Antibody-drug conjugate, method for preparing the same, and use thereof

Abstract

The present disclosure relates to a biologically active substance complex, a method for preparing the same, and use thereof, and specifically discloses an antibody-drug conjugate represented by Formula XV, a method for preparing the same, and use thereof in the prevention and / or treatment of diseases associated with abnormal cell activity, including but not limited to the prevention and / or treatment of tumor diseases. JPEG2025524637000392.jpg20109

Description

Technical Field

[0001] The present disclosure belongs to the field of pharmaceutical technology, and relates to various antibodies, antibody-drug conjugates and their preparation methods, and their use in the prevention and / or treatment of diseases related to abnormal cell activities, including but not limited to the prevention and / or treatment of tumor diseases.

Background Art

[0002] Chemotherapy that utilizes cytotoxicity is a standard treatment for cancer. However, cytotoxic molecules with high killing ability kill normal cells and cause severe toxic side effects. Since targeted anti-tumor drugs have both targeting and anti-tumor activities, they have become the focus of the current tumor research field. However, due to the target selectivity problem of targeted drugs, relatively large toxic side effects often occur, thereby limiting the therapeutic effect of targeted drugs. Biopharmaceuticals such as antibodies or antibody fragments have strong targeting, but their therapeutic effect on solid tumors is limited or they have no therapeutic effect at all. An ADC is a conjugate of an antibody and a small molecule drug, which fuses the targeting effect of the antibody and the activity of a biologically active molecule, becoming a biological missile and having very promising therapeutic effect and safety advantages. The antibody induces the ADC to bind to target cells and then be internalized into the cells. The small molecule drug is released by enzymatic degradation under the action of specific enzymes inside the cells to treat diseases.

[0003] In recent years, ADC drugs have developed very rapidly, and there are already 14 ADCs on the market. Many target ADCs, such as antibody / ADC drugs targeting NaPi2b, are all in the clinical research stage. The development of monoclonal antibodies or ADC drugs with discrimination, better quality, and better safety for many targets can provide tumor patients with broader and better drug options and has a broad market outlook.

Summary of the Invention

[0004] To improve the therapeutic effect of an antibody-drug conjugate (ADC), reduce the toxic side effects of the drug, and improve the therapeutic window, the present disclosure provides an antibody-drug conjugate represented by formula (XV) or a pharmaceutically acceptable salt or solvate thereof, which comprises a biologically active molecule (drug molecule), a linker, and a targeting moiety. The targeting moiety is linked to the linker via an active group (e.g., a thiol group) to form an antibody-drug conjugate. The present disclosure has also developed an antibody comprising a variable light chain domain (VL) and / or a variable heavy chain (VH) domain that contains anti-NaPi2b, and the antibody of the variable light chain domain and / or variable heavy chain domain is derived from a human antibody.

[0005] Therefore, in a first aspect of the present disclosure, the present disclosure provides an antibody-drug conjugate represented by formula XV,

Chemical formula

Chemical formula

[0006] It should also be noted that regarding "the 1-position of L1 is linked to Tb via an S atom", those skilled in the art can understand that the 1-position of L1 is linked to the thiol group contained in Tb itself (such as an antibody) after the disulfide bond is opened (for example, the reduction of the disulfide bond by the reducing agent TCEP can open the disulfide bond to generate thiol -SH), that is, the -S- between L1 and Tb is not a separately externally connected sulfur atom. For example,

Chemical formula

Chemical formula

[0007] The extension unit is a composition of an antibody-drug conjugate or a drug-linker conjugate or a linker that functions to link the Tb bound to the target to the remaining part of the antibody-drug conjugate or the remaining part of the linker. The extension unit can link the Tb unit to L2 (if present) or L3, and specific examples include

Chemical formula

[0008] In some embodiments, L1 is

Chemical formula

Chemical formula

Chemical formula

[0009] The linking unit is a composition of an antibody-drug conjugate or a drug linker conjugate or a linker that functions to link an extension unit to an amino acid residue or a fragment or short peptide consisting of 1 to 10 amino acid residues. When the linking unit is present, L1 can be linked to L3. Specific examples are

Chemical formula

[0010] In some embodiments, L2 is absent or present, and when L2 is present, L2 is

Chemical formula

Chemical formula

[0011] In some embodiments, L1 is

Chemical formula

[0012] In some embodiments, L1 is [Chemical formula] selected from, each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, a C6-10 aryl group, a 5-10 membered heteroaryl group and an amide group (preferably selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond), Rx and Ry are independently selected from H and C1-4 alkyl groups, each m is independently selected from 0, 1, 2, 3, 4, 5 and 6, y1 is selected from any integer between 1 and 6 (e.g., 4, 5, 6), each y2 is independently selected from any integer between 0 and 15 (e.g., 6 - 15), each y3 is independently selected from 1, 2 and 3, each y4 is independently selected from 0 and 1, the 1-position is linked to Tb via an S atom, and the 2-position is linked to L2 or L3.

[0013] In some embodiments, L1 is [Chemical formula] selected from, m is selected from 2, 3, 4, y1 is selected from any integer between 1 and 6 (e.g., 4, 5, 6), each y2 is independently selected from any integer between 0 and 10 (e.g., 6 - 10), each y3 is independently selected from 1 or 2, the 1-position is linked to Tb via an S atom, and the 2-position is linked to L2 or L3.

[0014] In some embodiments, L1 is [Chemical formula] selected from.

[0015] In some embodiments, L1 is [Chemical formula] selected from, the 1-position is linked to Tb via an S atom, and the 2-position is linked to L2 or L3.

[0016] In some embodiments, L2 is absent or present, and when L2 is present, L2 is [Chemical formula] selected from, y1 is selected from any integer between 1 and 6 (e.g., 4, 5, 6), each y2 is independently selected from any integer between 0 and 10 (e.g., 6 - 10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, the 1st position is linked to L1, and the 2nd position is linked to L3.

[0017] In some embodiments, L2 is absent or present, and when L2 is present, L2 is [Chemical formula] selected from, the 1st position is linked to L1, and the 2nd position is linked to L3.

[0018] In some embodiments, L2 is absent.

[0019] In some embodiments, L2 is [Chemical formula] selected from. In some embodiments, L3 is selected from an amino acid residue or a short peptide consisting of 2 to 10 amino acid residues, and the above amino acid residue is selected from a natural amino acid residue, a non - natural amino acid residue, or an amino acid residue shown by AA1 or its stereoisomer.

[0020] In some embodiments, L3 is an amino acid residue Val, D - Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, AA 1 or a short peptide consisting of 2 to 10 amino acid residues selected from Val, Cit, Phe, Lys, D - Val, Leu, Gly, Ala, Asn, Asp, AA 1 selected from.

[0021] In some embodiments, L3 is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, AA 1 , Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Ala-Ala, Val-AA 1 , Ala-AA 1 , Gly-AA 1 , AA 1 -Gly, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Asp, Val-AA 1 -Gly, Ala-AA 1 -Gly, Gly-AA 1 -Gly, Lys-Ala-Ala-Asn, Lys-Ala-Ala-Asp, Gly-Phe-Gly, Gly-Gly-Phe-Gly, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Phe, Val-Lys-Gly, Val-Lys-Gly-Gly, Val-Lys and Lys-Ala-Asn.

[0022] In some embodiments, L3 is AA 1 , AA 1 -Gly, Val-Cit, Val-AA 1 -Gly, AA 1 -Ala-Asn and Gly-Gly-Phe-Gly.

[0023] In some embodiments, L3 is AA 1 and Val-AA 1 -Gly.

[0024] In some embodiments, L3 is Val-AA 1 -Gly. In some embodiments, L3 is

Chemical Formula

[0025] In some embodiments, L3 is [Chemical formula] selected from, the 1-position is linked to L1 or L2, and the 2-position is linked to L4 or D.

[0026] In some embodiments, L3 is [Chemical formula] selected from, the 1-position is linked to L1 or L2, and the 2-position is linked to L4 or D.

[0027] In some embodiments, the structure of the amino acid residue shown in AA 1 is shown as follows [[ID=P38]] [Chemical formula] AA 1 wherein R a and R b are each independently H, [Chemical formula] selected from, and R a and R b are not both H at the same time, or, R a and R b together with the carbon atom to which they are commonly linked, form a 4- to 10-membered heterocyclic ring, and the above 4- to 10-membered heterocyclic ring is optionally substituted with one or more R 0 and r, r 1is independently selected from any integer of 0 to 20, R m1 and R n1 are each independently selected from H, a C1-6 alkyl group, a C3-6 cycloalkyl group and -COOR x1 and R x1 is selected from a C1-6 alkyl group, or R m1 and R n1 together with the nitrogen atom to which they are commonly linked form a 4- to 10-membered heterocyclic ring, and the above 4- to 10-membered heterocyclic ring is optionally substituted with one or more R 0’ ; R z is selected from a C1-6 alkyl group, R 0 and R 0’ are each independently selected from a C1-6 alkyl group, a C3-6 cycloalkyl group, -NR m2 R n2 and a 4- to 10-membered heterocyclic group optionally substituted with a C1-6 alkyl group, R m2 and R n2 are each independently selected from H and a C1-6 alkyl group.

[0028] In some embodiments, either one of R a and R b is H, and the other is

Chemical formula

[0029] In some embodiments, either one of R a and R b is H, and the other is

Chemical formula

[0030] In some embodiments, R a and R btogether with the carbon atoms commonly linked thereto, form a 5- to 6-membered heterocyclic ring substituted with R 0

[0031] In some embodiments, R a and R b together with the carbon atoms commonly linked thereto, form a piperidine ring or a piperazine ring substituted with R 0

[0032] In some embodiments, R a and R b together with the carbon atoms commonly linked thereto, form a piperidine ring substituted with R 0

[0033] In some embodiments, R a and R b together with the carbon atoms commonly linked thereto,

Chemical formula

[0034] In some embodiments, R a and R b together with the carbon atoms commonly linked thereto,

Chemical formula

[0035] In some embodiments, r, r 1 are each independently selected from 0, 1, 2, 3, 4, and 5.

[0036] In some embodiments, r, r 1 are each independently selected from 0 and 4. ​​​​​

[0037] In some embodiments, one of r and r 1 is 0 and the other is 4.

[0038] In some embodiments, R m1 , R n1 are each independently selected from H, a methyl group, an ethyl group, an n-propyl group, an n-butyl group, -COOCH3, -COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, -COOC(CH3)3, and -COOCH2CH2CH2CH3.

[0039] In some embodiments, R m1 , R n1 are each independently selected from H, a C1-6 alkyl group, a C3-6 cycloalkyl group, and a tert-butoxycarbonyl group.

[0040] In some embodiments, R m1 , R n1 are each independently selected from H and a C1-6 alkyl group.

[0041] In some embodiments, R m1 , R n1 are each independently selected from H, a methyl group, an ethyl group, and an n-propyl group.

[0042] In some embodiments, in r, r 1 when r is 4 and r 1 is 0, R m1 , R n1 are each independently selected from H and a C1-6 alkyl group (e.g., H, a methyl group), and when r is 0 and r 1 is 4, R m1 , R n1 are each independently selected from a C1-6 alkyl group (e.g., a methyl group, an ethyl group, an n-propyl group), preferably from a C2-6 alkyl group (e.g., an ethyl group, an n-propyl group).

[0043] In some embodiments, R m1 and R n1 together with the nitrogen atom commonly linked thereto optionally form a 5- to 6-membered heterocyclic ring substituted with R 0’ .

[0044] In some embodiments, R m1 and R n1 together with the nitrogen atom commonly linked thereto optionally form a piperidine ring or a piperazine ring substituted with R 0’ .

[0045] In some embodiments, R m1 and R n1 together with the nitrogen atom commonly linked thereto

Chemical formula

[0046] In some embodiments, R z is a methyl group.

[0047] In some embodiments, R 0 , R 0’ are each independently selected from a C1-6 alkyl group, -NR m2 R n2 and a 5- to 6-membered heterocyclic group optionally substituted with a C1-6 alkyl group.

[0048] In some embodiments, R 0 is selected from a C1-6 alkyl group and a 5- to 6-membered heterocyclic group substituted with a C1-6 alkyl group, and the 5- to 6-membered heterocyclic group is selected from a piperidinyl group and a piperazinyl group.

[0049] In some embodiments, R 0is selected from a methyl group, an ethyl group, and a 5- to 6-membered heterocyclic group substituted with a methyl group, and the 5- to 6-membered heterocyclic group is a piperidinyl group.

[0050] In some embodiments, R 0 is selected from a methyl group and a 5- to 6-membered heterocyclic group substituted with a methyl group, and the 5- to 6-membered heterocyclic group is a piperidinyl group.

[0051] In some embodiments, R 0 is a methyl group, an ethyl group, and

Chemical formula

[0052] In some embodiments, R 0 is a methyl group and

Chemical formula

[0053] In some embodiments, R 0’ is a C1-6 alkyl group and -NR m2 R n2 selected from.

[0054] In some embodiments, R 0’ is a methyl group and -NR m2 R n2 selected from.

[0055] In some embodiments, R m2 and R n2 are methyl groups.

[0056] In some embodiments, the amino acid residue represented by AA 1 is

Chemical formula

[0057] In some embodiments, AA 1 The amino acid residues shown in

Chemical formula

[0058] In some embodiments, AA 1 The amino acid residues shown in

Chemical formula

[0059] In some embodiments, L4 is absent or present, and when L4 is present, L4 is

Chemical formula

[0060] In some embodiments, L4 is absent.

[0061] In some embodiments, L4 is

Chemical formula

[0062] In some embodiments, L4 is

Chemical formula

[0063] In some embodiments,

Chemical formula

Table 1

[0064] In some embodiments, q is selected from any numerical value between 0.1 and 16.0, and in preferred embodiments, q is selected from any integer between 0.1 and 16.0.

[0065] In some embodiments, q is selected from any numerical value between 0.1 and 8.0, and in preferred embodiments, q is selected from any integer between 0.1 and 8.0.

[0066] In some embodiments, q is selected from any numerical value between 2 and 8.

[0067] In some embodiments, q is selected from any numerical value between 3 and 8.

[0068] In some embodiments, q is selected from any numerical value between 4 and 8.

[0069] In some embodiments, q is selected from any numerical value between 6 and 8.

[0070] In some embodiments, q is selected from any integer between 2 and 8.

[0071] In some embodiments, q is selected from any integer between 3 and 8.

[0072] In some embodiments, q is selected from any integer between 4 and 8.

[0073] In some embodiments, q is selected from any integer between 6 and 8.

[0074] In some embodiments, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0075] In some embodiments, q is selected from 2, 4, 6, and 8.

[0076] In the present disclosure, the above-mentioned biologically active molecular fragment refers to a part (fragment or group) that can form a drug having biological activity (for example, a small molecule cytotoxic drug containing a group after losing one atom or atomic group) or its derivative (for example, its precursor) after cleavage / degradation / enzyme cleavage of a linker between tumor tissues or within tumor cells in an antibody-drug conjugate (or called antibody-drug conjugate, ADC) known in the art. To avoid ambiguity, the "drug" does not only refer to the "pharmaceutical product" approved by the pharmaceutical regulatory agency, but further includes any molecule having potential therapeutic biological activity in clinical practice, or in development and academic research.

[0077] In some embodiments, D is a fragment having an anti-tumor biologically active molecule.

[0078] In some embodiments, D is a fragment having an anti-tumor biologically active molecule, wherein the biologically active molecule is selected from a cytotoxic agent or its derivative, such as a DNA topoisomerase inhibitor (for example, a camptothecin-based biologically active molecule, such as camptothecin, DXD, camptothecin with modified substituents, or DXD with modified substituents) or a tubulin inhibitor (for example, an MMAF-based tubulin inhibitor, an MMAE-based tubulin inhibitor).

[0079] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0080] Tb, L1, L2, L3 and q have the meanings provided by any of the embodiments described above and specifically described herein.

[0081] In some embodiments, R1, R2 are each independently selected from H, halogen, and C1-4 alkyl groups.

[0082] In some embodiments, R1 and R2 together with the carbon atom to which they are linked form a 5-6 membered heterocyclic ring, and the heterocyclic ring contains one, two or three O, S or N or any combination thereof.

[0083] In some embodiments, R1 is selected from H and halogen, and R2 is selected from H and C1-4 alkyl groups.

[0084] In some embodiments, R1 and R2, together with the carbon atom to which they are attached,

Chemical formula

[0085] In some embodiments, R1 is H or F, and R2 is H or a methyl group.

[0086] In some embodiments, R1 is F, R2 is a methyl group, or R1 and R2, together with the carbon atom to which they are attached,

Chemical formula

[0087] In some embodiments, R1 is F and R2 is a methyl group.

[0088] In some embodiments, R1 and R2, together with the carbon atom to which they are attached,

Chemical formula

[0089] In some embodiments, R3 is selected from H and C1-C4 alkyl groups.

[0090] In some embodiments, R3 and X, together with the carbon atom to which they are attached, form a 5- to 6-membered carbon ring.

[0091] In some embodiments, R3 is H, or R3 and X, together with the carbon atom to which they are attached,

Chemical formula

[0092] In some embodiments, R3 is H.

[0093] In some embodiments, W is absent or present, and when W is present, W is -O-, -S-, -NR4-,

Chemical formula

[0094] In some embodiments, W is absent or present, and when W is present, W is -O-, -S-, -NR4-,

Chemical formula

[0095] In some embodiments, W is -O-, -NR4- and

Chemical formula

[0096] In some embodiments, W is selected from -O- and -NR4-, the 1-position is linked to X, and the 2-position is linked to L4 or L3.

[0097] In some embodiments, X is optionally substituted -(CH2) n1 -,

Chemical formula

[0098] In some embodiments, X is optionally substituted [ka] wherein the 1-position is linked to the parent ring and the 2-position is linked to W or L4, and the above substituents are selected from one or two C1-4 alkyl groups (e.g., methyl groups), or two C1-4 alkyl groups (e.g., methyl groups) together with the carbon atoms linked thereto form a C3-6 cycloalkyl group (e.g., cyclopropyl group).

[0099] In some embodiments, X is [ka] wherein position 1 is linked to the parent ring and position 2 is linked to W or L4.

[0100] In some embodiments, X is [ka] wherein position 1 is linked to the parent ring and position 2 is linked to W.

[0101] In some embodiments, when W is absent, X is [ka] wherein position 1 is linked to the parent ring and position 2 is linked to L4; and when W is present, X is selected from: [ka] wherein position 1 is linked to the parent ring and position 2 is linked to W.

[0102] In some embodiments, W is selected from -O-, -NR4-, and

Chemical formula

Chemical formula

[0103] In some embodiments, R4 and R5 are each independently selected from H, a C1-4 alkyl group, and a C3-6 cycloalkyl group.

[0104] In some embodiments, each R4 is independently selected from H, a C1-4 alkyl group, and a C3-6 cycloalkyl group, and R5 is H.

[0105] In some embodiments, each R4 is independently selected from H, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a tert-butyl group, and a cyclopropyl group, and R5 is H.

[0106] In some embodiments, R 5a , R 5b are each independently selected from H and a C1-4 alkyl group.

[0107] In some embodiments, R 5a , R 5b are each independently selected from H and a methyl group.

[0108] In some embodiments, each R7 is independently selected from H and a C1-4 alkyl group.

[0109] In some embodiments, R7 is H.

[0110] In some embodiments, n is selected from 1, 2, and 3.

[0111] In some embodiments, n is 1.

[0112] In some embodiments, n1 is selected from 1, 2, 3, and 4.

[0113] In some embodiments, n2 is 1.

[0114] In some embodiments, n3 is 0.

[0115] In some embodiments, L3 is

Chemical formula

Chemical formula

[0116] In some embodiments, L3 is

Chemical formula

[0117] In some embodiments, L3 is

Chemical formula

[0118] In some embodiments, L4 is absent or present, and when L4 is present, L4 is

Chemical formula

[0119] In some embodiments, L4 does not exist.

[0120] In some embodiments, L4

Chemical formula

[0121] In some embodiments, L4

Chemical formula

[0122] It should be noted that, as described above, W does not exist or exists. Therefore, when W does not exist, the first position of L4 is connected to L3, and the second position is connected to X; when W exists, the first position of L4 is connected to L3, and the second position is connected to W. Hereinafter, the connection relationship of L4 can be understood with reference to the foregoing content.

[0123] In some embodiments,

Chemical formula

Table 2-1

Table 2-2

Table 2-3

[0124] In the formula, the first position is connected to Tb, and the second position is connected to W.

[0125] In some embodiments, D is

Chemical formula

Chemical formula

[0126] In some embodiments,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0127] In some embodiments, W is absent or present, and when W is present, W is -O-, -S-, -NR4-,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0128] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-1, [Chemical formula] In the formula, Tb, L1, L2, L3, L4, X, R1, R2, R3, R4 and q have the meanings provided by any of the embodiments described above and specifically described in this specification.

[0129] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-1A or I-1B,

Chemical formula

[0130] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-2,

Chemical formula

[0131] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-2A or I-2B,

Chemical formula

[0132] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-3,

Chemical formula

[0133] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-3A or I-3B,

Chemical formula

[0134] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-A,

Chemical formula

[0135] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-B,

Chemical formula

[0136] In some embodiments, the antibody-drug conjugate is selected from the following:

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

[0137] In some embodiments, Tb is an anti-NaPi2b antibody or an antigen-binding fragment thereof.

[0138] In some embodiments, the antibody or antigen-binding fragment thereof and the monoclonal antibody or antigen-binding fragment thereof include Fab, Fab’, F(ab’)2, Fd, Fv (e.g., scFv), dAb, complementarity-determining region fragment, non-human antibody, humanized antibody, chimeric antibody, fully human antibody, Probody, monoclonal antibody, bispecific antibody or multispecific antibody.

[0139] In some embodiments, Tb is an anti-NaPi2b antibody or an antigen-binding fragment thereof that has endocytosis, does not have endocytosis activity, or has relatively weak endocytosis.

[0140] In some embodiments, Tb is an anti-NaPi2b antibody or an antigen-binding fragment thereof that has endocytosis activity.

[0141] In some embodiments, Tb is an anti-NaPi2b antibody or an antigen-binding fragment thereof that does not have endocytosis activity or has relatively weak endocytosis activity.

[0142] In some embodiments, Tb is an anti-NaPi2b antibody or an antigen-binding fragment thereof.

[0143] In some embodiments, the anti-NaPi2b antibody is a non-human antibody, humanized antibody, chimeric antibody, or fully human antibody.

[0144] In some embodiments, the anti-NaPi2b antibody is a monoclonal antibody, bispecific antibody, or multispecific antibody.

[0145] In some embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0146] In some embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof is selected from lifastuzumab or antigen-binding fragment thereof, upifitamab or antigen-binding fragment thereof, and antibody C1.

[0147] In some preferred embodiments, the anti-NaPi2b antibody is selected from 16G5B3, 60A12B3, 22H9C4, 66C12D12 and 34F2A10 mouse antibodies, or humanized antibodies thereof.

[0148] In some preferred embodiments, among them, the anti-NaPi2b antibody is selected from 60A12B3-hz1 antibody, 22H9C4-hz1 antibody, 66C12D12-hz1 antibody and 34F2A10-hz1 antibody.

[0149] The antibody against Tb or antigen-binding fragment thereof can be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibody of the present disclosure are obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and then transfected into host cells. Next, the transfected host cells are cultured under specific conditions to express the antibody of the present disclosure.

[0150] In some preferred embodiments, in the above antibody-drug conjugate, Tb is the anti-NaPi2b antibody or antigen-binding fragment thereof described in the second aspect.

[0151] In the second aspect, the present disclosure provides an anti-NaPi2b antibody or antigen-binding fragment thereof.

[0152] In some embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): The HCDR1 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1, 17, 19, 34, 36, 52, 54, 71, 73, 74 or 33, or a variant of its sequence, the HCDR2 or a variant of its sequence, and the HCDR3 or a variant of its sequence, and / or The LCDR1 contained in the light chain variable region (VL) shown in SEQ ID NO: 2, 18, 20, 35, 37, 53, 55, 72, 75 or 51, or a variant of its sequence, the LCDR2 or a variant of its sequence, and the LCDR3 or a variant of its sequence.

[0153] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises the HCDR1 contained in the VH shown in SEQ ID NO: 1 or 17, or a variant of its sequence, the HCDR2 or a variant of its sequence, and the HCDR3 or a variant of its sequence, and / or the LCDR1 contained in the VL shown in SEQ ID NO: 2 or 18, or a variant of its sequence, the LCDR2 or a variant of its sequence, and the LCDR3 or a variant of its sequence.

[0154] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises the HCDR1 contained in the VH shown in SEQ ID NO: 19 or 34, or a variant of its sequence, the HCDR2 or a variant of its sequence, and the HCDR3 or a variant of its sequence, and / or the LCDR1 contained in the VL shown in SEQ ID NO: 20 or 35, or a variant of its sequence, the LCDR2 or a variant of its sequence, and the LCDR3 or a variant of its sequence.

[0155] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises the HCDR1 contained in the VH shown in SEQ ID NO: 36 or 52, or a variant of its sequence, the HCDR2 or a variant of its sequence, and the HCDR3 or a variant of its sequence, and / or the LCDR1 contained in the VL shown in SEQ ID NO: 37 or 53, or a variant of its sequence, the LCDR2 or a variant of its sequence, and the LCDR3 or a variant of its sequence.

[0156] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 54, 71 or 73, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 55 or 72.

[0157] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 74 or 33, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 75 or 51.

[0158] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 17, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 18.

[0159] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 34, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 35.

[0160] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in VH shown in SEQ ID NO: 52, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in VL shown in SEQ ID NO: 53.

[0161] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in VH shown in SEQ ID NO: 71 or 73, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in VL shown in SEQ ID NO: 72.

[0162] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in VH shown in SEQ ID NO: 33, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in VL shown in SEQ ID NO: 51.

[0163] In certain preferred embodiments, the variant of the above sequence is a CDR having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared to the CDR from which it is derived.

[0164] In certain preferred embodiments, the substitution is a conservative substitution.

[0165] In certain embodiments, the CDR is defined based on the AbM, Chothia, Kabat or IMGT numbering system.

[0166] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0167] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (a) A heavy chain variable region (VH) comprising three CDRs: HCDR1 of the sequence of SEQ ID NO: 3 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; HCDR2 of the sequence of SEQ ID NO: 4 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and HCDR3 of the sequence of SEQ ID NO: 6 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and / or, A light chain variable region (VL) comprising three CDRs: LCDR1 of the sequence of SEQ ID NO: 7 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; LCDR2 of the sequence of SEQ ID NO: 9 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and LCDR3 of the sequence of SEQ ID NO: 10 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto. (b) The heavy chain variable region (VH) comprising three CDRs: the HCDR1 of the sequence of SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR2 of the sequence of SEQ ID NO: 5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR3 of the sequence of SEQ ID NO: 6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and / or, The light chain variable region (VL) comprising three CDRs: the LCDR1 of the sequence of SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR2 of the sequence of SEQ ID NO: 9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR3 of the sequence of SEQ ID NO: 10, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; (c) The heavy chain variable region (VH) comprising three CDRs: the HCDR1 of the sequence of SEQ ID NO: 21, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR2 of the sequence of SEQ ID NO: 22, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR3 of the sequence of SEQ ID NO: 24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and / or, The light chain variable region (VL) comprising three CDRs: LCDR1 of the sequence with SEQ ID NO: 25, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 of the sequence with SEQ ID NO: 26, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and LCDR3 of the sequence with SEQ ID NO: 27, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto. (d) The heavy chain variable region (VH) comprising three CDRs: HCDR1 of the sequence with SEQ ID NO: 21, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; HCDR2 of the sequence with SEQ ID NO: 23, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and HCDR3 of the sequence with SEQ ID NO: 24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and / or The light chain variable region (VL) comprising three CDRs: LCDR1 of the sequence with SEQ ID NO: 25, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 of the sequence with SEQ ID NO: 26, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and LCDR3 of the sequence with SEQ ID NO: 27, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto. (e) The heavy chain variable region (VH) containing three CDRs: the HCDR1 of the sequence of SEQ ID NO: 38, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR2 of the sequence of SEQ ID NO: 39, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR3 of the sequence of SEQ ID NO: 41, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and / or, The light chain variable region (VL) containing three CDRs: the LCDR1 of the sequence of SEQ ID NO: 42, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR2 of the sequence of SEQ ID NO: 44, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR3 of the sequence of SEQ ID NO: 45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; (f) The heavy chain variable region (VH) containing three CDRs: the HCDR1 of the sequence of SEQ ID NO: 38, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR2 of the sequence of SEQ ID NO: 40, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR3 of the sequence of SEQ ID NO: 41, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and / or, The light chain variable region (VL) comprising three CDRs: LCDR1 of the sequence with accession number 42, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 of the sequence with accession number 44, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and LCDR3 of the sequence with accession number 45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto. (g) The heavy chain variable region (VH) comprising three CDRs: HCDR1 of the sequence with accession number 56, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; HCDR2 of the sequence with accession number 57, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and HCDR3 of the sequence with accession number 60, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and / or The light chain variable region (VL) comprising three CDRs: LCDR1 of the sequence with accession number 61, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 of the sequence with accession number 63, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and LCDR3 of the sequence with accession number 64, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto. (h) The heavy chain variable region (VH) comprising three CDRs: the HCDR1 of the sequence of SEQ ID NO: 56, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR2 of the sequence of SEQ ID NO: 58, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR3 of the sequence of SEQ ID NO: 60, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and / or The light chain variable region (VL) comprising three CDRs: the LCDR1 of the sequence of SEQ ID NO: 62, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR2 of the sequence of SEQ ID NO: 63, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR3 of the sequence of SEQ ID NO: 64, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, or (i) The heavy chain variable region (VH) comprising three CDRs: the HCDR1 of the sequence of SEQ ID NO: 56, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR2 of the sequence of SEQ ID NO: 59, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR3 of the sequence of SEQ ID NO: 60, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and / or The light chain variable region (VL) comprising three CDRs: LCDR1 of the sequence with accession number 62, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 of the sequence with accession number 63, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and LCDR3 of the sequence with accession number 64, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto.

[0168] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (a) The above VH comprises HCDR1 with the sequence of SEQ ID NO: 3, HCDR2 with the sequence of SEQ ID NO: 4, and HCDR3 with the sequence of SEQ ID NO: 6, and / or The above VL comprises LCDR1 with the sequence of SEQ ID NO: 7, LCDR2 with the sequence of SEQ ID NO: 9, and LCDR3 with the sequence of SEQ ID NO: 10. (b) The above VH comprises HCDR1 with the sequence of SEQ ID NO: 3, HCDR2 with the sequence of SEQ ID NO: 5, and HCDR3 with the sequence of SEQ ID NO: 6, and / or The above VL comprises LCDR1 with the sequence of SEQ ID NO: 8, LCDR2 with the sequence of SEQ ID NO: 9, and LCDR3 with the sequence of SEQ ID NO: 10. (c) The above VH comprises HCDR1 with the sequence of SEQ ID NO: 21, HCDR2 with the sequence of SEQ ID NO: 22, and HCDR3 with the sequence of SEQ ID NO: 24, and / or The above VL comprises LCDR1 with the sequence of SEQ ID NO: 25, LCDR2 with the sequence of SEQ ID NO: 26, and LCDR3 with the sequence of SEQ ID NO: 27. (d) The above VH comprises HCDR1 with the sequence of SEQ ID NO: 21, HCDR2 with the sequence of SEQ ID NO: 23, and HCDR*3 with the sequence of SEQ ID NO: 24, and / or The above VL includes LCDR1 with the sequence of SEQ ID NO: 25, LCDR2 with the sequence of SEQ ID NO: 26, and LCDR3 with the sequence of SEQ ID NO: 27. (e) The above VH includes HCDR1 with the sequence of SEQ ID NO: 38, HCDR2 with the sequence of SEQ ID NO: 39, and HCDR3 with the sequence of SEQ ID NO: 41, and / or The above VL includes LCDR1 with the sequence of SEQ ID NO: 42, LCDR2 with the sequence of SEQ ID NO: 44, and LCDR3 with the sequence of SEQ ID NO: 45. (f) The above VH includes HCDR1 with the sequence of SEQ ID NO: 38, HCDR2 with the sequence of SEQ ID NO: 40, and HCDR3 with the sequence of SEQ ID NO: 41, and / or The above VL includes LCDR1 with the sequence of SEQ ID NO: 43, LCDR2 with the sequence of SEQ ID NO: 44, and LCDR3 with the sequence of SEQ ID NO: 45. (g) The above VH includes HCDR1 with the sequence of SEQ ID NO: 56, HCDR2 with the sequence of SEQ ID NO: 57, and HCDR3 with the sequence of SEQ ID NO: 60, and / or The above VL includes LCDR1 with the sequence of SEQ ID NO: 61, LCDR2 with the sequence of SEQ ID NO: 63, and LCDR3 with the sequence of SEQ ID NO: 64. (h) The above VH includes HCDR1 with the sequence of SEQ ID NO: 56, HCDR2 with the sequence of SEQ ID NO: 58, and HCDR3 with the sequence of SEQ ID NO: 60, and / or The above VL includes LCDR1 with the sequence of SEQ ID NO: 62, LCDR2 with the sequence of SEQ ID NO: 63, and LCDR3 with the sequence of SEQ ID NO: 64, or (i) The above VH includes HCDR1 with the sequence of SEQ ID NO: 56, HCDR2 with the sequence of SEQ ID NO: 59, and HCDR3 with the sequence of SEQ ID NO: 60, and / or The above VL includes LCDR1 with the sequence of SEQ ID NO: 62, LCDR2 with the sequence of SEQ ID NO: 63, and LCDR3 with the sequence of SEQ ID NO: 64.

[0169] In certain embodiments, the anti-NaPi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (a) A heavy chain variable region (VH) comprising three CDRs: HCDR1 of the sequence of SEQ ID NO: 11 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; HCDR2 of the sequence of SEQ ID NO: 12 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and HCDR3 of the sequence of SEQ ID NO: 14 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and / or A light chain variable region (VL) comprising three CDRs: LCDR1 of the sequence of SEQ ID NO: 15 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; LCDR2 of the sequence of WAS or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and LCDR3 of the sequence of SEQ ID NO: 10 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto (b) A heavy chain variable region (VH) comprising three CDRs: HCDR1 of the sequence of SEQ ID NO: 11 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; HCDR2 of the sequence of SEQ ID NO: 13 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and HCDR3 of the sequence of SEQ ID NO: 14 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and / or The light chain variable region (VL) comprising three CDRs: LCDR1 of the sequence with accession number 15, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 of the sequence of WAS, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and LCDR3 of the sequence with accession number 10, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto. (c) The heavy chain variable region (VH) comprising three CDRs: HCDR1 of the sequence with accession number 28, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; HCDR2 of the sequence with accession number 29, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and HCDR3 of the sequence with accession number 31, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and / or The light chain variable region (VL) comprising three CDRs: LCDR1 of the sequence with accession number 32, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 of the sequence of YTS, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and LCDR3 of the sequence with accession number 27, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto. (d) The heavy chain variable region (VH) comprising three CDRs: the HCDR1 of the sequence of SEQ ID NO: 28, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR2 of the sequence of SEQ ID NO: 30, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR3 of the sequence of SEQ ID NO: 31, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and / or The light chain variable region (VL) comprising three CDRs: the LCDR1 of the sequence of SEQ ID NO: 32, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR2 of the sequence of YTS, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR3 of the sequence of SEQ ID NO: 27, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; (e) The heavy chain variable region (VH) comprising three CDRs: the HCDR1 of the sequence of SEQ ID NO: 46, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR2 of the sequence of SEQ ID NO: 47, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR3 of the sequence of SEQ ID NO: 49, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and / or The light chain variable region (VL) comprising three CDRs: the LCDR1 of the sequence with accession number 50, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR2 of the sequence which is SAS, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR3 of the sequence with accession number 45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto. (f) The heavy chain variable region (VH) comprising three CDRs: the HCDR1 of the sequence with accession number 46, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR2 of the sequence with accession number 48, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR3 of the sequence with accession number 49, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and / or The light chain variable region (VL) comprising three CDRs: the LCDR1 of the sequence with accession number 50, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR2 of the sequence which is SAS, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR3 of the sequence with accession number 45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto. (g) The heavy-chain variable region (VH) containing three CDRs: HCDR1 of the sequence with SEQ ID NO: 65, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; HCDR2 of the sequence with SEQ ID NO: 66, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; HCDR3 of the sequence with SEQ ID NO: 68, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and / or, The light-chain variable region (VL) containing three CDRs: LCDR1 of the sequence with SEQ ID NO: 69, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 of the sequence which is WAS, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR3 of the sequence with SEQ ID NO: 64, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; (h) The heavy-chain variable region (VH) containing three CDRs: HCDR1 of the sequence with SEQ ID NO: 65, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; HCDR2 of the sequence with SEQ ID NO: 67, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; HCDR3 of the sequence with SEQ ID NO: 68, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and / or, The light chain variable region (VL) comprising three CDRs: the LCDR1 of the sequence with accession number 69, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR2 of the sequence of WAS, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR3 of the sequence with accession number 64, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; or (i) The heavy chain variable region (VH) comprising three CDRs: the HCDR1 of the sequence with accession number 76, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR2 of the sequence with accession number 77, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the HCDR3 of the sequence with accession number 78, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and / or The light chain variable region (VL) comprising three CDRs: the LCDR1 of the sequence with accession number 79, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR2 of the sequence of HTS, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; the LCDR3 of the sequence with accession number 16, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto.

[0170] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (a) The VH includes an HCDR1 with the sequence of SEQ ID NO: 11, an HCDR2 with the sequence of SEQ ID NO: 12, and an HCDR3 with the sequence of SEQ ID NO: 14, and / or the VL includes an LCDR1 with the sequence of SEQ ID NO: 15, an LCDR2 with the sequence of WAS, and an LCDR3 with the sequence of SEQ ID NO: 10, (b) The VH includes an HCDR1 with the sequence of SEQ ID NO: 11, an HCDR2 with the sequence of SEQ ID NO: 13, and an HCDR3 with the sequence of SEQ ID NO: 14, and / or the VL includes an LCDR1 with the sequence of SEQ ID NO: 15, an LCDR2 with the sequence of WAS, and an LCDR3 with the sequence of SEQ ID NO: 10, (c) The VH includes an HCDR1 with the sequence of SEQ ID NO: 28, an HCDR2 with the sequence of SEQ ID NO: 29, and an HCDR3 with the sequence of SEQ ID NO: 31, and / or the VL includes an LCDR1 with the sequence of SEQ ID NO: 32, an LCDR2 with the sequence of YTS, and an LCDR3 with the sequence of SEQ ID NO: 27, (d) The VH includes an HCDR1 with the sequence of SEQ ID NO: 28, an HCDR2 with the sequence of SEQ ID NO: 30, and an HCDR3 with the sequence of SEQ ID NO: 31, and / or the VL includes an LCDR1 with the sequence of SEQ ID NO: 32, an LCDR2 with the sequence of YTS, and an LCDR3 with the sequence of SEQ ID NO: 27, (e) The VH includes an HCDR1 with the sequence of SEQ ID NO: 46, an HCDR2 with the sequence of SEQ ID NO: 47, and an HCDR3 with the sequence of SEQ ID NO: 49, and / or the VL includes an LCDR1 with the sequence of SEQ ID NO: 50, an LCDR2 with the sequence of SAS, and an LCDR3 with the sequence of SEQ ID NO: 45, (f) The VH includes an HCDR1 with the sequence of SEQ ID NO: 46, an HCDR2 with the sequence of SEQ ID NO: 48, and an HCDR3 with the sequence of SEQ ID NO: 49, and / or the VL includes an LCDR1 with the sequence of SEQ ID NO: 50, an LCDR2 with the sequence of SAS, and an LCDR3 with the sequence of SEQ ID NO: 45, (g) The VH described above includes an HCDR1 with the sequence of SEQ ID NO: 65, an HCDR2 with the sequence of SEQ ID NO: 66, and an HCDR3 with the sequence of SEQ ID NO: 68, and / or The VL described above includes an LCDR1 with the sequence of SEQ ID NO: 69, an LCDR2 with the sequence of WAS, and an LCDR3 with the sequence of SEQ ID NO: 64. (h) The VH described above includes an HCDR1 with the sequence of SEQ ID NO: 65, an HCDR2 with the sequence of SEQ ID NO: 67, and an HCDR3 with the sequence of SEQ ID NO: 68, and / or The VL described above includes an LCDR1 with the sequence of SEQ ID NO: 69, an LCDR2 with the sequence of WAS, and an LCDR3 with the sequence of SEQ ID NO: 64. Or (i) The VH described above includes an HCDR1 with the sequence of SEQ ID NO: 76, an HCDR2 with the sequence of SEQ ID NO: 77, and an HCDR3 with the sequence of SEQ ID NO: 78, and / or The VL described above includes an LCDR1 with the sequence of SEQ ID NO: 79, an LCDR2 with the sequence of HTS, and an LCDR3 with the sequence of SEQ ID NO: 16.

[0171] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure includes the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) has one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids, or any combination thereof) compared to the CDRs defined according to the Kabat or IMGT numbering system, and still includes mutations having the activity of binding to NaPi2b.

[0172] In certain preferred embodiments, the substitutions described in the present disclosure are conservative substitutions.

[0173] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof binds to human NaPi2b, monkey NaPi2b, and / or rat NaPi2b.

[0174] In certain embodiments, the VH of the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a framework region (FR) derived from the variable heavy chain region (VH) of a human immunoglobulin, and / or the VL of the antibody or antigen-binding fragment thereof comprises a framework region (FR) derived from the variable light chain region (VL) of a human immunoglobulin. Thus, in certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is fully human. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is humanized.

[0175] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain framework region of a human immunoglobulin or a variant thereof, wherein the variant has at most 20 conservative substitutions of amino acids (e.g., at most 20, at most 15, at most 10, or at most 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions of amino acids) compared to the amino acid sequence encoded by the germline antibody gene from which it is derived, and / or (b) a light chain framework region of a human immunoglobulin or a variant thereof, wherein the variant has at most 20 conservative substitutions of amino acids (e.g., at most 20, at most 15, at most 10, or at most 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions of amino acids) compared to the amino acid sequence encoded by the germline antibody gene from which it is derived.

[0176] In certain embodiments, the degree of humanization of the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0177] In certain embodiments, the anti-NaPi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise: (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence selected from: (i) the sequences shown in SEQ ID NO: 1, 17, 19, 34, 36, 52, 54, 71, 73, 74 or 33, (ii) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof) compared to the sequences shown in SEQ ID NO: 1, 17, 19, 34, 36, 52, 54, 71, 73, 74 or 33, or (iii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the sequences shown in SEQ ID NO: 1, 17, 19, 34, 36, 52, 54, 71, 73, 74 or 33, and / or (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence selected from: (iv) the sequences shown in SEQ ID NO: 2, 18, 20, 35, 37, 53, 55, 72, 75 or 51, (v) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof) compared to the sequences shown in SEQ ID NO: 2, 18, 20, 35, 37, 53, 55, 72, 75 or 51, or (vi) An array having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity when compared to the sequences shown in SEQ ID NO: 2, 18, 20, 35, 37, 53, 55, 72, 75 or 51.

[0178] In certain embodiments, the anti-NaPi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise a VH shown in SEQ ID NO: 1 or 17, and / or a VL shown in SEQ ID NO: 2 or 18.

[0179] In certain embodiments, the anti-NaPi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise a VH shown in SEQ ID NO: 19 or 34, and / or a VL shown in SEQ ID NO: 20 or 35.

[0180] In certain embodiments, the anti-NaPi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise a VH shown in SEQ ID NO: 36 or 52, and / or a VL shown in SEQ ID NO: 37 or 53.

[0181] In certain embodiments, the anti-NaPi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise a VH shown in SEQ ID NO: 54, 71 or 73, and / or a VL shown in SEQ ID NO: 55 or 72.

[0182] In certain embodiments, the anti-NaPi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise a VH shown in SEQ ID NO: 74 or 33, and / or a VL shown in SEQ ID NO: 75 or 51.

[0183] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure has a VH with at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the VH of any one of the above five form sets, and / or a VL with at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the VL of the set, or In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure has a VH with one or more amino acid substitutions, deletions or additions or any combination thereof, such as one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions, deletions or additions or any combination thereof compared to the VH of any one of the above five form sets, and / or a VL with one or more amino acid substitutions, deletions or additions or any combination thereof, such as one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions, deletions or additions or any combination thereof compared to the VL of the set. In a preferred embodiment, the above substitutions are conservative substitutions.

[0184] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) VH of the sequence shown in SEQ ID NO: 1 and VL of the sequence shown in SEQ ID NO: 2, (b) VH of the sequence shown in SEQ ID NO: 17 and VL of the sequence shown in SEQ ID NO: 18, (c) VH of the sequence shown in SEQ ID NO: 19 and VL of the sequence shown in SEQ ID NO: 20, (d) VH of the sequence shown in SEQ ID NO: 34 and VL of the sequence shown in SEQ ID NO: 35, (e) The VH of the sequence shown in SEQ ID NO: 36 and the VL of the sequence shown in SEQ ID NO: 37, (f) The VH of the sequence shown in SEQ ID NO: 52 and the VL of the sequence shown in SEQ ID NO: 53, (g) The VH of the sequence shown in SEQ ID NO: 54 and the VL of the sequence shown in SEQ ID NO: 55, (h) The VH of the sequence shown in SEQ ID NO: 71 and the VL of the sequence shown in SEQ ID NO: 72, (i) The VH of the sequence shown in SEQ ID NO: 73 and the VL of the sequence shown in SEQ ID NO: 72, (j) The VH of the sequence shown in SEQ ID NO: 74 and the VL of the sequence shown in SEQ ID NO: 75, (k) The VH of the sequence shown in SEQ ID NO: 33 and the VL of the sequence shown in SEQ ID NO: 51, (l) Independently, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the VH and VL described in any one set of (a) to (k) respectively, or (m) A heavy chain variable region (VH) and a light chain variable region (VL) having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or any combination thereof) compared to the VH and VL described in any one set of (a) to (k) respectively. In a preferred embodiment, the above substitutions are conservative substitutions.

[0185] In certain embodiments, the anti-NaPi2b antibodies of the present disclosure are chimeric antibodies, humanized antibodies or fully human antibodies. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is Fab, Fab’, (Fab’) 2、Selected from Fv fragments such as scFv or Fv (dsFv) linked by disulfide bonds, diabodies, and multispecific antibodies. In certain embodiments, the anti-NaPi2b antibody of the present disclosure is an scFv.

[0186] In certain embodiments, the heavy chain of the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, and the variant has at most 50 conservative substitutions of amino acids (e.g., at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions of amino acids) compared to the wild-type sequence from which it is derived. In certain embodiments, the light chain of the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof, and the variant has at most 50 conservative substitutions of amino acids (e.g., at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions of amino acids) compared to the wild-type sequence from which it is derived.

[0187] In some embodiments, the constant region is modified, e.g., mutated, to modify the properties of the anti-NaPi2b antibody molecule (e.g., to modify one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function). A functional modification can be effected by substituting at least one amino acid residue of the antibody constant region with a different residue, e.g., by modifying (e.g., decreasing) the effector function by modifying the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q). The Fc region of an antibody mediates several important effector functions, such as ADCC, antibody-dependent cell phagocytosis (ADCP), CDC, etc.

[0188] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure has a heavy chain constant region (CH) selected from, for example, heavy chain constant regions selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly, for example, heavy chain constant regions selected from IgG1, IgG2, IgG3, and IgG4, and more particularly a heavy chain constant region selected from IgG1 (e.g., human IgG1). In some embodiments, the human IgG1 heavy chain constant region is shown in SEQ ID NO: 70. In some embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure has a light chain constant region selected from, for example, κ or λ light chain constant regions, preferably a κ light chain constant region (e.g., human κ light chain constant region). In some embodiments, the light chain constant region has the sequence shown in SEQ ID NO: 80.

[0189] In some embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises the CH shown in SEQ ID NO: 70 or a variant thereof, wherein the variant has at most 20 conservative substitutions of amino acids (e.g., at most 20, at most 15, at most 10, or at most 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions of amino acids) compared to SEQ ID NO: 70, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO: 70.

[0190] In some embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises a light chain constant region or a variant thereof. In some embodiments, the light chain constant region comprises a κ light chain constant region. In some embodiments, the light chain constant region comprises the light chain constant region (CL) shown in SEQ ID NO: 80 or a variant thereof, and the variant has at most 20 conservative substitutions of amino acids (e.g., at most 20, at most 15, at most 10, or at most 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions of amino acids) compared to SEQ ID NO: 80, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO: 80.

[0191] In some embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) shown in SEQ ID NO: 70 and the light chain constant region (CL) shown in SEQ ID NO: 80.

[0192] In one embodiment, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises the following: (a) a heavy chain comprising an amino acid sequence selected from the following or consisting of the following: (i) a sequence comprising the VH sequence shown in SEQ ID NO: 71 and the CH sequence shown in SEQ ID NO: 70, (ii) a sequence having one or more amino acid substitutions, deletions, or additions or any combination thereof (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acid substitutions, deletions, or additions or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions or any combination thereof) compared to the sequence shown in (i), or (iii) An array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity when compared with the sequence shown in (i), and (b) A light chain comprising an amino acid sequence selected from the following: (iv) An array comprising the VL array shown in SEQ ID NO: 72 and the CL array shown in SEQ ID NO: 80, (v) An array having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof) when compared with the array shown in (iv), or (vi) An array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity when compared with the array shown in (iv).

[0193] In certain embodiments, the above substitutions in (ii) or (v) are conservative substitutions.

[0194] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) A heavy chain comprising or consisting of an amino acid sequence selected from the following: (i) An array comprising the VH array shown in SEQ ID NO: 73 and the CH array shown in SEQ ID NO: 70, (ii) An array having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acid substitutions, deletions, or additions, or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof) when compared to the array shown in (i), or (iii) An array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity when compared to the array shown in (i), and (b) A light chain comprising, or consisting of, an amino acid sequence selected from the following: (iv) An array comprising the VL array shown in SEQ ID NO: 72 and the CL array shown in SEQ ID NO: 80, (v) An array having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acid substitutions, deletions, or additions, or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof) when compared to the array shown in (iv), or (vi) An array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity when compared to the array shown in (iv).

[0195] In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.

[0196] In certain embodiments, the anti-NaPi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise: (a) a heavy chain comprising an amino acid sequence selected from the following, or consisting of the following: (i) a sequence comprising the VH sequence shown in SEQ ID NO: 17 and the CH sequence shown in SEQ ID NO: 70, (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions, or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof) compared to the sequence shown in (i), or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the sequence shown in (i), and (b) a light chain comprising an amino acid sequence selected from the following, or consisting of the following: (iv) a sequence comprising the VL sequence shown in SEQ ID NO: 18 and the CL sequence shown in SEQ ID NO: 80, (v) an array having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof) compared to the array shown in (iv), or (vi) an array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the array shown in (iv).

[0197] In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.

[0198] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the following sequences: (i) the sequence shown in SEQ ID NO: 81, (ii) an array having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof) compared to the array shown in (i), or (iii) An array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity when compared to the sequence shown in (i), and The light chain contains or consists of the following sequences: (iv) The sequence shown in SEQ ID NO: 82, (v) One or more amino acid substitutions, deletions or additions or any combination thereof (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof) when compared to the sequence shown in (iv), or (vi) An array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity when compared to the sequence shown in (iv).

[0199] In a preferred embodiment, in (ii) or (v), the substitution is a conservative substitution.

[0200] In one embodiment, the anti-NaPi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain and a light chain, The heavy chain contains or consists of the following sequences: (i) The sequence shown in SEQ ID NO: 83, (ii) An array having one or more amino acid substitutions, deletions or additions, or any combination thereof (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions, or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof) compared to the array shown in (i), or (iii) An array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the array shown in (i), and The light chain contains or consists of the following sequences: (iv) The sequence shown in SEQ ID NO: 84, (v) An array having one or more amino acid substitutions, deletions or additions, or any combination thereof (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions, or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof) compared to the array shown in (iv), or (vi) An array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the array shown in (iv).

[0201] In a preferred embodiment, in (ii) or (v), the substitution is a conservative substitution.

[0202] In certain embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain selected from the group consisting of: (a) a heavy chain comprising VH shown in SEQ ID NO: 71 and CH shown in SEQ ID NO: 70, and a light chain comprising VL shown in SEQ ID NO: 72 and CL shown in SEQ ID NO: 80; (b) a heavy chain comprising VH shown in SEQ ID NO: 73 and CH shown in SEQ ID NO: 70, and a light chain comprising VL shown in SEQ ID NO: 72 and CL shown in SEQ ID NO: 80, preferably comprising a heavy chain shown in SEQ ID NO: 81 and a light chain shown in SEQ ID NO: 82; (c) a heavy chain comprising VH shown in SEQ ID NO: 17 and CH shown in SEQ ID NO: 70, and a light chain comprising VL shown in SEQ ID NO: 18 and CL shown in SEQ ID NO: 80, preferably comprising a heavy chain shown in SEQ ID NO: 83 and a light chain shown in SEQ ID NO: 84.

[0203] In certain embodiments, the antibodies provided by the present disclosure are multispecific antibodies that bind to NaPi2b and one or more other antigens. In certain preferred embodiments, the multispecific antibody is a bispecific, trispecific, or tetravalent antibody. In some embodiments, the multispecific antibodies of the present disclosure comprise the anti-NaPi2b antibodies or antigen-binding fragments thereof described above, and another antibody or fragment thereof or antibody mimetic.

[0204] Antibody derivatives of the present disclosure The anti-NaPi2b antibodies or antigen-binding fragments thereof of the present disclosure can be derivatized, for example, conjugated to another molecule (such as another polypeptide or protein). Generally, derivatization of the antibody or its antigen-binding fragment (such as labeling) does not adversely affect its binding to NaPi2b. Accordingly, the anti-NaPi2b antibodies or antigen-binding fragments thereof of the present disclosure are also intended to include their derivatized forms. For example, the anti-NaPi2b antibodies or antigen-binding fragments thereof of the present disclosure can be conjugated (by chemical coupling, gene fusion, non-covalent linkage, or other methods) to one or more other molecular groups, such as another antibody (such as to form a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (such as avidin or a polyhistidine tag) that can mediate the binding of the antibody or its antigen-binding fragment to another molecule.

[0205] One type of derivatized antibody (such as a bispecific antibody) is produced by cross-linking two or more antibodies (belonging to the same type or different types). Methods for obtaining bispecific antibodies are known in the art and examples thereof include, but are not limited to, chemical cross-linking methods, cell engineering methods (hybridoma methods), or genetic engineering methods.

[0206] Another type of derivatized antibody is a labeled antibody. For example, the antibodies or antigen-binding fragments thereof of the present disclosure can be linked to a detectable label. The detectable labels described in the present disclosure may be any substance detectable by fluorescence, spectral, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such labels are known in the art, and examples thereof include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa750)), acridinium ester-based compounds, magnetic beads (e.g., Dynabeads®), calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the above marker-modified avidin (e.g., streptavidin), but are not limited thereto. Patents teaching the use of such markers include, but are not limited to, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all of which are incorporated herein by reference). The above detectable labels can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation computer, and fluorescent markers can be detected using a photodetector for detecting the emitted light. Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product resulting from the action of the enzyme on the substrate, and calorimetric markers are detected by simply visualizing the colored marker. In certain embodiments, such labels are applicable to immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.).In certain embodiments, to reduce potential steric hindrance, the detectable label can be linked to an antibody or antigen-binding fragment thereof of the present disclosure via linkers of different lengths.

[0207] Furthermore, an antibody or antigen-binding fragment thereof of the present disclosure can be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, such as increasing the serum half-life.

[0208] Preparation of Antibodies The antibodies of the present disclosure can be obtained by various methods known in the art, such as by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present disclosure are obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and then transfected into host cells. Next, the transfected host cells are cultured under specific conditions to express the antibodies of the present disclosure.

[0209] The antigen-binding fragments of the present disclosure can be obtained by hydrolyzing a complete antibody molecule (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). These antigen-binding fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab’ fragments can be obtained directly from host cells, and Fab’ fragments can be chemically coupled to form F(ab’)2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). Also, Fv, Fab or F(ab’)2 fragments can be obtained directly by separating them from recombinant host cell culture supernatants. Other techniques for preparing these antigen-binding fragments are well known to those skilled in the art.

[0210] In a third aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region, or one or more CDRs thereof, of the second aspect of the present disclosure. In certain embodiments, the nucleotide sequence can be substituted by codon degeneracy. In certain embodiments, the nucleotide sequence is codon optimized.

[0211] In certain embodiments, the isolated nucleic acid molecules described in the present disclosure comprise: (i) a first nucleic acid and a second nucleic acid encoding the heavy chain variable region and the light chain variable region, respectively, of an antibody or an antigen-binding fragment thereof of the second aspect of the present disclosure, or (ii) a first nucleic acid encoding the heavy chain variable region and the heavy chain constant region of an antibody or an antigen-binding fragment thereof of the second aspect of the present disclosure, and a second nucleic acid encoding the light chain variable region and the light chain constant region, or (iii) a first nucleic acid and a second nucleic acid encoding the heavy chain and the light chain, respectively, of an antibody or an antigen-binding fragment thereof of the second aspect of the present disclosure. In certain embodiments, the first nucleic acid and the second nucleic acid comprise degenerate sequences or nucleic acids of substantially the same sequence as the first nucleic acid and the second nucleic acid of any one of (i)-(iii) above. In certain embodiments, the degenerate sequence or substantially the same sequence has a sequence identity of at least about 85%, 90%, 95%, 99% or higher, or a sequence having one or more nucleotide substitutions, or a sequence that differs by less than 3, 6, 15, 30 or 45 nucleotides, compared to the nucleic acid molecules described in (i)-(iii).

[0212] In a fourth aspect, there is provided a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the third aspect of the present disclosure. In certain embodiments, the vector of the present disclosure is a cloning vector or an expression vector. In certain embodiments, the vector of the present disclosure is, for example, a plasmid, a cosmid, a phage, a lentivirus, etc. In certain embodiments, the vector can express the antibody or an antigen-binding fragment thereof of the present disclosure in a subject (e.g., a mammal, e.g., a human) in vivo.

[0213] In a fifth aspect, there is provided a host cell comprising the isolated nucleic acid molecule of the third aspect of the present disclosure or the vector of the fourth aspect of the present disclosure. The host cell may be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, SP2 / 0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the present disclosure is a mammalian cell, e.g., CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44).

[0214] In a sixth aspect, there is provided a method for preparing an antibody or an antigen-binding fragment thereof of the second aspect of the present disclosure, or a multispecific antibody of the present disclosure, comprising culturing the host cell of the present disclosure under conditions that allow expression of the antibody or an antigen-binding fragment thereof, or the multispecific antibody, and recovering the antibody or an antigen-binding fragment thereof from the cultured host cell culture.

[0215] Antibody-drug conjugate In a seventh aspect, there is provided an anti-NaPi2b antibody-drug conjugate, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the antibody is an antibody that binds to the above NaPi2b or an antigen-binding fragment thereof.

[0216] In some embodiments, the antibody-drug conjugate is

Chemical formula

Chemical formula

[0217] q is selected from any numerical value between 0.1 and 16.0, preferably any numerical value between 2 and 8. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some preferred embodiments, q is 2, 4, 6 or 8.

[0218] Preparation of Antibody-Drug Conjugates In an eighth aspect of the present disclosure, the present disclosure provides a method for preparing the above antibody-drug conjugate, Tb and the drug linker complex shown in Formula III

Chemical formula

[0219] Lg is a leaving group, and Lg is selected from halogen, sulfone group, tertiary amine base (Me3N + , Et3N + ), diazonium base, -OMs, MeSO2-, CF3SO3-.

[0220] In some embodiments, Lg is selected from F, Cl, MeSO2-.

[0221] In some embodiments, Lg is selected from F, MeSO2-.

[0222] In some embodiments, the method comprises coupling Tb with the drug linker complex shown in Formula III

Chemical formula

[0223] In some embodiments, the ratio of the amount of Tb to the amount of the drug linker complex is 1:(1 - 20), such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:(10 - 20), 1:(12 - 20), 1:(14 - 20), 1:(16 - 20) or 1:(18 - 20).

[0224] In some embodiments, the coupling reaction is carried out in water and / or an organic solvent.

[0225] In some embodiments, the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitrile-based (such as acetonitrile), alcohol-based (such as methanol, ethanol) or any combination thereof.

[0226] In some embodiments, the method further comprises purifying the composite product.

[0227] In some embodiments, the composite product is purified by chromatography.

[0228] In some embodiments, the chromatography method includes one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography or affinity chromatography.

[0229] In a ninth aspect of the present disclosure, the present disclosure provides a population of antibody-drug conjugates comprising the antibody-drug conjugates described in the present disclosure, their stereoisomers, their prodrugs, their pharmaceutically acceptable salts, or their pharmaceutically acceptable solvates, wherein the antibody-drug conjugates have one, two, or more q values.

[0230] In some embodiments, when the antibody-drug conjugate within the population has one q value, the q value is equal to the DAR value.

[0231] In some embodiments, when the antibody-drug conjugate within the population has two or more q values, among them, the proportion of the antibody-drug conjugate having one specific q value with respect to all the antibody-drug conjugates in the population is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%.

[0232] In some embodiments, the drug-to-antibody ratio (DAR) in the antibody-drug conjugate within the population is selected from an integer or a decimal between 1 and 10.

[0233] In some embodiments, the drug-to-antibody ratio (DAR) of the population is selected from 1.5 to 2.5, 3.5 to 4.5, 5.5 to 6.5, and 7.5 to 8.5. In some embodiments, the drug-to-antibody ratio (DAR) of the population is selected from approximately 2.0, 4.0, 6.0, and 8.0. In some embodiments, the drug-to-antibody ratio (DAR) in the antibody-drug conjugate within the population is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, and 9.

[0234] In some embodiments, the population comprises ADCs having a DAR distribution of 1 to 8, such as 1.5, 2, 4, 6, and 8 (i.e., drug-loading species of 1.5, 2, 4, 6, and 8). It should be noted that degradation products can be generated such that the population can include DARs of 1, 3, 5, and 7. Also, the population can have a DAR greater than 8. The antibody-drug conjugate is generated by interchain disulfide reduction and subsequent coupling. In some embodiments, the antibody-drug conjugate includes both the antibody-drug conjugate having a DAR of 4 or less (i.e., drug-loading species of 4 or less) and the antibody-drug conjugate having a DAR of 6 or more (i.e., drug-loading species of 6 or more).

[0235] In a tenth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate of the first aspect or the seventh aspect, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, the anti-NaPi2b antibody of the second aspect or its antigen-binding fragment, the nucleic acid described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, or the population of antibody-drug conjugates of the ninth aspect, and optionally one or more pharmaceutical adjuvants.

[0236] In certain embodiments, the dosage of the antibody-drug conjugate of the first aspect or the seventh aspect, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, the anti-NaPi2b antibody of the second aspect or its antigen-binding fragment, the nucleic acid described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, or the population of antibody-drug conjugates of the ninth aspect included in the pharmaceutical composition is any therapeutically effective amount.

[0237] In certain embodiments, the pharmaceutical composition includes the antibody-drug conjugate of the first aspect or the seventh aspect, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, and a pharmaceutically acceptable carrier and / or excipient.

[0238] In certain embodiments, the pharmaceutical composition of the present disclosure comprises a population of the antibody-drug conjugate of the ninth aspect, and a pharmaceutically acceptable carrier and / or excipient. In certain preferred embodiments, the pharmaceutical composition of the present disclosure comprises the above population of antibody-drug conjugates, or comprises a population of antibody-drug conjugates and a buffer. In certain further preferred embodiments, the pharmaceutical composition of the present disclosure further comprises an excipient and / or a surfactant.

[0239] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the anti-NaPi2b antibody of the present disclosure or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and / or excipient. In certain preferred embodiments, the pharmaceutical composition of the present disclosure comprises an anti-NaPi2b antibody or an antigen-binding fragment thereof, and a buffer. In certain further preferred embodiments, the pharmaceutical composition of the present disclosure further comprises an excipient and / or a surfactant.

[0240] In some embodiments, the buffer is a histidine buffer. In certain preferred embodiments, the buffer is a 20 mM histidine buffer having a pH of 6.0.

[0241] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the host cell of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient, wherein the host cell comprises the above isolated nucleic acid molecule or vector.

[0242] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the multispecific antibody of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient.

[0243] In some embodiments, the drug-to-antibody ratio (DAR) in the pharmaceutical composition is selected from an integer or a decimal between 1 and 10.

[0244] In some embodiments, the drug-to-antibody ratio (DAR) in the pharmaceutical composition is selected from 1.5 to 2.5, 3.5 to 4.5, 5.5 to 6.5, and 7.5 to 8.5. In some embodiments, the drug-to-antibody ratio (DAR) in the pharmaceutical composition is selected from about 2.0, 4.0, 6.0, and 8.0, In some embodiments, the drug-to-antibody ratio (DAR) in the antibody-drug conjugate in the pharmaceutical composition is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, and 9.

[0245] In a 11th aspect, there is provided the use of an antibody or antigen-binding fragment thereof of the present disclosure in the preparation of a reagent kit for detecting the presence or level of NaPi2b in a sample. In another aspect, the present disclosure provides a diagnostic or therapeutic reagent kit comprising one or more of the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, antibody-drug conjugate, population of antibody-drug conjugates, or pharmaceutical composition described in the present disclosure. Optionally, the diagnostic or therapeutic reagent kit further comprises an instruction manual.

[0246] In a 12th aspect of the present disclosure, there is provided the use of the antibody-drug conjugate composition or the pharmaceutical composition in the preparation of a drug for treating and / or preventing a disease associated with abnormal cell activity (e.g., a cancer disease). The antibody-drug conjugate composition or the pharmaceutical composition may be in a therapeutically effective amount.

[0247] In some examples, there is provided the use of an anti-NaPi2b antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or multispecific antibody of the present disclosure in the preparation of a drug for modulating (inhibiting or blocking) the activity of NaPi2b.

[0248] In some examples, there is provided the use of an anti-NaPi2b antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or multispecific antibody of the present disclosure in the preparation of a drug for treating or preventing a disease associated with the activity of NaPi2b.

[0249] In some embodiments, provided is the use of an anti-NaPi2b antibody or an antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, an antibody-drug conjugate, or a multispecific antibody of the present disclosure in the preparation of a drug for treating or preventing tumors associated with the activity of NaPi2b.

[0250] In some embodiments, provided is the use of an antibody-drug conjugate of the first or seventh aspect described in the present disclosure, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, an anti-NaPi2b antibody of the second aspect or an antigen-binding fragment thereof, a nucleic acid described in the third aspect, a vector described in the fourth aspect, a host cell described in the fifth aspect, a population of antibody-drug conjugates of the ninth aspect, or a pharmaceutical composition of the tenth aspect in the preparation of a drug for treating or preventing tumors.

[0251] In some embodiments, provided is the use of an antibody-drug conjugate of the first or seventh aspect described in the present disclosure, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, a population of antibody-drug conjugates of the ninth aspect, or a pharmaceutical composition of the tenth aspect in the preparation of a drug for treating or preventing tumors associated with a target of Tb.

[0252] In some embodiments, provided is the use of an antibody-drug conjugate of the first or seventh aspect described in the present disclosure, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, a population of antibody-drug conjugates of the ninth aspect, or a pharmaceutical composition of the tenth aspect in the preparation of a drug for treating or preventing tumors associated with NaPi2b.

[0253] In some preferred embodiments, provided is the use of the antibody-drug conjugate of the first aspect or the seventh aspect described in the present disclosure, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, the anti-NaPi2b antibody of the second aspect or its antigen-binding fragment, the nucleic acid described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, the population of antibody-drug conjugates of the ninth aspect, or the pharmaceutical composition of the tenth aspect, in the preparation of a drug for treating or preventing tumors related to NaPi2b.

[0254] In a thirteenth aspect of the present disclosure, the present disclosure provides a method of using the antibody-drug conjugate of the first aspect or the seventh aspect described above, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, the anti-NaPi2b antibody of the second aspect or its antigen-binding fragment, the nucleic acid described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, the population of antibody-drug conjugates of the ninth aspect, or the pharmaceutical composition of the tenth aspect, for the treatment and / or prevention of diseases related to abnormal cell activity (such as tumors).

[0255] In the above twelfth and thirteenth aspects, the above tumors are selected from esophageal cancer (such as esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumors, lung cancer (such as small cell lung cancer, non-small cell lung cancer or lung adenocarcinoma), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer (such as human colorectal adenocarcinoma), rectal cancer, colorectal cancer, liver cancer, kidney cancer, urothelial cancer, epidermal cancer, non-Hodgkin lymphoma, central nervous system tumors (such as glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0256] In some embodiments, the above tumors are tumors related to NaPi2b.

[0257] In some embodiments, the above tumors are tumors related to NaPi2b.

[0258] Unless otherwise defined, scientific and technical terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art. Also, the cell culture, molecular genetics, nucleic acid chemistry, and immunological laboratory procedures used in this specification are common procedures widely used in the art. At the same time, to better understand this disclosure, the following provides definitions and explanations of related terms.

[0259] As used herein, examples of the term "pharmaceutically acceptable salt" are organic acid addition salts formed from organic acids that form pharmaceutically acceptable anions, including, but not limited to, formates, acetates, propionates, benzoates, maleates, fumarates, succinates, tartrates, citrates, ascorbates, α-ketoglutarates, α-glycerophosphates, alkyl sulfonates, or aryl sulfonates. Preferably, the alkyl sulfonate is methanesulfonate or ethanesulfonate, and the aryl sulfonate is benzenesulfonate or p-toluenesulfonate. Suitable inorganic salts can also be formed, including, but not limited to, hydrochlorides, hydrobromides, hydroiodides, nitrates, bicarbonates and carbonates, sulfates, or phosphates.

[0260] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a vector and / or excipient that is pharmacologically and / or physiologically compatible with the active ingredient, which is well known in the art (see, for example, Remington’s Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, reagents for maintaining osmotic pressure, reagents for delaying absorption, and preservatives.

[0261] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with an appropriate acid that provides a pharmaceutically acceptable anion.

[0262] In the present disclosure, the above-mentioned pharmaceutical adjuvants refer to excipients and additives used in the preparation and formulation of pharmaceuticals, and in addition to the active ingredient, refer to substances that are reasonably evaluated in terms of safety and are included in pharmaceutical preparations. Pharmaceutical adjuvants act as excipients and carriers, and in addition to improving stability, they further have important functions such as solubilization, solubilization assistance, and sustained release, and are important components that can affect the quality, safety, and efficacy of pharmaceuticals. Depending on their origin, they can be divided into natural products, semi-synthetic products, and fully synthetic products. Depending on their actions and uses, they can be divided into solvents, propellants, solubilizing agents, solubilization aids, emulsifiers, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow promoters, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, moisturizing agents, absorbents, diluents, aggregating agents and anti-aggregating agents, filter aids, release retardants, etc. Depending on the administration route, they can be divided into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ophthalmic administration, etc. The same pharmaceutical adjuvant can be used in pharmaceutical preparations with different administration routes and has different actions and uses.

[0263] The above-mentioned pharmaceutical composition can be prepared into various appropriate dosage forms according to the administration route. For example, tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder aerosols, sprays, etc. Among them, the above-mentioned pharmaceutical composition or appropriate dosage form may contain 0.01 mg to 1000 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or complex, appropriately 0.1 mg to 800 mg, preferably 0.5 to 500 mg, more preferably 0.5 to 350 mg, and particularly preferably 1 to 250 mg.

[0264] The above pharmaceutical composition can be administered in the form of an injection, including an injection, a sterile powder for injection, and a concentrated solution for injection. Among them, carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterilized non-volatile oils, such as glycerides or diglycerides, can also be used as solvents or suspension media.

[0265] As used herein, the term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. Such an effect may be prophylactic, based on the complete or partial prevention of a disease or its symptoms, and / or may be therapeutic, based on the partial or complete stabilization or cure of a disease, and / or on the side effects caused by the disease. "Treatment" as used herein includes (a) preventing a disease or symptom from developing in a patient who is susceptible to, but not yet diagnosed with, the disease or symptom, (b) inhibiting the symptoms of a disease, i.e., preventing its progression, or (c) alleviating the symptoms of a disease, i.e., causing a deterioration of the disease or symptom, and encompasses any treatment of a patient's disease.

[0266] In the present disclosure, the term "individual" includes humans or non-human animals. Exemplary human individuals include human individuals (referred to as patients) suffering from a disease (e.g., a disease described herein) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domestic animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0267] In the present disclosure, the term "effective dose" refers to the amount of a compound that, after administration, alleviates to some extent one or more symptoms of the disorder being treated.

[0268] In the present disclosure, the terms "biologically active substance", "biologically active molecule" or "drug molecule" refer to substances that inhibit or prevent the functions of cells and / or cause cell death or destruction. In some embodiments of the present disclosure, the biologically active substance, biologically active molecule or drug molecule in the complex is a molecule having antitumor biological activity. For example, At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212and radioisotopes such as radioisotopes of Lu; metal complexes such as, for example, metal platinum complexes, metal gold complexes, oxaliplatin; glycopeptide antibiotics such as, for example, bleomycin, norpomycin; DNA topoisomerase inhibitors such as, for example, topoisomerase I inhibitors, camptothecin, hydroxycamptothecin, 9 - aminocamptothecin, SN - 38, irinotecan, topotecan, belotecan, rubitecan, topoisomerase II inhibitors, actinomycin D, doxorubicin, doxorubicin, docardamycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide; drugs that interfere with DNA synthesis such as, for example, methotrexate, 5 - fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine; drugs that act on structural proteins such as, for example, tubulin inhibitors, vinca alkaloids, vincristine, vinca alkaloids, paclitaxel, docetaxel, cabazitaxel; tumor signal pathway inhibitors such as, for example, serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartic acid kinase inhibitors or histidine acid kinase inhibitors, and are proteasome inhibitors, histone deacetylase inhibitors, tumor angiogenesis inhibitors, cell cycle protein inhibitors, maytansine derivatives, calicheamicin derivatives, auristatin derivatives, Pyrrolobenzodiazepines (PBD) derivatives, melphalan, mitomycin C, chlorambucil, or other active substances that inhibit the growth of tumor cells and promote apoptosis and necrosis of tumor cells; enzymes such as ribozymes and fragments thereof; antibiotics; small molecule toxins or enzyme - active toxins of bacterial, fungal, plant or animal origin, fragments and / or variants thereof, etc. toxins; growth inhibitors; and further comprising a drug module. The term "toxin" refers to a substance that can have a harmful effect on the growth or proliferation of cells.

[0269] In the present disclosure, the term "linker" refers to a fragment that links a biologically active molecule (drug molecule) to a target moiety.

[0270] In the present disclosure, the term "target moiety" refers to a moiety of a complex that can specifically bind to a target (or a moiety of a target) on the cell surface. Through the interaction between the target moiety and the target, the complex can be delivered to a specific cell population.

[0271] In the present disclosure, an antibody or an antigen-binding fragment thereof includes a derivatized antibody or an antigen-binding fragment thereof, such as an antibody or an antigen-binding fragment thereof having a thiol group, wherein, through the above derivatization, the antibody has a group or ability to react with a drug linker complex. The above thiol group -SH can be derivatized by ring-opening a disulfide bond (for example, reduced by a reducing agent TCEP).

[0272] As used herein, the terms "cancer" and "tumor" are used interchangeably.

[0273] As used herein, the term "gene" includes not only DNA, but also its mRNA, its cDNA, and its cRNA.

[0274] As used herein, the term "polynucleotide" is used interchangeably with nucleic acid, and also includes DNA, RNA, probes, oligonucleotides, and primers.

[0275] As used herein, the terms "polypeptide" and "protein" are used interchangeably.

[0276] As used herein, the term "cell" includes cells within an animal individual and cultured cells.

[0277] NaPi2b involved in the anti-NaPi2b antibody described in the present disclosure may be a normal NaPi2b in the art, and naturally also represents NaPi2b variants.

[0278] The term "NaPi2b": NaPi2b is encoded by the SLC34A2 gene consisting of 690 amino acids, structurally traverses the membrane 8 times, and has 4 extracellular loops. Sodium-dependent phosphate transporter 2B (NaPi2b) selectively transports divalent Pi (HPO4 2- ) to maintain the in vivo Pi homeostasis. Human NaPi2b has 85.3% homology with rat and 95.8% homology with cynomolgus monkey. NaPi2b shows high similarity with the NaPi2a / 2c amino acid sequences of the family (having 51.29% homology with NaPi2a and 44.64% homology with NaPi2c), and shows >80% homology especially in the transmembrane region.

[0279] KD refers to the dissociation constant obtained from the ratio of Kd (dissociation rate of the interaction between a specific binding molecule and a target protein) and Ka (binding rate of the interaction between a specific binding molecule and a target protein) (or Kd / Ka expressed in molar concentration (M)). The KD value can be measured using methods well established in the art. A preferred method for measuring the KD of a binding molecule is to use a biosensor system such as surface plasmon resonance, for example, the Biacore TM (GE Healthcare Life Sciences) system.

[0280] As used herein, the percent homology between two amino acid sequences is equal to the percent identity between the two sequences. The percent sequence identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100), and it is necessary to introduce an optimal contrast of the two sequences considering the number of gaps and the length of each gap. Using methods generally known in the art, sequence comparison and determination of percent identity between sequences can be performed, and using mathematical algorithms, such sequence comparison and determination of percent identity can be performed. For example, the algorithm of Meyers and Miller, 1988 Comput. Appl. Biosci. 4:11-17 (incorporated in the ALIGN program (version 2.0)) can be used to determine the percent identity between amino acid sequences and / or between nucleotide sequences. Further, the GAP program in the GCG software package obtained online from Accelrys can be used (using its default parameters) to determine the percent identity between amino acid sequences or between nucleotide sequences. In one embodiment, the two sequences are of equal length.

[0281] The term "epitope" refers to a part of an antigen polypeptide or protein that has antigenic or immunogenic activity in an animal, preferably a mammal. The epitope of an antibody or an antigen-binding fragment thereof of the present disclosure can be measured by existing techniques such as synthetic peptide methods, immunoinformatics prediction, measurement of polypeptide activity, epitope peptide scanning methods, phage display techniques, X-ray diffraction and nuclear magnetic resonance analysis, antibody homology modeling protein docking prediction methods, and the like. The phrase "antibodies that bind to the same epitope" as used herein refers to different antibodies that bind to a common epitope. When a second antibody binds to a part of the peptide or a part of the tertiary structure to which the first antibody binds, it can be confirmed that the first antibody and the second antibody bind to the same epitope.

[0282] In the present disclosure, the term "antibody" is taken in its broadest interpretation to include full monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two full antibodies, provided they have the desired biological activity. In the present disclosure, "antibody" and "immunoglobulin" can be used interchangeably. The "antibody molecule" or "antibody" described herein refers to an immunoglobulin molecule and an immunologically active portion of the immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that immunospecifically binds to an antigen. Thus, the term antibody broadly encompasses not only full antibody molecules but also fragments of the above antibodies, as well as variants (including derivatives) of the above antibodies and antibody fragments. When "antibody molecule" or "antibody" is used in the same context as antigen-binding fragment, "antibody molecule" or "antibody" refers to a full antibody molecule or a full-length antibody. The term antibody molecule described herein includes, but is not limited to, for example, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab')2, Fv linked by disulfide bonds (sdFv), Fv, and full antibodies or full-length antibodies. The term "single-chain Fv" or "scFv" refers to a polypeptide containing the VL domain of an antibody linked to the VH domain of the antibody. For example, an antibody that immunospecifically binds to NaPi2b can cross-react with other antigens. Preferably, an antibody that immunospecifically binds to NaPi2b does not cross-react with other antigens. Immunospecific binding can be identified, for example, by immunoassays or other methods known to those skilled in the art. A "full" antibody or "full-length" antibody refers to a protein containing two heavy chains (H) and two light chains (L) linked to each other by disulfide bonds, and the protein includes (1) with respect to the heavy chain, a variable region (abbreviated herein as "VH") and a heavy chain constant region containing three domains CH1, CH2, and CH3, and (2) with respect to the light chain, a light chain variable region (abbreviated herein as "VL") and a light chain constant region containing one domain CL. The antibodies of the present disclosure include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies of the antibodies of the present disclosure), and epitope-binding fragments of any of the above.The immunoglobulin molecules of the present disclosure may be of any type of immunoglobulin (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Preferably, the antibodies of the present disclosure have a VH domain, VH CDR (generally represented as HCDR herein), VL domain, or VL CDR (generally represented as LCDR herein) having any one of the amino acid sequences or fragments or variants thereof described in the sequence and its specific information table, or consist of them.

[0283] In the present disclosure, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, i.e., each antibody constituting the population is identical, except for minor natural variations that may be present. Monoclonal antibodies have high specificity for one determinant (epitope) of an antigen, whereas the corresponding polyclonal antibodies contain different antibodies against different determinants (epitopes). The advantage of monoclonal antibodies is, in addition to specificity, that they are not contaminated by other antibodies during synthesis. Here, the modifier "monoclonal" means that the antibody is characterized by being derived from a substantially homogeneous population of antibodies, but it should not be understood to require production by a specific method.

[0284] In some embodiments of the present disclosure, the monoclonal antibody further includes, in particular, a chimeric antibody, i.e., a part of the heavy chain and / or light chain is identical or homologous to an antibody of a certain type, class, or subclass as long as they have the desired biological activity (see, for example, US 4,816,567 and Morrison et al., 1984, PNAS, 81: 6851-6855), and the remaining part is identical or homologous to an antibody of another type, class, or subclass. The chimeric antibodies used in the present disclosure include primatized antibodies containing variable region antigen-binding sequences derived from non-human primates (e.g., old world monkeys, orangutans, etc.) and human constant region sequences.

[0285] The term "antigen-binding fragment" refers to a part of an antibody, preferably the antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fd, Fv, dAb and complementarity-determining region fragments, diabodies, linear antibodies, and single-chain antibody molecules. The term "antigen-binding fragment" as used herein refers to a partial fragment of an antibody having antigen-binding activity, among which the above fragments include, but are not limited to, single-chain Fv (scFv), Fab, Fab’, F(ab’)2, Fv linked by disulfide bonds (sdFv), Fv, di-scFv, etc., and have the complete or partial function of an antibody. The term also includes Fab’, which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab’)2 under reducing conditions. However, the term is not limited to these molecules as long as the above fragments have binding affinity for the antigen. Furthermore, these functional fragments include not only fragments obtained by treating the full-length molecule of an antibody protein with an appropriate enzyme, but also proteins produced in an appropriate host cell using a genetically modified antibody gene.

[0286] The term "Fab’" as used herein refers to a monovalent fragment of the variable region of an antibody obtained by treating F(ab’)2 under reducing conditions as described above. However, the Fab’ of the present disclosure also includes Fab’ produced using a genetically modified antibody gene.

[0287] As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains linked via a linker or directly (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure of NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated sequences of the GGGGS amino acid sequence or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, or variants thereof may be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers used in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may be present between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by the linkage of two scFvs.

[0288] As used herein, in the above antibody or antigen-binding fragment thereof, even if it contains a variant, amino acid substitution, deletion or addition, it still has the activity of binding to the above antigen.

[0289] The term "bispecific antibody" is also referred to as "bifunctional antibody complex", and refers to a complex in which a first antibody (fragment) and a second antibody (fragment) are formed via a complex arm, and the complex has bifunctionality and bispecificity in order to retain the activities of the respective antibodies.

[0290] The term "multispecific antibody" includes, for example, a trispecific antibody which is an antibody having three different antigen-binding specificities, and a tetravalent antibody which is an antibody having four different antigen-binding specificities.

[0291] The term "complete antibody" or "full-length antibody" refers to an antibody containing an antigen-binding variable region and a light chain constant region (CL), and heavy chain constant regions (CH1, CH2, and CH3). The constant region may be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. The complete antibody is preferably a complete antibody having one or more effector functions.

[0292] The term "Probody" is a modified antibody that includes an antibody or antibody fragment that can specifically bind to its target and can bind to a masking group, where the masking group has a dissociation constant for the binding ability of the antibody or antibody fragment to its target that is at least 100-fold, 1000-fold, or 10,000-fold greater than the dissociation constant for the binding ability of the antibody or antibody fragment not bound to the masking group to its target.

[0293] In the present disclosure, a "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which the hypervariable region residues of the human recipient immunoglobulin have been replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues having the desired specificity, affinity, and functionality (donor antibody). In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Moreover, the humanized antibody may further contain residues not present in the recipient antibody or the donor antibody. These modifications are for further optimizing the performance of the antibody. A humanized antibody generally contains at least one, usually two variable regions, of which all or substantially all of the hypervariable loops correspond to the non-human immunoglobulin and the FRs are completely or substantially completely of human immunoglobulin sequence. A humanized antibody can further contain at least a portion of the immunoglobulin constant region (Fc, usually the human immunoglobulin Fc). Relevant details can be found, for example, in Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr Op Struct Biol 2:593-596.

[0294] Complete antibodies can be divided into different "classes" according to the amino acid sequence of the heavy chain constant region. The five main classes are IgA, IgD, IgE, IgG, and IgM, and some of these classes can also be divided into different "subclasses" (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of the different classes of antibodies are designated α, β, ε, γ, and μ, respectively. The different subunit structures and three-dimensional conformations of immunoglobulins are well known in the art.

[0295] As used herein, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure are preferably determined according to the Kabat, Chothia, AbM, or IMGT numbering systems.

[0296] As used herein, the term "framework residue" or "FR residue" refers to an amino acid residue within the antibody variable region other than the CDR residues defined above.

[0297] As used herein, the term "germline antibody gene" is a gene that encodes an immunoglobulin expressed in non-lymphocytes that has not undergone the genetic rearrangement and maturation processes that result in the expression of a specific immunoglobulin. The advantages provided by various embodiments of the present disclosure arise from the recognition that the amino acid sequence encoded by a germline antibody gene retains more of the important amino acid sequence structures characteristic of an individual of the animal species than the amino acid sequence encoded by a mature antibody gene. Thus, when therapeutically applied to the species, there is less recognition as a foreign substance by the species.

[0298] As amino acid substitutions, conservative amino acid substitutions are preferred herein. Conservative amino acid substitutions represent substitutions that occur within a set of amino acids related to the amino acid side chain. Preferred sets of amino acids are the acidic set (aspartic acid and glutamic acid), the basic set (lysine, arginine, and histidine), the nonpolar set (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), and the uncharged polar family (glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine). More preferred sets of amino acids are the aliphatic hydroxyl (serine and threonine), the amide-containing set (asparagine and glutamine), the aliphatic set (alanine, valine, leucine, and isoleucine), and the aromatic set (phenylalanine, tryptophan, and tyrosine). Such amino acid substitutions are preferably made within a set that does not impair the properties of the substance having the original amino acid sequence.

[0299] Furthermore, it is known that the lysine residue at the carboxy terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705:129-134 (1995)), and that two amino acid residues (glycine and lysine) at the carboxy terminus of the heavy chain of an antibody produced in cultured mammalian cells are deleted and the proline residue newly located at the carboxy terminus is amidated (Analytical Biochemistry, 360:75-83 (2007)). However, deletions and modifications such as those in the heavy chain sequence do not affect the antigen-binding affinity and effector functions (complement activation, antibody-dependent cell cytotoxicity, etc.) of the antibody.

[0300] The description method of the 20 conventional amino acids related to this specification follows the conventional usage. For example, refer to Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this specification, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Also, in the present disclosure, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala, arginine can be represented by R or Arg, glycine can be represented by G or Gly, and glutamine can be represented by Q or Gln.

[0301] As used herein, the term "prevent" refers to a method implemented to prevent or delay the onset of a disease or medical condition or symptom (e.g., tumors and infectious diseases) in a subject. As used herein, the term "treat" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of the present disclosure, a beneficial or desired clinical outcome includes, but is not limited to, alleviation of symptoms, reduction in the scope of the disease, stabilization of the disease state (i.e., not worsening), delay or mitigation of the progression of the disease, improvement or alleviation of the disease state, and remission (either partial or complete) of symptoms, whether detectable or undetectable. Further, "treat" may refer to an extended survival period compared to the expected survival period (if not receiving treatment).

[0302] As used herein, the term "subject" refers to a mammal, such as a primate, such as a non-human primate or a human. In certain embodiments, the subject (e.g., a human) has or is at risk of having tumors and infectious diseases.

[0303] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor and an infectious disease) refers to an amount sufficient to prevent, inhibit, or delay the occurrence of the disease (e.g., a tumor and an infectious disease), and an effective amount for treating a disease refers to an amount sufficient to cure, or at least partially inhibit, the disease and its complications in a patient suffering from the disease. Determination of such an effective amount is within the ability of one of ordinary skill in the art. For example, an amount effective for therapeutic use depends on the severity of the disease being treated, the overall state of the patient's own immune system, general patient circumstances such as age, weight, gender, the mode of drug administration, and other treatments administered simultaneously.

[0304] As used herein, the term "effector function" is attributable to the biological activity of the antibody Fc region (natural sequence Fc region or amino acid sequence variant Fc region) and refers to that which varies depending on the antibody isotype.

[0305] The term "pharmaceutically acceptable" means that a molecular entity, fragment, or composition does not produce adverse, allergic, or other harmful reactions when properly administered to an animal or human. Specific examples of some substances that can be a pharmaceutically acceptable carrier or a composition thereof include saccharides (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, and the like.

[0306] The term "population of antibody-drug conjugates" refers to a group or population of a mixture consisting of the antibody-drug conjugates of the present invention, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, its tautomers, or its pharmaceutically acceptable solvates, among which the q of the antibody-drug conjugates may be the same or different. Further, it is also referred to as an "antibody-drug conjugate mixture".

[0307] Using experimental animals, for example, by administering an antibody to a nude mouse transplanted with a tumor cell line expressing NaPi2b and measuring any changes in the cancer cells, the in vivo therapeutic effect of the antibody and antibody-drug conjugate against cancer can be determined.

[0308] In the present disclosure, amino acid substitutions in the antibody are, in most cases, substituted with L-amino acids, but are not limited thereto. In some embodiments, the antibody peptide chain may contain one or more D-amino acids. Peptides containing D-amino acids are more stable and less likely to degrade in the oral cavity, intestinal tract, or plasma than peptides containing only L-amino acids.

[0309] The monoclonal antibodies used in the present disclosure can be produced by many methods. For example, the monoclonal antibodies used in the present disclosure can be obtained by the hybridoma method using many species (including mouse, hamster, rat, and human cells) (see, for example, Kohler et al., 1975, Nature, 256:495), or can be produced by recombinant DNA technology (see, for example, US4,816,567), or can be isolated from a phage antibody library (see, for example, Clackson et al., 1991, Nature, 352:624-628, and Marks et al., 1991, Journal of Molecular Biology, 222:581-597).

[0310] As used herein, unless otherwise clearly indicated, the descriptive methods "each... is independently selected" and "... are each independently selected" employed throughout this specification are interchangeable with each other and should both be understood broadly, and may mean that the specific options represented between the same or different reference numerals do not affect each other in different groups, or may mean that the specific options represented between the same or different reference numerals do not affect each other in the same group.

[0311] As used herein, the term "direct bond" means that the groups on both sides thereof are directly bonded. For example, in the compound represented by Formula II [Chemical formula] when X is a direct bond, the structure is [Chemical formula] The rest of the direct bonds can be understood by referring to the foregoing content.

[0312] As used herein, the term "absent" means that the group is absent. For example, in the compound represented by Formula II [Chemical formula] when W is absent, the structural formula is [Chemical formula] That is.

[0313] In the compound represented by Formula II [Chemical formula] R1 and R2 together with the carbon atoms to which they are attached [Chemical formula] form, and the "dashed" bond indicates the position where the above heterocyclic ring is condensed with the benzene ring. For example, [Chemical formula] is formed.

[0314] As used herein, the drug linker complex represented by Formula III [Chemical formula] wherein L4 is [Chemical formula] When it is the case, the symbols 1 and 2 in the formula indicate the connection positions to the remaining groups of L4. Specifically, the 1-position is connected to L3, and the 2-position is connected to the drug molecule

Chemical formula

Chemical formula

[0315] In this specification, the compound represented by formula II

Chemical formula

Chemical formula

Chemical formula

[0316] In this specification, the definition of X is, for example, "X is optionally substituted

Chemical formula

Chemical formula

[0317] In this specification, the definition of X is, for example, "X is optionally substituted [Chemical formula] selected from, and the above substituents are selected from C3-C6 cycloalkyl groups (such as cyclopropyl groups) formed by two C1-C4 alkyl groups (such as methyl groups) together with the carbon atom to which they are simultaneously attached", and X is, for example [Chemical formula] may also be. Other similar definitions of X can be understood by referring to the foregoing content.

[0318] AA 1 the structure of the amino acid residue shown in [Chemical formula] In, when r is 0, those skilled in the art will understand that the structure of the amino acid residue shown in AA 1 is [Chemical formula] will change.

[0319] AA 1 the structure of the amino acid residue shown in [Chemical formula] In, R a and R b together with the carbon atom to which they are commonly attached form a 4- to 10-membered heterocyclic ring, and the above 4- to 10-membered heterocyclic ring is optionally substituted by one or more R 0 , wherein the term "the above 4- to 10-membered heterocyclic ring is optionally substituted by one or more R 0 " means that the above 4- to 10-membered heterocyclic ring may not be substituted, or may be substituted by one or more R 0 , and in the above plurality of R 0 , each R 0The definitions may be the same or different. Other similar definitions can be understood by referring to the foregoing content.

[0320] In this specification, for example, when L3 is selected from Lys, Val-Cit, Ala-Ala-Asn, Ala-Ala-Asp, Gly-Gly-Phe-Gly, Val-Lys-Gly, Val-Ala, Lys-Ala-Asn, "the distal amino group of the above lysine (Lys) is optionally substituted with one, two or three substituents selected from a tert-butoxycarbonyl group, a C1-6 alkyl group (preferably a methyl group), and O" means that the distal amino group of Lys in each option of the above L3 is optionally substituted with one, two or three substituents selected from a tert-butoxycarbonyl group, a C1-6 alkyl group (preferably a methyl group), and O. In the formula, the "distal amino group of Lys" refers to the exposed amino group -NH2 of the lysine residue

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0321] In each part of this specification, the substituents of the compounds of the present disclosure are disclosed according to the type or range of the groups. In particular, the present disclosure includes each independent sub - combination of each member of these group types and ranges. For example, the term "C1 - 6 alkyl group" specifically refers to the independently disclosed methyl group, ethyl group, C3 alkyl group, C4 alkyl group, C5 alkyl group, and C6 alkyl group.

[0322] In this specification, the term "C1 - 6 alkyl group" refers to a straight - chain or branched - chain alkyl group containing 1 to 6 carbon atoms, including, for example, "C1 - 3 alkyl group" or "C1 - 4 alkyl group", methyl group, ethyl group, etc. Specific examples include, but are not limited to, methyl group, ethyl group, n - propyl group, isopropyl group, n - butyl group, isobutyl group, sec - butyl group, tert - butyl group, pentyl group, hexyl group.

[0323] In this specification, the term "C 1~4 alkyl group" refers to a straight - chain or branched - chain alkyl group containing 1 to 4 carbon atoms, including, for example, "C 1~3 alkyl group", methyl group, ethyl group, etc. Specific examples include, but are not limited to, methyl group, ethyl group, n - propyl group, isopropyl group, n - butyl group, isobutyl group, sec - butyl group, tert - butyl group, etc.

[0324] In this specification, the term "C 2~6"Alkenyl group" refers to a linear, branched or cyclic alkenyl group having at least one double bond and 2 to 6 carbon atoms. For example, " 2~4 "Alkenyl group", etc. are included. Examples thereof include, but are not limited to, vinyl group, 1-propenyl group, 2-propenyl group, 1-butenyl group, 2-butenyl group, 1,3-butadienyl group, 1-pentenyl group, 2-pentenyl group, 3-pentenyl group, 1,3-pentadienyl group, 1,4-pentadienyl group, 1-hexenyl group, 2-hexenyl group, 3-hexenyl group, 1,4-hexadienyl group, cyclopentenyl group, 1,3-cyclopentadienyl group, cyclohexenyl group, 1,4-cyclohexadienyl group, etc.

[0325] In this specification, the term " 2~6 "Alkynyl group" refers to a linear or branched alkynyl group having at least one triple bond and 2 to 6 carbon atoms. For example, " 2~4 "Alkynyl group", etc. are included. Examples thereof include, but are not limited to, ethynyl group, propynyl group, 2-butynyl group, 2-pentynyl group, 3-pentynyl group, 4-methyl-2-pentynyl group, 2-hexynyl group, 3-hexynyl group, 5-methyl-2-hexynyl group, etc.

[0326] In this specification, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0327] In this specification, the term "3- to 6-membered cycloalkyl group" or " 3~6 "Cycloalkyl group" refers to a saturated cyclic alkyl group containing 3 to 6 carbon atoms, and includes cyclopropylalkyl group (i.e., cyclopropyl group), cyclobutylalkyl group (i.e., cyclobutyl group), cyclopentylalkyl group (i.e., cyclopentyl group), cyclohexyl group.

[0328] In this specification, the term "3- to 7-membered carbon cycloalkyl group" or " 3~7The term "cycloalkyl group" refers to a saturated cyclic alkyl group containing 3 to 7 carbon atoms, including cyclopropylalkyl group, cyclobutylalkyl group, cyclopentylalkyl group, cyclohexyl group, and cycloheptyl group.

[0329] As used herein, the term "C1-6 alkoxy group" refers to the alkyl group defined above that is linked to the parent molecular moiety through an oxygen atom. Specific examples include, but are not limited to, methoxy group, ethoxy group, propoxy group, isopropoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, tert-butoxy group, pentyloxy group, hexyloxy group, etc.

[0330] As used herein, the term "C 1~4 alkoxy group" refers to the alkyl group defined above that is linked to the parent molecular moiety through an oxygen atom. Specific examples include, but are not limited to, methoxy group, ethoxy group, propoxy group, isopropoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, tert-butoxy group, etc.

[0331] As used herein, the term "4- to 10-membered heterocyclic group" refers to a cyclic group containing 4 to 10 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom. The term "4- to 6-membered heterocyclic group" refers to a cyclic group containing 4 to 6 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom. Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) in the cyclic structure may be substituted with oxo groups. The "4- to 8-membered heterocyclic group" includes, for example, "4- to 8-membered nitrogen-containing heterocyclic group", "4- to 8-membered oxygen-containing heterocyclic group", "4- to 7-membered heterocyclic group", "4- to 7-membered oxygen-containing heterocyclic group", "4- to 7-membered heterocyclic group", "4- to 6-membered heterocyclic group", "5- to 7-membered heterocyclic group", "5- to 6-membered heterocyclic group", "5- to 6-membered nitrogen-containing heterocyclic group", and specific examples include, but are not limited to, oxocyclobutane group, pyrrolidinyl group, tetrahydrofuranyl group, piperidinyl group, piperazinyl group, tetrahydropyranyl group, homopiperazinyl group, etc.

[0332] As used herein, the term "4- to 10-membered heterocycle" refers to a ring containing 4 to 10 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom. The term "5- to 6-membered heterocyclic ring" refers to a ring containing 5 to 6 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom, and includes rings such as pyrrolidine, tetrahydrofuran, piperidine, piperazine, tetrahydropyran, etc., but is not limited thereto.

[0333] As used herein, the term "aryl group" refers to a monocyclic or polycyclic hydrocarbon group having aromaticity, such as a 6- to 10-membered aryl group, a 5- to 8-membered aryl group, etc. Specific examples include, but are not limited to, phenyl group, naphthyl group, anthracenyl group, phenanthryl group, etc. The above-mentioned "6- to 10-membered aryl group" refers to an aryl group containing 6 to 10 ring atoms. The above-mentioned "C6-10 aryl group" refers to an aryl group containing 6 to 10 carbon atoms.

[0334] As used herein, the term "heteroaryl group" refers to an aromatic cyclic group in which at least one ring atom is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure may be substituted with oxo groups. Specific examples include 5- to 10-membered heteroaryl groups such as furanyl group, thienyl group, pyrrolyl group, thiazolyl group, isothiazolyl group, thiadiazolyl group, oxazolyl group, isoxazolyl group, oxadiazolyl group, imidazolyl group, pyrazolyl group, 1, 2,3-triazolyl group, 1,2,4-triazolyl group, 1,2,3-oxadiazolyl group, 1,2,4-oxadiazolyl group, 1,2,5-oxadiazolyl group, 1,3,4-oxadiazolyl group, pyridyl group, 2-pyridonyl group, 4-pyridonyl group, pyrimidinyl group, 1,4-dioxyheterocyclohexenyl group, 2H-1,2-oxazinyl group, 4H-1,2-oxazinyl group, 6H-1,2-oxazinyl group, 4H-1,3-oxazinyl group, 6H-1,3-oxazinyl group, 4H-1,4-oxazinyl group, pyridazinyl group, pyrazinyl group, 1,2,3-triazinyl group, 1,3,5-triazinyl group, 1,2,4,5-tetrazinyl group, azacycloheptatrienyl group, 1,3-diazacycloheptatrienyl group, azacyclooctatetraenyl group, etc., 5- to 6-membered heteroaryl groups, 5- to 10-membered nitrogen-containing heteroaryl groups, 6- to 10-membered oxygen-containing heteroaryl groups, 6- to 8-membered nitrogen-containing heteroaryl groups, 5- to 8-membered oxygen-containing heteroaryl groups, etc., but are not limited thereto.

[0335] As used herein, the bond in the structural formula represented by the wavy line "~~" is intended to represent that the structure represents a cis or trans isomer, or a mixture of cis and trans isomers in any ratio.

[0336] The term "drug-to-antibody ratio" or "DAR" refers to the ratio of drug to antibody in a population (or mixture) or composition or ADC molecule, for example, a small molecule toxin conjugated to the antibody of an ADC. The DAR of an ADC may be in the range of 1 to 16, but higher loads (e.g., 20) are also possible depending on the number of binding sites on the antibody. The term DAR can be used when referring to the number of drugs loaded on a single antibody, or alternatively, when referring to the average or mean DAR of a set of ADCs. It should be understood that the latter is usually referred to as the average DAR. In the process of measuring the DAR value by mass spectrometry, the antibody is reduced to isolated heavy and light chains, DAR1 represents a complex containing a light or heavy chain and coupled to one toxin molecule, DAR2 represents a complex containing a light or heavy chain and coupled to two toxin molecules, and DAR3 represents a complex containing a light or heavy chain and coupled to three toxin molecules.

Advantages of the Invention

[0337] (Beneficial effects of the invention) By extensively studying a large number of target antibody-drug conjugates (ADCs), the present disclosure achieves enrichment of the tumor microenvironment, linker-specific in vivo enzymatic cleavage characteristics and coupling methods with target moieties, and screening and verification of numerous in vivo and in vitro drug efficacy bindings, thereby obtaining novel antibody biological active molecule conjugates. The conjugates obtained by the above method can achieve numerous surprising technical effects as follows.

[0338] The conjugates obtained by the above method have better solubility and excellent chemical stability such that the reversible Michael addition reaction caused by the maleimide binding means in conventional ADCs does not occur. Therefore, a high drug-antibody ratio can be achieved, and in some embodiments, the DAR value of the above conjugate can reach 6 to 8.

[0339] It has extremely high coupling efficiency, and in some embodiments, the above coupling efficiency can reach 90% or more.

[0340] Numerous studies have found linkers that are highly stable in plasma but can be cleaved in the tumor microenvironment (both intracellular and extracellular to tumor cells), and thus can exhibit good anti-tumor effects in tumors with low antigen expression or no antigen expression.

[0341] The conjugate (ADC) obtained by the above method can increase the exposure of the overall ADC molecule in the relatively acidic tumor environment by modulating the physicochemical properties of the linker and the overall ADC molecule. Therefore, the ADC has better tumor tissue targeting, i.e., the ability to enrich in the tumor microenvironment, increases the tumor-to-blood concentration ratio, and reduces the toxicity associated with the mechanism of the ADC molecule (toxicity caused by the binding and endocytosis of the ADC to cell surface antigens in non-tumor tissues, or also called "on-target toxicity"), and thus has a higher therapeutic index.

[0342] The conjugate obtained by the above method has high stability in in vivo circulation, reduces the shedding of drug molecules in non-target tissues, and reduces the "off-target" toxicity caused by the shedding of toxins in non-target tissues.

[0343] Since the biologically active molecule of the above conjugate has higher anti-tumor cell activity, it has an excellent by-stander effect, and the ADC can more effectively kill tumor cells with high antigen expression and tumor cells with low or no antigen expression in tumor tissues.

[0344] The toxin-linker of the present disclosure can utilize the extracellular cleavage ability of its linker in the tumor microenvironment to form an antibody-drug conjugate with an antibody that does not have the ability of cell endocytosis, and such an antibody-drug conjugate still has high anti-tumor activity.

[0345] The toxin-linker of the present disclosure utilizes the extracellular cleavage ability of the linker and its enrichment ability in the tumor microenvironment, and can form an antibody-drug conjugate with an antibody that does not have the ability of cellular endocytosis and an antibody that does not have antigen-binding ability outside tumor cells. Such an antibody-drug conjugate still has high antitumor activity.

[0346] The anti-NaPi2b antibody provided by the present disclosure has a very high degree of humanization or is a fully human antibody, and thus can be safely administered to a human subject without inducing an immunogenic response.

[0347] In summary, the ADC of the present invention has significant clinical value.

Brief Description of the Drawings

[0348]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4

Figure 5

Figure 6

Figure 7

Modes for Carrying Out the Invention

[0349] The present disclosure will be further described below by way of specific embodiments, which do not limit the present disclosure. Those skilled in the art can make various modifications and improvements based on the suggestions of the present disclosure without departing from the basic idea and scope of the present disclosure. When the reagents or equipment used are not specified by the manufacturer, they may all be ordinary commercially available products.

[0350] The abbreviations in the present disclosure have the following meanings:

Table 4-1

Table 4-2

Table 4-3

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

[0351] Hereinafter, the present disclosure will be described with reference to examples that are intended to illustrate (but not limit) the present disclosure.

[0352] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present disclosure are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Current Protocols in Molecular Biology, Third Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art know that the examples are intended to illustrate the present disclosure in an exemplary manner and are not intended to limit the scope of the claims of the present disclosure.

[0353] Preparation method The structures of the compounds described in the following examples were identified by nuclear magnetic resonance ( 1 1H NMR) or mass spectrometry (MS).

[0354] For the measurement of nuclear magnetic resonance ( 1 1H NMR), a Bruker 400 MHz nuclear magnetic resonance spectrometer was used, the measurement solvent was deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterodimethyl sulfoxide (DMSO-d6), and the internal standard substance was tetramethylsilane (TMS).

[0355] The abbreviations of the nuclear magnetic resonance (NMR) spectra used in the examples are shown below.

[0356] s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfoxide. The δ value is represented by ppm value.

[0357] For the measurement instrument for mass spectrometry (MS), an Agilent (ESI) mass spectrometer with model number Agilent 6120B was used.

[0358] I. Synthesis of biologically active molecules and intermediates used in the synthesis process of "drug-linker compounds" A. Synthesis of biologically active molecules Example A1.1: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(1H-pyrazol-4-yl)-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.1)

Chemical Structure

[0359] Step 2: Dissolve (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizino-3,6,10(4H)-trione (2.63 g), (2-amino-4-fluoro-5-methylphenyl)(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanone (3.03 g), and p-toluenesulfonic acid (1.74 g) in dichloromethane (50 mL). Then, remove the solvent, heat the mixture to 120 °C under nitrogen gas protection, and react for 4 hours. Dissolve the mixture in ethyl acetate (300 mL), wash the organic phase with water (100 mL × 2), dry it, remove the organic solvent, and separate the residue by silica gel column chromatography to obtain the target product (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(1H-pyrazol-4-yl)-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.1). ESI-MS (m / z): 447 [M+H] + 。

[0360] Example A1.2: Synthesis of (S)-7-ethyl-7-hydroxy-14-(1H-pyrazol-4-yl)-10,13-dihydro-11H-[1,3]dioxino[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.2) [Chemical formula] Using the same method and reaction conditions as in Example A1.1, replace methyl 2-amino-4-fluoro-5-methylbenzoate with methyl 6-aminobenzene[d][1,3]dioxolane-5-carboxylate to obtain the target product (S)-7-ethyl-7-hydroxy-14-(1H-pyrazol-4-yl)-10,13-dihydro-11H-[1,3]dioxino[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.2). ESI-MS (m / z): 459 [M+H] + .

[0361] Example A1.3: Synthesis of (S)-14-(3-aminophenyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.3) [Chemical formula] Compound (A1.3-A) (1.4 g), compound (A1.3-B) (2.2 g), Pd2(DBA)3 (300 mg), tricyclohexylphosphine (300 mg), and potassium acetate (1.1 g) were sequentially added to a mixed solvent of dioxane (30 mL) and water (5 mL). The mixture was heated at 100 °C under nitrogen gas protection and stirred for 12 hours to react. After cooling, ethyl acetate (200 mL) was added, washed with water (100 mL), dried, and separated by silica gel column chromatography to obtain the target product (S)-14-(3-aminophenyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.3). ESI-MS (m / z): 484 [M+H] + 。

[0362] Example A1.4: Synthesis of (S)-14-(4-aminophenyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.4)

Chemical formula

[0363] Example A1.5: Synthesis of (S)-14-(3-aminopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.5)

Chemical formula

Chemical formula

[0364] Step 2: Synthesis of (S)-14-(3-azidopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

Chemical Structure

[0365] Step 3: Synthesis of (S)-14-(3-aminopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

Chemical Structure

[0366] Example A1.6: Synthesis of (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.6)

Chemical Structure

Chemical formula

[0367] Step 2: Synthesis of (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.6)

Chemical formula

[0368] Example A1.7: Synthesis of (S)-N-methyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.7) [Chemical formula] Step 1: Preparation of compound methylbenzyl (3-(6-nitrobenzo[d][1,3]dioxol-5-yl)-3-oxypropyl) carbamate [Chemical formula] Compound 1-(6-nitrobenzo[d][1,3]dioxin-5-yl)ethane-1-one (A1.7-A, 500 mg), methylamine hydrochloride (1.6 g) and paraformaldehyde (714 mg) were dissolved in ethanol (8 mL), and subjected to a reflux reaction at 100 °C for 16 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the obtained residue was dissolved in dichloromethane (80 mL). The organic phase was extracted with water (50 mL × 3). The obtained aqueous phase was adjusted to pH = 9 with sodium hydrogen carbonate, and then benzyl chloroformate (513 mg, 3.0 mmol) was added, followed by reaction at room temperature for 16 hours. The reaction solution was extracted with ethyl acetate (30 mL × 3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, suction filtered, concentrated to obtain a crude product, and the crude product was separated and purified by C18 (acetonitrile / water containing 0.05% formic acid = 5 - 95%) to obtain the target compound (A1.7-B, 180 mg, yield 23%). LCMS (ESI) [M+H] + : 387.1. 1 H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.35 (m, 5H), 7.32 (m, 1H), 6.17 (s, 2H), 5.13 (s, 2H), 3.71 (m, 2H), 3.03 (m, 3H), 2.99 - 2.86 (m, 2H).

[0369] Step 2: Preparation of compound benzyl (3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxypropyl)(methyl)carbamate (A1.7-C) [Chemical formula] Compound A1.7-B Methylbenzyl (3-(6-nitrobenzo[d][1,3]dioxol-5-yl)-3-oxypropyl) carbamate (180 mg, 0.47 mmol) was dissolved in a mixed solution of saturated aqueous ammonium chloride (8 mL) and ethanol (8 mL). Next, iron powder (130 mg) was added to the reaction solution, and the reaction was stirred at 80 °C for 2 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a solid. The crude product was separated and purified by TLC (petroleum ether:ethyl acetate = 2 / 1) to obtain the target compound A1.7-C (76 mg). LCMS (ESI) [M+H] + : 357.0。

[0370] Step 3: Preparation of Benzyl (S)-(2-(7-ethyl-7-hydroxy-8,11-dioxy-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-14-yl)ethyl)(methyl)carbamate (A1.7-D)

Chemical Structure

[0371] Step 4: Preparation of (S)-7-ethyl-7-hydroxy-14-(2-(methylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.7-E)

Chemical Structure

[0372] Step 5: Preparation of (S)-2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizin-14-yl)ethyl)(methyl)amino)-2-oxyethyl acetate (A1.7-F)

Chemical Structure

[0373] Step 6: Preparation of compound (S) - N - (2 - (7 - ethyl - 7 - hydroxy - 8,11 - dioxo - 7,8,11,13 - tetrahydro - 10H - [1,3] dioxolo[4,5 - g] pyrano[3’,4’:6,7] indolizino[1,2 - b] quinolin - 14 - yl) ethyl) - 2 - hydroxy - N - methylacetamide (A1.7)

Chemical formula

[0374] Example A1.8: Synthesis of (S)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.8) [Chemical formula] Step 1: Preparation of isopropylbenzyl (3-(6-nitrobenzo[d][1,3]dioxin-5-yl)-3-oxypropyl) carbamate (A1.8-B) [Chemical formula] To a dichloromethane solution (10 mL) of compound (A1.8-A) 3-(isopropylamino)-1-(6-nitrobenzo[d][1,3]dioxin-5-yl)propan-1-one (900 mg), triethylamine (1.8 g) and benzyloxycarbonyl chloride (734 mg, 4.3 mmol) were added in sequence, and the reaction was carried out at room temperature for 2 hours. Water (50 mL) was added to the reaction solution for dilution, and the mixture was extracted with dichloromethane (50 mL × 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and the crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 3 / 1) to obtain the target compound (A1.8-B) (600 mg). LCMS (ESI) [M+H] + : 414.9; 1 H NMR (400 MHz, DMSO-d6) δ 7.72 (s, 1H), 7.34 (br s, 6H), 6.31 (s, 2H), 5.08 (s, 2H), 4.14 - 4.10 (m, 1H), 3.48 (d, J = 7.9 Hz, 2H), 3.03 (s, 2H), 1.13 - 1.11 (m, 6H).

[0375] Step 2: Preparation of compound benzyl (3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxypropyl)(isopropyl)carbamate (A1.8-C) [Chemical formula] Compound (A1.8-B) isopropylbenzyl (3-(6-nitrobenzo[d][1,3]dioxin-5-yl)-3-oxypropyl)carbamate (200 mg, 0.48 mmol) was dissolved in a mixed solution of saturated ammonium chloride (3 mL) and ethanol (3 mL). Next, iron powder (135 mg) was added to the reaction solution, and the reaction was stirred at room temperature for 2 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a solid. The crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 3 / 1) to obtain the target compound (A1.8-C) (65 mg). LCMS (ESI) [M+H] + : 395.2.

[0376] Step 3: Preparation of (S)7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.8-D) [Chemical formula] At room temperature, compound (A1.8-C) benzyl (3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxypropyl)(isopropyl)carbamate (65 mg), compound (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (45 mg) and p-toluenesulfonic acid (33 mg, 0.17 mmol) were dissolved in dichloromethane (10 mL) solution. After clarifying the solution and mixing it uniformly, it was concentrated under reduced pressure, evacuated with an oil pump, and the reaction was carried out at 120 °C under vacuum for 2 hours. The reaction solution was cooled to room temperature, water (50 mL) was added, and it was extracted with dichloromethane (30 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate in sequence, filtered, the filtrate was concentrated under reduced pressure, and the crude product was separated and purified by column chromatography (dichloromethane / methanol = 10 / 1) to obtain the target compound (A1.8-D) (40 mg). LCMS (ESI) [M+H] + :478.0; 1 1H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.67 (s, 1H), 7.57 (s, 1H), 7.27 (s, 1H), 6.52 (s, 1H), 6.33 (s, 2H), 5.44 (s, 2H), 5.35 (s, 2H), 3.41 (br s, 2H), 3.23 (br s, 3H), 1.94 - 1.78 (m, 2H), 1.25 (d, J = 6.4 Hz, 6H), 0.88 (t, J = 7.3 Hz, 3H).

[0377] Step 4: Preparation of compound ((S)-2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxino[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)amino)-2-oxoacetic acid ethyl ester (A1.8-E)

Chemical Structure

[0378] Step 5: Preparation of compound (S)-N-(2-(7-ethyl-7-hydroxy-8,1,1-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxino[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N-isopropylacetamide (A1.8)

Chemical formula

[0379] Example A1.9: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.9) [ka] The compound (S)-7-ethyl-7-hydroxy-14-(3-chloropropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (100 mg, 0.213 mmol) was dissolved in 10% sulfuric acid (5 mL) solution and reacted at 110 ° C for 48 hours. Saturated sodium bicarbonate (30 mL) solution was added to the reaction solution, extracted with dichloromethane (10 mL × 5), dried over anhydrous sodium sulfate, suction filtered, and concentrated under reduced pressure to obtain a crude product. Purification by high-performance liquid preparative separation (acetonitrile / water containing 0.05% formic acid) gave (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.9, 1.78 mg). LCMS (ESI) [M+H] + :451.0; 11H NMR (400 MHz, DMSO-d6) δ 7.63 (s, 1H), 7.50 (s, 1H), 7.24 (s, 1H), 6.48 (s, 1H), 6.28 (s, 2H), 5.47 - 5.37 (m, 2H), 5.32 - 5.19 (m, 2H), 3.51 - 3.46 (m, 2H), 3.17 - 3.13 (m, 2H), 1.92 - 1.76 (m, 4H), 0.90 - 0.84 (m, 3H).

[0380] Example A1.10: Synthesis of (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(3-hydroxypropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.10) [Chemical Structure] Step 1: At 0 °C, 1 mol / L boron trichloride (96 mL) and 4-chlorobutyronitrile (9.9 g) were added dropwise to a solution of compound A1.10-A (10 g) in 1,2-dichloroethane (200 mL). The mixture was stirred at 80 °C for 2 hours to allow the reaction to proceed. The reaction solution was cooled to room temperature, 2 mol / L hydrochloric acid (90 mL) was added, and the mixture was refluxed at 80 °C and stirred for 0.5 hour. The reaction solution was cooled to room temperature, diluted with a small amount of water, extracted with dichloromethane (200 mL × 3), the organic phase was dried over anhydrous sodium sulfate, filtered by suction, concentrated to obtain a crude product, and the crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 10 / 1) to obtain the target compound A1.10-B (4 g). LCMS (ESI) [M+H] + : 230.0.

[0381] Step 2: To (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (35 mg) and p-toluenesulfonic acid (41.4 mg) monohydrate were added to Compound A1.10-B (50 mg), and they were dissolved in a dichloromethane (30 mL) solution. After clarifying the solution and mixing it uniformly, it was concentrated under reduced pressure, evacuated with an oil pump, and the reaction was carried out under vacuum at 120 °C for 3 hours. It was shown by LCMS that the reaction was complete. The reaction solution was cooled to room temperature, water (20 mL) was added, and it was extracted with dichloromethane (20 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate in sequence, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (dichloromethane / methanol = 20 / 1) to obtain the target compound A1.10-C (80 mg), which was a white solid. LCMS (ESI) [M+H] + : 457.0.

[0382] Step 3: Compound A1.10-C (75 mg) was dissolved in hexamethylphosphoric triamide, pure water (0.8 mL) was added, and the reaction solution was stirred at 100 °C for 72 hours. The completion of the reaction was detected by LCMS. It was purified by preparative chromatography (0.01% TFA in water, MeCN) to obtain the target compound (10 mg). LCMS (ESI) [M+H] + : 439.2; 1 H NMR (400 MHz, DMSO-d6) δ 8.22 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 6.50 (s, 1H), 5.43 (s, 2H), 5.30 (s, 2H), 4.67 (t, J = 4.9 Hz, 1H), 3.55 - 3.47 (m, 2H), 3.28 - 3.20 (m, 2H), 2.51 (s, 3H), 1.93 - 1.81 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H).

[0383] Example A1.11: Synthesis of (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(3-aminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.11)

Chemical formula

[0384] Step 2: Compound A1.11-A (93 mg) was dissolved in tetrahydrofuran (5 mL), triphenylphosphine (78 mg) was added, and the reaction was carried out at room temperature for 4 hours. Then hydrochloric acid (4 M, 1 mL) was added to the reaction mixture, the temperature of the reaction mixture was raised to 55 °C, and the reaction was carried out for 16 hours. The completion of the reaction was monitored by LCMS. The reaction mixture was directly concentrated, and the crude product was produced by a reverse-phase column (mobile phase A was 0.05% aqueous formic acid solution, B was acetonitrile) to obtain the target product A1.11 (28 mg, yield: 36%), which was a white solid. LCMS (ESI) [M+H] + : 438.4; 11H NMR (400 MHz, CD3OD) δ 8.52 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 10.7 Hz, 1H), 7.63 (s, 1H), 5.49 (ABq, J = 78.9, 16.3 Hz, 2H), 5.31 (br s, 2H), 3.35 - 3.32 (m, 2H), 3.22 - 3.10 (m, 2H), 2.55 (s, 3H), 2.13 - 2.11 (m, 2H), 1.96 - 1.94 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H).

[0385] Example A1.12: Synthesis of (S,E)-14-(3-amino-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.12)

Chemical Structure

[0386] Step 2: A solution of compound (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propen-1-yl)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-7-yl acetate (A1.12-C, 50 mg, 0.085 mmol) in methanol (15 mL) was added with sodium methoxide (9.2 mg, 0.17 mmol), and stirred at 50 °C for 2 hours for reaction. It was shown by LCMS that the reaction was complete. The reaction solution was concentrated to obtain the crude product of the target compound tert-butyl (S,E)-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)allyl)carbamate (A1.12-D, 50 mg), which was a brown solid. LCMS (ESI) [M+H] + = 548;

[0387] Step 3: Trifluoroacetic acid (1 mL) was added to a dichloromethane solution (2 mL) of compound tert-butyl (S,E)-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)allyl)carbamate (A1.12-D, 50 mg, 0.091 mmol), and stirred at room temperature for 30 minutes for reaction. It was shown by LCMS that the reaction was complete. The reaction solution was concentrated, and the crude product was purified by high performance liquid fractionation (acetonitrile / aqueous solution of 0.05% formic acid) to obtain the target compound (S,E)-14-(3-amino-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[… LCMS (ESI) [M+H] + = 448.3; 1 1H NMR (400 MHz, DMSO-d6) δ 8.20 (s, 2H), 7.72 (s, 1H), 7.54 (s, 1H), 7.40 (d, J = 16.5 Hz, 1H), 7.27 (s, 1H), 6.52 - 6.46 (m, 2H), 6.31 (s, 2H), 5.42 (s, 2H), 5.27 (s, 2H), 3.86 (br s, 2H), 1.90 - 1.83 (m, 2H), 0.88 (t, J = 7.1 Hz, 3H).

[0388] Example A1.13: Synthesis of (S,E)-14-(3-hydroxy-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.13) [Chemical formula] Step 1: Compound A1.9 (200 mg, 0.444 mmol) was dissolved in dimethyl sulfoxide (2 mL), IBX (311 mg, 1.11 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Then, IBX (186 mg, 0.666 mmol) was added to the reaction solution, and further pyrrolidine (6.3 mg, 0.089 mmol) and acetonitrile (3 mL) were added to the reaction solution. The reaction solution was stirred at room temperature overnight. Completion of the reaction was detected by LCMS. The mixture was extracted with ethyl acetate (20 mL × 3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, suction filtered, rotary evaporated to obtain a crude product. The crude product was purified by silica gel column (DCM:MeOH = 50:1~10:1) to obtain the target compound (S,E)-14-(3-oxo-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.13-A, 100 mg, purity 50.0%, yield 25.0%), which was a yellow solid. LCMS (ESI) [M+H] + = 447.1。

[0389] Step 2: Compound A1.13-A (40 mg, 0.09 mmol) was dissolved in tetrahydrofuran (1 mL), then sodium cyanoborohydride (28 mg, 0.448 mmol) was added to the reaction solution, and the mixture was stirred at room temperature overnight. Completion of the reaction was indicated by LCMS. The reaction solution was concentrated to obtain a crude product. The crude product was purified by prep-HPLC (HCl in water / MeCN) to obtain the target compound (3.56 mg, purity 93.6%, yield 9.0%), which was a pale yellow solid. LCMS (ESI) [M+H] + = 449.2; 1H NMR (400 MHz, DMSO-d6) δ 7.61 (s, 1H), 7.50 (s, 1H), 7.24 (s, 1H), 7.20 (d, J = 16.4 Hz, 1H), 6.69 - 6.59 (m, 1H), 6.49 (s, 1H), 6.30 (s, 2H), 5.42 (s, 2H), 5.26 (s, 2H), 5.16 (br s, 1H), 4.34 (br s, 2H), 1.93 - 1.81 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0390] Example A1.14: Synthesis of (S,E)-4-Ethyl-8-fluoro-4-hydroxy-11-(3-hydroxy-1-propen-1-yl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.14)

Chemical Structure

[0391] Step 2: In a 50 mL three-necked flask, compound (S,E)-3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)acrylaldehyde (A1.14-A, 40 mg, 0.228 mmol) and anhydrous THF (5 mL) were added. The temperature was lowered to -78 °C under N2 protection, and a THF solution of lithium tri-sec-butylborohydride (0.11 mL, 1 N) was slowly added dropwise while maintaining the temperature at -78 °C, and stirring was continued for 1 hour. After completion of the reaction, saturated aqueous ammonium chloride solution was added to quench the reaction, the temperature was raised to room temperature, diluted with saturated brine (50 mL), extracted with ethyl acetate (20 mL × 3), the organic phases were combined, washed with water, dried, filtered, and the solvent was evaporated to dryness under reduced pressure to obtain (S,E)-4-ethyl-8-fluoro-4-hydroxy-11-(3-hydroxy-1-propen-1-yl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.14, 28 mg), which was a pale white solid. LCMS (ESI) [M+H] + = 437.1.

[0392] Example A1.15: Synthesis of (S)-7-ethyl-7-hydroxy-14-(2-(n-propylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.15a) and (S)-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N-n-propylacetamide (A1.15b)

Chemical Structure

[0393] Step 2: Compound A1.15-B (1.5 g, 3.63 mmol) was dissolved in a mixed solution of saturated ammonium chloride (20 mL) and ethanol (20 mL). Next, iron powder (1.02 g, 18.1 mmol) was added to the reaction solution, and the reaction was stirred at 80 °C for 1 hour. Completion of the reaction was indicated by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to a solid. The crude product was separated and purified by C18 column reverse phase (acetonitrile / aqueous solution of 0.05% FA: 5% - 55%) to obtain the target compound A1.15-C (600 mg, yield 43.0%), which was a yellow solid. LCMS (ESI) [M+H] + = 385.2, t R = 1.329 min.

[0394] Step 3: At room temperature, compound A1.15-C (350 mg, 0.91 mmol), compound (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (A1.15-D, 218 mg, 0.83 mmol) and p-toluenesulfonic acid (170 mg, 0.87 mmol) were dissolved in dichloromethane (5 mL) solution. After clarifying the solution and mixing it uniformly, it was concentrated under reduced pressure, evacuated with an oil pump, and the reaction was carried out at 120 °C in vacuo for 2 hours. It was shown by LCMS that the reaction was complete. The reaction solution was cooled to room temperature, water (50 mL) was added, and it was extracted with dichloromethane (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate in turn, filtered, and the filtrate was concentrated under reduced pressure to obtain compound A1.15-E (390 mg, yield 76%), which was a brown solid. LCMS (ESI) [M+H] + = 612.0。

[0395] Step 4: At room temperature, compound A1.15-E (390 mg, 0.638 mmol) was dissolved in dichloromethane (5 mL) solution. Trimethyl iodoxysilane (510 mg, 2.55 mmol) was added dropwise under nitrogen gas protection at 0 °C, and the reaction system was stirred at room temperature for 2 hours. Ethyl ether (2 mL) and concentrated hydrochloric acid (4 mL) were added to the reaction solution, and it was stirred for 30 minutes. The reaction system was adjusted to pH = 9 with saturated sodium bicarbonate, and it was extracted with dichloromethane (50 mL × 5). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. It was purified by preparative high-performance liquid chromatography (acetonitrile / water containing 0.05% formic acid) to obtain compound A1.15a (200 mg, yield 66.1%), which was an off-white solid. LCMS (ESI) [M+H] + = 478.2; 1H NMR (400 MHz, DMSO-d6) δ 8.30 (s, 1H, HCO2H), 7.67 (s, 1H), 7.51 (s, 1H), 7.23 (s, 1H), 6.62 - 6.41 (m, 1H), 6.29 (s, 2H), 5.42 (s, 2H), 5.28 (s, 2H), 3.27 - 3.25 (m, 2H), 2.91 - 2.79 (m, 2H), 2.58 - 2.56 (m, 2H), 1.91 - 1.79 (m, 2H), 1.44 - 1.42 (m, 2H), 0.90 - 0.84 (m, 6H).

[0396] Step 5: At room temperature, compound A1.15a (120 mg, 0.251 mmol) was dissolved in dichloromethane (5 mL), and at 0 °C, compound acetoxyacetyl chloride (167 mg, 1.25 mmol) and triethylamine (133 mg, 1.25 mmol) were added dropwise. The reaction was stirred at 0 °C for 30 minutes to react. It was detected by LCMS that the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain the crude product, compound A1.15-F (120 mg, yield 82.8%), which was a yellow solid. LCMS (ESI) [M+H] + = 578.3.

[0397] Step 6: At room temperature, compound A1.15-F (120 mg, 0.207 mmol) was dissolved in ethanol (4 mL), and then concentrated hydrochloric acid (2 mL) was added to the reaction solution. The reaction was stirred at 70 °C for 1 hour to react. It was detected by LCMS that the reaction was complete. Water (20 mL) was added to the reaction solution, and it was extracted with dichloromethane (30 mL × �). The organic phases were combined, dried over anhydrous sodium sulfate in sequence, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. It was purified by preparative high-performance liquid chromatography (acetonitrile / water containing 0.05% formic acid) to obtain compound A1.15b (20 mg, yield 18.1%), which was an off-white solid. LCMS (ESI) [M+H] + = 536.2; 1H NMR (400 MHz, DMSO-d6) δ 7.93 (s, 0.7H), 7.69 (s, 0.3H), 7.51 (s, 1H), 7.24 (s, 1H), 6.49 (s, 1H), 6.30 (s, 2H), 5.42 (s, 2H), 5.35 (s, 1.5H), 5.29 (s, 0.5H), 4.65 - 4.62 (m, 1H), 4.13 - 3.97 (m, 2H), 3.51 - 3.49 (m, 2H), 3.33 - 3.30 (m, 2H), 3.24 - 3.20 (m, 2H), 1.86 - 1.84 (m, 2H), 1.60 - 1.52 (m, 2H), 0.89 - 0.86 (m, 6H).

[0398] Example A1.16: Synthesis of (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(3-isopropylaminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.16) [Chemical formula] To a DMF solution (10 mL) of A1.10-C (200 mg, 0.439 mmol) and isopropylamine (50 mg, 0.877 mmol), diisopropylethylamine (170 mg, 1.32 mmol) and sodium iodide (99 mg, 0.659 mmol) were added. The reaction was sealed at 50 °C and reacted for 2 hours. Ethyl acetate was added to the reaction solution, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, suction filtered, concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by pre-TLC (dichloromethane:methanol = 10:1), and then a yellow solid was obtained. The compound was further purified by high-speed liquid fractionation (acetonitrile / water containing 0.05% formic acid) to obtain the target compound (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(3-isopropylaminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.16, 2.03 mg). LCMS (ESI) [M+H] + = 480.2; 1 1H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 8.0 Hz, 1H), 8.27 (s, 1H, HCO2H) 7.92 (d, J = 11.0 Hz, 1H), 7.33 (s, 1H), 6.53 (s, 1H), 5.45 (s, 2H), 5.30 (s, 2H), 3.30 - 3.22 (m, 3H), 3.18 - 3.10 (m, 2H), 2.54 (s, 3H), 2.06 - 1.94 (m, 2H), 1.93 - 1.80 (m, 2H), 1.26 - 1.20 (m, 6H), 0.88 (t, J = 7.3 Hz, 3H).

[0399] Example A1.17: Synthesis of (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(3-cyclopropylaminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.17)

Chemical Structure

[0400] Example A1.18: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-(4-aminophenyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.18)

Chem.

[0401] Step 2: Compound A1.18B (620 mg, 2.28 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (600 mg, 2.28 mmol), and p-toluenesulfonic acid (560 mg, 2.96 mmol) were added to a reaction flask, dissolved uniformly with dichloromethane, the dichloromethane was rotary dried, evacuated, heated to 120 °C under vacuum, maintained at this temperature for 6 hours, and after completion of the reaction was detected by LCMS, methanol (10 mL) was added to the system, followed by further addition of water (80 mL), a large amount of precipitate was deposited, and after the precipitate was filtered and dried, the target compound (A1.18C, 800 mg, yield 80.0%) was obtained as a yellow solid. LCMS (ESI) [M+H] + = 502.2; 1 H NMR (400 MHz, DMSO) δ 8.52 (d, J = 8.2 Hz, 2H), 8.13 - 7.89 (m, 3H), 7.64 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 13.4 Hz, 1H), 6.54 (s, 1H), 5.41 (s, 2H), 5.06 (m, 2H), 2.40 (s, 3H), 1.94 - 1.84 (m, 2H), 0.87 (m, 3H).

[0402] Step 3: Raney nickel (609 mg, 10.5 mmol) was added to a mixed solution of compound A1.18C (1.0 g, 2.1 mmol) in methanol (25 mL) and tetrahydrofuran (50 mL), and the reaction solution was stirred at room temperature in a hydrogen gas atmosphere for 5 hours. Complete reaction was detected by LCMS, the reaction solution was filtered, the filtrate was rotary dried, and the target compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(4-aminophenyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.18, 650 mg, yield 67%) was obtained as a yellow solid. LCMS (ESI) [M+H] + = 472.3; 1 1H NMR (400 MHz, DMSO) δ 7.93 - 7.81 (m, 2H), 7.40 - 7.25 (m, 3H), 6.81 (d, J = 8.2 Hz, 2H), 6.51 (s, 1H), 5.62 (s, 2H), 5.41 (s, 2H), 5.10 (s, 2H), 2.41 (s, 3H), 1.93 - 1.80 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H).

[0403] B. Synthesis of Intermediate Containing Biologically Active Molecular Fragment Example B1.1: Synthesis of (S)-2-Amino-N-((4-(4-Ethyl-8-Fluoro-4-Hydroxy-9-Methyl-3,14-Dioxo-3,4,12,14-Tetrahydro-1H-Pyrano[3’,4’:6,7]Indolizino[1,2-b]Quinolin-11-Yl)Butoxy)Methyl)Acetamide (B1.1) [Chemical Structure] Compound (B1.1-A) (368 mg), compound (B1.1-B) (452 mg) and pyridinium p-toluenesulfonate (PPTS) (25 mg) were refluxed in dichloromethane (20 mL) for 20 h, then washed with aqueous sodium bicarbonate solution and aqueous hydrochloric acid solution respectively, and the organic solvent was removed under reduced pressure. The residue was dissolved in DMF (5 mL), piperidine (1 mL) was added, the compound was stirred for 20 min, most of the low-boiling components were removed under reduced pressure, and the residue was separated by preparative HPLC to obtain the target product (S)-2-Amino-N-((4-(4-Ethyl-8-Fluoro-4-Hydroxy-9-Methyl-3,14-Dioxo-3,4,12,14-Tetrahydro-1H-Pyrano[3’,4’:6,7]Indolizino[1,2-b]Quinolin-11-Yl)Butoxy)Methyl)Acetamide (B1.1). ESI-MS (m / z): 539 [M+H] + .

[0404] Example B1.2: Synthesis of (S)-2-((2-aminoacetamido)methoxy)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)acetamide (B1.2) [Chemical formula] Step 1: Compound B1.2-A (274 mg), diisopropylethylamine (334 mg) and HBTU (369 mg) were successively added to N,N-dimethylformamide (10 mL), and then compound B1.2-B (300 mg) was added. The reaction mixture was stirred at room temperature for 2 hours. Ethyl acetate (50 mL) was added to the reaction mixture, and it was washed with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, suction filtered, and the filtrate was concentrated under reduced pressure to obtain a solid. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound B1.2-C (300 mg). LCMS (ESI) [M+H] + : 830.2; 1 H NMR (400 MHz, DMSO-d6) δ 8.74 (s, 1H), 7.91 - 7.84 (m, 2H), 7.80 - 7.67 (m, 2H), 7.65 - 7.54 (m, 2H), 7.53 - 7.46 (m, 1H), 7.45 - 7.37 (m, 2H), 7.36 - 7.27 (m, 2H), 7.25 - 7.23 (m, 1H), 6.57 - 6.44 (m, 1H), 6.29 (s, 2H), 5.50 - 5.19 (m, 4H), 4.71 - 4.57 (m, 2H), 4.32 - 3.97 (m, 7H), 3.80 - 3.53 (m, 4H), 3.20 - 3.14 (m, 2H), 1.92 - 1.80 (m, 2H), 1.28 - 1.22 (m, 3H), 0.87 - 0.82 (m, 3H).

[0405] Step 2: Piperidine (1 mL) was added to a solution of compound B1.2-C (300 mg) in N,N-dimethylformamide (4 mL), and the reaction mixture was stirred at room temperature for 20 minutes. After removing the low-boiling components from the reaction mixture, the target product was obtained and used directly in the synthesis of the next step. LCMS (ESI) [M+H] + = 608.0.

[0406] Example B1.3: Synthesis of (S)-2-amino-N-((2-(((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)amino)-2-oxoethoxy)methyl)acetamide (B1.3)

Chemical formula

[0407] Example B1.4: Synthesis of (S)-2-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (B1.4)

Chemical formula

[0408] Example B1.5: Synthesis of (S)-2-amino-N-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)acetamide (B1.5)

Chemical Structure

[0409] Example B1.6: Synthesis of (S)-2-amino-N-((4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)-1H-pyrazol-1-yl)methyl)acetamide (B1.6)

Chemical Structure

[0410] Example B1.7: Synthesis of (S)-2-amino-N-((2-(((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)amino)-2-oxoethoxy)methyl)acetamide (B1.7)

Chem.

[0411] Example B1.8: Synthesis of (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.8)

Chem.

[0412] Example B1.9: Synthesis of (S)-2-amino-N-(4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.9)

Chemical formula

[0413] Compound A1.4 (60 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (3 mL), and (tert-butoxycarbonyl)glycine (26 mg, 0.15 mmol), HATU (56 mg, 0.15 mmol), and N,N-diisopropylethylamine (48 mg, 0.37 mmol) were added in sequence. The mixture was stirred at room temperature for 1 hour, and the completion of the reaction was detected by TLC. Next, TFA (1.0 mL) was directly added to the above reaction solution. Stirring was continued at room temperature for 1 hour, and the completion of the reaction was detected by LCMS. The reaction solution was concentrated to remove trifluoroacetic acid, and a crude product was obtained. The crude product was purified by preparative chromatography (0.01% aqueous trifluoroacetic acid, acetonitrile) to obtain the target product (S)-2-amino-N-(4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]-dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.9) (26.3 mg, yield 38%), which was a yellow solid. ESI-MS (m / z): 541 [M+H] + ; 1 H NMR (400 MHz, DMSO) δ 10.71 (s, 1H), 8.16 (s, 2H), 7.86 (d, J = 8.6 Hz, 2H), 7.67-7.58 (m, 3H), 7.29 (s, 1H), 7.04 (s, 1H), 6.50 (s, 1H), 6.28 (s, 2H), 5.40 (s, 2H), 5.05 (s, 2H), 3.87 (s, 2H), 1.93-1.81 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0414] Example B1.10: Synthesis of (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]-dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)propyl)acetamide (B1.10)

Chemical Structure

[0415] Step 2: Trifluoroacetic acid (2 mL) was added to a solution of compound B1.10-A (100 mg) in dichloromethane (4 mL), and the mixture was stirred at room temperature for 1 hour for reaction. The reaction solution was concentrated, N,N-dimethylformamide solution (3 mL) was added to the crude product, and the mixture was further purified by a C18 column (acetonitrile / aqueous solution of 0.05% formic acid: 5% - 60%) to obtain the target compound (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]-dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)propyl)acetamide (B1.10, 80 mg). LCMS (ESI) [M+H] + : 507; 11H NMR (400 MHz, DMSO-d6) δ 8.26 (s, 1H), 7.64 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.51 (s, 1H), 6.29 (s, 2H), 5.42 (s, 2H), 5.23 (s, 2H), 3.31 (s, 2H), 3.30 - 3.27 (m, 2H), 3.13 - 3.07 (m, 2H), 2.04 - 1.76 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0416] Example B1.11: Synthesis of (S)-2-((2-aminoacetamido)methoxy)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)acetamide (B1.11)

Chemical Structure

[0417] Step 2: Preparation of (S)-2-((2-aminoacetamido)methoxy)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)acetamide To a solution of compound B1.11B (220 mg, 0.261 mmol) in N,N-dimethylformamide (4 mL) was added piperidine (1 mL), and the reaction was stirred at room temperature for 20 minutes. It was detected by LCMS that the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain the target compound (220 mg), which was a brown solid. The above product was used directly in the next step of synthesis without purification. LCMS (ESI) [M+H] + : 622.1

[0418] Example B1.12: Synthesis of (S)-2-amino-N-((3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)propoxy)methyl)acetamide (B1.12)

Chemical Structure

[0419] Step 2: B1.12-A (240 mg) was dissolved in DMF (5 mL), piperidine (1 mL) was added, the compound was stirred for 20 minutes, and the low-boiling components were removed under reduced pressure. The residue was used directly in the next step of the synthesis. ESI-MS (m / z): 525.2 [M+H] + 。 A small amount of the crude product was purified by reverse-phase chromatography (acetonitrile / aqueous solution of 0.05% FA: 5% - 50%), and then the target compound was obtained. ESI-MS (m / z): 525.1 [M+H] + ; 1 H NMR (400 MHz, DMSO) δ 9.13 (t, J = 6.6 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H), 8.02 (brs, 2H), 7.89 (d, J = 10.8 Hz, 1H), 7.32 (s, 1H), 6.54 (s, 1H), 5.44 (s, 2H), 5.28 (s, 2H), 4.66 (d, J = 6.5 Hz, 2H), 3.64 (s, 2H), 3.53 (t, J = 6.1 Hz, 2H), 3.25 - 3.18 (m, 2H), 2.52 (s, 3H), 1.98 - 1.84 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H).

[0420] Example B1.13: Synthesis of (S)-2-Amino-N-(3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)propyl)acetamide (B1.13)

Chemical formula

[0421] Step 2: Trifluoroacetic acid (2 mL) was added to a solution of compound B1.13-A (100 mg) in dichloromethane (4 mL), and the mixture was stirred at room temperature for 1 hour to react. The reaction solution was concentrated, N,N-dimethylformamide solution (3 mL) was added to the crude product, and the mixture was further purified by a C18 column (acetonitrile / aqueous solution of 0.05% formic acid: 5% - 60%) to obtain the target compound (S)-2-amino-N-(3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)propyl)acetamide (B1.13, 88 mg). LCMS (ESI) [M+H] + : 495.

[0422] Example B1.14: Synthesis of (S)-2-amino-N-((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)propoxy)methyl)acetamide (B1.14)

Chemical Structure

[0423] Step 2: B1.14-A (240 mg) was dissolved in DMF (5 mL), piperidine (1 mL) was added, and the compound was stirred for 20 minutes. The low-boiling components were removed under reduced pressure, and the residue was directly used for the next-step synthesis. A small amount of the crude product was purified by reverse-phase chromatography (acetonitrile / aqueous solution of 0.05% FA: 5% - 50%) to obtain the target compound. ESI-MS (m / z): 537.4 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 9.13 (t, 1H), 8.04 (br, 2H), 7.58 (s, 1H), 7.51 (s, 1H), 7.25 (s, 1H), 6.29 (s, 2H), 5.43 (S, 2H), 5.21 (s, 2H), 4.65 (d, 2H), 3.63 (m, 2H), 3.53 (m, 2H), 3.11 (m, 2H), 1.87 (m, 4H), 0.88 (t, 3H).

[0424] Example B1.15: (S,E)-2-Amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)allyl)acetamide

Chemical formula

[0425] Step 2: At room temperature, compound B1.15-A (130 mg, 0.215 mmol) was dissolved in a 1,4-dioxane solution of hydrochloric acid (5 mL), and the mixture was reacted at room temperature for 30 minutes. It was shown by LCMS that the reaction was complete. The reaction solution was purified by reverse phase to obtain the target compound (60 mg, yield: 52%) as a brown solid. LCMS (ESI) [M+H] + =505.2; 1 H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.15 (s, 2H), 7.70 (s, 1H), 7.54 (s, 1H), 7.26 (s, 1H), 7.21 (d, J = 16.2 Hz, 1H), 6.51 (d, J = 16.2 Hz, 1H), 6.31 (s, 2H), 5.42 (s, 2H), 5.28 (s, 2H), 4.19 (s, 2H), 1.86 - 1.82 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0426] Example B1.16: (S,E)-2-Amino-N-(((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)allyl)oxy)methyl)acetamide [Chemical Structure] Step 1: Under nitrogen gas protection, raw material A1.13 (150 mg, 0.335 mmol) was dissolved in toluene (3 mL), (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)methyl acetate (B1.1-A, 308 mg, 0.84 mmol) was added, and then zinc acetate (123 mg, 0.67 mmol) was added to the reaction solution. The reaction solution was stirred at 100 °C overnight. Completion of the reaction was detected by LCMS. The reaction solution was directly purified by reverse-phase chromatography (acetonitrile / aqueous solution of 0.05% formic acid: 5% - 50%) to obtain the target compound (9H-fluoren-9-yl)methyl (S,E)-(2-(((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)allyl)oxy)methyl)amino)-2-oxoethyl)carbamate (B1.16-A, 80 mg, yield 31%), which was a white solid. LCMS (ESI) [M+H] + = 757.4.

[0427] Step 2: Compound B1.16-A (80 mg, 0.11 mmol) was dissolved in N,N-dimethylformamide (2 mL), and then piperidine (0.05 mL) was added to the reaction solution, followed by stirring at room temperature for 1 hour. Completion of the reaction was indicated by LCMS. The reaction solution was concentrated to obtain a crude product, and the crude product was separated and purified by C18 column reverse-phase (acetonitrile / aqueous solution of 0.05% ammonia: 5% - 50%) to obtain the target compound (35 mg, yield 62%), which was a brown solid. LCMS (ESI) [M+H]+ = 535.2, tR = 1.070 min.

[0428] Example B1.17: (S,E)-2-Amino-N-(((3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-oxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)allyl)oxy)methyl)acetamide

Chemical Structure

[0429] Step 2: Compound 4 (25 mg, 0.034 mmol) was dissolved in N,N-dimethylformamide (2 mL), then diethylamine (0.2 mL) was added. After the addition was complete, the reaction mixture was stirred at room temperature for 1 hour. After the completion of the reaction was detected by LCMS, the reaction mixture was directly concentrated under reduced pressure to evaporate the solvent to dryness, and the target compound (S,E)-2-amino-N-(((3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-oxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)allyl)oxy)methyl)acetamide, a yellow viscous product, was obtained. LCMS (ESI) [M+H]+ = 523.2; LCMS (ESI) [M+H] + = 523.1, t R = 0.446 min, 1.311 min. 11H NMR (400 MHz, DMSO) δ 9.26 (t, J = 6.5 Hz, 1H), 8.23 (d, J = 8.2 Hz, 1H), 8.06 (s, 2H), 7.91 (m, 1H), 7.40 (d, J = 16.3 Hz, 1H), 7.34 (s, 1H), 6.70 - 6.60 (m, 1H), 6.54 (s, 1H), 5.44 (s, 2H), 5.35 (s, 2H), 4.80 (d, J = 6.6 Hz, 2H), 4.38 (d, J = 3.9 Hz, 2H), 3.68 (d, 2H), 2.52 (s, 3H), 1.90 - 1.84 (m, 2H), 0.88 (m, 3H).

[0430] C. Synthesis of Intermediate Containing Coupling Molecular Fragment Example C1.1: N 6 -(tert-Butoxycarbonyl)-N 2 -((1-(2-(Methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoyl)-L-valyl)-L-lysine (Compound C1.1)

Chemical Structure

[0431] Step 2: tert-butyl 29-amino-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate Palladium carbon catalyst (Pd / C, 10%, 100 mg) was added to a solution of tert-butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate (1.16 g) in ethyl acetate (20 mL). The above solution was stirred in a hydrogen gas atmosphere for 5 hours, then the palladium carbon was filtered off, the solvent was removed under reduced pressure, and the target product was obtained. ESI-MS (m / z): 528 [M + H] + 。

[0432] Step 3: 1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoic acid tert-Butyl 29-amino-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate (527 mg) and 2-methylthio-pyrimidine-5-carboxylic acid (170 mg) were added to dry DMF (10 mL), then cooled in an ice bath, and DIPEA (0.2 mL) and HBTU (420 mg) were sequentially added to the above solution. The above mixture was stirred at room temperature for 20 hours. Then it was diluted with ethyl acetate (100 mL), washed with water (100 mL × 4), the organic phase was dried over anhydrous sodium sulfate, and then the organic solvent was removed under reduced pressure. The residue was dissolved in DCM (10 mL), then TFA (10 mL) was added, the mixture was stirred at room temperature for 1 hour, then the low-boiling components were removed under reduced pressure, and the residue was separated by preparative HPLC to obtain 1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoic acid. ESI-MS (m / z): 624 [M + H] + 。

[0433] Step 4: N 6 -(tert-Butoxycarbonyl)-N 2 -((1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoyl)-L-valyl)-L-lysine 1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoic acid (623 mg) was dissolved in DMF (10 mL), and DIPEA (300 μL) and HBTU (420 mg) were added at 0 °C. The mixture was stirred for 30 minutes, then valyl-(Boc)lysine dipeptide compound (345 mg) was added. The reaction was stirred for 20 hours, then ethyl acetate (100 mL) was added, washed with dilute hydrochloric acid (20 mL × 3), the organic phase was dried, and then the organic solvent was removed under reduced pressure. The crude product was separated by preparative HPLC to obtain the target compound N 6 -(tert-Butoxycarbonyl)-N 2-((1-(2-(Methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoyl)-L-valyl)-L-lysine (Compound C1.1) was obtained. ESI-MS (m / z): 951 [M + H] + 。

[0434] Example C1.2: N 6 -(tert-Butoxycarbonyl)-N 2 -((29-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoyl)-L-valyl)-L-lysine (Compound C1.2)

Chemical formula

[0435] Step 2: 29-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoic acid tert-Butyl 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate (1 g) was dissolved in dichloromethane (10 mL), and then TFA (5 mL) was added. The mixture was allowed to stand at room temperature for 1 hour, and then concentrated under reduced pressure to remove low-boiling components, obtaining the target product 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoic acid. MS (m / z): 648 [M + H] + 。

[0436] Step 3: N 6 -(tert-Butoxycarbonyl)-N 2 -((29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoyl)-L-valyl)-L-lysine 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoic acid (647 mg) was dissolved in DMF (10 mL), and DIPEA (300 μL) and HBTU (420 mg) were added at 0 °C. The mixture was stirred for 30 minutes, and then the valyl-lysine dipeptide compound (345 mg) was added. The reaction was stirred for 20 hours, then ethyl acetate (100 mL) was added, and the mixture was washed with dilute hydrochloric acid (20 mL × 3). The organic phase was dried and then concentrated under reduced pressure to remove the organic solvent. The crude product was separated by preparative HPLC to obtain the target compound N 6 -(tert-Butoxycarbonyl)-N 2-((29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoyl)-L-valyl)-L-lysine (compound C1.2) was obtained. ESI-MS (m / z): 975 [M + H] + .

[0437] Example C1.3: (1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoyl)-glycyl-glycyl-L-phenylalanine (compound C1.3) [ka] Using the same method and reaction conditions as in Step 4 of Example C1.1, and using the corresponding starting materials, the desired product (1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoyl)-glycyl-glycyl-L-phenylalanine (compound C1.3) was obtained. ESI-MS (m / z): 885 [M+H] + .

[0438] Example C1.4: (36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-30-aza-hexatriacontan-(35-yne)-acyl)glycylglycylphenylalanine (compound C1.4) [ka] Using the same method and reaction conditions as in Step 4 of Example C1.1, and using the raw materials shown in the reaction formula, the target product (36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-30-aza-hexacontan-(35-yn)-acyl)glycylglycylphenylalanine (Compound C1.4) was obtained. ESI-MS (m / z): 951 [M+H] + 。

[0439] Example C1.5: N 6 -(tert-Butoxycarbonyl)-N 2 -((36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-30-aza-hexacontan-(35-yn)-acyl)-L-valyl)-L-lysine (C1.5)

Chemical formula

[0440] Example C1.6: N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(methylthio)pyrimidin-5-yl)-hex-5-ynoyl)-L-valyl)-L-lysine (Compound C1.6)

Chemical formula

[0441] Example C1.7:N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)-hex-5-ynoyl)-L-valyl)-L-lysine (Compound C1.7)

Chemical Structure

[0442] Example C1.8:N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(Methylthio)pyrimidine-5-formamido)hexanoyl)-L-valyl)-L-lysine (Compound C1.8)

Chemical formula

[0443] Example C1.9:N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(4-(2-(Methylthio)pyrimidine-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (Compound C1.9)

Chemical formula

[0444] Step 2: N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine Compound 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (370 mg) and N-hydroxysuccinimide (152 mg) were dissolved in dichloromethane (5 mL), stirred, and further dicyclohexylcarbodiimide (273 mg) was added. Then the reaction mixture was stirred at room temperature for 1 hour. 10 mL of water was added to the reaction mixture, and it was extracted with ethyl acetate (20 mL × 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. This crude product was dissolved in N,N-dimethylformamide (10 mL), stirred, and compound N 2 -L-valyl-N 6 -(Boc)-L-lysine was added, and the reaction mixture was reacted at room temperature for 16 hours. Citric acid was gradually added to the reaction mixture to adjust the pH to about 5, water was added, and it was further extracted with ethyl acetate (3 × 10 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated to remove ethyl acetate, and the target product N 6 -(tert-butoxycarbonyl)-N 2 -((6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (Compound C1.9, 400 mg) was obtained. ESI-MS (m / z): 635.3 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 12.46 (s, 1H), 9.06 (s, 2H), 9.06 (s, 1H), 8.70 (s, 1H), 8.07 (d, J = 7.4 Hz, 1H), 7.77 (d, J = 9.0 Hz, 1H), 6.76 (t, J = 5.6 Hz, 1H), 4.41 (t, J = 7.0 Hz, 2H), 4.19 (dd, J = 8.9, 7.0 Hz, 1H), 4.14 - 4.05 (m, 1H), 2.92 - 2.85 (m, 2H), 2.56 (s, 3H), 2.24 - 2.10 (m, 2H), 1.97 - 1.81 (m, 3H), 1.71 - 1.64 (m, 1H), 1.61 - 1.50 (m, 3H), 1.36 (s, 9H), 1.28 - 1.22 (m, 6H), 0.88 - 0.76 (m, 6H).

[0445] Example C1.10: (6-(2-(Methylthio)pyrimidin-5-yl)hexa-(5-yn)-acyl)glycylglycyl-L-phenylalanine (Compound C1.10)

Chemical Structure

[0446] Using the same method and reaction conditions as in Example C1.6 and different reaction raw materials, the target products in the following table were obtained.

[0447]

Table 6

[0448] Example C1.13: (29-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa29alkanoyl)glycylglycyl-L-phenylalanine (Compound C1.13)

Chemical Structure

[0449] Example C1.14: N 6 -(tert-Butoxycarbonyl)-N 2 -((39-(2-(Methylthio)pyrimidin-5-yl)-5,34-dioxo-3,9,12,15,18,21,24,27,30-nonaoxa-6,33-diazahentriacontan-38-inoyl)-L-valyl)-L-lysine (C1.14)

Chemical Structure

[0450] Step 2: Compound C1.14-B (300 mg) was dissolved in anhydrous methanol (5 mL), Pd / C (10%, 60 mg) was added, replaced with hydrogen gas three times, stirred overnight at room temperature for reaction, suction filtered, concentrated, and the target compound C1.14-C (293 mg) was obtained, which was a colorless oil. LCMS (ESI) [M+H] + : 856.1.

[0451] Step 3: 6-(2-(Methylthio)pyrimidin-5-yl)hex-5-ynoic acid (84 mg) was dissolved in N,N-dimethylformamide (3 mL), then N,N-diisopropylethylamine (61 mg) and HATU (108 mg) were added in sequence. The reaction solution was stirred at room temperature for 20 minutes, then compound C1.14-C (196 mg) was added, stirred at 40 °C for 3 hours for reaction, and the crude product was separated and purified by a C18 column reverse phase (acetonitrile / aqueous solution of 0.01% FA: 5% - 65%) to obtain the target compound N 6 -(tert-Butoxycarbonyl)-N 2 -((39-(2-(Methylthio)pyrimidin-5-yl)-5,34-dioxo-3,9,12,15,18,21,24,27,30-nonaoxa-6,33-diazahentriacontan-38-inoyl)-L-valyl)-L-lysine (C1.14) (170 mg). LCMS (ESI) [M+H] + : 1074.0; 11H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 2H), 8.23 (t, J = 5.7 Hz, 1H), 8.13 (d, J = 8.9 Hz, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.43 (d, J = 6.5 Hz, 1H), 6.69 (s, 1H), 4.09 (dd, J = 8.8, 6.5 Hz, 1H), 4.02 (s, 2H), 3.98 (d, J = 2.9 Hz, 2H), 3.74 - 3.70 (m, 1H), 3.50 (s, 30H), 3.45 (d, J = 6.2 Hz, 2H), 3.39 (d, J = 5.8 Hz, 2H), 3.26 (d, J = 6.1 Hz, 2H), 3.20 (q, J = 5.9 Hz, 2H), 2.81 (t, J = 6.6 Hz, 2H), 2.52 (s, 3H), 2.24 (t, J = 7.4 Hz, 2H), 2.12 - 2.06 (m, 1H), 1.79 - 1.74 (m, 2H), 1.67 - 1.59 (m, 1H), 1.50 (d, J = 5.3 Hz, 1H), 1.36 (s, 9H), 1.24 (s, 2H), 1.20 - 1.09 (m, 2H), 0.85 (t, J = 6.3 Hz, 6H).

[0452] Example C1.15:N 6 -(tert-Butoxycarbonyl)-N 2 -((32-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanoyl)-L-valyl)-L-lysine (C1.15)

Chemical formula

[0453] Example C1.16: N 6 ,N 6 -dimethyl-N 2 -((6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine

Chemical Structure

[0454] Step 2: Compound C1.16-B (3.0 g) and sodium acetate (1.90 g) were dissolved in a methanol (100 mL) solution, reacted at room temperature for 10 minutes, paraformaldehyde (2.8 g) was added to the reaction solution, stirred at room temperature for 30 minutes to react, and then sodium cyanoborohydride (1.0 g) was added to the reaction solution. The reaction was stirred at room temperature for 16 hours. After filtering the reaction solution, the filtrate was separated and purified by a C18 column reverse phase (acetonitrile / 0.05% aqueous formic acid solution: 5% - 55%) to obtain the target compound C1.16-C (1.70 g). LCMS (ESI) [M+H] + : 408.1; 1 1H NMR (400 MHz, DMSO) δ 7.86 (d, J = 7.3 Hz, 1H), 7.40 - 7.26 (m, 6H), 5.03 (s, 2H), 4.08 - 4.06 (m, 1H), 3.90 - 3.85 (m, 1H), 2.50 - 2.45 (m, 2H), 2.35 (s, 6H), 2.05 - 1.93 (m, 1H), 1.73 - 1.55 (m, 2H), 1.51 - 1.40 (m, 2H), 1.33 - 1.22 (m, 2H), 0.85 (dd, J = 16.5, 6.8 Hz, 6H).

[0455] Step 3: At room temperature, compound C1.16-C (1.6 g) was dissolved in methanol (80 mL), and then Pd / C (10%, 0.16 g) was added to the reaction solution. The mixture was stirred at room temperature for 12 hours under hydrogen gas to cause a reaction. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C1.16-D (680 mg). LCMS (ESI) [M+H] + : 274.2.

[0456] Step 4: 6-(2-(Methylthio)pyrimidin-5-yl)hex-5-ynoic acid (944 mg) was dissolved in N,N-dimethylformamide (30 mL), and then N,N-diisopropylethylamine (1.3 g) and 2-(7-azabenzotriazole)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU, 1.8 g) were added in sequence. The reaction solution was stirred at room temperature for 20 minutes, and then compound C1.16-D (1.1 g) was added. The mixture was stirred at 40 °C for 3 hours to cause a reaction, and the crude product was separated and purified by a C18 column reverse phase (acetonitrile / aqueous solution of 0.01% formic acid: 5% - 65%) to obtain the target compound N 6 ,N 6 -dimethyl-N 2 -((6-(2-(Methylthio)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine C1.16 (1.2 g). LCMS (ESI) [M+H] + : 492.1; 11H NMR (400 MHz, DMSO) δ 8.68 (s, 2H), 8.00 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 4.20 (t, 1H), 4.08 (dd, J = 12.7, 7.7 Hz, 1H), 2.47 - 2.40 (m, 4H), 2.37 - 2.31 (m, 2H), 2.30 (s, 6H), 2.01 - 1.92 (m, 1H), 1.82 - 1.73 (m, 2H), 1.71 - 1.56 (m, 2H), 1.49 - 1.37 (m, 2H), 1.33 - 1.23 (m, 2H), 0.88 - 0.82 (m, 6H).

[0457] Example C1.17: N 6 , N 6 -Dimethyl-N 2 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine C1.17

Chemical Structure

[0458] Example C1.18:N 2 -(tert-Butoxycarbonyl)-N 6 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine

Chemical Structure

[0459] Step 2: At room temperature, compound C1.18-C (4.7 g) was dissolved in methanol (80 mL), then Pd / C (10%, 0.6 g) was added to the reaction solution, and the mixture was stirred with hydrogen gas at room temperature for 12 hours to react. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C1.18-D (3.4 g). LCMS (ESI) [M+H] + : 346.2.

[0460] Step 3: Dissolve 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (2.68 g) in N,N-dimethylformamide (50 mL), then add triethylamine (3 mL) and HBTU (3.8 g) further. Stir the mixture at room temperature for 10 minutes, then add ethyl acetate (200 mL). Wash the mixture with saturated sodium bicarbonate (20 mL×2), hydrochloric acid (0.1 M, 50 mL×2), water (50 mL×2), dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a crude product intermediate. Dissolve the above crude product in DMF (30 mL), add DIPEA (1.6 mL), then add C1.18-D (3.4 g). Stir the reaction solution at room temperature for 3 hours, then add ethyl acetate (200 mL). Wash with hydrochloric acid (0.1 M, 50 mL×2), water (50 mL×2), dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a crude product. Separate and purify by silica gel column chromatography to obtain the target compound N 2 -(tert-Butoxycarbonyl)-N 6 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (C1.18, white solid, 3.8 g) was obtained. LCMS (ESI) [M+H] + : 596.4.

[0461] Example C1.19: N 6 ,N 6 -Dimethyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine

Chemical formula

[0462] Step 2: Compound 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (300 mg, 0.88 mmol) was dissolved in N,N-dimethylformamide (6 mL), HATU (337 mg, 0.88 mmol) and DIPEA (286 mg, 2.21 mmol) were added, and the reaction was stirred at room temperature for 30 minutes. Then, dipeptide C1.16-D (243 mg, 0.88 mmol) was added, and the reaction was stirred at room temperature for 2 hours. The completion of the reaction was detected by LCMS, and the reaction solution was separated and purified by a C18 column (acetonitrile / aqueous solution of 0.01% FA: 5% - 50%) to obtain the target compound N 6 ,N 6 -dimethyl-N 2-((6-(4-(2-(Methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (300 mg, yield 57%) was obtained as a white solid. LCMS (ESI) [M+H] + = 595.5, t R = 2.024 min. 1 H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 2H), 9.01 (s, 1H), 7.85 (br s, 1H), 7.84 (d, J = 8.9 Hz, 1H), 4.48 (t, J = 6.8 Hz, 2H), 4.17 - 4.15 (m, 1H), 4.02 (br s, 1H), 3.44 (s, 3H), 2.42 (br s, 2H), 2.30 (s, 6H), 2.18 - 2.14 (m, 2H), 1.99 - 1.96 (m, 1H), 1.88 (dd, J = 14.6, 7.1 Hz, 2H), 1.67 (br s, 1H), 1.59 - 1.53 (m, 2H), 1.43 (br s, 2H), 1.28 - 1.26 (m, 5H), 0.83 - 0.89 (m, 6H).

[0463] Example C1.20:N 6 ,N 6 -diethyl-N 2 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine

Chemical formula

[0464] Step 2: At room temperature, compound C1.20-A (1.6 g, 3.68 mmol) was dissolved in methanol (80 mL). Next, Pd / C (0.16 g) was added to the reaction solution, and the mixture was stirred with hydrogen gas at room temperature for 12 hours to react. LCMS indicated the completion of the reaction. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C1.20-B (900 mg, yield 82%), which was an off-white solid. 11H NMR (400 MHz, DMSO-d6) δ 8.04 (br s, 1H), 4.02 - 3.99 (m, 1H), 3.10 (d, J = 4.5 Hz, 1H), 2.65 (q, J = 7.1 Hz, 4H), 2.55 - 2.51 (m, 2H), 2.06 - 1.93 (m, 1H), 1.73 - 1.54 (m, 2H), 1.47 - 1.38 (m, 2H), 1.30 - 1.21 (m, 2H), 1.01 (t, J = 7.1 Hz, 6H), 0.89 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H).

[0465] Step 3: Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg, 1 mmol) was dissolved in DMF (8 mL), and HATU (380 mg, 1 mmol) and triethylamine (322 mg, 2.5 mmol) were added sequentially. After stirring at room temperature for 20 minutes, compound C1.20-B (301 mg, 1 mmol) was added, and stirring was continued at room temperature for 30 minutes. After completion of the reaction was detected by LCMS, the reaction solution was directly purified by a C18 column reverse phase (acetonitrile and 0.05% aqueous formic acid system) to obtain the target compound (280 mg, yield 51%), which was a white solid. LCMS (ESI) [M+H] + = 552.3; 11H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 2H), 7.95 (d, J = 8.9 Hz, 1H), 7.86 (d, J = 7.2 Hz, 1H), 4.18 (dd, J = 8.8, 6.8 Hz, 1H), 4.02 (dd, J = 12.8, 7.2 Hz, 1H), 3.41 (s, 3H), 2.74 - 2.69 (m, 4H), 2.62 - 2.52 (m, 4H), 2.44 - 2.29 (m, 2H), 2.04 - 1.94 (m, 1H), 1.86 - 1.77 (m, 2H), 1.72 - 1.54 (m, 2H), 1.51 - 1.39 (m, 2H), 1.33 - 1.23 (m, 2H), 1.02 (t, J = 7.2 Hz, 6H), 0.87 - 0.82 (m, 6H).

[0466] Example C1.21:N 6 ,N 6 -Diethyl-N 2 -((6-(4-(2-(Methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine

Chemical Structure

[0467] Example C1.22: N 6 ,N 6 -Dipropyl-N 2 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine

Chemical formula

[0468] Step 2: At room temperature, compound C1.22-A (2.0 g, 4.32 mmol) was dissolved in methanol (80 mL). Next, Pd / C (0.16 g) was added to the reaction solution, and the mixture was stirred with hydrogen gas at room temperature for 12 hours to react. LCMS indicated the completion of the reaction. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C1.22-B (1.2 g, yield 85.5%), which was a white solid.

[0469] Step 3: Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100 mg, 0.373 mmol) was dissolved in N,N-dimethylformamide (1 mL), then HATU (142 mg, 0.373 mmol) and N,N-diisopropylethylamine (120 mg, 0.93 mmol) were added, and the system was stirred for 30 minutes. Next, compound C1.22-B (122 mg, 0.371 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. After the completion of the reaction was detected by LCMS, the reaction solution was directly purified by a C18 column reverse phase (acetonitrile and 0.05% formic acid aqueous solution system), and the target compound N 6 ,N 6 -dipropyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (50 mg, yield 28%) was obtained as a pale yellow solid. LCMS (ESI) [M+H] + = 580.0; 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 7.98 - 7.93 (m, 2H), 4.24 - 4.16 (m, 1H), 4.10 (d, J = 5.2 Hz, 1H), 3.41 (s, 3H), 2.79 - 2.64 (m, 6H), 2.55 (t, J = 7.1 Hz, 2H), 2.45 - 2.26 (m, 2H), 2.06 - 1.91 (m, 1H), 1.89 - 1.78 (m, 2H), 1.76 - 1.66 (m, 1H), 1.64 - 1.57 (m, 1H), 1.57 - 1.42 (m, 6H), 1.37 - 1.24 (m, 2H), 0.93 - 0.78 (m, 12H).

[0470] Example C1.23: N 6 ,N 6 -dipropyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine [Chemical formula] The compound 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (500 mg, 1.475 mmol) was dissolved in N,N-dimethylformamide (10 mL), then HATU (560 mg, 1.475 mmol) and N,N-diisopropylethylamine (476 mg, 3.688 mmol) were added. The system was stirred for 30 minutes, then compound C1.22-B (485 mg, 1.475 mmol) was added. The reaction solution was stirred at room temperature for 1 hour. After the completion of the reaction was detected by LCMS, the reaction solution was directly purified by a C18 column reverse phase (acetonitrile and 0.05% aqueous formic acid system) to obtain the target compound N 6 ,N 6 -dipropyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (320 mg, yield 33%) was obtained as a white solid. LCMS (ESI) [M+H] + = 651.5; 1 H NMR (400 MHz, DMSO) δ 9.49 (s, 2H), 9.00 (s, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.79 (d, J = 8.9 Hz, 1H), 4.47 (t, J = 6.9 Hz, 2H), 4.21 - 4.11 (m, 1H), 4.09 - 4.01 (m, 1H), 3.44 (s, 3H), 2.45 - 2.35 (m, 6H), 2.23 - 2.09 (m, 2H), 1.97 - 1.86 (m, 3H), 1.72 - 1.64 (m, 1H), 1.61 - 1.50 (m, 3H), 1.43 - 1.34 (m, 6H), 1.32 - 1.22 (m, 4H), 0.86 - 0.77 (m, 12H).

[0471] Example C1.24: tert-Butyl ((S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynamide)-6-oxohexyl)carbamate

Chem.

[0472] Step 2: Compound C1.24-B (7.00 g, 5.60 mmol), diisopropylethylamine (10.7 mL, 60.4 mmol), and HBTU (8.60 g, 22.7 mmol) were dissolved in N,N-dimethylformamide (100 mL), and then p-aminobenzyl alcohol (3.71 g, 30.2 mmol) was added. The reaction was allowed to proceed at room temperature for 2 hours. Complete reaction was detected by LCMS. Ethyl acetate (300 mL) was added to the reaction mixture, and the mixture was washed with saturated brine (100 mL × 3), dried over anhydrous sodium sulfate, filtered by suction, and the filtrate was concentrated under reduced pressure to give a solid. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound tert-butyl (S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynamide)-6-oxohexyl)carbamate (6.00 g, yield: 69.9%), which was a yellow oil. LCMS (ESI) [M+H] + = 570.1; 1 H NMR (400 MHz, CDCl3) δ 9.24 (s, 1H), 8.46 (s, 2H), 7.51 - 7.47 (m, 2H), 7.31 - 7.23 (m, 3H), 7.02 - 6.95 (m, 1H), 4.83 (br s, 1H), 4.63 - 4.60 (m, 2H), 3.19 - 2.96 (m, 4H), 2.57 (s, 3H), 2.52 - 2.48 (m, 2H), 2.47 - 2.41 (m, 2H), 1.99 - 1.92 (m, 2H), 1.87 - 1.77 (m, 2H), 1.76 - 1.63 (m, 2H), 1.43 (s, 9H).

[0473] Example C1.25: tert-butyl (S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamide)-6-oxohexyl)carbamate [Chemical] Step 1: Compound C1.25-A (1.60 g, 5.21 mmol), diisopropylethylamine (2.68 mL, 20.8 mmol) and HBTU (2.97 g, 7.82 mmol) were dissolved in N,N-dimethylformamide (30 mL), and the reaction was carried out at room temperature for 30 minutes. Subsequently, lysine protected with Boc (1.28 g, 5.21 mmol) was added, and the reaction was carried out at room temperature for 2 hours. It was detected by LCMS that the reaction was complete. An aqueous citric acid solution (30 mL) was added to the reaction solution to adjust the pH to 5, and the aqueous solution was extracted with dichloromethane (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, suction filtered, concentrated under reduced pressure, and the target compound C1.25-B (3.0 g, crude product) was obtained, which was a yellow oil. LCMS (ESI) [M+H] + = 536.0;

[0474] Step 2: Compound C1.25-B (3.00 g, 5.60 mmol), diisopropylethylamine (2.89 g, 22.4 mmol) and HBTU (3.19 g, 8.40 mmol) were dissolved in N,N-dimethylformamide (30 mL). Subsequently, p-aminobenzyl alcohol (1.38 g, 11.2 mmol) was added, and the reaction was carried out at room temperature for 2 hours. It was detected by LCMS that the reaction was complete. Ethyl acetate (200 mL) was added to the reaction solution, washed with saturated brine (80 mL × 3), dried over anhydrous sodium sulfate, suction filtered, and the filtrate was concentrated under reduced pressure to obtain a solid. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound (S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamide)-6-oxohexyl)carbamic acid tert-butyl (2.90 g), which was a yellow oil. LCMS (ESI) [M+H] + = 641.1; 11H NMR (400 MHz, CDCl3) δ 9.13 (s, 1H), 8.95 (s, 2H), 7.94 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 6.83 - 6.77 (m, 1H), 4.93 - 4.80 (m, 1H), 4.64 - 4.58 (m, 2H), 4.40 - 4.29 (m, 2H), 3.12 - 2.97 (m, 4H), 2.60 (s, 3H), 2.31 - 2.23 (m, 2H), 1.96 - 1.83 (m, 4H), 1.76 - 1.61 (m, 4H), 1.41 (s, 9H), 1.37 - 1.29 (m, 4H).

[0475] Example C1.26:N 6 ,N 6 -Dibutyl-N 2 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine

Chemical Structure

Claims

1. An anti-NaPi2b antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment is (a) HCDR1, HCDR2 and HCDR3 included in the heavy chain variable region VH shown in Sequence IDs 1, 17, 19, 34, 36, 52, 54, 71, 73, 74 or 33, and / or (b) LCDR1, LCDR2 and LCDR3 included in the light chain variable region VL shown in Sequence IDs 2, 18, 20, 35, 37, 53, 55, 72, 75 or 51 This includes the complementarity determination region CDR, Anti-NaPi2b antibody or its antigen-binding fragment.

2. The antibody or its antigen-binding fragment is (1) VH and / or VL as defined in accordance with the IMGT numbering system, (a) The VH includes HCDR1 whose sequence is sequence number 11, HCDR2 whose sequence is sequence number 12, HCDR3 whose sequence is sequence number 14, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 15, LCDR2 whose sequence is WAS, and LCDR3 whose sequence is sequence number 10. (b) The VH includes HCDR1 whose sequence is sequence number 11, HCDR2 whose sequence is sequence number 13, HCDR3 whose sequence is sequence number 14, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 15, LCDR2 whose sequence is WAS, and LCDR3 whose sequence is sequence number 10. (c) The VH includes HCDR1 whose sequence is sequence number 28, HCDR2 whose sequence is sequence number 29, HCDR3 whose sequence is sequence number 31, and / or The VL includes LCDR1 whose sequence is sequence number 32, LCDR2 whose sequence is YTS, and LCDR3 whose sequence is sequence number 27. (d) The VH includes HCDR1 whose sequence is sequence number 28, HCDR2 whose sequence is sequence number 30, HCDR3 whose sequence is sequence number 31, and / or The VL includes LCDR1 whose sequence is sequence number 32, LCDR2 whose sequence is YTS, and LCDR3 whose sequence is sequence number 27. (e) The VH includes HCDR1 whose sequence is sequence number 46, HCDR2 whose sequence is sequence number 47, HCDR3 whose sequence is sequence number 49, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 50, LCDR2 whose sequence is SAS, and LCDR3 whose sequence is sequence number 45. (f) The VH includes HCDR1 whose sequence is sequence number 46, HCDR2 whose sequence is sequence number 48, HCDR3 whose sequence is sequence number 49, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 50, LCDR2 whose sequence is SAS, and LCDR3 whose sequence is sequence number 45. (g) The VH includes HCDR1 whose sequence is sequence number 65, HCDR2 whose sequence is sequence number 66, HCDR3 whose sequence is sequence number 68, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 69, LCDR2 whose sequence is WAS, and LCDR3 whose sequence is sequence number 64. (h) The VH includes HCDR1 whose sequence is sequence number 65, HCDR2 whose sequence is sequence number 67, HCDR3 whose sequence is sequence number 68, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 69, LCDR2 whose sequence is WAS, and LCDR3 whose sequence is sequence number 64. Or (i) The VH includes HCDR1 whose sequence is sequence number 76, HCDR2 whose sequence is sequence number 77, HCDR3 whose sequence is sequence number 78, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 79, LCDR2 whose sequence is HTS, and LCDR3 whose sequence is sequence number 16. (2) VH and / or VL as defined in accordance with the kabat numbering system, (a) The VH includes HCDR1 whose sequence is sequence number 3, HCDR2 whose sequence is sequence number 4, HCDR3 whose sequence is sequence number 6, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 7, LCDR2 whose sequence is sequence number 9, and LCDR3 whose sequence is sequence number 10. (b) The VH includes HCDR1 whose sequence is sequence number 3, HCDR2 whose sequence is sequence number 5, HCDR3 whose sequence is sequence number 6, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 8, LCDR2 whose sequence is sequence number 9, and LCDR3 whose sequence is sequence number 10. (c) The VH includes HCDR1 whose sequence is sequence number 21, HCDR2 whose sequence is sequence number 22, HCDR3 whose sequence is sequence number 24, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 25, LCDR2 whose sequence is sequence number 26, and LCDR3 whose sequence is sequence number 27. (d) The VH includes HCDR1 whose sequence is sequence number 21, HCDR2 whose sequence is sequence number 23, HCDR3 whose sequence is sequence number 24, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 25, LCDR2 whose sequence is sequence number 26, and LCDR3 whose sequence is sequence number 27. (e) The VH includes HCDR1 whose sequence is sequence number 38, HCDR2 whose sequence is sequence number 39, HCDR3 whose sequence is sequence number 41, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 42, LCDR2 whose sequence is sequence number 44, and LCDR3 whose sequence is sequence number 45. (f) The VH includes HCDR1 whose sequence is sequence number 38, HCDR2 whose sequence is sequence number 40, HCDR3 whose sequence is sequence number 41, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 43, LCDR2 whose sequence is sequence number 44, and LCDR3 whose sequence is sequence number 45. (g) The VH includes HCDR1 whose sequence is sequence number 56, HCDR2 whose sequence is sequence number 57, HCDR3 whose sequence is sequence number 60, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 61, LCDR2 whose sequence is sequence number 63, and LCDR3 whose sequence is sequence number 64. (h) The VH includes HCDR1 whose sequence is sequence number 56, HCDR2 whose sequence is sequence number 58, HCDR3 whose sequence is sequence number 60, and / or The VL includes LCDR1 whose sequence is sequence number 62, LCDR2 whose sequence is sequence number 63, and LCDR3 whose sequence is sequence number 64, or (i) The VH includes HCDR1 whose sequence is sequence number 56, HCDR2 whose sequence is sequence number 59, HCDR3 whose sequence is sequence number 60, and / or The aforementioned VL includes LCDR1 whose sequence is sequence number 62, LCDR2 whose sequence is sequence number 63, and LCDR3 whose sequence is sequence number 64. Includes, Preferably, the anti-NaPi2b antibody or its antigen-binding fragment binds to human NaPi2b, monkey NaPi2b and / or rat NaPi2b. The antibody or antigen-binding fragment thereof according to claim 1.

3. The anti-NaPi2b antibody or its antigen-binding fragment is (a) VH as shown in Sequence ID No. 1 or 17, and / or VL as shown in Sequence ID No. 2 or 18, (b) VH as shown in Sequence ID No. 19 or 34, and / or VL as shown in Sequence ID No. 20 or 35, (c) VH as shown in Sequence ID No. 36 or 52, and / or VL as shown in Sequence ID No. 37 or 53, (d) VH as shown in Sequence ID No. 54, 71 or 73, and / or VL as shown in Sequence ID No. 55 or 72, (e) VH as shown in Sequence ID No. 74 or 33, and / or VL as shown in Sequence ID No. 75 or 51, (f) A VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one VH of (a) to (e), and / or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one VL of (a) to (e), or (g) A VH having one or more amino acid substitutions, deletions or additions or any combination thereof compared to any one VH of (a) to (e), for example, one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions, deletions or additions or any combination thereof, and / or a VL having one or more amino acid substitutions, deletions or additions or any combination thereof compared to any one VL of (a) to (e), for example, one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions, deletions or additions or any combination thereof, This includes, preferably, the substitution is a conservative substitution. The anti-NaPi2b antibody or antigen-binding fragment thereof according to claim 1.

4. The anti-NaPi2b antibody or its antigen-binding fragment is (a) VH of the sequence shown in Sequence ID 1 and VL of the sequence shown in Sequence ID 2, (b) VH of the sequence shown in sequence number 17 and VL of the sequence shown in sequence number 18, (c) VH of the sequence shown in Sequence ID No. 19 and VL of the sequence shown in Sequence ID No. 20, (d) VH of the sequence shown in sequence number 34 and VL of the sequence shown in sequence number 35, (e) VH of the sequence shown in sequence number 36 and VL of the sequence shown in sequence number 37, (f) VH of the sequence shown in sequence number 52 and VL of the sequence shown in sequence number 53, (g) VH of the sequence shown in Sequence ID No. 54 and VL of the sequence shown in Sequence ID No. 55, (h) VH of the sequence shown in sequence number 71 and VL of the sequence shown in sequence number 72, (i) VH of the sequence shown in sequence number 73 and VL of the sequence shown in sequence number 72, (j) VH of the sequence shown in sequence number 74 and VL of the sequence shown in sequence number 75, (k) VH of the sequence shown in sequence number 33 and VL of the sequence shown in sequence number 51, VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to VH and VL in any one of the groups (l) (a) to (k), and / or VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, or VH having one or more amino acid substitutions, deletions or additions or any combination thereof, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof, compared to VH and VL in any one of the groups (m)(a) to (k), and / or VL having one or more amino acid substitutions, deletions or additions or any combination thereof, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof, This includes, preferably, the substitution is a conservative substitution. The anti-NaPi2b antibody or antigen-binding fragment thereof according to claim 1.

5. The antibody or its antigen-binding fragment is a chimeric antibody, a humanized antibody, or a fully human antibody, Selectively, the antibody or its antigen-binding fragment is selected from scFv, Fab, Fab', (Fab')2, Fv fragment, Fv linked by disulfide bonds (dsFv), diabody, and multispecific antibodies. For example, the multispecific antibody is a bispecific antibody, a triplicate antibody, or a quadruplicate antibody. The anti-NaPi2b antibody or antigen-binding fragment thereof according to claim 1.

6. The antibody or its antigen-binding fragment is (a) heavy chain constant region CH of human immunoglobulin or its variants, and / or (b) further comprising the light chain constant region CL of human immunoglobulin or a variant thereof, Of these, the mutant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the wild-type sequence from which it is derived, or the mutant has one or more amino acid substitutions, deletions, or additions or any combination thereof compared to the wild-type sequence from which it is derived, for example, at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acid substitutions, deletions, or additions or any combination thereof, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions or any combination thereof, preferably the substitutions are conservative substitutions. Preferably, the heavy chain steady region is an IgG heavy chain steady region, for example, an IgG1, IgG2, IgG3, or IgG4 heavy chain steady region, and / or the light chain steady region is a κ or λ light chain steady region. More preferably, the antibody or its antigen-binding fragment comprises the human IgG1 heavy chain constant region, and / or the antibody or its antigen-binding fragment comprises the human κ light chain constant region. The anti-NaPi2b antibody or antigen-binding fragment thereof according to claim 1.

7. The heavy chain constant region comprises CH shown in SEQ ID NO: 70 or a variant thereof, wherein the variant has at least 20 conserved amino acid substitutions compared to SEQ ID NO: 70, for example, at least 20, at least 15, at least 10, or at least 5 conserved amino acid substitutions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO: 70, and / or The light chain constant region includes CL shown in SEQ ID NO: 80 or a variant thereof, wherein the variant has at least 20 conserved amino acid substitutions compared to SEQ ID NO: 80, for example, at least 20, at least 15, at least 10, or at least 5 conserved amino acid substitutions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO:

80. Preferably, the antibody or its antigen-binding fragment includes a heavy chain constant region CH shown in SEQ ID NO: 70 and a light chain constant region CL shown in SEQ ID NO:

80. The anti-NaPi2b antibody or its antigen-binding fragment according to claim 6.

8. The antibody is (a) A heavy chain containing VH shown in SEQ ID NO: 71 and CH shown in SEQ ID NO: 70, and a light chain containing VL shown in SEQ ID NO: 72 and CL shown in SEQ ID NO: 80, (b) A heavy chain containing VH shown in SEQ ID NO: 73 and CH shown in SEQ ID NO: 70, and a light chain containing VL shown in SEQ ID NO: 72 and CL shown in SEQ ID NO: 80, preferably a heavy chain shown in SEQ ID NO: 81 and a light chain shown in SEQ ID NO: 82, (c) A heavy chain containing VH shown in SEQ ID NO: 17 and CH shown in SEQ ID NO: 70, and a light chain containing VL shown in SEQ ID NO: 18 and CL shown in SEQ ID NO: 80, preferably a heavy chain shown in SEQ ID NO: 83 and a light chain shown in SEQ ID NO: 84, including, The anti-NaPi2b antibody or antigen-binding fragment thereof according to claim 1.

9. The antibody-drug conjugate represented by formula XV, 【Chemistry 1】 Formula XV or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, During the ceremony, Tb is an anti-NaPi2b antibody or its antigen-binding fragment. q is the drug-antibody coupling ratio, D is a biologically active molecular fragment, L １ It is an extension unit, L ２ It does not exist, or it is a connecting unit. L ３ is Val-AA １ -Gly, Val-AA １ , Ala-AA １ , Gly-AA １ , AA １ -Gly, Ala-AA １ -Gly, Gly-AA １ -Gly, AA １ -Ala-Asn, AA １ selected from, and the structure of the amino acid residue shown in said AA １ is shown as follows: 【Chemistry 2】 AA １ In the formula, R ａ , R ｂ Each of these is independently H, 【Transformation 3】 Selected from and R ａ , R ｂ It is not H at the same time, Or, R ａ and R ｂ These, together with carbon atoms commonly linked to them, form a 4- to 10-membered heterocycle, and the 4- to 10-membered heterocycle can optionally have one or more R ０ Replaced by, r, r １ Each of these is independently selected from any integer between 0 and 20. R ｍ１ , R ｎ１ These are H, C1-6 alkyl groups, C3-6 cycloalkyl groups, and -COOR, respectively, independently. ｘ１ Selected from, R ｘ１ It is selected from C1-6 alkyl groups, Or, R ｍ１ and R ｎ１ These, together with a nitrogen atom commonly linked to them, form a 4- to 10-membered heterocycle, and the 4- to 10-membered heterocycle may optionally have one or more R atoms. ０’ Replaced by, R ｚ It is selected from C1-6 alkyl groups, R ０ , R ０’ These are, independently, C1-6 alkyl groups, C3-6 cycloalkyl groups, and -NR ｍ２ R ｎ２ and optionally selected from 4-10 membered heterocyclic groups substituted with C1-6 alkyl groups, R ｍ２ , R ｎ２ Each is independently selected from H and C1-6 alkyl groups, L ４ L is either nonexistent or exists. ４ If L ４ teeth, 【Chemistry 4】 The winner was selected from the group, and the first place went to L. ３ It connects to D, and the second place connects to D. Antibody-drug conjugates, stereoisomers of said antibody-drug conjugates, their prodrugs, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable solvates thereof.

10. (1) The antibody or antigen-binding fragment described in Tb includes Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, non-human antibody, humanized antibody, chimeric antibody, fully human antibody, probody, monoclonal antibody, bispecific antibody, or multispecific antibody. (2) q is selected from any number between 0.1 and 16.0, (3) D is a molecular fragment that has antitumor biological activity. A feature that satisfies one or more of the following conditions: An antibody-drug conjugate according to claim 9, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

11. (1) q is selected from 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and any number in between. (2) In the biologically active molecular fragment, the biologically active molecule is a DNA topoisomerase inhibitor or a tubulin inhibitor. (3) L １ teeth, 【Transformation 5】 Each Z is independently selected from direct bonds, carbon-carbon triple bonds, carbon-carbon double bonds, C6-10 aryl groups, 5-10 heteroaryl groups, and amide groups; Rx and Ry are independently selected from H and C1-4 alkyl groups, respectively; each m is independently selected from 0, 1, 2, 3, 4, 5, and 6; y1 is independently selected from any integer between 1 and 6; each y2 is independently selected from any integer between 0 and 15; each y3 is independently selected from 1, 2, and 3; each y4 is independently selected from 0 and 1; the 1st position is linked to Tb via an S atom; the 2nd position is L ２ or L ３ Connect to, (4) L ２ L is either nonexistent or exists. ２ If L ２ teeth, 【Transformation 6】 y1 is selected from any integer between 1 and 6, each y2 is independently selected from any integer between 0 and 10, each y3 is independently selected from 1 and 2, each y4 is independently selected from 0 and 1, and the first is L １ L is linked to 2nd place. ３ Connect to, (5) AA １ In the amino acid residue, R ａ , R ｂ One of them is H, and the other is 【Transformation 7】 Selected from, Or, AA １ In the amino acid residue, R ａ and R ｂ R ０ It forms a 5-6 member heterocycle substituted with, (6) AA １ In the amino acid residues, r, r １ Each of these is independently selected from 0, 1, 2, 3, 4, and 5. (7) AA １ In the amino acid residue, R ｍ１ , R ｎ１ Each is independently selected from H, methyl group, ethyl group, n-propyl group, n-butyl group, -COOCH3, -COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, -COOC(CH3)3 and -COOCH2CH2CH2CH3, or R ｍ１ and R ｎ１ R ０’ It forms a 5-6 member heterocycle substituted with, (8) AA １ In the amino acid residue, R ｚ It is a methyl group, (9) AA １ In the amino acid residue, R ０ , R ０’ These are, independently, C1-6 alkyl groups and -NR ｍ２ R ｎ２ and optionally selected from 5-6 membered heterocyclic groups substituted with C1-6 alkyl groups, A feature that satisfies one or more of the following conditions: An antibody-drug conjugate according to claim 10, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

12. (1) q is selected from 0.1, 1, 2, 3, 4, 5, 6, 7, 8 and any number in between. (2) When the biologically active molecule in the biologically active molecule fragment is a DNA topoisomerase inhibitor, the DNA topoisomerase inhibitor is a camptothecin-based biologically active molecule, (3) When the biologically active molecule in the biologically active molecule fragment is a tubulin inhibitor, the tubulin inhibitor is an MMAF-type tubulin inhibitor or an MMAE-type tubulin inhibitor. (4) L １ In this, each Z is independently selected from a direct bond, a carbon-carbon triple bond, and a carbon-carbon double bond. (5) L １ In this case, y1 is 4, 5, or 6. (6) L １ In this case, each y2 is independently selected from any integer between 6 and 10. (7) L ２ L is either nonexistent or exists. ２ If L ２ teeth, 【Transformation 8】 The winner was selected from the group, and the first place went to L. １ L is linked to 2nd place. ３ Connect to, (8) AA １ In the amino acid residue shown, R ａ , R ｂ One of them is H, and the other is 【Chemistry 9】 Selected from, or R ａ and R ｂ R ０ It forms a 5-6 member heterocycle substituted with R ０ A piperidine ring or piperazine ring substituted with, (9) AA １ In the amino acid residues, r, r １ These are independently selected from 0 and 4, (10) AA １ In the amino acid residue of, R ｍ１ , R ｎ１ is independently selected from H, a C1-6 alkyl group, a C3-6 cycloalkyl group, and a tert-butoxycarbonyl group, or R ｍ１ and R ｎ１ together with the nitrogen atom commonly linked thereto, form a piperidine ring or a piperazine ring substituted with R ０’ ​ (11) AA １ In the amino acid residue, R ０ This is selected from C1-6 alkyl groups and 5-6 membered heterocyclic groups substituted with C1-6 alkyl groups, and the 5-6 membered heterocyclic group is selected from piperidinyl groups and piperazinyl groups. (12) AA １ In the amino acid residue of, R ０’ is a C1-6 alkyl group and -NR ｍ２ R ｎ２ is selected from, (13) AA １ In the amino acid residue, R ｍ２ , R ｎ２ It is a methyl group, (14) L ４ L is either nonexistent or exists. ４ If L ４ teeth, 【Chemistry 10】 And the first place is L ３ It connects to D, and the second place connects to D. A feature that satisfies one or more of the following conditions: An antibody-drug conjugate according to claim 11, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

13. (1) q is selected from 2, 3, 4, 5, 6, 7, 8 and any number in between. (2) L １ teeth, 【Chemistry 11】 m is selected from 2, 3 and 4, y1 is selected from any integer between 1 and 6, each y2 is independently selected from any integer between 0 and 10, each y3 is independently selected from 1 or 2, the first position is linked to Tb via an S atom, and the second position is L ２ or L ３ Connect to, (3) L ３ AA １ AA １ -Gly, Val-AA １ -Gly, AA １ - Selected from Ala-Asn, (4) AA １ In the amino acid residue shown, R ａ and R ｂ R ０ It forms a 5-6 member heterocycle substituted with R ０ A piperidine ring substituted with, (5) AA １ In the amino acid residues, r, r １ One of them is 0, and the other is 4. (6) AA １ In the amino acid residue, R ｍ１ , R ｎ１ Each is independently selected from H and C1-6 alkyl groups, or R ｍ１ and R ｎ１ R ０’ Forms a piperidine ring substituted with, (7) AA １ In the amino acid residue, R ０ is selected from a methyl group, an ethyl group, and a 5-6 membered heterocyclic group substituted with a methyl group, wherein the 5-6 membered heterocyclic group is a piperidinyl group, and preferably R ０ These are methyl groups, ethyl groups and 【Chemistry 12】 Selected from, (8) AA １ In the amino acid residue, R ０’ is a methyl group and -NR ｍ２ R ｎ２ Selected from, (9) When the biologically active molecule in the biologically active molecule fragment is a DNA topoisomerase inhibitor, and the DNA topoisomerase inhibitor is a camptothecin-based biologically active molecule, the camptothecin-based biologically active molecule is camptothecin, DXD, substituted camptothecin, or substituted DXD. A feature that satisfies one or more of the following conditions: An antibody-drug conjugate according to claim 12, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

14. (1) q is selected from 3, 4, 5, 6, 7, 8 and any number in between, preferably q is selected from 4, 5, 6, 7, 8 and any number in between, preferably q is selected from 6, 7, 8 and any number in between, (2) L １ teeth, 【Chemistry 13】 Selected from, the 1st position is linked to Tb via an S atom, and the 2nd position is L ２ or L ３ Connect to, (3) L ２ It does not exist, or 【Chemistry 14】 And, (4) L ３ AA １ Val-AA １ - Selected from Gly, preferably L ３ Val-AA １ - Selected from Gly, (5) AA １ In the amino acid residue shown, R ａ and R ｂ Along with the carbon atoms commonly bonded to them, 【Chemistry 15】 It forms, and the first carbon atom is R ａ and R ｂ It is a carbon atom that is linked together with it, (6) r, r １ In this case, r is 4, r １ If R is 0, ｍ１ , R ｎ１ Each is independently selected from H and C1-6 alkyl groups, where r is 0, r １ If R is 4, ｍ１ , R ｎ１ Each is independently selected from C1-6 alkyl groups, or R ｍ１ and R ｎ１ Along with the nitrogen atom commonly linked to them, 【Chemistry 16】 It forms, and the first carbon atom is R ａ and R ｂ The carbon atoms that are bonded together are A feature that satisfies one or more of the following conditions: An antibody-drug conjugate according to claim 13, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

15. AA １ The amino acid residues shown are 【Chemistry 17】 Selected from, Most preferably, AA １ The amino acid residues shown are [Chemistry 18] Characterized by being selected from, An antibody-drug conjugate according to claim 13, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

16. L ３ teeth, 【Chemistry 19】 Selected from, X － These include halogen ions, carboxylate ions, sulfate ions, bisulfate ions, and OH － The winner was selected from the group, and the first place went to L. １ or L ２ L is linked to 2nd place. ４ Alternatively, connect to D, Most preferably, L ３ teeth, 【Chemistry 20】 The winner was selected from the group, and the first place went to L. １ or L ２ L is linked to 2nd place. ４ Alternatively, characterized by being connected to D, An antibody-drug conjugate according to claim 13, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. 【Request Item 17】 【Chemistry 21】 The structure is selected from the following structural fragments: Table 1 、 Preferably, 【Chemistry 22】 The structure is selected from the following: Table 2-1 Table 2-2 Table 2-3 In the formula, the first position is connected to Tb, and the second position is connected to D. An antibody-drug conjugate according to claim 13, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

18. The antibody-drug conjugate has the structure shown in formula I, 【Chemistry 23】 Equation I In the formula, the definitions of Tb, L1, L2, L3, L4 and q are as described in claim 9. R １ , R ２ Each of these is independently selected from H, halogen, -OH, optionally substituted C1-6 alkyl groups, and optionally substituted C1-6 alkoxy groups, or R １ and R ２ Together with the carbon atoms linked thereto, these form a 5-7 membered carbon ring or a 5-7 membered heterocycle, and the heterocycle contains one or more O, S, N, carbonyl groups, sulfoxide groups or sulfone groups, or any combination thereof. R ３ H, halogen, -OH, -NH ２ , selected from optionally substituted C1-6 alkyl groups and optionally substituted C1-6 alkoxy groups, or R ３ And X, together with the carbon atoms linked thereto, form a 5-7 membered carbon ring or a 5-7 membered heterocycle, and the heterocycle contains one or more O, S, N, carbonyl groups, sulfoxide groups or sulfone groups, or any combination thereof, or R ３ and R ２ Together with the carbon atoms linked thereto, these form a 5-7 membered carbon ring or a 5-7 membered heterocycle, and the heterocycle contains one or more O, S, N, carbonyl groups, sulfoxide groups or sulfone groups, or any combination thereof. W is either nonexistent or exists, and if W exists, W is -O-, -S-, -NR ４ - 【Chemistry 24】 The winner is selected from the following, the first place winner is linked to X, and the second place winner is linked to L. ４ or L ３ Connect to, X is directly bonded, or optionally substituted -O-(CH ２ ) ｎ３ -, -NR ４ - (CH ２ ) ｎ３ -, -S-(CH ２ ) ｎ３ -, carbonyl- (CH ２ ) ｎ３ -, -SO ２ - (CH ２ ) ｎ３ -, - (CH ２ ) ｎ１ - 【Chemistry 25】 Selected from C3-6 cycloalkyl groups, C6-10 aryl groups, 5-10 membered heteroaryl groups, and 4-10 membered heterocyclic groups, with the 1st position linked to the parent ring and the 2nd position being W or L. ４ The substituents are selected from one or more C1-4 alkyl groups and C3-6 cycloalkyl groups, or multiple C1-4 alkyl groups, together with carbon atoms simultaneously linked to them, form a C3-6 cycloalkyl group. Each M is independently directly bonded and -CR ５ａ R ５ｂ - Selected from, R ４ , R ５ , R ５ａ , R ５ｂ , R ６ , R ７ Each is independently selected from H, an optionally substituted C1-4 alkyl group, an optionally substituted C1-4 alkoxy group, and an optionally substituted C3-6 cycloalkyl group. n, n', n1, n2, and n3 are each independently selected from any integer between 0 and 6. The first position is linked to the camptothecin nucleus, and the second position is W or L. ４ Characterized by being connected to, An antibody-drug conjugate according to claim 17, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

19. (1) R １ , R ２ Each is independently selected from H, halogen, and C1-4 alkyl groups, or R １ and R ２ Together with the carbon atoms linked to them, they form a 5-6 membered heterocycle, and the heterocycle contains one, two, or three O, S, or N atoms, or any combination thereof. (2) R ３ is selected from H, C1-4 alkyl groups, or R ３ And X, together with the carbon atoms linked to them, form a 5-6 membered carbon ring. (3) W is either nonexistent or exists, and if W exists, W is -O-, -S-, -NR ４ - 【Chemistry 26】 The winner is selected from the following, the first place winner is linked to X, and the second place winner is linked to L. ４ or L ３ Connect to, (4) X is optionally substituted with -(CH ２ ) ｎ１ - 【Chemistry 27】 Selected from C6-10 aryl groups, 5-10 membered heteroaryl groups, and 4-10 membered heterocyclic groups, with the 1st position linked to the parent ring and the 2nd position being W or L. ４ The substituents are selected from one or two C1-4 alkyl groups, or two C1-4 alkyl groups, together with carbon atoms simultaneously linked to them, form a C3-6 cycloalkyl group. (5) Note ４ , R ５ Each is independently selected from H, C1-4 alkyl groups, and C3-6 cycloalkyl groups. (6) R ５ａ , R ５ｂ Each is independently selected from H and C1-4 alkyl groups, (7) Each R ７ These are independently selected from H and C1-4 alkyl groups. (8) n is selected from 1, 2 and 3, (9) n1 is selected from 1, 2, 3 and 4, (10) n2 is 1, (11) n3 is 0, A feature that satisfies one or more of the following conditions: An antibody-drug conjugate according to claim 18, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

20. (1) R １ is selected from H and halogen, R ２ is selected from H and C1-4 alkyl groups, or R １ and R ２ Along with the carbon atoms that are linked to them, 【Chemistry 28】 The dashed line indicates the position where the heterocycle condenses with the benzene ring. (2) R ３ is H, or R ３ And X, along with the carbon atoms linked to them, 【Chemistry 29】 The dashed line indicates the position where the carbon ring condenses with the benzene ring and the pyridine ring. (3) W is either nonexistent or exists, and if W exists, W is -O-, -S-, -NR ４ - 【Transformation 30】 The winner is selected from the following, the first place winner is linked to X, and the second place winner is linked to L. ４ or L ３ Connect to, (4) Each R ４ R is independently selected from H, C1-4 alkyl groups and C3-6 cycloalkyl groups, ５ H is, (5) Note ５ａ , R ５ｂ These are independently selected from H and methyl groups, (6) R ７ H is, (7) n is 1, A feature that satisfies one or more of the following conditions: An antibody-drug conjugate according to claim 18, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

21. (1) R １ is H or F, and R ２ is H or a methyl group, or R １ and R ２ Along with the carbon atoms that are linked to them, 【Chemistry 31】 The dashed line indicates the position where the heterocycle condenses with the benzene ring. (2) W is -O-, -NR ４ - and 【Chemistry 32】 The winner is selected from the following, the first place winner is linked to X, and the second place winner is linked to L. ４ or L ３ Connect to, (3) Each R ４ R is independently selected from H, methyl group, ethyl group, n-propyl group, isopropyl group, tert-butyl group and cyclopropyl group, ５ H is, (4) X is optionally replaced 【Transformation 33】 Selected from the options, the first position is linked to the parent ring, and the second position is W or L ４ The substituent is selected from one or two C1-4 alkyl groups, or two C1-4 alkyl groups, together with carbon atoms simultaneously linked to them, form a C3-6 cycloalkyl group, where the C1-4 alkyl group is, for example, a methyl group, and the C3-6 cycloalkyl group is, for example, a cyclopropyl group. A feature that satisfies one or more of the following conditions: An antibody-drug conjugate according to claim 18, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

22. (1) W is -O- and -NR ４ - Selected from, (2) X is, 【Transformation 34】 Selected from the options, the first position is linked to the parent ring, and the second position is W or L ４ Characterized by being connected to, An antibody-drug conjugate according to claim 18, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

23. If W does not exist, X is 【Chemistry 35】 Selected from, characterized in that the first position is connected to the parent ring and the second position is connected to W, An antibody-drug conjugate according to claim 18, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. 【Request Item 24】 【Chemistry 36】 The structure is selected from the following: 【Chemistry 37-1】 【Chemistry 37-2】 【Chemistry 37-3】 During the ceremony, the winner was L ４ Connected to L ４ If there is no such thing, the first place goes to L ３ Characterized by being connected to, An antibody-drug conjugate according to claim 18, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

25. The antibody-drug conjugate has a structure represented by formula I-1, formula I-2, or formula I-3. 【Transformation 38】 In the formula, Tb, L １ , L ２ , L ３ , L ４ X, R １ , R ２ , R ３ , R ４ And q are as defined in claim 18, 【Chemistry 39】 In the formula, Tb, L １ , L ２ , L ３ , L ４ X, R １ , R ２ , R ３ And q are as defined in claim 18, 【Chemistry 40】 In the formula, Tb, L １ , L ２ , L ３ , L ４ X, R １ , R ２ , R ３ , R ４ , R ５ n and q are as defined in claim 18, An antibody-drug conjugate according to claim 18, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

26. The antibody-drug conjugate has a structure represented by formula I-1A, formula I-1B, formula I-2A, formula I-2B, formula I-3A, or formula I-3B. 【Chemistry 41】 In the formula, Tb, L ２ , L ３ , L ４ X, R １ , R ２ , R ３ , R ４ And q are as defined in claim 18, 【Chemistry 42】 In the formula, Tb, L ２ , L ３ , L ４ X, R １ , R ２ , R ３ And q are as defined in claim 18, 【Chemistry 43】 In the formula, Tb, L ２ , L ３ , L ４ X, R １ , R ２ , R ３ , R ４ and q are as defined in claim 18, An antibody-drug conjugate according to claim 18, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

27. The antibody-drug conjugate has a structure represented by formula I-A or formula I-B, 【Chemistry 44】 In the formula, Tb, X, R １ , R ２ , R ３ , R ａ , R ｂ And q are as defined in claim 18, 【Chemistry 45】 In the formula, Tb, X, R １ , R ２ , R ３ , R ａ , R ｂ and q are as defined in claim 18, An antibody-drug conjugate according to claim 18, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

28. The antibody-drug conjugate is Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Characterized by being selected from, An antibody-drug conjugate according to claim 9, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

29. Tb is an anti-NaPi2b antibody or its antigen-binding fragment, and preferably the anti-NaPi2b antibody is selected from lifastuzumab, upifitamab, 60A12B3-hz1, 22H9C4-hz1, 66C12D12-hz1, 34F2A10-hz1, C1, 16G5B3-hz2 and 16G5B3-hz1 antibodies or their antigen-binding fragments. An antibody-drug conjugate according to claim 9, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

30. Tb is characterized by being selected from the anti-NaPi2b antibody or its antigen-binding fragment according to any one of claims 1 to 8. An antibody-drug conjugate according to any one of claims 9 to 29, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

31. The antibody-drug conjugate is 【Chemistry 46-1】 【Chemistry 46-2】 Selected from, In the formula, Tb １ The antibodies are lifastuzumab, upifitamab, 60A12B3-hz1, 22H9C4-hz1, 66C12D12-hz1, 34F2A10-hz1, C1, 16G5B3-hz2, and 16G5B3-hz1 antibodies, and q is selected from any numerical value between 0.1 and 16.0, preferably any numerical value between 2 and 8, and more preferably q is 2, 4, 6, or 8. An antibody-drug conjugate according to claim 30, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

32. A method for preparing an antibody-drug conjugate according to claim 30, Tb and the drug linker complex shown in formula III 【Chemistry 47】 This includes coupling the two in an appropriate solvent and under suitable conditions. During the ceremony, L １ , L ２ , L ３ , L ４ , Tb is as defined in claim 30, R １ , R ２ , R ３ X and W are as defined in claim 30, Lg is a leaving group, and Lg is selected from halogens, sulfone groups, tertiary amine bases (Me3N+, Et3N+), diazonium bases, -OMs, MeSO2-, and CF3SO3-. method.

33. The above method involves Tb and a drug linker complex represented by formula III. 【Chemistry 48】 The step includes a coupling reaction between and under appropriate solvents and conditions to form a C-S bond, Preferably, the ratio of the amount of Tb to the amount of the drug linker complex substance is 1:(1 to 20), Preferably, the coupling reaction is carried out in water and / or an organic solvent, preferably the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, a nitrile (e.g., acetonitrile), an alcohol (e.g., methanol, ethanol), or any combination thereof, wherein the nitrile may be acetonitrile, and the alcohol may be methanol or ethanol. Preferably, the method further comprises a step of purifying the complex product, preferably the complex product being purified by a chromatographic method, preferably the chromatographic method comprising one or more of ion exchange chromatography, hydrophobic chromatography, reversed-phase chromatography, or affinity chromatography. The method according to claim 32.

34. Encoding the anti-NaPi2b antibody or its antigen-binding fragment as described in claim 1, Isolated nucleic acid molecules.

35. A vector comprising an isolated nucleic acid molecule as described in claim 34, preferably the vector being a cloning vector or an expression vector. vector.

36. A vector comprising the isolated nucleic acid molecule described in claim 34 or the vector described in claim 35, host cell.

37. A method for preparing an anti-NaPi2b antibody or an antigen-binding fragment thereof according to claim 1, comprising: culturing the host cells according to claim 36 under conditions that enable the expression of the antibody or the antigen-binding fragment thereof; and recovering the antibody or the antigen-binding fragment thereof from the cultured host cell culture. method.

38. An antibody-drug conjugate wherein the antibody is an anti-NaPi2b antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 8, linked to a complex portion via a linker, the complex portion being selected from a detectable label, a radioisotope, a fluorescent substance, a luminescent substance, a coloring substance, an enzyme, polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide having binding activity, a protein, a receptor, a ligand, and other active substances that inhibit tumor cell growth and promote apoptosis or necrosis of tumor cells. Antibody-drug conjugate.

39. A group of antibody-drug conjugates, comprising the antibody-drug conjugate described in claim 30, wherein the antibody-drug conjugate has one, two or more q values. A group of antibody-drug conjugates.

40. A pharmaceutical composition comprising the antibody-drug conjugate described in claim 30, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, preferably further comprising a pharmaceutically acceptable carrier and / or excipient. Pharmaceutical composition.

41. A pharmaceutical composition comprising an anti-NaPi2b antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 8, and preferably further comprising a pharmaceutically acceptable carrier and / or excipient. Pharmaceutical composition.

42. The drug-to-antibody ratio (DAR) is selected from an integer or decimal between 1 and 10. Preferably, the drug-to-antibody ratio (DAR) of the population is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, and 7.5-8.

5. Preferably, the drug-to-antibody ratio (DAR) of the population is selected from approximately 2.0, 4.0, 6.0, and 8.

0. Preferably, the drug-to-antibody ratio (DAR) of the population is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, and 9. The group described in claim 39.

43. The use of an antibody-drug conjugate according to claim 30, or a stereoisomer of an antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, in the preparation of a drug for treating and / or preventing a disease associated with abnormal cellular activity, Selectively, the disease associated with the abnormal cellular activity may be a cancerous disease. Preferably, the cancerous disease is selected from esophageal cancer, brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, kidney cancer, urothelial carcinoma, epidermal carcinoma, non-Hodgkin lymphoma, central nervous system tumor, prostate cancer, or thyroid cancer, where the esophageal cancer is, for example, esophageal adenocarcinoma or esophageal squamous cell carcinoma, the lung cancer is, for example, small cell lung cancer, non-small cell lung cancer, or lung adenocarcinoma, the central nervous system tumor is, for example, glioma, glioblastoma multiforme, glioma, or sarcoma, and the colon cancer is, for example, human colon adenocarcinoma. Preferably, the cancerous disease is selected from colon cancer, colorectal cancer, colon adenocarcinoma, small cell lung cancer, and ovarian cancer. More preferably, the cancerous disease is a cancerous disease related to NaPi2b. use.

44. Use of an anti-NaPi2b antibody or its antigen-binding fragment according to any one of claims 1 to 8 in the preparation of a drug for treating and / or preventing a disease related to abnormal cell activity, Selectively, the disease associated with the abnormal cellular activity may be a cancerous disease. Preferably, the cancerous disease is selected from esophageal cancer, brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, kidney cancer, urothelial carcinoma, epidermal carcinoma, non-Hodgkin lymphoma, central nervous system tumor, prostate cancer, or thyroid cancer, where the esophageal cancer is, for example, esophageal adenocarcinoma or esophageal squamous cell carcinoma, the lung cancer is, for example, small cell lung cancer, non-small cell lung cancer, or lung adenocarcinoma, the central nervous system tumor is, for example, glioma, glioblastoma multiforme, glioma, or sarcoma, and the colon cancer is, for example, human colon adenocarcinoma. Preferably, the cancerous disease is selected from colon cancer, colorectal cancer, colon adenocarcinoma, small cell lung cancer, and ovarian cancer. More preferably, the cancerous disease is a cancerous disease related to NaPi2b. use.

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