TWI876009B - Antibody-drug conjugates and medical use thereof - Google Patents

Abstract

The present invention relates a kind of antibody-drug conjugates and medical use thereof. Concretely, the present invention relates a kind of anti-BCMA antibody-drug conjugates and medical use thereof. Furtherly, the present invention relates antibody-drug conjugates containing anti-BCMA antibody or its antigen-binding fragment, their medicinal salts or solvates thereof, the use in the preparation of medicine for the treatment of BCMA mediated diseases or conditions thereof, and the use in tumor detection and diagnosis.

Description

Antibody-drug conjugates and their medical uses

The present invention belongs to the field of biomedicine. Specifically, the present invention relates to anti-BCMA antibody conjugates and their medical uses.

B cells are lymphocytes that play an important role in adaptive humoral immunity and the production of antibodies that specifically recognize antigens. The three subclasses of B cells are naive B cells, memory B cells, and plasma cells. In the process of VDJ recombination, in which two or three fragments of DNA are selected from a genomic library and recombined to produce a combinatorial array of antibody variable domains, further changes in the variable domains encoded by B cells of different lineages lead to up to 10 ⁹ unique B cell lineages, which produce antibodies that are specific to unique targets. B cells are involved in a variety of diseases, and malignant transformation of B cells leads to cancer, including some lymphomas, such as multiple myeloma and Hodgkin's lymphoma. B cells are also involved in autoimmune diseases, including systemic lupus erythematosus (SLE) and IgA nephropathy. Cancers and autoimmune diseases involving B cells can be considered to be abnormal B cell function, so a possible strategy to control these diseases is to use antibodies that target the pathological B cells.

BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily and is a non-glycosylated intrinsic membrane receptor for the ligands BAFF (B cell activating factor) and APRIL (proliferation inducing ligand). BCMA and its corresponding ligands have different roles in regulating humoral immunity, B cell development and homeostasis. BCMA is expressed by tonsil memory B cells and by germinal center B cells, and can be detected in the spleen, lymph nodes, thymus, adrenal glands and liver, and analysis of multiple B cell lines has shown that BCMA expression levels increase after maturation.

Antibodies against BCMA are described, for example, in Gras M-P. et al. Int Immunol. 7 (1995) 1093-1106, WO200124811, WO200124812, WO2010104949 and WO2012163805. Antibodies against BCMA and their use for treating lymphoma and multiple myeloma are described, for example, in WO2002066516 and WO2010104949. WO2013154760 relates to a chimeric antigen receptor comprising a BCMA recognition portion and a T-cell activation portion.

US9273141 provides an antigen binding protein capable of internalization, specifically binding to BCMA and inhibiting the binding of BAFF and/or APRIL to BCMA, and provides a complex comprising the antigen binding protein and a cytotoxic agent.

The purpose of the present invention is to provide an anti-BCMA antibody-drug conjugate, or a pharmaceutically acceptable salt or solvent compound thereof, which is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof represented by the general formula (A),

Ab-(L ₂ -L ₁ -D) _y (A)

in,

D is a cytotoxic drug;

L1 _{and L2} are connector units;

y is a number from 1 to 20;

Ab is an anti-BCMA antibody or an antigen-binding fragment thereof, wherein the anti-BCMA antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and an antibody light chain variable region, wherein the antibody heavy chain variable region comprises at least one HCDR selected from the following sequences: SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and the antibody light chain variable region comprises at least one LCDR selected from the following sequences: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

In a preferred embodiment of the present invention, the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof, wherein the heavy chain variable region of the anti-BCMA antibody or its antigen-binding fragment comprises: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the light chain variable region of the anti-BCMA antibody or its antigen-binding fragment comprises: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-BCMA antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region of the anti-BCMA antibody or its antigen-binding fragment comprises: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region comprises: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, the CDR sequence of the anti-BCMA antibody or its antigen-binding fragment may undergo 1 to 3 amino acid mutations that optimize antibody activity, antibody stability or reduce immunogenicity compared to the original CDR sequence.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, the anti-BCMA antibody or its antigen-binding fragment is a murine antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a human antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, the anti-BCMA antibody or its antigen-binding fragment further comprises a heavy chain constant region of human IgG1 or its variant, IgG2 or its variant, IgG3 or its variant or IgG4 or its variant.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention, the Ab antibody or its antigen-binding fragment further comprises a human IgG1, IgG2 or IgG4 heavy chain constant region.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises a human IgG1 heavy chain constant region with enhanced ADCC toxicity after amino acid mutation.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises a heavy chain constant region as shown in SEQ ID NO: 22.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, the anti-BCMA antibody or its antigen-binding fragment further comprises a light chain constant region derived from a human antibody κ chain, λ chain or a variant thereof. Preferably, the anti-BCMA antibody or its antigen-binding fragment further comprises a light chain constant region derived from a human antibody κ chain.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, the anti-BCMA antibody or its antigen-binding fragment further comprises a light chain constant region as shown in SEQ ID NO: 23.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-BCMA antibody or its antigen-binding fragment comprises a heavy chain variable region selected from the following sequences: SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-BCMA antibody or its antigen-binding fragment comprises a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the following sequences: SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

In a preferred embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment comprises a light chain variable region selected from the following sequences: SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-BCMA antibody or its antigen-binding fragment comprises a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the following sequences: SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.

In a preferred embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment contains a heavy chain shown in the following sequence: SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.

In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-BCMA antibody or its antigen-binding fragment comprises a heavy chain having at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.

In a preferred embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment contains a light chain selected from the following sequences: SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

In a preferred embodiment of the present invention, such as the anti-BCMA antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-BCMA antibody or its antigen-binding fragment comprises a light chain having at least 80%, 85%, 90%, 95% or 99% identity with the following sequences: SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

In a better embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 9 and the light chain variable region shown in SEQ ID NO: 12.

In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is selected from SEQ ID NO: 9, or has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 9, and the light chain variable region is selected from SEQ ID NO: 12, or has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 12.

In a better embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 10 and the light chain variable region shown in SEQ ID NO: 13.

In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is selected from SEQ ID NO: 10, or has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 10, and the light chain variable region is selected from SEQ ID NO: 13 or has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 13.

In a better embodiment of the present invention, the anti-BCMA antibody comprises the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 14.

In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is selected from SEQ ID NO: 11, or has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 11, and the light chain variable region is selected from SEQ ID NO: 14, or has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 14.

In a better embodiment of the present invention, the anti-BCMA antibody comprises the heavy chain shown in SEQ ID NO: 15 and the light chain shown in SEQ ID NO: 18.

The anti-BCMA antibody comprising the heavy chain shown in SEQ ID NO: 15 and the light chain shown in SEQ ID NO: 18 is Ab1.

In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain is selected from SEQ ID NO: 15, or has at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 15, and the light chain is selected from SEQ ID NO: 18, or has at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 18.

In a better embodiment of the present invention, the anti-BCMA antibody comprises the heavy chain shown in SEQ ID NO: 16 and the light chain shown in SEQ ID NO: 19.

The anti-BCMA antibody comprising the heavy chain shown in SEQ ID NO: 16 and the light chain shown in SEQ ID NO: 19 is Ab2.

In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain is selected from SEQ ID NO: 16, or has at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 16, and the light chain is selected from SEQ ID NO: 19, or has at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 19.

In a better embodiment of the present invention, the anti-BCMA antibody comprises the heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 20.

The anti-BCMA antibody comprising the heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 20 is Ab3.

In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain is selected from SEQ ID NO: 17, or has at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 17, and the light chain is selected from SEQ ID NO: 20, or has at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 20.

In one embodiment of the present invention, the cytotoxic drug is selected from toxins, chemotherapeutic drugs, antibiotics, radioisotopes and nucleolytic enzymes.

In a preferred embodiment of the present invention, the cytotoxic drug is selected from tubulin inhibitors or DNA topoisomerase inhibitors that inhibit cell division; preferably DM1, DM3, DM4, camptothecin, SN-38, MMAF or MMAE; more preferably tubulin inhibitors MMAE or MMAF, or DNA topoisomerase inhibitor SN-38.

In a further preferred embodiment of the present invention, the cytotoxic drug is selected from:

In a preferred embodiment of the present invention, the cytotoxic drug is selected from camptothecin derivatives, preferably ixotecan:

In a better embodiment of the present invention, the cytotoxic drug is an isotecan derivative, preferably, the isotecan derivative is compound 2-A:

In some embodiments of the present invention, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound represented by general formula (I),

in,

L1 _{and L2} are connector units;

y is an integer selected from 1 to 8, preferably from 2 to 4;

Ab is selected from anti-BCMA antibodies or antigen-binding fragments thereof as defined above.

In some embodiments of the present invention, according to the antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, it is a compound as shown in general formula (I) or its pharmaceutically acceptable salt or solvent compound,

in,

_L1 and _L2 are connector units:

y is a number selected from 1 to 8, preferably a number from 2 to 8, further a number from 2 to 6, and more preferably a number from 3 to 6;

Ab is selected from anti-BCMA antibodies or antigen-binding fragments thereof as defined above.

In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, it is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as shown in the general formula (I-A):

In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, it is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as shown in the general formula (I-B):

According to the anti-BCMA antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound of the present invention, it is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as shown in the general formula (II):

in,

L1 _{and L2} are connector units;

y is a number selected from 1 to 8, preferably a number from 2 to 4;

Ab is selected from BCMA antibodies or antigen-binding fragments thereof as defined above.

In a preferred embodiment of the present invention, the anti-BCMA antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound according to the present invention is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as shown in the general formula (II-1):

In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, it is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as shown in the general formula (II-A):

In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, it is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as shown in the general formula (II-B):

According to the anti-BCMA antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound of the present invention, it is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as shown in the general formula (III),

in,

L1 _{and L2} are connector units;

y is a number selected from 1 to 10, preferably a number from 2 to 8, and more preferably a number from 4 to 8;

Ab is selected from BCMA antibodies or antigen-binding fragments thereof as defined above.

In some embodiments of the present invention, the L ₁ is as shown in the general formula (B):

in,

_{M1 is -CR1R2-} ;

_R1 and _R2 are the same or different, and _R1 and _R2 are each independently selected from hydrogen, alkyl, halogen, hydroxyl or amine;

n is an integer from 0 to 5, preferably 1, 2 or 3.

In a preferred embodiment of the present invention, the heterocyclic end of _L1 is connected to _L2 .

In some embodiments of the present invention, the L ₂ is represented by the following general formula (C):

in,

_{M2 is -CR4R5-} ;

_R3 is selected from hydrogen, halogen, hydroxyl, amino, alkyl, alkoxy and cycloalkyl:

_R4 and _R5 are the same or different, and _R4 and _R5 are each independently selected from hydrogen, alkyl, halogen, hydroxyl or amine;

m is an integer from 0 to 5, preferably 1, 2 or 3.

In a preferred embodiment of the present invention, the S end of _L2 is connected to the connector unit _L1 .

In a further preferred embodiment of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent thereof as shown in formula (I), formula (IA) or formula (IB) has a linker unit _L1 as defined in the above formula (B) and a linker unit _L2 as defined in the above formula (C).

In a further preferred embodiment of the present invention, the antibody-drug conjugate represented by the general formula (II-A) or a pharmaceutically acceptable salt or solvent thereof has a linker unit L ₂ as defined in the above general formula (C).

In some embodiments of the present invention, the L ₂ is as shown in the general formula (D):

-K ¹ -K ² -K ³ -K ⁴ - (D)

in,

，s選自2至8的整數，進一步選為4至8的整數，更較佳為4至6的整數； K ¹ is

, s is selected from an integer from 2 to 8, further selected from an integer from 4 to 8, and more preferably selected from an integer from 4 to 6;

^K2 is ^-NR1 ( _CH2CH2O ) _{pCH2CH2C(O )} -, _-NR1 ( _CH2CH2O ) _pCH2C ( ^O )-, _-S ( _CH2 ) _pC ( _O )- or a single bond, p is an integer selected from 1 to 20, preferably an integer from 1 to 6 _;

^R1 is selected from hydrogen, deuterium, hydroxyl, amine, alkyl, halogen, halogenated alkyl, deuterated alkyl and hydroxyalkyl;

^K3 is a tetrapeptide residue, preferably, the tetrapeptide residue is a peptide residue formed by two or more amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamate, and aspartic acid; more preferably, it is a tetrapeptide residue of GGFG;

K ⁴ is -NR ² (CR ³ R ⁴ )t-, R ² , R ³ or R ⁴ are each independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, halogenated alkyl, deuterated alkyl and hydroxyl alkyl, t is selected from 1 or 2,

The ^K1 end of the linker unit _-L2- is connected to Ab, and the ^K4 end is connected to _L1 .

，s為5； In a more preferred embodiment of the present invention, ^K1 is

, s is 5;

K ² is the key;

K ³ is the tetrapeptide residue of GGFG;

K ⁴ is -NR ² (CR ³ R ⁴ )t-, R ² , R ³ or R ⁴ are each independently hydrogen, deuterium, hydroxyl, amino, C _1-6 alkyl, halogen, C _1-6 halogenated alkyl, C _1-6 deuterated alkyl and C _1-6 hydroxyl alkyl, t is 1 or 2,

The connector unit -L ₂ - has its K ¹ end connected to Ab and its K ⁴ end connected to L ₁ .

In some embodiments of the invention, _L1 is selected from a bond, ^-O- ( ^{CRaRb )} _m - ^{CR5R6 -C} (O)-, -O- ^CR5R6- ( ^{CRaRb )} _m- , ^-O - ^CR5R6- , -NH-( ^{CRaRb )} _m- ^CR5R6 - ^C (O)- or -S-( ^{CRaRb )} _m- ^CR5R6 - ^C (O)-;

^Ra and ^Rb are each independently selected from hydrogen, deuterium, halogen or alkyl;

^R5 is a halogen group or a cycloalkyl group;

^R6 is selected from hydrogen, halogen alkyl or cycloalkyl;

Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a cycloalkyl group;

m is selected from 0, 1, 2, 3 or 4.

Preferably, the O end of _L1 is connected to the connector unit _L2 .

In a preferred embodiment of the present invention, L ₁ is represented by the general formula (E):

wherein R ⁵ is selected from a halogen alkyl group or a cycloalkyl group, and R ⁶ is selected from a hydrogen group, a halogen alkyl group or a cycloalkyl group, or R ⁵ and R ⁶ together with the carbon atom to which they are connected form a cycloalkyl group;

Preferably, R ⁵ is selected from C _1-6 haloalkyl or C _3-6 cycloalkyl, R ⁶ is selected from hydrogen, C _1-6 haloalkyl or cycloalkyl, or R ⁵ and R ⁶ together with the carbon atom to which they are attached form a C _3-6 cycloalkyl;

m is an integer selected from 0 to 4,

Preferably, the general formula (E) is selected from the following substituents:

In a preferred embodiment of the present invention, the -L ₂ -L ₁ - is selected from the following structures:

K ² is the key;

K ³ is the tetrapeptide residue of GGFG;

^R5 is a halogen group or a _C3-6 cycloalkyl group;

^R6 is selected from hydrogen, halogen alkyl or _C3-6 cycloalkyl;

Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

R ² , R ³ or R ⁴ are each independently selected from hydrogen or alkyl;

s is selected from an integer from 2 to 8; preferably, s is selected from an integer from 4, 5 or 6;

m is an integer selected from 0 to 4;

Preferably, -L ₂ -L ₁ - is selected from the following structures:

In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, it is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as shown in the general formula (IV):

in,

W is selected from _C1-8 alkyl, _C1-8 alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, optionally, wherein the _C1-8 alkyl, cycloalkyl and straight chain heteroalkyl are each independently further substituted with one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

^K2 is selected from ^-NR1 ( _CH2CH2O ) _p1CH2CH2C (O _{) -} , _-NR1 ( _CH2CH2O ) _p1CH2C (O ₎ -, ^-S ( _CH2 ) _p1C (O)- or a bond, ^R1 is selected from hydrogen, alkyl, halogenated alkyl, deuterated alkyl and hydroxyalkyl, and _p1 is selected from an integer from 1 to 20 _;

^K3 is a peptide residue consisting of 2 to 7 amino acids, the amino acids may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available attachment point, the substituent being one or more independently selected from halogen, hydroxyl, cyano, amine, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

^R2 is selected from hydrogen, alkyl, halogenated alkyl, deuterated alkyl and hydroxyalkyl;

^R3 and ^R4 are each independently selected from hydrogen, halogen, alkyl, halogenated alkyl, deuterated alkyl and hydroxyalkyl;

^R5 is selected from halogen, halogenalkyl, deuterated alkyl, cycloalkyl, heterocyclo, aryl or heteroaryl;

^R6 is selected from hydrogen, halogen, halogenated alkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

Alternatively, R ⁵ and R ⁶ together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclic group;

m is an integer selected from 0 to 4;

y is a number selected from 1 to 10, y is a decimal or an integer;

Ab is selected from anti-BCMA antibodies or antigen-binding fragments thereof as defined above.

In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, it is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as shown in the general formula (IV-A):

In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvent compound, it is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as shown in the general formula (IV-A), wherein,

^R5 is selected from halogen alkyl or _C3-6 cycloalkyl;

^R6 is selected from hydrogen, halogen alkyl or _C3-6 cycloalkyl;

Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

R ² , R ³ or R ⁴ are each independently selected from hydrogen or alkyl;

s is an integer selected from 2 to 8;

m is an integer from 0 to 4.

In a better embodiment of the present invention, according to the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound of the present invention, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound represented by the general formula (IV-A) is selected from the following structures:

According to some embodiments of the present invention, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound is selected from the following compounds,

In a preferred embodiment of the present invention, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound is selected from the following compounds:

Wherein, y is selected from 2 to 10, preferably 4 to 8, more preferably 6 to 8, further preferably 7 to 8, and most preferably 6 or 8; or y is selected from 1 to 10, preferably 2 to 10, further preferably 2 to 6, more preferably 3 to 6, and most preferably 6.

In a preferred embodiment, the present invention relates to a method for preparing an antibody-drug conjugate as shown in general formula (IV) or a pharmaceutically acceptable salt or solvate thereof, which comprises the following steps:

After Ab is reduced, it is coupled with the general formula (F) to obtain the compound represented by the general formula (IV);

in,

Ab is an anti-BCMA antibody or an antigen-binding fragment thereof as defined above;

W, K ² , K ³ , R ² to R ⁶ , m and y are as defined in the general formula (IV).

In a preferred embodiment, the general formula (F) is a compound represented by the general formula (F-1):

or its tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or its pharmaceutically acceptable salts,

wherein K ² , K ³ , R ² to R ⁶ , s and m are as defined above in -L ₂ -L ₁ -.

In a preferred embodiment, the compound represented by general formula (F) or general formula (F-1) is selected from:

On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound, and one or more pharmaceutically acceptable excipients, diluents or carriers.

On the other hand, the present invention provides a medical use, which relates to the use of an anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate or a pharmaceutical composition thereof for treating or preventing BCMA-mediated diseases or conditions.

On the other hand, the present invention provides a medical use, which relates to the use of an anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate or a pharmaceutical composition thereof in the preparation of a drug for treating or preventing a BCMA-mediated disease or condition.

In a preferred embodiment of the present invention, the BCMA-mediated disease or disorder is cancer or an autoimmune disease, wherein the cancer is preferably a cancer expressing BCMA, more preferably lymphoma, leukemia or myeloma, and most preferably multiple myeloma; the autoimmune disease is selected from lupus erythematosus, IgA nephropathy and rheumatoid arthritis.

The antibody-drug conjugate and its pharmaceutically acceptable salt or solvent compound of the present invention have obvious tumor inhibition effects in vitro and in vivo, low toxicity to normal cells or tissues, good safety, and good competitive inhibition effects on related ligands and soluble BCMA in serum; they have good drug stability in vitro, which is convenient for long-term storage and transportation; at the same time, they have good metabolic activity in vivo, long-term in vivo efficacy, and broad prospects for clinical application.

Description of invention

1. Terminology

In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by a person of ordinary skill in the art to which the present invention belongs.

The three-letter codes and single-letter codes of amino acids used in the present invention are as described in J.Biol.Chem, 243, p3558 (1968).

The term "antibody" mentioned in the present invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the heavy chain of immunoglobulin are different, so its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain respectively. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4. Light chains are classified into kappa chains or lambda chains according to the difference in the constant region. Each of the five types of Ig can have a kappa chain or a lambda chain.

In the present invention, the antibody light chain variable region described in the present invention may further include a light chain constant region, and the light chain constant region includes a human or mouse κ, λ chain or its variants.

In the present invention, the antibody heavy chain variable region described in the present invention may further include a heavy chain constant region, and the heavy chain constant region includes human or mouse IgG1, IgG2, IgG3, IgG4 or its variants.

The sequences of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region). The variable region includes 3 hypervariable regions (HVR) and 4 relatively conserved framework regions (FR). The 3 hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions, and the order from the amino end to the carboxyl end is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; The three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues in the VL region and VH region of the antibody or antigen-binding fragment of the present invention conform to the known Kabat numbering rules and Kabat or ABM definition rules (http://bioinf.org.uk/abs/).

The term "antigen presenting cell" or "APC" is a cell that displays foreign antigens complexed with MHC on its surface. T cells recognize this complex using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes, B lymphoblasts, and monocyte-derived dendritic cells.

The term "antigen presentation" refers to the process by which APCs capture antigens and make them available for recognition by T cells, for example as components of MHC-I/MHC-II conjugates.

The term "BCMA" includes any variant or isoform of BCMA naturally expressed by cells. The antibodies of the present invention may cross-react with BCMA obtained from non-human species. Alternatively, the antibody may also be human BCMA-specific and may not show cross-reactivity with other species. BCMA or any variant or isoform thereof may be isolated from cells or tissues that naturally express them, or produced by recombinant technology using techniques commonly used in the art and those described herein. Preferably, the anti-BCMA antibody targets human BCMA with a normal glycosylation pattern.

The term "recombinant human antibody" includes human antibodies prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as:

1. Antibodies isolated from transgenic or transchromosomal animals (such as mice) containing human immunoglobulin genes or fusion tumors prepared therefrom;

2. Antibodies isolated from host cells transformed to express antibodies, such as transfectomas;

3. Antibodies isolated from recombinant human antibody libraries; and

4. Antibodies prepared, expressed, created or isolated by splicing human immunoglobulin gene sequences to other DNA sequences.

Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes but also include subsequent rearrangements and mutations that occur, for example, during antibody maturation.

The term "mouse antibody" in the present invention refers to a monoclonal antibody against human BCMA prepared according to the knowledge and skills in this field. During preparation, the test subject is injected with BCMA antigen, and then the fusion tumor expressing the antibody with the desired sequence or functional properties is isolated. In a preferred embodiment of the present invention, the murine BCMA antibody or its antigen-binding fragment may further contain the light chain constant region of the murine κ, λ chain or its variant, or further contain the heavy chain constant region of the murine IgG1, IgG2, IgG3 or IgG4 or its variant.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic cell mutagenesis in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (i.e., "humanized antibodies").

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the human antibody variable region framework. Humanized antibodies can overcome the disadvantage of chimeric antibodies that they carry a large amount of mouse protein components, thus inducing a strong immune response. In order to avoid a decrease in activity while reducing immunogenicity, the variable region of the human antibody can be subjected to minimal reverse mutations to maintain activity.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, a fusion tumor that secretes mouse-specific monoclonal antibodies must be selected, and then the variable region gene is cloned from the mouse fusion tumor cells, and the constant region gene of the human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are connected to form a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising the heavy chain constant region of human IgG1, IgG2 or IgG4, or the heavy chain constant region of IgG1 with enhanced ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogs, which generally include at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of a parental antibody. Antibody fragments retain at least some of the binding specificity of the parental antibody. Typically, when activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23: 1126-1136.

The "Fab fragment" consists of a light chain and the CH1 and variable region of a heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The "Fc" region contains two heavy chain fragments that comprise the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

A "Fab' fragment" contains a light chain and a portion of a heavy chain including the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

A "F(ab')2 fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by the disulfide bonds between the two heavy chains.

The "Fv region" contains variable regions from both the heavy and light chains, but lacks a constant region.

The term "multispecific antibody" is used in its broadest sense to cover antibodies with multiple epitope specificities. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has multiple epitope specificities; antibodies having two or more VL and VH regions, each VH-VL unit binds to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binds to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments covalently or non-covalently linked together, etc.

The term "single-chain antibody" refers to a single-chain recombinant protein composed of the heavy chain variable region VH and the light chain variable region VL of the antibody connected by a linker peptide. It is the smallest antibody fragment with a complete antigen binding site.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment can target the same or different antigens.

The term "binding to BCMA" in the present invention means being able to interact with human BCMA.

The term "antigen binding site" of the present invention refers to the three-dimensional spatial site recognized by the antibody or antigen binding fragment of the present invention.

The term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. Epitopes generally include at least 3 to 15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

The terms "specific binding" and "selective binding" used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when human BCMA is used as an analyte and an antibody is used as a ligand, the antibody binds to the predetermined antigen with an equilibrium dissociation constant ( _KD ) of approximately less than ^10-7 M or even less when measured in an instrument by surface plasmon resonance (SPR) technology, and its affinity for binding to the predetermined antigen is at least twice its affinity for binding to non-specific antigens other than the predetermined antigen or closely related antigens (such as BSA, etc.). The term "antibody that recognizes an antigen" can be used interchangeably with the term "specifically binding antibody" herein.

The term "cross-reactivity" refers to the ability of an antibody of the invention to bind to BCMA from a different species. For example, an antibody of the invention that binds to human BCMA may also bind to BCMA from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (e.g., SPR and ELISA), or binding or functional interaction with cells that physiologically express BCMA. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of a ligand preferably reduces or alters the normal level or type of activity that occurs when the ligand binds in the absence of inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in ligand binding affinity when contacted with an anti-BCMA antibody compared to the ligand not contacted with an anti-BCMA antibody.

The term "inhibit growth" (e.g. with respect to cells) is intended to include any measurable decrease in cell growth.

The terms "induced immune response" and "enhanced immune response" are used interchangeably and refer to the stimulation of an immune response (i.e., passive or adaptive) to a specific antigen. The term "induced" with respect to induced CDC or ADCC refers to the stimulation of a specific direct cell killing mechanism.

The "ADCC" mentioned in the present invention, namely antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of target cells coated with antibodies by cells expressing Fc receptors by recognizing the Fc segment of antibodies. The ADCC effector function of antibodies can be enhanced, reduced or eliminated by modifying the Fc segment on IgG. The modification refers to mutations in the heavy chain constant region of the antibody.

Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimental Techniques, Chapters 5-8 and 15. For example, mice can be immunized with human BCMA or fragments thereof, and the resulting antibodies can be coated, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments of the invention use genetic engineering methods to add one or more human FR regions to the non-human CDR region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001 ISBN012441351.

The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified by general methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, the mammalian expression system will lead to glycosylation of the antibody, especially at the highly conserved N-terminus of the FC region. Stable pure strains are obtained by expressing antibodies that specifically bind to human antigens. Positive pure strains are expanded and cultured in a serum-free medium in a bioreactor to produce antibodies. The culture fluid secreting the antibody can be purified and collected by general techniques. The antibody can be filtered and concentrated by general methods. Soluble mixtures and polymers can also be removed by general methods, such as molecular screening and ion exchange. The obtained product needs to be immediately frozen, such as -70℃, or freeze-dried.

The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibody (mAb) described in the present invention refers to an antibody obtained from a single pure cell line, which is not limited to a eukaryotic, prokaryotic or phage pure cell line. Monoclonal antibodies or antigen-binding fragments can be recombined using techniques such as fusion tumor technology, recombination technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.

"Administering," "giving," and "treating" as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" may refer to, for example, treatment, pharmacokinetic, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with cells, and contact of an agent with a fluid, wherein the fluid is in contact with cells. "Administering," "administering," and "treating" also mean in vitro and ex vivo manipulation of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," when applied to human, veterinary, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

"Treatment" means administering an oral or topical therapeutic agent, such as one comprising any of the antibodies of the invention, to a patient who has one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the patient or population being treated, either by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be assessed by any clinical test method commonly used by doctors or other professional health care personnel to assess the severity or progression of the symptom. Although the embodiments of the present invention (e.g., treatment methods or products) may not be effective in alleviating the target disease symptom in every patient, they should reduce the target disease symptom in a statistically significant number of patients as determined by any statistical test method known in the art, such as Student's t test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

The term "consisting essentially of..." or variations thereof used throughout the specification and application means including all the elements or groups of elements described, and optionally including other elements of similar or different properties to the element, which other elements do not significantly change the basic or novel properties of the specified dosage regimen, method or composition.

The term "naturally occurring" as applied to a certain object in the present invention refers to the fact that the object can be found in nature. For example, a polypeptide sequence or polynucleotide sequence that exists in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by humans in a laboratory is naturally occurring.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's general health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

"Exogenous" refers to substances produced outside of an organism, cell, or human body, depending on the context.

"Endogenous" refers to substances that are produced in cells, organisms or the human body in some context.

"Identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percent identity.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include their progeny. Thus, the words "transformants" and "transformed cells" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all progeny may not be exactly the same in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as that screened in the original transformed cell are included. "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur. For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a specific sequence may but need not be present.

"Pharmaceutical composition" means a composition containing one or more antibodies or antigen-binding fragments thereof, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thereby exert biological activity.

"Pharmaceutically acceptable salt" refers to a salt of the antibody-drug conjugate of the present invention, which is safe and effective when used in mammals and has the desired biological activity. The antibody-drug conjugate of the present invention contains at least one amine group, and thus can form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrosulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrophosphate, dihydrogen phosphate, salicylate, hydrocitrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

"Solvent compound" refers to the antibody-drug conjugate compound of the present invention and one or more solvent molecules to form a pharmaceutically acceptable solvent compound. Non-limiting examples of solvent molecules include: water, ethanol, acetonitrile, isopropanol, ethyl acetate.

"Cytotoxic drug" as used in the present invention refers to a substance that inhibits the function of cells and/or causes cell death or destruction.

"Tubulin inhibitors" refer to a class of compounds that interfere with the cell mitosis process by inhibiting the polymerization of tubulin or promoting the assembly of tubulin, thereby exerting an anti-tumor effect. Non-limiting examples include: maytansine, calicheamicin, taxanes, vincristine, colchicine, pyraclostrobin/auritasin/monomethyl auristatin E (MMAE)/monomethyl auristatin F (MMAF).

"Linker" refers to a chemical module comprising a covalent bond or chain of atoms that covalently attaches the antibody to the drug. Non-limiting examples of linkers include: arylene, heteroarylene, PEG, polymethyleneoxy, succinate, succinamide, diglycolate, malonate, and hexamidine.

"Drug loading" (DAR) is represented by y, which is the average number of cytotoxic drugs per antibody in general formula (A). The drug loading in the present invention can range from 1 to 20 cytotoxic drugs (D) per antibody. The antibody-drug conjugate of general formula (A) is a collection of antibodies conjugated with a certain range (1 to 20) of cytotoxic drugs. The drug loading (DAR) in the antibody-drug conjugate from the conjugation reaction can be characterized by general means, such as mass spectrometry, HPLC and ELISA. By these means, the quantitative distribution of the antibody-drug conjugate at the y value can be determined.

2. Abbreviation

MC is 6-maleimidohexanoyl

MMAF is a variant of monomethyl auristatin E with phenylalanine (MW 731.5) at the C-terminus of the molecule

The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention. The experimental methods in the examples of the present invention that do not specify specific conditions are usually carried out according to general conditions, such as the Cold Spring Harbor Antibody Technology Experimental Manual and the Molecular Cloning Manual; or according to the conditions recommended by the raw material or product manufacturer. Reagents that do not specify the specific source are general reagents purchased on the market.

Example 1 Antigen preparation

The protein encoding the extracellular region of human BCMA with a His tag (BCMA-His) was synthesized by SinoBiologics (Cat No.: 10620-H08H).

BCMA-His sequence:

SEQ ID NO：21

SEQ ID NO: 21

Example 2 Obtaining Mouse Fusion Tumor and Antibody Sequences

Animal immunization was performed with human antigen BCMA-His, a total of 5 Balb/c and 5 A/J mice, female, 10 weeks old, using Sigma complete Freund's adjuvant (CFA) and Sigma incomplete Freund's adjuvant (IFA). The immunogen and immune adjuvant were fully mixed and emulsified at a ratio of 1:1 to make a stable "oil-in-water" liquid; the injection dose was 25μg/200μL/mouse.

Table 1. Immunization schedule

The serum of the immunized mice was evaluated for serum titer and ability to bind to cell surface antigens using the indirect ELISA method as described in Example 3, and cell fusion was initiated based on the titer detection (greater than 100,000-fold dilution). The immunized mice with strong serum titer, affinity and FACS binding were selected for one final immunization and then killed. The spleen cells and SP2/0 myeloma cells were fused and plated to obtain fusion tumors. The target fusion tumors were screened by indirect ELISA, and single cell lines were established by limiting dilution. The positive antibody strains obtained were further screened by indirect ELISA to select fusion tumors that bind to recombinant proteins. The confluent tumor cells in logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript ^TM Reverse Transcriptase, Takara #2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-primer set (Novagen, TB326 Rev.B 0503) and then sequenced, and the sequence of mouse antibody M1 was finally obtained.

The heavy chain and light chain variable region sequences of mouse monoclonal antibody M1 are as follows:

M1 HCVR

SEQ ID NO：1

SEQ ID NO: 1

M1 LCVR

SEQ ID NO：2

SEQ ID NO: 2

Table 2. Heavy chain and light chain variable region CDR sequences of mouse monoclonal antibody M1

Example 3 In vitro binding activity detection method of antibodies

(1) In vitro indirect ELISA binding experiment:

BCMA His protein (Sino Biological Inc., cat# 10620-H08H) was diluted to 1 μg/ml with pH 7.4 PBS and added to a 96-well high-affinity ELISA plate at a volume of 100 μl/well. The plate was incubated overnight (16-20 hours) in a 4°C refrigerator. The plate was washed 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), and then 150 μl/well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST was added and incubated at room temperature for 1 hour for blocking. After the blocking was completed, the blocking solution was discarded and the plate was washed 4 times with PBST buffer.

Dilute the antibody to be tested with PBST containing 3% BSA, starting at 1μM, 10-fold gradient, 10 doses, add 100μl/well to the ELISA plate, and incubate at room temperature for 1 hour. After incubation, wash the plate 4 times with PBST, add 100μl/well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225) diluted with PBST containing 3% BSA, and incubate at room temperature for 1 hour. After washing the plate 4 times with PBST, add 100μl/well TMB colorimetric substrate (Cell Signaling Technology, cat#7004S), incubate at room temperature for 1 minute in the dark, add 100μl/well stop solution (Cell Signaling Technology, cat#7002S) to terminate the reaction, read the absorbance at 450nm with an enzyme labeler (BioTek, model Synergy H1), and analyze the data. The concentration signal value curve analysis results are shown in the following table:

Table 3. Affinity of mouse antibodies to human BCMA antigen (EC ₅₀ values)

(2) In vitro cell binding experiment:

Collect the cultured BCMA-high-expressing cells (HEK-293T cells overexpressing BCMA and tumor cells expressing BCMA, NCI-H929), adjust the cell density and spread them in 96-well U-bottom plates, with 1×10 ⁵ to 2×10 ⁵ cells per well. Centrifuge at 1200g for 5 minutes, remove the supernatant, add 100ul of gradient diluted antibody solution or mouse immune serum, and incubate at 4℃ for 60min; Centrifuge at 1200g for 5 minutes, remove the supernatant, wash the cells twice with PBS, add 100ul of fluorescent-labeled secondary antibody (PE-GAM: goat anti-mouse monoclonal antibody; or PE-GAH: goat anti-human monoclonal antibody) per well, and incubate at 4℃ for 60min. Centrifuge at 1200g for 5 minutes and remove the supernatant. After washing the cells twice with PBS, resuspend them in PBS and use a flow cytometer to detect the signal and analyze the results by concentration curve.

Table 4. Affinity of mouse antibodies for cells expressing BCMA (EC ₅₀ values)

Example 4 Mouse Antibody Humanization Experiment

The humanization of mouse anti-human BCMA monoclonal antibody is carried out as disclosed in many literatures in this field. In short, the human constant domain is used to replace the constant domain of the parent (mouse antibody), and the human antibody sequence is selected based on the identity of the mouse antibody and the human antibody. The present invention humanizes the mouse antibody M1.

Based on the typical structure of the mouse antibody VH/VL CDR, the heavy and light chain variable region sequences were compared with the human antibody germline database to obtain a human germline template with high identity.

The CDR region of the mouse antibody M1 was transplanted onto the selected corresponding humanized template. Then, based on the three-dimensional structure of the mouse antibody, the embedded residues, the residues that directly interacted with the CDR region, and the residues that had an important impact on the conformation of VL and VH were reverted to mutation, and the chemically unstable amino acid residues in the CDR region were optimized. After expression testing and comparison of the number of reverted mutations, the sequence of the humanized heavy chain variable region HCVR was selected and designed. The sequence is as follows:

HCVR1

SEQ ID NO：9

SEQ ID NO: 9

HCVR2

SEQ ID NO：10

SEQ ID NO: 10

HCVR3

SEQ ID NO：11

SEQ ID NO: 11

The sequence of the humanized light chain variable region LCVR was selected and designed, and the sequence is as follows:

LCVR1

SEQ ID NO：12

SEQ ID NO: 12

LCVR2

SEQ ID NO：13

SEQ ID NO: 13

LCVR3

SEQ ID NO：14

SEQ ID NO: 14

The designed heavy chain and light chain variable region sequences are connected to the human IgG1 heavy chain and human antibody light chain constant region sequences, respectively. Exemplary heavy chain and light chain constant region sequences are shown below:

IgG1 C

SEQ ID NO：22

SEQ ID NO: 22

Ig kappa C

SEQ ID NO：23

SEQ ID NO: 23

The heavy chain and light chain sequences are as follows:

Ab1 HC

SEQ ID NO：15

SEQ ID NO: 15

Ab2 HC

SEQ ID NO：16

SEQ ID NO: 16

Ab3 HC

SEQ ID NO：17

SEQ ID NO: 17

Ab1 LC

SEQ ID NO：18

SEQ ID NO: 18

Ab2 LC

SEQ ID NO：19

SEQ ID NO: 19

Ab3 LC

SEQ ID NO：20

SEQ ID NO: 20

Table 5. Sequence numbers of antibodies and their heavy chain, light chain, and variable regions

According to the amino acid sequences of the light and heavy chains of the above humanized antibodies, cDNA fragments were synthesized and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2 and incubated in a _CO2 incubator for 4 to 5 days. The cell culture medium was collected, centrifuged and filtered, and then loaded onto an antibody purification affinity column. The column was washed with phosphate buffer, flushed with glycine hydrochloride buffer (pH 2.7 0.1M Gly-HCl), neutralized with 1M Tris hydrochloric acid pH 9.0, and dialyzed against phosphate buffer to obtain the humanized antibody protein of the present invention.

Example 5 In vitro binding affinity and kinetics experiments

The affinity (EC ₅₀ ) of each humanized antibody for human BCMA antigen determined using the in vitro indirect ELISA binding experiment described in Example 3(1) is shown in the following table:

Table 6. Affinity of each humanized antibody for human BCMA antigen (EC ₅₀ )

The affinity (EC ₅₀ ) of each humanized antibody to NCI-H929 tumor cells determined using the in vitro cell binding assay described in Example 3(2) is shown in the following table:

Table 7. Affinity of each humanized antibody to NCI-H929 tumor cells (EC ₅₀ )

Example 6 Antibody endocytosis

After binding to BCMA, whether the antibody of the present invention can be internalized into cells together with human BCMA was tested using NCI-H929 (ATCC Accession No. CRL-9068). NCI-H929 cells were digested with trypsin (first washed with PBS, 37°C, about 2 minutes), the cells were collected and resuspended with pre-cooled FACS buffer, and the cell concentration was adjusted to 1×10 ⁶ cells/mL. Take the EP tube, add 1mL of cell suspension, centrifuge at 1500rpm for 5 minutes, remove the supernatant, add 1mL of the prepared antibody to be tested to resuspend the cells, the final concentration of the antibody is 20μg/ml, incubate at 4 degrees for 1h, centrifuge and discard the supernatant (4℃, 1500rpm×5min), wash twice with FACS buffer, and remove the supernatant. Add 100μL of fluorescent secondary antibody working solution to each tube to resuspend the cells, incubate at 4℃ for 30min, centrifuge and discard the supernatant (4℃, 1500rpm×5min), wash twice with FACS buffer, and remove the supernatant. Add 1.0 mL of pre-warmed NCI-H929 complete cell culture medium to each tube to re-suspend the cells and mix them. Divide into 4 tubes, 200 μL in each tube, which are 0 min group, blank group, 30 min group and 2 h group. Take out 0 min and blank and put them on ice. Put the rest in a 37°C incubator and internalize for 30 min and 2 h respectively. Take out the EP tubes at the corresponding time points and put them on ice for pre-cooling for 5 min. Centrifuge all treatment groups and discard the supernatant (4°C, 1500 rpm×5 min), wash once with FACS buffer and discard the supernatant. 250 μL stripping buffer was added to the EP tubes of all treatment groups except the 0min group, incubated at room temperature for 8min, centrifuged and discarded the supernatant (4°C, 1500rpm×5min), washed twice with FACS buffer, and discarded the supernatant. 100 μL immunostaining fixative was added to all treatment groups, placed at 4°C for more than 30min, and detected by flow cytometer DxFlex. BCMA antibody internalization percentage = (fluorescence intensity value at each time point - average fluorescence intensity value of blank group) / average fluorescence intensity value at zero point - average fluorescence intensity value of blank group. The results are shown in the table below:

Table 8. Antibody internalization in NCI-H929 tumor cells (EC ₅₀ )

The results showed that compared with the anti-BCMA antibody J6M0 (described in U.S. Patent 9,273,141), the antibody of the present invention has higher endocytosis efficiency and can be internalized quickly.

Example 7 Antibody conjugated to MC-MMAF

The antibody of the present invention has cell affinity activity and cell endocytosis activity, making the antibody of the present invention suitable for coupling with drugs to form antibody-drug conjugates for the treatment of BCMA-mediated diseases. The coupling process is shown in the following formula, where Ab represents Ab2 or Ab3 antibody:

In the first step, dissolve S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) in 0.9 mL acetonitrile solution for use. Add the above-prepared acetonitrile solution of S-(3-hydroxypropyl)thioacetate to the antibody pH=4.3 acetic acid/sodium acetate buffer (10.35 mg/mL, 9.0 mL, 0.97 mol), then drop 1.0 mL of sodium cyanoborohydride (14.1 mg, 224 mol) aqueous solution, and shake the reaction at 25°C for 2 hours. After the reaction is completed, desalt and purify with Sephadex G25 gel column (extraction phase: 0.05 M PBS solution at pH 6.5), and obtain the product 1f solution, which is concentrated to 10 mg/mL and directly proceeds to the next step.

In the second step, 0.35 mL of 2.0 M carboxyammonium hydrochloride solution was added to the 1f solution (11.0 mL), and the reaction was shaken at 25°C for 30 minutes. The reaction solution was desalted and purified using a Sephadex G25 gel column (extraction phase: 0.05 M PBS solution at pH 6.5) to obtain the product 2f solution (concentration 6.17 mg/mL, 14.7 mL).

In the third step, the compound MC-MMAF (1.1 mg, 1.2 mol, prepared by the method disclosed in PCT patent W02005081711) was dissolved in 0.3 mL of acetonitrile, added to the 2f solution (concentration 6.17 mg/mL, 3.0 mL), and after shaking for 4 hours at 25°C, the reaction solution was desalted and purified using a Sephadex G25 gel column (extraction phase: 0.05 M PBS solution at pH 6.5), and then filtered under sterile conditions to obtain the product Ab-MC-MMAF. The average DAR value y of the product ADC2 (Ab2-MC-MMAF) was determined to be 4 using HIC-HPLC, and the PBS buffer solution of the antibody-drug conjugate (3.7 mg/mL, 4.7 mL) was refrigerated at 4°C. The product ADC3 (Ab3-MC-MMAF) was prepared by the above method. The average DAR value y of the product ADC3 (Ab3-MC-MMAF) was determined to be 4.1 by HIC-HPLC. The PBS buffer solution (3.5 mg/mL, 5.0 mL) of the antibody-drug conjugate was refrigerated at 4°C.

Example 8 Antibody-coupled SN-38

The antibody-drug conjugate is prepared by the following conjugation process, where Ab represents Ab2:

In the first step, dissolve S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) in 0.9 mL acetonitrile solution for use. Add the above-prepared acetonitrile solution of S-(3-hydroxypropyl)thioacetate to the antibody pH=4.3 acetic acid/sodium acetate buffer (10.35 mg/mL, 9.0 mL, 0.97 mol), then drop 1.0 mL of sodium cyanoborohydride (14.1 mg, 224 mol) aqueous solution, and shake the reaction at 25°C for 2 hours. After the reaction is completed, desalt and purify with Sephadex G25 gel column (extraction phase: 0.05 M PBS solution at pH 6.5), and obtain a 1h solution of the product, which is concentrated to 10 mg/mL and directly proceeds to the next step.

In the second step, 0.35 mL of 2.0 M carboxyammonium hydrochloride solution was added to the 1h solution (11.0 mL). After shaking the reaction at 25°C for 30 minutes, the reaction solution was desalted and purified using a Sephadex G25 gel column (extraction phase: 0.05 M PBS solution at pH 6.5) to obtain the product 2h solution (concentration 6.2 mg/mL, 15.0 mL). After concentrating to about 10 mg/mL, it was used in the next reaction.

In the third step, the compound MC-SN-38 (1.3 mg, 1.2 mol) was dissolved in 0.3 ml of acetonitrile and added to the 2h solution (concentration 6.2 mg/mL, 3.0 mL). After shaking for 4 hours at 25°C, the reaction solution was desalted and purified using a Sephadex G25 gel column (extraction phase: 0.05 M PBS solution at pH 6.5), and then filtered under sterile conditions to obtain the product Ab-SN-38 antibody-drug conjugate PBS buffer solution (3.7 mg/mL, 4.7 mL), which was refrigerated at 4°C. The average value y was determined by ultraviolet method. Place the cuvette containing sodium succinate buffer in the reference absorption cell and the sample measurement absorption cell respectively, deduct the solvent blank, and then place the cuvette containing the test solution in the sample measurement absorption cell to measure the absorbance at 280nm and 370nm.

Data processing:

By establishing a standard curve, measuring the absorption at a wavelength of 280nm, the antibody content Cmab is determined, and measuring the absorption at a wavelength of 370nm, the small molecule content CDrug is determined.

The average drug loading value y=CDrug/Cmab.

The DAR average value y of the Ab2-SN-38 antibody-drug conjugate was determined by the above method to be 3.9.

Example 9 Antibody-conjugated Exetecan Derivatives

In the first step, 2a (2 g, 17.2 mmol) was dissolved in 75 mL of acetonitrile, and potassium carbonate (9.27 g, 67.2 mmol), benzyl bromide (20 mL, 167.2 mmol) and tetrabutylammonium iodide (620 mg, 1.68 mmol) were added in sequence. The reaction solution was stirred at room temperature for 48 hours, filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate (20 ml). The combined filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography with a developing agent system C to obtain product 5a (3.2 g, yield: 90.1%).

In the second step, 5a (181.3 mg, 0.879 mmol) and 4b (270 mg, 0.733 mmol) were added to the reaction bottle, 6 mL of tetrahydrofuran was added, the atmosphere was replaced with hydrogen three times, the temperature was cooled to 0 to 5°C in an ice-water bath, potassium tert-butoxide (164 mg, 1.46 mmol) was added, the ice bath was removed, the temperature was raised to room temperature and stirred for 40 minutes, 15 mL of ice water was added, ethyl acetate (40 mL×2) and chloroform (20 mL×5) were extracted, the organic phases were combined and concentrated. The residue was dissolved in 6 mL of dioxane, 3 mL of water was added, sodium bicarbonate (73.8 mg, 0.879 mmol) and 9-fluorenylmethyl chloroformate (190 mg, 0.734 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Add 30 mL of water, extract with ethyl acetate (20 mL × 3), wash the organic phase with saturated sodium chloride solution (30 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the resulting residue by silica gel column chromatography with a developing agent system C to obtain product 5b 10-cyclopropyl-1-( 9H -fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester (73 mg, yield: 19.4%).

MS m/z(ESI): 515.0[M+1].

In the third step, 5b (30 mg, 0.058 mmol) was dissolved in 6.75 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V=2:1), palladium carbon (18 mg, 10% content, dry type) was added, the hydrogen atmosphere was replaced three times, and the reaction was stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain a crude product 5c 10-cyclopropyl-1-( 9H -fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid (20 mg), and the product was directly used for the next step without purification.

MS m/z(ESI): 424.9[M+1].

Step 4: Add 1b (15 mg, 28.2 μmol) to the reaction bottle, add 1.5 mL N , N -dimethylformamide, replace the atmosphere with hydrogen three times, cool to 0 to 5°C in an ice-water bath, add a drop of triethylamine, add crude 5c (20 mg, 47.1 μmol), add 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylchlorophenoxylate (25.4 mg, 86.2 μmol), stir in an ice bath for 40 minutes. Add 15 mL of water, extract with ethyl acetate (20 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (20 mL × 2), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The resulting residue was purified by thin layer chromatography with developing solvent System B to give the title product 5d ( 9H -fluoren-9-yl)methyl (2-(((1-cyclopropyl-2-((( 1S , 9S )-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro- 1H , 12H -benzo[ de ]pyrano[3',4':6,7]indolizino[1,2- b ]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (23.7 mg, yield: 78.9%).

MS m/z(ESI):842.1[M+1].

Step 5: Dissolve 5d (30 mg, 35.7 μmol) in 3 mL of dichloromethane, add 1.5 mL of diethylamine, and stir at room temperature for 2 hours. The reaction solution is concentrated under reduced pressure, 1.5 mL of toluene is added and concentrated under reduced pressure, and the process is repeated twice. Add 4.5 mL of n-hexane to the residue for slurry, and after standing, pour off the supernatant and retain the solid. The solid residue was concentrated under reduced pressure and dried by oil pump to give the crude product 5e 2-((2-aminoacetamido)methoxy)-2-cyclopropyl- N -(( 1S , 9S )-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dihydro-2,3,9,10,13,15-hexahydro- 1H , 12H -benzo[ de ]pyrano[3',4':6,7]indolizino[1,2- b ]quinolin-1-yl)acetamide (23 mg), which was used directly in the next reaction without purification.

MS m/z(ESI): 638.0[M+18].

In the sixth step, the crude product 5e (20 mg, 32.3 μmol) was dissolved in 1 mL N , N -dimethylformamide, replaced with hydrogen three times, cooled to 0 to 5°C in an ice-water bath, 4 g (31.8 mg, 67.3 μmol) of 0.5 mL N , N -dimethylformamide solution was added, and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylchlorophenoxylate (27.8 mg, 94.3 μmol) was added. The reaction was stirred in an ice bath for 10 minutes, the ice bath was removed, and the temperature was raised to room temperature and stirred for 1 hour to generate compound 5 . The reaction solution was purified by HPLC (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain products 5-A and 5-B (3.6mg, 2.6mg).

MS m/z(ESI): 1074.4[M+1].

Single configuration compound 5-A (short retention time):

UPLC analysis: retention time 1.14 minutes, purity: 85% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH ₄ OAc), B-acetonitrile).

¹ H NMR (400MHz, DMSO- d ₆ ): δ 8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.2 6-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,2H),4.54-4. 40(m,2H),3.76-3.64(m,4H),3.62-3.48(m,2H),3.20-3.07(m,2H),3.04-2.94(m,2H),2.80-2.62(m,2H),2.45-2.30(m,3H),2 .25-2.15(m,2H),2.15-2.04(m,2H),1.93-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.87(t,3H),0.64-0.38(m,4H).

Single configuration compound 5-B (longer retention time):

UPLC analysis: retention time 1.16 minutes, purity: 89% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH ₄ OAc), B-acetonitrile).

¹ H NMR (400MHz, DMSO- d ₆ ): δ 8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26- 7.13(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.60-5.50(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.78-4.68(m,1H),4.60-4.40(m,2H) ,3.76-3.58(m,4H),3.58-3.48(m,1H),3.20-3.10(m,2H),3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13(m,2H) ,2.13-2.04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.91-0.79(m,3H),0.53-0.34(m,4H).

For the preparation methods of other intermediates, please refer to Intermediate 5.

At 37°C, add the prepared aqueous solution of tri(2-carboxyethyl)phosphine (10mM, 0.347mL, 3.47μmol) to the PBS buffer solution of antibody Ab2 (pH=6.5 0.05M PBS buffer solution; 7.3ml, 13.8mg/ml, 0.681μmol), place in a water bath oscillator, oscillate at 37°C for 3 hours, stop the reaction; cool the reaction solution to 25°C with a water bath, dilute to 14.0ml, and take out 3.3ml of the solution for further reaction.

Compound 5-A (3.0 mg, 3.72 μmol) was dissolved in 0.15 mL DMSO, added to the above 3.3 ml solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (extraction phase: 0.05 M PBS buffer solution with a pH of 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (1.35 mg/mL, 13 mL) of the exemplary product ADC1 (Compound 26) of the Ab-Ixitectin derivative, which was stored frozen at 4° C.

The average value y was determined by ultraviolet method. After placing the cuvette containing sodium succinate buffer in the reference absorption cell and the sample measurement absorption cell respectively, after deducting the solvent blank, the cuvette containing the test solution was placed in the sample measurement absorption cell to measure the absorbance at 280nm and 370nm.

Data processing:

By establishing a standard curve, measuring the absorption at a wavelength of 280nm, the antibody content Cmab is determined, and measuring the absorption at a wavelength of 370nm, the small molecule content CDrug is determined.

The average drug loading value y=CDrug/Cmab.

The exemplary product ADC1 (compound 26, 7.6) was determined by the above method. Samples of ADC1-1 (y=4), ADC1-2 (y=6), and ADC1-3 (y=8) were obtained by UV-HPLC purification.

For the preparation methods of other antibody-drug conjugates, please refer to ADC1.

Example 10 Killing effect of antibody-drug conjugate on NCI-H929 tumor cells

To detect the killing effect of the antibody-drug conjugate of the present invention on tumor cells, NCI-H929 cells (ATCC accession number CRL-9068) were used for evaluation. NCI-H929 cells were collected, counted by centrifugation, and the cell density was adjusted to 0.44×10 ⁶ cells/mL with complete culture medium, and plated in the middle 60 wells of a white 96-well plate, 90 μL per well, with a cell number of 40,000. 100 μL PBS was added to the remaining wells, and the cell plate was placed in a 37°C, 5% CO ₂ incubator for overnight culture. On the second day of the experiment, the antibody-drug conjugate solution was prepared in a 96-well V-bottom plate with PBS, starting at a concentration of 15μg/mL, diluted 3 times, and 9 concentrations. After preparation, it was added to a white 96-well plate, 10μL per well, duplicate wells, and the cell plate was placed in a 37°C, 5% CO ₂ incubator for 72 hours. On the fifth day of the experiment, the test readings were taken out: the cell culture plate was taken out, equilibrated to room temperature, and 50μL CTG solution (Promega G7573) was added to each well. After shaking and mixing, it was placed in the dark for 10 minutes, and then the luminescence program of the enzyme labeler was used for detection. The EC50 value was calculated using GraphPad Prims software. The experimental results are shown in the following table:

Table 9. Antibody-drug conjugate killing activity against NCI-H929 tumor cells (EC ₅₀ )

Example 11 Anti-tumor effect of antibody-drug conjugates

To further study the killing effect of antibody-drug conjugates on tumors formed in vivo, the anti-tumor effect of the antibody-drug conjugates of the present invention was evaluated after forming transplanted tumors in mice with NCI-H929 cells. 9x10 ⁶ NCI-929 cells were injected subcutaneously into 8-week-old immunodeficient nude mice (NOD-SCID). After 8 days, antibody-drug conjugates ADC2 (Ab2-MC-MMAF, Example 7, y=4) and ADC1-3 (Example 9, y=8) were injected intravenously once every week at a dose of 1 mg/kg. Human IgG1 protein was used as a control at a dose of 1 mg/kg. There were 5 mice in each control group or drug group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate = 100% - (tumor volume of the drug group on day 14 - tumor volume of the drug group on day 0) / (tumor volume of the control group on day 14 - tumor volume of the control group on day 0). The experimental results are shown in Table 10. Antibody-drug conjugates ADC2 (Ab2-MC-MMAF, Example 7, y=4) and ADC1-3 (Example 9, y=8) both showed tumor inhibition activity.

Table 10. Antitumor effects of antibody-drug conjugates

Example 12 In vivo tumor killing effect of antibody-drug conjugates

To further study the killing effect of antibody-drug conjugates on tumors formed in vivo, the anti-tumor effect of the antibody-drug conjugates of the present invention was evaluated after forming transplanted tumors in mice with NCI-H929 cells. 9x10 ⁶ NCI-929 cells were injected subcutaneously into 8-week-old immunodeficient nude mice (NOD-SCID). After 8 days, antibody-drug conjugates ADC2 (Example 7, y=4) and ADC3 (Example 7, y=4.1) were injected intravenously twice every week at a dose of 3 mg/kg. Human IgG1 protein was used as a control at a dose of 3 mg/kg. There were 5 mice in each control group or drug group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate TGI = 100% - (tumor volume of the drug group on day 14 - tumor volume of the drug group on day 0) / (tumor volume of the control group on day 14 - tumor volume of the control group on day 0). The experimental results are shown in Table 11. Both ADC2 (Example 7, y = 4) and ADC3 (Example 7, y = 4.1) showed tumor-killing effects.

Table 11. Antibody-drug conjugates' tumor-killing activity in vivo

Example 13 Antibody-drug conjugate tumor cell killing activity

To detect the killing effect of the antibody-drug conjugate of the present invention on tumor cells, the BCMA high expression level cell line NCI-H929 (ATCC deposit number CRL-9068) and the BCMA low expression level cell line RPMI-8226 (ATCC, cat: CCL-155) were used for evaluation. NCI-H929 and RPMI-8226 cells were collected, centrifuged and counted, and the cell density was adjusted to 1×10 ⁵ /mL with complete culture medium, and the cells were plated in the middle 60 wells of a white 96-well plate, 100 μl per well, and the cell number was 10,000 cells/well. 100 μl/well DPBS was added to the edge wells, and the cell plate was placed in a 37°C, 5% CO ₂ incubator for overnight culture. The next day, the antibody-drug conjugate working solution was prepared in a 96-well V-bottom plate with complete medium, starting at 133uM, 5-fold dilution, 9 concentrations, and added to a white 96-well plate after preparation, 80μl per well, duplicate wells, and the cell plate was placed in a 37°C, 5% CO ₂ incubator for 72 hours. On the fifth day of the experiment, the test reading was taken out: the cell culture plate was taken out, equilibrated to room temperature, and 90μl CellTiter-Glo® cell viability assay reagent (Promega, Cat#: G7573) was added to each well, and after vortexing and mixing, it was placed in the dark for 10 minutes, and then the luminescence program of the enzyme labeler was used for detection. The IC ₅₀ value was calculated using GraphPad Prims software. The experimental results are shown in the following table:

Table 12. Antibody-drug conjugates' killing effects on tumor cells

The experimental results showed that the in vitro killing activity of BCMA-ADC against tumor cells was positively correlated with the expression level of BCMA on the cell surface, and the killing effect on NCI-H929 was significantly stronger than that on RPMI-8226; as the amount of coupled small molecule toxins increased, the killing activity against tumor cells increased accordingly.

Example 14 PK study of antibody-drug conjugates

The BALB/c mouse model was used to evaluate the drug metabolism of the anti-BCMA drug conjugate BCMA-ADC in mice. Human BALB/c transgenic mice (Shanghai Xipuer-Bike Laboratory Animal Co., Ltd.) with an average body weight of 18 to 22g and an age of 18 to 22 weeks were randomly divided into 2 groups, with 3 animals in each group. The tested BCMA-drug conjugates were all administered at 10mpk, IV, in a single dose. PBS solvent was used as a negative control group. Blood was collected at 1, 2, 4, 8, 24, 48, 96, 144, and 240 hours, and plasma was separated and stored in a -20°C refrigerator. After that, the recombinant human BCMA protein was used to coat the high-affinity 96-well bottle bottom plate, and the diluted plasma sample to be tested was added. The HRP-labeled secondary antibody was then used to detect the BCMA-ADC concentration in the mouse plasma, and the WinNonlin software non-compartmental model and the formula for intravascular administration were used to analyze its PK parameters. The experimental results are shown in the table below:

Table 13. Metabolic kinetic parameters of antibody-drug conjugates in mice

Considering parameters such as half-life t _1/2 , exposure, and time to peak blood concentration, ADC1-2 showed good in vivo metabolic activity.

Example 15 Cytotoxicity of Antibody Drug Conjugates

In order to detect the cytotoxic effects of the antibody-drug conjugate (ADC) and exotecan derivative (Example 9, compound ( 5-A )) disclosed herein on tumor cells, the BCMA high expression level cell line NCI-H929 (ATCC Accession No. CRL-9068) was used for evaluation.

Collect NCI-H929 cells, count by centrifugation, and adjust the cell density to 2×10 ⁵ cells/mL with complete culture medium. Plate the cells in the middle 60 wells of a white 96-well plate with 50 μL per well and 10,000 cells/well. Add 100 μL/well DPBS to the edge wells and incubate the cell plate in a 37°C, 5% CO ₂ incubator overnight. The next day, the antibody-drug conjugate working solution was prepared in a 96-well V-bottom plate using complete culture medium, and the exotecan derivative compound ( 5-A ) and ADC were diluted in equal molar amounts, with the highest working concentration being 300 nM, 5-fold dilution, and 9 concentrations. After preparation, it was added to a white 96-well plate, 100 μL per well, in duplicate, and the cell plate was placed in a 37°C, 5% CO ₂ incubator for a further 72 hours. On the fifth day of the experiment, the test was read: the cell culture plate was removed and equilibrated to room temperature. 75 μL CellTiter-Glo® Cell Viability Assay Reagent (Promega, Cat#: G7573) was added to each well. After vortexing and mixing, the plate was placed in the dark for 10 minutes, and then the luminescence program of the enzyme marker was used for detection. The IC ₅₀ value was calculated using GraphPad Prims software. The antibody-drug conjugate (ADC1-4, y=4) of the negative antibody IgG1 and the exotecan derivative compound ( 5-A ) was used as a negative control. The experimental results are shown in the following table:

Table 14. Cytotoxic effects of test compounds on tumor cells

By comparing the cytotoxic cytotoxicity IC ₅₀ , ADC1-1 (y=4) is about 1/115 (0.88/101) times that of compound (5-A). The experimental results show that the in vitro cytotoxic activity of the antibody-drug conjugate of the present invention on tumor cells is significantly stronger than the killing effect of the small molecule toxin compound (5-A) on NCI-H929.

Example 16 Preparation of Exotecan Derivatives

2 mL of ethanol and 0.4 mL of N,N-dimethylformamide were added to 2e (4 mg, 7.53 μmol), the atmosphere was replaced with hydrogen three times, the mixture was cooled to 0 to 5°C in an ice-water bath, 0.3 mL of N-methylmorpholine was added dropwise, and the mixture was stirred until the reaction liquid became clear. 2-cyclopropyl-2-hydroxyacetic acid 1e (2.3 mg, 19.8 μmol, prepared by the method disclosed in patent application "WO2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol) were added to the reaction liquid in sequence, and the mixture was stirred at 0 to 5°C for 1 hour. Remove the ice-water bath, heat to 30°C and stir for 2 hours. The reaction solution was concentrated under reduced pressure, and the crude compound 2-C was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc), B-acetonitrile, gradient extraction, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (2-A: 1.5mg, 2-B: 1.5mg).

MS m/z(ESI): 534.0[M+1].

Single configuration compound 2-B (short retention time)

UPLC analysis: retention time 1.06 minutes, purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

¹ HNMR (400MHz, DMSO-d6): δ 8.37(d,1H),7.76(d,1H),7.30(s,1H),6.51(s,1H),5.58-5.56(m,1H) ,5.48(d,1H),5.41(s,2H),5.32-5.29(m,2H),3.60(t,1H),3.19-3.13( m,1H),2.38(s,3H),2.20-2.14(m,1H),1.98(q,2H),1.87-1.83(m,1H) ,1.50-1.40(m,1H),1.34-1.28(m,1H),0.86(t,3H),0.50-0.39(m,4H).

Single configuration compound 2-A (longer retention time)

UPLC analysis: retention time 1.10 minutes, purity: 86% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

¹ HNMR (400MHz, DMSO-d6): δ8.35(d,1H),7.78(d,1H),7.31(s,1H),6.52(s,1 H),5.58-5.53(m,1H),5.42(s,2H),5.37(d,1H),5.32(t,1H),3.62(t,1H),3 .20-3.15(m,2H),2.40(s,3H),2.25-2.16(m,1H),1.98(q,2H),1.87-1.82(m ,1H),1.50-1.40(m,1H),1.21-1.14(m,1H),0.87(t,3H),0.47-0.35(m,4H).

Example 17 In vitro proliferation inhibition test of ixetemcan derivatives on tumor cells

Compounds 2-A and 2-B were tested for their inhibitory activity on the proliferation of U87MG cells (Chinese Academy of Sciences Cell Bank, Catalog #TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Catalog No. HTB-30) in vitro. Cells were treated with different concentrations of compounds in vitro. After 6 days of culture, the proliferation of cells was tested using CTG (Luminescent Cell Viability Assay, Promega, Catalog No.: G7573) reagent, and the in vitro activity of the compound was evaluated based on the IC50 value.

U87MG and SK-BR-3 cells were cultured with EMEM medium containing 10% FBS (GE, catalog number SH30024.01) and McCoy's 5A medium containing 10% FBS (Gibco, catalog number 16600-108), respectively.

Take U87MG and SK-BR-3 cells in logarithmic growth phase, wash them once with PBS (phosphate buffer, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3ml trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies) to digest for 2 to 3 minutes, and after the cells are completely digested, add 10 to 15ml cell culture medium, wash the digested cells, centrifuge at 1000rpm for 5 minutes, discard the supernatant, and then add 10 to 20ml cell culture medium to resuspend the cells to make a single cell suspension.

Mix the U87MG and SK-BR-3 single cell suspensions, adjust the live cell density to 2.75×103cells/ml and 8.25×103cells/ml respectively with cell culture medium, mix the cell suspensions after density adjustment, and add 180μl/well to a 96-well cell culture plate. Add only 200ul of culture medium to the peripheral wells of the 96-well plate. Incubate the culture plate in an incubator for 24 hours (37°C, 5% CO ₂ ).

Dissolve the compound in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) and prepare a stock solution with an initial concentration of 10mM.

The starting concentration of small molecule compounds is 500nM, and the dosage method is as follows:

Add 30ul of different samples to be tested to the first column of a 96-well U-bottomed drug plate, with a sample concentration of 100uM; add 20ul DMSO to each well from the second to the 11th columns. Take 10ul of the sample from the first column and add it to the 20ul DMSO in the second column, mix well, take 10ul to the third column, and so on to the 10th column. Take 5ul of the drug from each well of the drug plate and add it to 95ul EMEM culture medium, mix well, and set aside.

Sample addition: Add 20 μl of the test sample of different concentrations to the culture plate, with each sample in duplicate. Incubate the culture plate in an incubator for 6 days (37°C, 5% CO ₂ ).

Color development: Take out the 96-well cell culture plate, add 90μl CTG solution to each well, and incubate at room temperature for 10 minutes.

Plate reading: Take out the 96-well cell culture plate, place it in an enzyme marker (BMG labtech, PHERAstar FS), and use the enzyme marker to measure the chemiluminescence.

Data analysis: Microsoft Excel and Graphpad Prism 5 were used to process and analyze the data. The experimental results are shown in Table 15.

Table 15. The killing effect of exitecan derivatives on tumor cells

<110> Shanghai Hansoh Biomedical Technology Co., Ltd. (SHANGHAI HANSOH BIOMEDICAL CO.,LTD) Jiangsu Hansoh Pharmaceutical Group Co., Ltd. (JIANGSU HANSOH PHARMACEUTICAL GROUP CO.,LTD.)

<120> ANTIBODY-DRUG CONJUGATES AND MEDICAL USE THEREOF

<130> 721030CPCT

<150> CN 202010224992.1

<151> 2020-03-26

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<151> 2020-07-29

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<151> 2021-01-29

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<223> Heavy chain variable region of mouse monoclonal antibody M1

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<223> The light chain variable domain of mouse monoclonal antibody M1

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<223> HCDR1 of mouse monoclonal antibody M1

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<213> Artificial Sequence

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<221> Structural domain

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<223> HCDR2 of mouse monoclonal antibody M1

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<221> Structural domain

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<221> Structural domain

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<223> LCDR1 of mouse monoclonal antibody M1

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<213> Artificial Sequence

<220>

<221> Structural domain

<222> (1)..(7)

<223> LCDR2 of mouse monoclonal antibody M1

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<210> 8

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<212> PRT

<213> Artificial Sequence

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<221> Structural domain

<222> (1)..(9)

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<221> Structural domain

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<223> Humanized heavy chain variable region HCVR1

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<211> 121

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<213> Artificial Sequence

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<221> Structural domain

<222> (1)..(121)

<223> Humanized heavy chain variable region HCVR2

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<210> 11

<211> 121

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<213> Artificial Sequence

<220>

<221> Structural domain

<222> (1)..(121)

<223> Humanized heavy chain variable region HCVR3

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<210> 12

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<213> Artificial Sequence

<220>

<221> Structural domain

<222> (1)..(106)

<223> Humanized light chain variable region LCVR1

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<211> 106

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<213> Artificial Sequence

<220>

<221> Structural domain

<222> (1)..(106)

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<210> 14

<211> 106

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<213> Artificial Sequence

<220>

<221> Structural domain

<222> (1)..(106)

<223> Humanized light chain variable region LCVR3

<400> 14

<210> 15

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> Ab1 HC

<400> 15

<210> 16

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> Ab2 HC

<400> 16

<210> 17

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> Ab3 HC

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<210> 18

<211> 213

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<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(213)

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<210> 19

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<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(213)

<223> Ab2 LC

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<211> 213

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<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(213)

<223> Ab3 LC

<400> 20

<210> 21

<211> 65

<212> PRT

<213> Artificial Sequence

<220>

<221> Peptide

<222> (1)..(65)

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<211> 330

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<213> Artificial Sequence

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<221> Structural domain

<222> (1)..(330)

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<213> Artificial Sequence

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<221> Structural domain

<222> (1)..(107)

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Claims (40)

An antibody-drug conjugate represented by general formula (A) or a pharmaceutically acceptable salt or solvent compound thereof,

wherein D is a cytotoxic drug; _L1 and _L2 are linker units; y is a number selected from 1 to 20; Ab is an anti-BCMA antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 1, wherein the anti-BCMA antibody or its antigen-binding fragment is selected from a murine antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a human antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 2, wherein the anti-BCMA antibody or its antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, and the anti-BCMA antibody or its antigen-binding fragment further comprises a light chain constant region of a human antibody κ chain, λ chain or a variant thereof. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 3, wherein the anti-BCMA antibody or its antigen-binding fragment further comprises an IgG1 heavy chain constant region with enhanced ADCC toxicity after amino acid mutation, and the anti-BCMA antibody or its antigen-binding fragment further comprises a light chain constant region of a human antibody κ chain. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 3, wherein the anti-BCMA antibody or its antigen-binding fragment further comprises a heavy chain constant region as shown in SEQ ID NO: 22, and the anti-BCMA antibody or its antigen-binding fragment further comprises a light chain constant region as shown in SEQ ID NO: 23. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 2, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the following sequences, or a heavy chain variable region having at least 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; and/or, a light chain variable region selected from the following sequences, or a light chain variable region having at least 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 6, wherein the anti-BCMA antibody or its antigen-binding fragment comprises: a heavy chain variable region shown in SEQ ID NO: 9 and a light chain variable region shown in SEQ ID NO: 12; or, a heavy chain variable region shown in SEQ ID NO: 10 and a light chain variable region shown in SEQ ID NO: 13; or, a heavy chain variable region shown in SEQ ID NO: 11 and a light chain variable region shown in SEQ ID NO: 14. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 6, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain selected from the following sequences, or a heavy chain having at least 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17; and/or, a light chain selected from the following sequences, or a light chain having at least 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 8, wherein the anti-BCMA antibody or its antigen-binding fragment comprises: the heavy chain shown in SEQ ID NO: 15 and the light chain shown in SEQ ID NO: 18; or, the heavy chain shown in SEQ ID NO: 16 and the light chain shown in SEQ ID NO: 19; or, the heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 20. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 1, wherein the cytotoxic drug is selected from toxins, chemotherapeutic drugs, antibiotics, radioactive isotopes and nucleolytic enzymes. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 10, wherein the cytotoxic drug is selected from tubulin inhibitors or DNA topoisomerase inhibitors that inhibit cell division. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 10, wherein the cytotoxic drug is selected from DM1, DM3, DM4, camptothecin, camptothecin derivatives, SN-38, MMAF or MMAE. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 10, wherein the cytotoxic drug is selected from MMAE or MMAF, or SN-38:

Alternatively, exotecan or an exotecan derivative:

The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 10 is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof represented by the general formula (III):

Wherein, L ₁ and L ₂ are linker units; y is a number selected from 1 to 10; and Ab is selected from the anti-BCMA antibody or antigen-binding fragment thereof described in any one of claims 1 to 9. In the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 14, y is a number selected from 2 to 8. In the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 14, y is a number selected from 4 to 8. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent thereof as described in claim 14, wherein L ₂ is represented by the general formula (D): -K ¹ -K ² -K ³ -K ⁴ -(D) wherein K ¹ is

, s is selected from an integer from 2 to 8; K ² is selected from -NR ¹ (CH ₂ CH ₂ O) _p CH ₂ CH ₂ C(O)-, -NR ¹ (CH ₂ CH ₂ O) _p CH ₂ C(O)-, -S(CH ₂ ) _p C(O)- or a single bond, and p is selected from an integer from 1 to 20; R ¹ is selected from hydrogen, deuterium, hydroxyl, amine, alkyl, halogen, halogenated alkyl, deuterated alkyl and hydroxyl alkyl; K ³ is a tetrapeptide residue; K ⁴ is -NR ² (CR ³ R ⁴ ) _t -, R ² , R ³ or R ⁴ are each independently hydrogen, deuterium, hydroxyl, amine, alkyl, halogen, halogenated alkyl, deuterated alkyl and hydroxyl alkyl, t is selected from 1 or 2, and the K of the linker unit L ₂ is The ¹ end is connected to Ab, and the ⁴ end of K is connected to L ₁ . The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 17, wherein p is selected from an integer from 1 to 6; the tetrapeptide residue is a peptide residue formed by two or more amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamine, and aspartic acid. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 17, wherein the tetrapeptide residue is GGFG. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 17, wherein L ₁ is selected from a bond, -O-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-, -O-CR ⁵ R ⁶ -(CR ^a R ^b ) _m -, -O-CR ⁵ R ⁶ -, -NH-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)- or -S-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-, wherein ^Ra and R ^b are each independently selected from hydrogen, deuterium, halogen or alkyl; R 5 is halogenalkyl or cycloalkyl; R ⁶ is selected from hydrogen, halogenalkyl or cycloalkyl; or, R ^{5 and R 6 together} with the carbon atom to which they are attached form a cycloalkyl; m is selected from 0, 1, 2, 3 or 4, and L The O terminal of ₁ is connected to the connector unit _L2 . The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 20, wherein _L1 is represented by the general formula (E):

wherein R ⁵ is selected from halogenalkyl or cycloalkyl, R ⁶ is selected from hydrogen, halogenalkyl or cycloalkyl, or R ⁵ and R ⁶ together with the carbon atom to which they are connected form a cycloalkyl; and m is selected from an integer from 0 to 4. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 21, wherein ^R5 is selected from _C1-6 haloalkyl or _C3-6 cycloalkyl, ^R6 is selected from hydrogen, _C1-6 haloalkyl or _C3-6 cycloalkyl, or ^R5 and ^R6 together with the carbon atoms to which they are attached form a _C3-6 cycloalkyl. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 21, wherein the general formula (E) is selected from the following substituents:

,

,

The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in any one of claims 1 to 9 and 11 to 23, wherein -L ₂ -L ₁ - is selected from the following structures:

^K2 is a bond; ^K3 is the tetrapeptide residue of GGFG; ^R5 is a halogenalkyl group or a _C3-6 cycloalkyl group; ^R6 is selected from hydrogen, a halogenalkyl group or a _C3-6 cycloalkyl group; or, ^R5 and ^R6 together with the carbon atom to which they are connected form a _C3-6 cycloalkyl group; ^R2 , ^R3 or ^R4 are each independently selected from hydrogen or an alkyl group; s is selected from an integer from 2 to 8; m is selected from an integer from 0 to 4. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 24, wherein -L2-L1- is selected from the following structures:

The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as described in claim 14 is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof represented by the general formula (IV):

wherein W is selected from C _1-8 alkyl, C _1-8 alkyl-cycloalkyl or a straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, optionally, the C _1-8 alkyl, cycloalkyl and straight chain heteroalkyl are each independently further substituted with one or more substituents selected from halogen, hydroxyl, cyano, amine, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; K ² is selected from -NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ CH ₂ C (O) -, -NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ C (O) -, -S (CH ₂ ) _p1 C (O) - or a bond, R ¹ is selected from a hydrogen atom, an alkyl, a halogenated alkyl, a deuterated alkyl and a hydroxyl alkyl, p _{R 1} is an integer selected from 1 to 20; K ³ is a peptide residue consisting of 2 to 7 amino acids, which may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available attachment point, and the substituent is one or more independently selected from halogen, hydroxyl, cyano, amine, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; R ² is selected from hydrogen atom, alkyl, halogenalkyl, deuterated alkyl and hydroxyalkyl; R ³ and R ⁴ are each independently selected from hydrogen atom, halogen, alkyl, halogenalkyl, deuterated alkyl and hydroxyalkyl; R ⁵ is selected from halogen, halogenalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl; R ^{R 6} is selected from a hydrogen atom, a halogen, a halogenated alkyl, a deuterated alkyl, a cycloalkyl, a heterocyclic group, an aryl group or a heteroaryl group; or, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group; m is selected from an integer from 0 to 4; y is selected from a number from 1 to 10, and y is a decimal or an integer; Ab is an anti-BCMA antibody or an antigen-binding fragment thereof. The antibody-drug conjugate as described in claim 26 is an antibody-drug conjugate represented by the general formula (IV-A) or a pharmaceutically acceptable salt or solvent compound thereof:

R ² , R ³ , R ⁴ , R ⁵ or R ⁶ are as defined in claim 26; s is an integer selected from 2 to 8; m is an integer selected from 0 to 4; y is a number selected from 1 to 10, and y is a decimal or an integer; Ab is an anti-BCMA antibody or an antigen-binding fragment thereof. The antibody-drug conjugate as described in claim 27, wherein the general formula (IV-A) is selected from the following structures:

The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound represented by the general formula (A) as described in claim 1, wherein the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound is selected from the following structures:

Wherein, y is selected from 2 to 10. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 29, wherein y is selected from 4 to 8. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 29, wherein y is selected from 6 to 8. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound as described in claim 29, wherein y is selected from 6 or 8. A method for preparing an antibody-drug conjugate as represented by general formula (IV) or a pharmaceutically acceptable salt or solvate thereof comprises the following steps:

After reduction, Ab is coupled with general formula (F) to obtain a compound represented by general formula (IV); wherein Ab is an anti-BCMA antibody or an antigen-binding fragment thereof, as defined in claim 1; W, K ² , K ³ , R ² to R ⁶ , m and y are as defined in claim 26. A pharmaceutical composition comprising an antibody-drug conjugate as described in any one of claims 1 to 32 or a pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate, and one or more pharmaceutically acceptable excipients, diluents or carriers. Use of an antibody-drug conjugate as described in any one of claims 1 to 32 or a pharmaceutical composition as described in claim 34 in the preparation of a drug for treating or preventing a BCMA-mediated disease or condition. The use as described in claim 35, wherein the BCMA-mediated disease or condition is cancer or autoimmune disease. The use as described in claim 36, wherein the cancer is a cancer expressing BCMA. The use as described in claim 36, wherein the cancer is lymphoma, leukemia or myeloma. The use as described in claim 36, wherein the cancer is multiple myeloma. The use as described in claim 36, wherein the autoimmune disease is selected from lupus erythematosus, IgA nephropathy and rheumatic arthritis.