%.

[0770] Step 3: The second intermediate, multispecific bioconjugate scaffold 4, was reacted with Boc-Gly-Amine* in a ratio of 1:1.2 using DMF (N,N-dimethylformamide) as solvent and a catalytic amount of TEA (triethylamine) as catalyst at 25°C for 2 hours. The solvent was removed by rotary evaporation and then purified by HPLC (high performance liquid chromatography). The purified product was dissolved in DCM (dichloromethane), followed by the addition of 20% by volume of TFA (trifluoroacetic acid) and reacted at room temperature for 5 minutes to remove the Boc (tert-butyloxycarbonyl) group. The final product, multispecific bioconjugate scaffold 3, was then purified by HPLC (high performance liquid chromatography) in a 46% yield.

[0771] use 1 The prepared Scaffold 3 and Scaffold 4 were characterized by H-NMR and liquid chromatography-mass spectrometry (ESI-MS). Figure 22-25 The results shown are analyzed as follows:

[0772] Scaffold3 1 The H-NMR characterization results are: 1 H NMR (CD3OD, 400 MHz) δ (ppm): 10.20 (s, 1H), 8.17-8.15 (d, 2H), 8.08-8.06 (m, 3H), 7.34-7.32 (d, 2H), 3.83 (s, 2H), 3.66 (s, 2H), 3.44-3.37 (m, 12H), 2.05 (s, 2H). ESI-MS characterization results are: C29H34N14O6, 674.3 (m / z); [M+H] + :675.2,[M+Na]+:697.3(m / z).

[0773] 1H-NMR characterization results for Scaffold 4 were: 1H NMR (CD3OD, 400 MHz) δ (ppm): 10.29 (s, 1H), 8.69 (s, 1H), 8.55-8.44 (m, 4H), 7.53-7.51 (m, 2H), 7.49-7.47 (d, 2H), 7.41-7.39 (m, 2H), 3.91 (s, 2H), 3.57-3.46 (m, 8H), 2.94 (s, 4H), 2.67 (s, 2H). ESI-MS characterization results were: C29H28N12O8, 672.2 (m / z); [M+H]+: 673.1, [M+Na]+: 695.1 (m / z).

[0774] The intermediate N3-NHS-NHS* in the synthesis of Scaffold 3 and Scaffold 4 was characterized by ESI-MS. Figure 26 The results shown were analyzed as follows: C21H19N7O10, 529.1 (m / z); [M+H]+: 530.2, [M+Na]+: 552.1 (m / z).

[0775] The multispecific bioconjugate linker (T-Linker) contained in this application can be prepared and characterized by similar methods and steps as above.

[0776] Example 2 Preparation of multispecific conjugates

[0777] EGFR-CD3-FITC

[0778] A multispecific conjugate, such as the multispecific conjugate EGFR-CD3-FITC, is synthesized by using the multispecific bioconjugate linker (T-Linker) provided in this application to prepare a multispecific conjugate (T-Body). The specific reaction formula is shown below:

[0779]

[0780] The preparation method comprises first reacting the multispecific bioconjugate Scaffold 2 with FITC-Amine. FITC-Amine (final concentration of 10 mM) is added to a 1 mM solution of Scaffold 2 in DMF (N,N-dimethylformamide). The mixture reacts at room temperature for 2 hours, followed by purification by HPLC to obtain N3-TZ-FITC. N3-TZ-FITC is then reacted with CD3-BCN at a 1:1 molar ratio. The reaction is carried out at a concentration of 100 μM using PBS (phosphate buffered saline) as the solvent. After reacting for 1 hour at room temperature, N3-CD3-FITC is purified by size exclusion chromatography. Finally, N3-CD3-FITC is reacted with EGFR-DBCO at a 1:1 molar ratio. The reaction is carried out at a concentration of 100 μM using PBS (phosphate buffered saline) as the solvent. After reacting for 2 hours at room temperature, EGFR-CD3-FITC is purified by size exclusion chromatography.

[0781] EGFR-CD3-FITC was characterized by SDS-PAGE and Coomassie blue staining. Figure 6 As shown, its purity is >90%.

[0782] The results of EGFR-CD3-FITC characterization by LC-MS are as follows Figure 7 shown.

[0783] EGFR-CD3-LCFA

[0784] Using a similar method as described above, a multispecific conjugate (T-Body) was prepared by using a multispecific bioconjugate linker (T-Linker) provided in this application. For example, a multispecific conjugate EGFR-CD3-LCFA (LCFA is a long-chain fatty acid) was synthesized. The preparation method of EGFR-CD3-LCFA can be the same as that of the example EGFR-CD3-FITC, except that FITC is replaced with LCFA. The specific reaction formula is as follows:

[0785]

[0786] EGFR-CD3-LCFA was characterized by SDS-PAGE and Coomassie blue staining. Figure 27 As shown in Figure 2, its purity is >90%. The results of LC-MS characterization of EGFR-CD3-LCFA are shown in Figure 2. Figure 28 shown.

[0787] Example 3 Preparation of multispecific conjugates

[0788] EGFR-CD3-CPG

[0789] A multispecific conjugate, such as the multispecific conjugate EGFR-CD3-CPG, is synthesized by using the multispecific bioconjugate linker (T-Linker) provided in this application to prepare a multispecific conjugate (T-Body). The specific reaction formula is shown below:

[0790]

[0791] The preparation method comprises first reacting the multispecific bioconjugate Scaffold 2 with CPG-Amine. 1 millimole of Scaffold 2 is added to a PBS (phosphate buffered saline) solution containing 100 micromolar CPG-Amine. The reaction is allowed to proceed at room temperature for 2 hours, followed by purification by HPLC to obtain N3-TZ-CPG. Next, N3-TZ-CPG is reacted with CD3-BCN at a 1:1 molar ratio. The reaction is carried out at a concentration of 100 micromolar using PBS (phosphate buffered saline) as the solvent. After reacting for 1 hour at room temperature, the N3-CD3-CPG is purified by size exclusion chromatography. Finally, N3-CD3-CPG is reacted with EGFR-DBCO at a 1:1 molar ratio. The reaction is carried out at a concentration of 100 micromolar using PBS (phosphate buffered saline) as the solvent. After reacting for 2 hours at room temperature, the EGFR-CD3-CPG is purified by size exclusion chromatography.

[0792] EGFR-CD3-CPG was characterized by SDS-PAGE and Coomassie blue staining. Figure 8 As shown, its purity is >90%.

[0793] The results of EGFR-CD3-CPG characterization by LC-MS are as follows Figure 9 shown.

[0794] EGFR-CD3-ASO

[0795] Using a similar method as described above, a multispecific conjugate (T-Body) was synthesized by using the multispecific bioconjugate linker (T-Linker) provided in this application, such as the multispecific conjugate EGFR-CD3-ASO. For example, an antisense oligonucleotide ASO for STAT3 can be used, and its sequence can be Me C*T*A *T*T*T*G*G*A*T*G*T* Me C* A*G* Me CSEQ ID NO: 13 (underlined representations of locked nucleic acid (LNA), for example, whose nucleoside may include a bicyclic sugar bridge connecting the 4'-position and the 2'-position, MeC represents methyl C, and * represents a phosphorothioate internucleoside linkage). The preparation method of EGFR-CD3-ASO can be the same as that of Example EGFR-CD3-FITC, except that FITC is replaced with ASO. The specific reaction formula is shown below:

[0796]

[0797] EGFR-CD3-ASO was characterized by SDS-PAGE and Coomassie blue staining. Figure 29 As shown in Figure 2, its purity is >90%. The results of LC-MS characterization of EGFR-CD3-ASO are shown in Figure 2. Figure 30 shown.

[0798] Example 4 Preparation of multispecific conjugates

[0799] Multispecific conjugates, such as the multispecific conjugates EGFR-CD3-CMV, EGFR-CD3-neo2, EGFR-CD3-IFNα and EGFR-CD3-M1, were synthesized by the method of preparing multispecific conjugates (T-Body) using the multispecific bioconjugate linker (T-Linker) provided in this application.

[0800] EGFR-CD3-CMV

[0801] The preparation method of EGFR-CD3-CMV is to first react the above-mentioned multi-specific bioconjugated linker 1 (Scaffold 1) with a branched peptide containing CMV antigen. 1 millimole of linker 1 (Scaffold 1) is added to a PBS (phosphate buffered saline) solution containing 100 micromoles of branched peptide, and then the above-mentioned 5 micromoles of SrtA ligase is added. After reacting at room temperature for 2 hours, size exclusion purification is used to obtain N3-TZ-CMV. Then, N3-TZ-CMV reacts with CD3-BCN at a molar ratio of 1 / 1. The reaction concentration is 100 micromoles, PBS (phosphate buffered saline) is used as a solvent, and after reacting at room temperature for 1 hour, size exclusion chromatography is used to purify N3-CD3-CMV. Finally, N3-CD3-CMV reacts with EGFR-DBCO at a molar ratio of 1 / 1. The reaction concentration is 100 micromoles, PBS (phosphate buffered saline) is used as a solvent, and after reacting at room temperature for 2 hours, size exclusion chromatography is used to purify EGFR-CD3-CMV. The specific reaction formula is as follows Figure 19 shown.

[0802] EGFR-CD3-CMV was characterized by SDS-PAGE and Coomassie blue staining. Figure 10 As shown, its purity is >90%.

[0803] The results of EGFR-CD3-CMV characterization by LC-MS are as follows Figure 11 shown.

[0804] EGFR-CD3-neo2

[0805] The preparation method of EGFR-CD3-neo2 is as follows: first, the multispecific bioconjugate Scaffold 1 described above is reacted with neo2 having the sequence shown in SEQ ID NO: 8 of the present application. 1 millimole of Scaffold 1 is added to a 100 micromolar branched peptide solution in PBS (phosphate buffered saline), followed by the addition of 5 micromolar SrtA ligase. After reacting at room temperature for 2 hours, the product is purified by size exclusion chromatography to obtain N3-TZ-neo2. Next, N3-TZ-neo2 is reacted with CD3-BCN at a 1:1 molar ratio. The reaction is carried out at a concentration of 100 micromolar using PBS (phosphate buffered saline) as the solvent. After reacting at room temperature for 1 hour, the product is purified by size exclusion chromatography to obtain N3-CD3-neo2. Finally, N3-CD3-neo2 is reacted with EGFR-DBCO at a 1:1 molar ratio. The reaction concentration was 100 μM, PBS (phosphate buffered saline) was used as the solvent, and after reacting at room temperature for 2 hours, EGFR-CD3-neo2 was purified by size exclusion chromatography.

[0806] EGFR-CD3-neo2 was characterized by SDS-PAGE and Coomassie blue staining. Figure 12 As shown, its purity is >90%.

[0807] The results of LC-MS characterization of EGFR-CD3-neo2 are as follows Figure 13 shown.

[0808] EGFR-CD3-IFNα

[0809] The method for preparing EGFR-CD3-IFNα is as follows: first, the multispecific bioconjugate Scaffold 1 described above is reacted with the IFNα sequence set forth in SEQ ID NO: 9 of the present application. One millimole of Scaffold 1 is added to a 100 micromolar branched peptide solution in PBS (phosphate buffered saline). Then, 5 micromolar SrtA ligase is added. The reaction is allowed to react at room temperature for 2 hours, followed by size exclusion purification to obtain N3-TZ-IFNα. Next, N3-TZ-IFNα is reacted with CD3-BCN at a 1:1 molar ratio. The reaction is carried out at a concentration of 100 micromolar, using PBS (phosphate buffered saline) as the solvent. After reacting at room temperature for 1 hour, the reaction is followed by size exclusion chromatography to obtain N3-CD3-IFNα. Finally, N3-CD3-IFNα is reacted with EGFR-DBCO at a 1:1 molar ratio. The reaction concentration was 100 μM, PBS (phosphate buffered saline) was used as the solvent, and after reacting at room temperature for 2 hours, EGFR-CD3-IFNα was purified by size exclusion chromatography.

[0810] EGFR-CD3-IFNα was characterized by SDS-PAGE and Coomassie blue staining. Figure 14 As shown, its purity is >90%.

[0811] The results of EGFR-CD3-IFNα characterization by LC-MS are as follows Figure 15 shown.

[0812] EGFR-CD3-M1

[0813] Using a similar preparation method as described above, the multi-specific bioconjugate linker 1 (Scaffold 1) and the sequence of M1 shown in SEQ ID NO: 14 of the present application (M1 is a short peptide that penetrates the blood-brain barrier) are reacted to obtain EGFR-CD3-M1. The specific reaction formula is as follows: Figure 31 shown.

[0814] EGFR-CD3-M1 was characterized by SDS-PAGE and Coomassie blue staining. Figure 32 As shown, its purity is >90%.

[0815] The results of LC-MS characterization of EGFR-CD3-M1 are as follows Figure 33 shown.

[0816] EGFR-CD3-PDL1

[0817] Using a similar preparation method as described above, the multispecific bioconjugate linker 1 (Scaffold 1) and the PDL1 sequence shown in SEQ ID NO: 15 of the present application (PDL1 is an anti-PDL1 VHH antibody) are reacted to obtain EGFR-CD3-PDL1. The specific reaction formula is as follows: Figure 34 shown.

[0818] EGFR-CD3-PDL1 was characterized by SDS-PAGE and Coomassie blue staining. Figure 35 As shown, its purity is >90%.

[0819] The results of EGFR-CD3-PDL1 characterization by LC-MS are as follows Figure 36 shown.

[0820] Example 5 Preparation of multispecific conjugates

[0821] EGFR-CD3-4-1BB

[0822] The multispecific conjugate (T-Body) was synthesized by the method of preparing a multispecific conjugate (T-Body) using a multispecific bioconjugate linker (T-Linker) provided in the present application, for example, a trispecific VHH nanobody EGFR-CD3-4-1BB, and the specific reaction formula is as follows: Figure 20 shown.

[0823] The preparation method is to first react the multi-specific bioconjugated linker 1 (Scaffold 1) with the VHH nanobody targeting 4-1BB. 1 millimole of linker 1 (Scaffold 1) is added to a PBS (phosphate buffered saline) solution containing 100 micromoles of VHH nanobody, and then the above-mentioned 5 micromoles of SrtA ligase is added. After reacting for 2 hours at room temperature, size exclusion purification is used to obtain N3-TZ-4-1BB. Then, N3-TZ-4-1BB is reacted with CD3-BCN at a molar ratio of 1 / 1. The reaction concentration is 100 micromoles, PBS (phosphate buffered saline) is used as a solvent, and after reacting for 1 hour at room temperature, size exclusion chromatography is used to obtain N3-CD3-4-1BB. Finally, N3-CD3-4-1BB is reacted with EGFR-DBCO at a molar ratio of 1 / 1. The reaction concentration was 100 μM, PBS (phosphate buffered saline) was used as the solvent, and after reacting for 2 hours at room temperature, EGFR-CD3-4-1BB was purified by size exclusion chromatography.

[0824] EGFR-CD3-4-1BB was characterized by SDS-PAGE and Coomassie blue staining. Figure 16 As shown, its purity is >90%.

[0825] The results of LC-MS characterization of EGFR-CD3-4-1BB are as follows Figure 17 shown.

[0826] HER2-CD3-PDL1

[0827] The multispecific conjugates were synthesized by the method for preparing multispecific conjugates using multispecific bioconjugate linkers provided in the present application, such as the trispecific VHH nanobody HER2-CD3-PDL1. The specific reaction formula is as follows: Figure 37 shown.

[0828] The preparation method is to first react the multi-specific bioconjugated linker 1 (Scaffold 1) with the VHH nanobody targeting PDL1. 1 millimole of linker 1 (Scaffold 1) is added to a PBS (phosphate buffered saline) solution containing 100 micromoles of VHH nanobody, and then the above-mentioned 5 micromoles of SrtA ligase is added. After reacting for 2 hours at room temperature, size exclusion purification is used to obtain N3-TZ-PDL1. Then, N3-TZ-PDL1 is reacted with CD3-BCN at a molar ratio of 1 / 1. The reaction concentration is 100 micromoles, PBS (phosphate buffered saline) is used as the solvent, and after reacting for 1 hour at room temperature, size exclusion chromatography is used to purify N3-CD3-PDL1. At the same time, HER2-DBCO is prepared. Its preparation method is the same as EGFR-DBCO, except that the VHH nanobody targeting EGFR is replaced with HER2 binding protein. Finally, N3-CD3-PDL1 and HER2-DBCO are reacted at a molar ratio of 1 / 1. The reaction concentration was 100 μM, PBS (phosphate buffered saline) was used as a solvent, and after reacting for 2 hours at room temperature, HER2-CD3-PDL1 was purified by size exclusion chromatography.

[0829] The results of HER2-CD3-PDL1 characterization by LC-MS are as follows Figure 38 shown.

[0830] Example 6 Multispecific conjugate EGFR-CD3-4-1BB inhibits tumor cell growth

[0831] The application of the EGFR-CD3-4-1BB prepared above in anti-tumor applications specifically involves EGFR-CD3-4-1BB-mediated PBMCs killing of tumor cells. PBMCs were obtained from Shanghai Ausells Biotechnology Co., Ltd., and the tumor cells used were A431 cells obtained from ATCC. Specific experimental details are as follows:

[0832] On day 1, A431 cells were seeded in 96-well plates at a density of 10,000 cells per well in a 100 μl volume. The culture medium used was DMEM supplemented with 20% FBS (fetal bovine serum) and 10% penicillin-streptomycin. Culture conditions were in a 37°C incubator with 5% carbon dioxide.

[0833] On the second day, the old culture medium in the 96-well plate was aspirated and 100 microliters of new culture medium was added. Then, 100 microliters of 1640 culture medium (containing 20% FBS (fetal bovine serum), 10% double antibody (penicillin-streptomycin mixture)) containing 150,000 PBMCs was added to each well. Then, different concentrations of EGFR-CD3-4-1BB were added to the experimental group, the corresponding concentrations of EGFR-TZ-4-1BB or EGFR-CD3-Gly were added to the control group, and the corresponding volume of PBS (phosphate buffered saline) was added to the blank group. The preparation method of the control group EGFR-TZ-4-1BB and EGFR-CD3-Gly was referred to the above embodiment and as shown in the following example. Figure 21 shown.

[0834] Specifically, 1 millimole of the connecting arm (Scaffold1) was added to a PBS (phosphate buffered saline) solution containing 100 micromolar VHH nanoantibody targeting 4-1BB, and then the above-mentioned 5 micromolar SrtA ligase was added. After reacting for 2 hours at room temperature, size exclusion purification was used to obtain N3-TZ-4-1BB. Then, N3-TZ-4-1BB and EGFR-DBCO were reacted at a molar ratio of 1 / 1. The reaction concentration was 100 micromolar, PBS (phosphate buffered saline) was used as a solvent, and EGFR-TZ-4-1BB was purified by size exclusion chromatography after reacting for 2 hours at room temperature.

[0835] Specifically, the linker (Scaffold 1) was reacted with CD3-BCN at a molar ratio of 1:1. The reaction concentration was 100 micromolar, PBS (phosphate buffered saline) was used as a solvent, and after reacting for 1 hour at room temperature, size exclusion chromatography was used to purify N3-CD3-Gly. Then, N3-CD3-Gly was reacted with EGFR-DBCO at a molar ratio of 1:1. The reaction concentration was 100 micromolar, PBS (phosphate buffered saline) was used as a solvent, and after reacting for 2 hours at room temperature, size exclusion chromatography was used to purify EGFR-CD3-Gly.

[0836] On the third day, the LDH lactate dehydrogenase cytotoxicity assay was used to test the killing effect of the above experimental groups on tumor cells. The kit used was KTA1030LDH Cytotoxicity Assay Kit LDH Cytotoxicity Assay Kit (Abbkine), and the specific operation steps were carried out according to the instructions provided by the kit. The results are as follows Figure 18 shown.

[0837] Example 7 Binding Ability of Multispecific Conjugates to Multiple Targets

[0838] use Octet RED96 was used to measure the binding of EGFR-CD3-PDL1 multispecific antibody to EGFR, CD3, and PDL1 receptors. First, EGFR, CD3, and PDL1 receptor proteins with Biotin tags were prepared (purchased from Sino-Bio). The above proteins were dissolved in PBS solution to a concentration of 100ug / ml. 50nM, 100nM, 200nM, 400nM, and 800nM EGFR-PDL1-CD3 multispecific antibody PBS solutions were prepared. The binding force was then measured using a streptavidin-coupled Biosensor sensor. Octet RED96 universal binding assay. The assay consists of three main steps: immobilization, binding, and dissociation. Following the assay, the instrument's built-in multi-concentration kinetic analysis software is used to fit the Kon, Kdis, and final KD values for the binding of the EGFR-PDL1-CD3 multispecific antibody to different receptors.

[0839] Binding characterization of EGFR-PDL1-CD3 multispecific antibody to EGFR, CD3, and PDL1 receptor proteins Figure 39 As shown in A-39B, Figure 39 (A) shows the binding curve of EGFR-CD3-PDL1 with EGFR, CD3, and PDL1 proteins. The binding height in the curve from low to high corresponds to EGFR-CD3-PDL1 concentrations of 50nM, 100nM, 200nM, 400nM, and 800nM; Figure 39 (B) shows the binding affinity of EGFR-CD3-PDL1 to different targets. The results show that the multispecific conjugate (T-Body) of the present application can simultaneously maintain the activity of multiple functional domains, such as receptor binding activity.

[0840] The present application uses the above-mentioned examples to illustrate the multispecific bioconjugate linker provided herein and its synthesis, the multispecific conjugate prepared using the bioconjugate linker, and their preparation methods and uses. However, the present application is not limited to the above-mentioned process steps, that is, it does not mean that the present application must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvements to the present application, equivalent replacement of raw materials used in the present application, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application. Sequence Listing <110> Beijing University <120> Multispecific bioconjugate linker and synthesis method thereof <130> 0198-PA-007CN <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 148 <212> PRT <213> Artificial Sequence <220> <223> 4-1BB VHH <400> 1 Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser 20 25 30 Asn Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly 35 40 45 Val Ala Val Ile Cys Thr Gly Gly Gly Ser Pro Ser Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Asp Leu Leu Arg Ala Gly Thr Pro Leu Ser Ser Tyr Glu 100 105 110 Phe Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Leu Pro Glu Thr Gly Gly Ala Ala Ala Leu Glu His His 130 135 140 His His His His 145 <210> 2 <211> 148 <212> PRT <213> Artificial Sequence <220> <223> EGFR VHH <400> 2 Met Asp Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Thr 1 5 10 15 Gly Gly Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg 20 25 30 Ser Tyr Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Asp Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly His His {130 135 140} His His His His 145 <210> 3 <211> 148 <212> PRT <213> Artificial Sequence <220> <223> CD3 VHH <400> 3 Met Asp Glu Val Gln Leu Val Glu Ser Gly Gly Gly Pro Val Gln Ala 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Tyr Arg 20 25 30 Gly Tyr Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Val Trp Ser Gly Gly Asn Thr Tyr Tyr Glu Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Met Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Ser Ala Thr Tyr 85 90 95 Tyr Cys Ala Ala Lys Ile Arg Pro Tyr Ile Phe Lys Ile Ala Gly Gln 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly His His 130 135 140 His His His His 145 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CPG - amino <220> <221> misc_feature <222> (1)..(1) <223> C12 amino modification <400> 4 tccatgacgt tcctgacgtt 20 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CMV1 / 2 <400> 5 Asn Leu Val Pro Met Val Ala Thr Val Pro Arg Gly Leu Arg Lys 1 5 10 15 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CMV3 <400> 6 Lys Gly Gly Asn Gln Leu Pro Glu Thr Gly Gly 1 5 10 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> SrtA target sequence <400> 7 Leu Pro Glu Thr Gly 1 5 <210> 8 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> neo2 <400> 8 Pro Lys Lys Lys Ile Gln Leu His Ala Glu His Ala Leu Tyr Asp Ala 1 5 10 15 Leu Met Ile Leu Asn Ile Val Lys Thr Asn Ser Pro Pro Ala Glu Glu 20 25 30 Lys Leu Glu Asp Tyr Ala Phe Asn Phe Glu Leu Ile Leu Glu Glu Ile 35 40 45 Ala Arg Leu Phe Glu Ser Gly Asp Gln Lys Asp Glu Ala Glu Lys Ala 50 55 60 Lys Arg Met Lys Glu Trp Met Lys Arg Ile Lys Thr Thr Ala Ser Glu 65 70 75 80 Asp Glu Gln Glu Glu Met Ala Asn Ala Ile Ile Thr Ile Leu Gln Ser 85 90 95 Trp Ile Phe Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Leu Pro 100 105 110 Glu Thr Gly Gly His His His His His His 115 120 <210> 9 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> IFN alpha <400> 9 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu Leu Pro Glu Thr Gly His His His His His His 165 170 175 <210> 10 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> Her2 Ab <400> 10 Met Val Asp Asn Lys Phe Asn Lys Glu Met Arg Asn Ala Tyr Trp Glu<000419o>1 5 10 15 Ile Ala Leu Leu Pro Asn Leu Asn Asn Gln Gln Lys Arg Ala Phe Ile 20 25 30 Arg Ser Leu Tyr Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu 35 40 45 Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Gly Leu Pro Glu Thr 50 55 60 Gly Gly Leu Glu His His His His His His 65 70 <210> 11 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> CD47 Ab <400> 11 Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro It should be noted that there may be a misspelling in the original text where "<000419o>" is likely " ". This has been maintained as is in the translation. 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 [[ID=##**##]]Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser Gly Gly Gly Ser Gly Gly Gly Ser Leu Pro 115 120 125 Glu Thr Gly Gly His His His His His His 130 135 <210> 12 <211> 144 <212> PRT <213> Artificial Sequence <220> <223> CD20 Ab <400> 12 Met Asp Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser 20 25 30 Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser 50 55 60 Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly His His His His His His 130 135 140 <210> 13 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> ASO <220> <221> misc_feature <222> (1)..(16) <223> Phosphorothioate internucleoside linkages <220> <221> misc_feature <222> (1)..(1) <223> 5-methylcytosine <220> <221> misc_feature <222> (13) <223> 5-methylcytosine <220> <221> misc_feature <222> (16) <223> 5-methylcytosine <220> <221> misc_feature <222> (1)..(3) <223> Locked Nucleic Acid (LNA) <220> <221> misc_feature <222> (14)..(16) <223> Locked Nucleic Acid (LNA) <400> 13 ctatttggat gtcagc 16 <210> 14 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> M1 <400> 14 Thr Phe Tyr Gly Gly Arg Pro Lys Arg Asn Asn Phe Leu Arg Gly Ile 1 5 10 15 Arg Ser Arg Gly Asp Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30 Ser Ala Leu Pro Glu Thr Gly Gly 35 40 <210> 15 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> PDL1 VHH <400> 15 Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser 20 25 30 Arg Arg Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Arg Val Ala Lys Leu Leu Thr Thr Ser Gly Ser Thr Tyr Leu Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Ser Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr 100 105 110 Ser Ser Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Pro Glu Thr 130 135 140 Gly Gly His His His His His His 145 150 <210> 16 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> SrtA enzyme <400> 16 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Lys Pro His Ile Asp Asn Tyr Leu His Asp Lys Asp Lys Asp 35 40 45 Glu Lys Ile Glu Gln Tyr Asp Lys Asn Val Lys Glu Gln Ala Ser Lys 50 55 60 Asp Lys Lys Gln Gln Ala Lys Pro Gln Ile Pro Lys Asp Lys Ser Lys 65 70 75 80 Val Ala Gly Tyr Ile Glu Ile Pro Asp Ala Asp Ile Lys Glu Pro Val 85 90 95 Tyr Pro Gly Pro Ala Thr Arg Glu Ala Leu Asn Arg Gly Val Ser Phe 100 105 110 Ala Lys Glu Asn Gln Ser Leu Asp Asp Gln Asn Ile Ser Ile Ala Gly 115 120 125 His Thr Phe Ile Gly Arg Pro Asn Tyr Gln Phe Thr Asn Leu Lys Ala 130 135 140 Ala Lys Lys Gly Ser Met Val Tyr Phe Lys Val Gly Asn Glu Thr Arg 145 150 155 160 Lys Tyr Lys Met Thr Ser Ile Arg Asn Val Lys Pro Thr Ala Val Gly 165 170 175 Val Leu Asp Glu Gln Lys Gly Lys Asp Lys Gln Leu Thr Leu Ile Thr 180 185 190 Cys Asp Asp Leu Asn Arg Glu Thr Gly Val Trp Glu Thr Arg Lys Ile 195 200 205 Leu Val Ala Thr Glu Val Lys 210 215

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, having the structure shown in Formula Ia: in, for The W is Said A, B and C are each independently selected from the following groups: where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 14-membered heteroaryl, represents the junction site; Among them, A, B and C, Cannot Existing at the same time, Among them, A, B and C, Cannot Existing at the same time, Among them, A, B and C, Cannot Existing at the same time, When A, B and C, When both exist, the R 12 is not an alkynyl group, When A, B and C, When both exist, the R 12 Not amino, A, B and C are not the same The La is -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -; The Lb is -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -; The Lc is -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -; in, X 1 , X 2 and X 3 are each independently selected from the group consisting of: -NH- and -O-, (K 1 ) n1 , (K 2 ) n2 and (K 3 ) n3 are each independently selected from the group consisting of -(CH2)-(CH2-O-CH2)3-(CH2)- and -(CH2)2-(CH2-O-CH2)3-(CH2)2-, or n1, n2 or n3 are each independently 0, (Y 1 ) p1 ,(Y 2 ) p2 and (Y 3 ) p3 Each independently is -NR 1 -C(=O)-,R 1 selected from the group consisting of hydrogen, protium, deuterium and tritium; or p1, p2 or p3 are each independently 0, (L 1 ) q1 ,(L 2 ) q2 and (L 3 ) q3 Each is independently selected from the following group: -CH2-aryl-, -(CH2)2- and -CH2-, or q1, q2 and q3 are each independently 0, wherein the aryl group is a 6-membered to 14-membered aryl group.

2. The compound according to claim 1, wherein: (1) A is B is C is or (2) A is B is C is or (3) A is B is C is or (4) A is B is C is or (5) A is B is C is or (6) A is Or B is C is or (7) A is B is C is or where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 14-membered heteroaryl, represents the junction site; When A, B and C, When both exist, the R 12 Not an alkynyl group; When A, B and C, When both exist, the R 12 Not amino.

3. The compound according to claim 1, wherein: (1) A is B is C is or (2) A is B is C is or (3) A is B is C is where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 14-membered heteroaryl, represents the junction site; When A, B and C, When both exist, the R 12 Not amino.

4. The compound according to claim 1, wherein: (1) A is B is C is or (2) A is B is C is or (3) A is B is C is where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 14-membered heteroaryl, represents the junction site; When A, B and C, When both exist, the R 12 Not amino.

5. The compound according to claim 1, wherein: (1) A is B is C is or (2) A is B is C is or (3) A is B is C is where R 12 Selected from the group consisting of hydrogen, protium, deuterium, tritium, halogens and C1-C 12 Alkyl, Represents the junction site.

6. The compound according to claim 1, wherein: A is B is C is where R 12 Selected from the group consisting of hydrogen, protium, deuterium, tritium, halogens and C1-C 12 Alkyl, Represents the junction site.

7. The compound according to claim 1, having the following structure:

8. A compound, or a pharmaceutically acceptable salt thereof, having a structure as shown in Formula IIa, wherein: in, for The W is The A 2 , B 2 and C 2 Each independently selected from the following group: and covalent bonds; where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 14-membered heteroaryl, Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect; The La is -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -; The Lb is -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -; The Lc is -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -; in, X 1 , X 2 and X 3 are each independently selected from the group consisting of: -NH- and -O-, (K 1 ) n1 , (K 2 ) n2 and (K 3 ) n3 are each independently selected from the group consisting of -(CH2)-(CH2-O-CH2)3-(CH2)- and -(CH2)2-(CH2-O-CH2)3-(CH2)2-, or n1, n2 or n3 are each independently 0, (Y 1 ) p1 ,(Y 2 ) p2 and (Y 3 ) p3 Each independently is -NR 1 -C(=O)-,R 1 is selected from the group consisting of hydrogen, protium, deuterium and tritium; or p1, p2 or p3 are each independently 0, (L 1 ) q1 ,(L 2 ) q2 and (L 3 ) q3 are each independently selected from the following group: -CH2-aryl-, -(CH2)2- and -CH2-, or q1, q2 and q3 are each independently 0, wherein the aryl group is a 6-membered to 14-membered aryl group, Among them, P 1 , P 2 and P 3 Each is independently selected from the group consisting of lipids, proteins, nucleic acids, small molecules and polysaccharides, or a combination thereof.

9. The compound according to claim 8, wherein: (1)A 2 for B 2 for C 2 for or covalent bond; or (2)A 2 for B 2 for C 2 for or covalent bond; or (3)A 2 for B 2 for C 2 for or covalent bond; or (4) A 2 for B 2 for C 2 for or covalent bond; or (5)A 2 for B 2 for C 2 for or covalent bond; or (6)A 2 for or covalent bond, B 2 is a covalent bond, C 2 is a covalent bond; or (7)A 2 for B 2 for C 2 for or covalent bond; or where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 14-membered heteroaryl, Represents the junction site, The A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

10. The compound according to claim 8, wherein: (1)A 2 for B 2 for C 2 for or covalent bond; or (2)A 2 for B 2 for C 2 for or covalent bond; or (3)A 2 for B 2 for C 2 for or covalent bonds; where R 12 , R 13 and R 14 Each is independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 14-membered heteroaryl, Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

11. The compound according to claim 8, wherein: (1)A 2 for B 2 for C 2 for or covalent bond; or (2)A 2 for B 2 for C 2 for or covalent bond; or (3)A 2 for B 2 for C 2 for or covalent bonds; where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 14-membered heteroaryl, Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

12. The compound according to claim 8, wherein: (1)A 2 for B 2 for C 2 for or covalent bond; or (2)A 2 for B 2 for C 2 for or covalent bond; or (3)A 2 for B 2 for C 2 for or covalent bonds; where R 12 Selected from the group consisting of hydrogen, protium, deuterium, tritium, halogens and C1-C 12 Alkyl, Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

13. The compound according to claim 8, wherein: A 2 for B 2 for C 2 for or covalent bond; where R 12 Selected from the group consisting of hydrogen, protium, deuterium, tritium, halogens and C1-C 12 Alkyl, Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

14. The compound according to claim 8, wherein: A 2 for B 2 for, C 2 for or covalent bond; where R 12 selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the connection site, the A 2 The two linking sites are randomly connected with Wc and P 1 Connect the B 2 The two linking sites are randomly connected with Wc and P 2 Connect the C 2 The two linking sites are randomly connected with Wc and P 3 connect.

15. The compound according to claim 8, wherein P 1 , P 2 and P 3 Each is independently selected from the following group: a nucleic acid molecule, a dye molecule, a cytokine, an antigen, and an antibody or an antigen-binding fragment thereof, or any combination thereof.

16. The compound of claim 15, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and a nanobody.

17. The compound of claim 15, wherein the antibody targets a target selected from the group consisting of 4-1BB, EGFR, CD3, Her2, CD47, and CD20.

18. The compound of claim 15, wherein the amino acid sequence of the antibody is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:

12.

19. A method for preparing the compound according to any one of claims 1 to 7, comprising: reacting the compound represented by formula (Ia-I) with reactant 1, reactant 2, and reactant 3; or reacting the compound represented by formula (Ia-I) with reactant 1 and reactant 2; or reacting the compound represented by formula (Ia-I) with reactant 1; The reactant 1 is HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -A, The reactant 2 is HX 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -B, The reactant 3 is HX 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -C, The W is X 1 , X 2 and X 3 are each independently selected from the group consisting of: -NH- and -O-, (K 1 ) n1 , (K 2 ) n2 and (K 3 ) n3 are each independently selected from the group consisting of -(CH2)-(CH2-O-CH2)3-(CH2)- and -(CH2)2-(CH2-O-CH2)3-(CH2)2-, or n1, n2 or n3 are each independently 0, (Y 1 ) p1 ,(Y 2 ) p2 and (Y 3 ) p3 Each independently is -NR 1 -C(=O)-,R 1 selected from the group consisting of hydrogen, protium, deuterium and tritium; or p1, p2 or p3 are each independently 0, (L 1 ) q1 ,(L 2 ) q2 and (L 3 ) q3 are each independently selected from the group consisting of -CH2-aryl-, -(CH2)2- and -CH2-, or q1, q2 and q3 are each independently 0, wherein the aryl group is a 6-membered to 14-membered aryl group, A, B and C are each independently selected from the following group: where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 14-membered heteroaryl; Among them, A, B and C, Cannot exist simultaneously; Among them, A, B and C, Cannot exist simultaneously; Among them, A, B and C, Cannot exist simultaneously; When A, B and C, When both exist, the R 12 Not an alkynyl group; When A, B and C, When both exist, the R 12 Not amino, A, B and C are not the same Among them, when A, B, or C is When the compound represented by the formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, Replace with After the compound represented by formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, an acid is added to remove Boc.

20. The preparation method according to claim 19, wherein the order of adding reactant 1, reactant 2 and reactant 3 is determined according to the order of introducing A, B and C, and the order of introducing A, B and C from earliest to latest is: where R 12 , R 13 and R 14 Each is independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 10-membered heteroaryl.

21. A method for preparing the compound according to any one of claims 8 to 18, comprising: reacting the compound of formula Ia according to any one of claims 1 to 7 with reactant 4, reactant 5 and reactant 6; The reactant 4 is A 1 -P 1 , The reactant 5 is B 1 -P 2 , The reactant 6 is C 1 -P 3 , Among them, A of reactant 4 1 React with A of the compound described in formula Ia, B of reactant 5 1 With the compound B of formula Ia, C of reactant 6 1 Reaction with C of the compound described in Formula Ia; When at least one of A, B or C is When A 1 , B 1 or C 1 At least one for When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for When at least one of A, B or C is When the corresponding A 1 , B 1 or C 1 At least one for where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 10-membered heteroaryl.

22. The preparation method according to claim 21, in, Cu was added to the reaction + As a catalyst.

23. The preparation method according to claim 21, in, 1-100% equivalent of SrtA ligase was added to the reaction as a catalyst.

24. A pharmaceutical composition comprising the compound according to any one of claims 8 to 18, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

25. A kit comprising the compound according to any one of claims 8 to 18, or a pharmaceutically acceptable salt thereof, and / or the pharmaceutical composition according to claim 24.

26. Use of a compound according to any one of claims 8 to 18, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition according to claim 24 and / or the kit according to claim 25 in the preparation of a medicament for treating and / or preventing a tumor, wherein the tumor is selected from tumors associated with the expression of the following group: 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

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