RU2826119C1 - Conjugate of anti-claudin antibody and medicinal agent and pharmaceutical use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology and can be used in medicine. Invention discloses an anti-claudin antibody conjugate with at least one exatecan fragment (see compound of general formula (Pc-L-Y-D)), where Pc is an antibody to claudin 18.2 or its antigen-binding fragment, and L, Y and n are as defined in the present description.

EFFECT: invention can be used in preparing a drug for treating cancer, the cells of which are characterized by high expression of claudin 18.2.

31 cl, 16 ex, 17 tbl, 5 dwg

Description

This application claims priority from Chinese patent application (Application No. CN201911273041.7) filed on December 12, 2019 and Chinese patent application (Application No. CN202011060513.3) filed on September 30, 2020.

AREA OF TECHNOLOGY

The present invention relates to a conjugate of an antibody to claudin and a drug, and in particular to a conjugate of an antibody to claudin 18.2 and an analogue of exatecan, a method for producing the same, a pharmaceutical composition containing the conjugate of the antibody and the drug, and their use for producing a drug for treating a disease or condition mediated by claudin 18.2, in particular, use for producing an anti-cancer drug.

PRIOR ART

The information set forth in this document is intended to provide general information relating to the present invention and does not necessarily constitute prior art.

Claudin-18 (CLDN18), a protein encoded by the claudin 18 gene in humans, belongs to the tight junction protein family and can control the passage of molecules between cell layers. The claudin protein contains four transmembrane regions and two extracellular loops in its structure, with its N-terminus and C-terminus in the cytoplasm. There are two splice variants of claudin-18, claudin 18.1 and claudin 18.2, which differ in sequence by eight amino acids in the first extracellular loops. Claudin 18.1 and claudin 18.2 differ in their expression distribution. Claudin 18.1 is selectively expressed in normal lung cells, while claudin 18.2 expression is highly restricted in normal cells, but it is frequently ectopically activated and overexpressed in various tumors (e.g., gastric cancer, lung cancer, and pancreatic cancer). Claudin 18.2 is considered a potential therapeutic target in gastric cancer and other cancers, and the discovery of such a target also provides a new option for the treatment of gastric cancer.

An antibody-drug conjugate (ADC) binds a monoclonal antibody or antibody fragment to a biologically active cytotoxin by a stable chemical linker compound, making full use of the specificity of binding of the antibody to the surface antigens of normal cells and tumor cells and the high potency of the cytotoxic substance, and eliminating the disadvantage of the former that it has a low therapeutic effect and the disadvantage of the latter that it has serious toxic side effects, etc. This means that the antibody-drug conjugate can bind to tumor cells more accurately and has a reduced effect on normal cells compared with conventional chemotherapeutic drugs known in the past.

Currently, some claudin 18.2-targeted antibodies and ADC drugs have been described in patents such as WO2020200196A1, WO2016166122 and WO2016165762. However, there is still a need to develop more effective and safe anti-claudin 18.2 antibody-drug conjugates for better application in the treatment of claudin 18.2-related tumors.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to ADCs of antibodies to claudin 18.2 and their use and provides an ADC medicament in which an antibody to claudin 18.2 or an antigen-binding fragment is conjugated to an exatecan analogue, a cytotoxic substance.

Accordingly, the present invention is intended to provide a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof:

Where:

Y is selected from the group consisting of -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-, -O-CR ¹ R ² -(CR ^a R ^b ) _m -, -O-CR ¹ R ² -, -NH-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)- and -S-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-;

R^a And R^bare identical or different and each is independently selected from the group consisting of hydrogen, deuterium, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl and heterocyclyl; or R^aand R^btogether with the carbon atoms to which they are attached, they form cycloalkyl or heterocyclyl;

R¹selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; R²selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; or R¹ And R²together with the carbon atoms to which they are attached, they form cycloalkyl or heterocyclyl;

or R ^a and R ² together with the carbon atoms to which they are attached form cycloalkyl or heterocyclyl;

m is an integer between 0 and 4;

n is a decimal or integer number between 1 and 10;

L represents a linker unit;

Pc is an antibody to claudin 18.2 or its antigen-binding fragment.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, the claudin 18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

i) the heavy chain variable region comprises HCDR (heavy chain complementarity determining region) 1, HCDR2 and HCDR3 having sequences identical to the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region set forth in SEQ ID NO: 3, and the light chain variable region comprises LCDR (light chain complementarity determining region) 1, LCDR2 and LCDR3 having sequences identical to the sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region set forth in SEQ ID NO: 4; or

ii) the variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3 having sequences identical to the sequences of HCDR1, HCDR2 and HCDR3 of the variable region of the heavy chain set forth in SEQ ID NO: 5, and the variable region of the light chain comprises LCDR1, LCDR2 and LCDR3 having sequences identical to the sequences of LCDR1, LCDR2 and LCDR3 of the variable region of the light chain set forth in SEQ ID NO: 6.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, the claudin 18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the sequences set forth in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; or

iv) the variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3 having the sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the variable region of the light chain comprises LCDR1, LCDR2 and LCDR3 having the sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

In some embodiments of the present invention, in the ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments, the antibody to claudin 18.2 is a murine antibody, a chimeric antibody or a humanized antibody.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, the claudin 18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

(1) the variable region of the heavy chain has the amino acid sequence set forth in SEQ ID NO: 3 or has at least 90% identity thereto, and the variable region of the light chain has the amino acid sequence set forth in SEQ ID NO: 4 or has at least 90% identity thereto;

(2) the variable region of the heavy chain has the amino acid sequence set forth in SEQ ID NO: 24 or has at least 90% identity thereto, and the variable region of the light chain has the amino acid sequence set forth in SEQ ID NO: 21 or has at least 90% identity thereto;

(3) the variable region of the heavy chain has the amino acid sequence set forth in SEQ ID NO: 5 or has at least 90% identity thereto, and the variable region of the light chain has the amino acid sequence set forth in SEQ ID NO: 6 or has at least 90% identity thereto; or

(4) the variable region of the heavy chain has the amino acid sequence set forth in SEQ ID NO: 31 or has at least 90% identity thereto, and the variable region of the light chain has the amino acid sequence set forth in SEQ ID NO: 28 or has at least 90% identity thereto.

In some embodiments of the present invention, in the ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, the antibody to claudin 18.2 is a humanized antibody comprising a framework region derived from a human antibody or a framework region variant thereof, wherein said framework region variant has back mutations of up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids in the light chain framework region and/or the heavy chain framework region of a human antibody;

preferably the framework variant comprises mutations selected from (a) or (b):

(a) one or more amino acid backmutations are optionally selected from the group consisting of 22S, 85I and 87H contained in the variable region of the light chain; and/or one or more amino acid backmutations are optionally selected from the group consisting of 48I, 82T and 69M contained in the variable region of the heavy chain; or

(b) one or more amino acid backmutations are optionally selected from the group consisting of 4L and 22S contained in the variable region of the light chain; and/or one or more amino acid backmutations are optionally selected from the group consisting of 38K, 40R, 48I, 66K, 67A, 69L, 71L and 73K contained in the variable region of the heavy chain;

preferably, the framework variant comprises mutations selected from the group consisting of:

(a-1) back mutations of amino acids 22S, 85I, and 87H contained in the variable region of the light chain and back mutations of amino acids 48I and 82T contained in the variable region of the heavy chain; or

(b-1) a reverse mutation of an amino acid selected from 4L contained in the variable region of the light chain.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, the claudin 18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region shown below:

(vii) a heavy chain variable region having the sequence set forth in SEQ ID NO: 3 and a light chain variable region having the sequence set forth in SEQ ID NO: 4;

(viii) a heavy chain variable region having the sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27 and a light chain variable region having the sequence set forth in SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23;

(ix) a heavy chain variable region having the sequence set forth in SEQ ID NO: 5 and a light chain variable region having the sequence set forth in SEQ ID NO: 6; or

(x) a heavy chain variable region having the sequence set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34 and a light chain variable region having the sequence set forth in SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30;

Preferably, the claudin 18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as shown below:

(xi) a heavy chain variable region having the sequence set forth in SEQ ID NO: 31 and a light chain variable region having the sequence set forth in SEQ ID NO: 29; or

(xii) a heavy chain variable region having the sequence set forth in SEQ ID NO: 26 and a light chain variable region having the sequence set forth in SEQ ID NO: 23.

In some embodiments of the present invention, in the ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention, the anti-claudin 18.2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region of an antibody; preferably, the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions and their normal variants, and the light chain constant region is selected from the group consisting of human κ and λ chain constant regions and their normal variants; more preferably, the antibody comprises a heavy chain constant region having the sequence indicated in SEQ ID NO: 7 and a light chain constant region having the sequence indicated in SEQ ID NO: 8; most preferably, the antibody comprises: a heavy chain having at least 90% identity to a heavy chain having the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 42, and a light chain having at least 90% identity to a light chain having the amino acid sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 39; or

a heavy chain having at least 90% sequence identity to a heavy chain having the amino acid sequence set forth in SEQ ID NO: 37 or SEQ ID NO: 49, and a light chain having at least 90% sequence identity to a light chain having the amino acid sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 46.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments, the claudin 18.2 antibody or antigen-binding fragment thereof comprises:

(c) a heavy chain having the sequence set forth in SEQ ID NO: 35 and a light chain having the sequence set forth in SEQ ID NO: 36;

(d) a heavy chain having the sequence set forth in SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45 and a light chain having the sequence set forth in SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO: 41;

(e) a heavy chain having the sequence set forth in SEQ ID NO: 37 and a light chain having the sequence set forth in SEQ ID NO: 38; or

(f) a heavy chain having the sequence set forth in SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 52 and a light chain having the sequence set forth in SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 48.

In some embodiments of the present invention, in the ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, the antibody to claudin 18.2 is selected from the group consisting of:

h1901-11, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain having the sequence set forth in SEQ ID NO: 41; and

h1902-5, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 49 and a light chain having the sequence set forth in SEQ ID NO: 47.

In some embodiments of the present invention, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (diabody), and disulfide-stabilized V region (dsFv).

In some embodiments of the present invention, in the ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, n may be an integer or decimal number from 1 to 10, and n may be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. n is a decimal or integer from 2 to 8, preferably a decimal or integer from 3 to 8, more preferably a decimal or integer from 5 to 9, or preferably a decimal or integer from 2 to 7. In some embodiments, n is a decimal or integer from 3.5 to 4.5.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention

Y represents -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-;

R^a And R^bare identical or different and each is independently selected from the group consisting of hydrogen, deuterium, halogen and alkyl;

R¹is a haloalkyl or C₃-₆cycloalkyl;

R²selected from the group consisting of hydrogen, haloalkyl and C₃-₆cycloalkyl;

or R¹ And R²together with the carbon atoms to which they are attached, they form C₃-₆cycloalkyl;

m represents 0 or 1.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, Y is selected from the group consisting of:

And ;

where the O-terminus of Y is attached to the linker unit L.

In some embodiments, the present invention provides a ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt or solvate thereof, wherein the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -.

In some embodiments, L ¹ is selected from the group consisting of -(succinimidyl-3-yl-N)-WC(O ₎ -, -CH ₂ -C(O)-NR ³ -WC(O _{)-, and -C(O)-WC(O)-, wherein W is selected from the group consisting of C 1-8 alkyl, C 1-8 alkyl -} C _3-6 cycloalkyl, and linear heteroalkyl having from 1 to 8 chain atoms, said heteroalkyl comprising from _{1 to 3 heteroatoms selected from the group consisting of N, O, and S, wherein said C 1-8 alkyl} , C _1-8 alkyl- _{C 3-6 cycloalkyl} , or linear heteroalkyl having from 1 to 8 chain atoms is independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl.

In some embodiments of the invention, L²selected from the group consisting of -NR⁴(CH₂CH₂O)p¹CH₂CH₂C(O)-, -NR⁴(CH₂CH₂O)p¹CH₂C(O)-, -S(CH₂)p¹C(O)- and chemical bonds, where p¹represents an integer from 1 to 20.

In some embodiments of the invention, L³is a peptide residue consisting of 2-7 amino acids, wherein the amino acids are selected from the group consisting of amino acid residues formed from the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl.

In some embodiments, L ⁴ is selected from the group consisting of -NR ⁵ (CR ⁶ R ⁷ ) _t -, -C(O)NR ⁵ -, -C(O)NR ⁵ (CH ₂ ) _t - and a chemical bond, where t is an integer from 1 to 6.

In some embodiments of the invention, R³, R⁴ And R⁵are identical or different and each is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl.

In some embodiments, R ⁶ and R ⁷ are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt or solvate thereof according to any of the above embodiments, the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, wherein

L ¹ is selected from the group consisting of -(succinimidyl-3-yl-N)-WC(O)-, -CH ₂ -C( _O )-NR ³ -WC(O)- and -C(O)-WC(O)-, wherein W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkyl cycloalkyl and linear heteroalkyl having from 1 to 8 chain atoms, said heteroalkyl _containing from ₁ to 3 heteroatoms selected from the group consisting of N, O and S, wherein said C _1-8 alkyl, cycloalkyl or linear heteroalkyl are each independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

L ² is selected from the group consisting of -NR ⁴ (CH ₂ CH ₂ O) p ¹ CH ₂ CH ₂ C(O)-, -NR ⁴ (CH ₂ CH ₂ O) p ¹ CH ₂ C(O)-, -S(CH ₂ ) p ¹ C(O)- and a chemical bond, where p ¹ is an integer from 1 to 20;

L ³ is a peptide residue consisting of 2-7 amino acids, wherein the amino acids are selected from the group consisting of amino acid residues formed from the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

L ⁴ is selected from the group consisting of -NR ⁵ (CR ⁶ R ⁷ ) _t -, -C(O)NR ⁵ -, -C(O)NR ⁵ (CH ₂ ) _t - and a chemical bond, where t is an integer from 1 to 6;

R³, R⁴ And R⁵are identical or different and each is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl;

R⁶ And R⁷are identical or different and each is independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention, the linker unit -L- is -L¹-L²-L³-L⁴-, Where

L ¹ is , and s ¹ is an integer from 2 to 8;

L ² is a chemical bond;

L ³ is a tetrapeptide residue, preferably a GGFG tetrapeptide residue (SEQ ID NO: 55);

L⁴represents -NR⁵(CR⁶R⁷)t-, where R⁵, R⁶ And R⁷are identical or different and each independently represents hydrogen or alkyl, and t represents 1 or 2;

and L¹-the end is attached to Pc, and L⁴-end attached to Y.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention, -L- is:

.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, -L-Y- is optionally selected from the group consisting of:

In some embodiments of the present invention, a ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention is a ligand-drug conjugate of the general formula (Pc-L _a -YD) or a pharmaceutically acceptable salt thereof,

Where:

W, L², L³, R⁵, R⁶ And R⁷are as defined in the above-mentioned linker unit -L-;

Pc, n, R¹, R²and m are as defined in the general formula (Pc-L-Y-D).

In some embodiments of the present invention, a ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention is a ligand-drug conjugate of the general formula (Pc-L _b -YD) or a pharmaceutically acceptable salt thereof,

Where:

s ¹ is an integer between 2 and 8;

Pc, R¹, R², R⁵-R⁷, m and n are as defined in the general formula (Pc-L_a-Y-D).

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, the ligand-drug conjugate is selected from the group consisting of:

where Pc and n are as defined in the general formula (Pc-L-Y-D).

In some embodiments of the present invention, a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof is provided, wherein the ligand-drug conjugate is selected from the group consisting of:

And

,

where n is as defined in the general formula (Pc-L-Y-D), and antibodies h1902-5 and h1901-11 are as defined previously.

The present invention further provides a method for producing a ligand-drug conjugate of the general formula (Pc-L _a -YD) or a pharmaceutically acceptable salt thereof, comprising the following steps:

subjecting Pc' and a compound of the general formula (L _a -YD) to a coupling reaction to obtain a compound of the general formula (Pc-L _a -YD);

Where:

Pc is an antibody to claudin 18.2 or an antigen-binding fragment thereof described above, and Pc' is obtained by reduction of Pc;

W, L², L³, R¹, R², R⁵-R⁷, m and n are as defined in the general formula (Pc-L_a-Y-D).

The present invention further provides a method for producing an antibody-drug conjugate of the general formula (Pc-L'-D), comprising the following step:

subjecting the reduced Pc to a coupling reaction with the general formula (L'-D) to obtain a compound where:

Pc is an antibody to claudin 18.2 or its antigen-binding fragment described above;

n is as defined in the general formula (Pc-L-Y-D).

In another aspect, the present invention relates to a pharmaceutical composition comprising a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention and one or more pharmaceutically acceptable excipients, diluents or carriers.

In another aspect, the present invention relates to the use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above embodiments or a pharmaceutical composition comprising them, as a medicament. In some embodiments, the medicament is for treating a disease or condition mediated by claudin 18.2; said disease or condition mediated by claudin 18.2 is preferably a cancer with high expression of claudin 18.2. In some embodiments, the medicament is for treating cancer. In some embodiments, the cancer is preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, neuroglioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, tumor Krukenberg, myeloproliferative tumor, squamous cell carcinoma, Ewing sarcoma, systemic light chain amyloidosis, or Merkel cell carcinoma; more preferably, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

In another aspect, the present invention relates to the use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention, or a pharmaceutical composition comprising them, for the preparation of a medicament for the treatment of a disease or condition mediated by claudin 18.2, wherein the disease or condition mediated by claudin 18.2 is a cancer with high expression of claudin 18.2. In some embodiments, the disease is preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, neuroglioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, tumor Krukenberg, myeloproliferative tumor, squamous cell carcinoma, Ewing sarcoma, systemic light chain amyloidosis, or Merkel cell carcinoma; more preferably, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

In another aspect, the present invention relates to the use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention or a pharmaceutical composition comprising them for the preparation of a medicament for the treatment or prevention of a tumor, wherein the tumor and cancer is preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, neuroglioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krukenberg tumor, myeloproliferative tumor, squamous cell carcinoma, Ewing sarcoma, systemic light chain amyloidosis, or Merkel cell carcinoma; more preferably, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

In another aspect, the present invention also relates to a method of treating and/or preventing a tumor, comprising administering to a subject in need thereof a therapeutically or prophylactically effective dose of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention or a pharmaceutical composition containing them, wherein the tumor is preferably a cancer associated with high expression of claudin 18.2.

In another aspect, the present invention also relates to a method for treating or preventing cancer, comprising administering to a subject in need thereof a therapeutically or prophylactically effective dose of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention or a pharmaceutical composition comprising them, wherein the tumor and cancer is preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, neuroglioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krukenberg tumor, myeloproliferative tumor, squamous cell carcinoma, Ewing sarcoma, systemic light chain amyloidosis, or Merkel cell carcinoma; more preferably, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

The active compound (e.g., a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to the present invention) may be formulated in a form suitable for administration by any suitable route, preferably in a unit dose form or in a single dose form that can be self-administered by a subject. The unit dose of the present invention may be in a tablet, capsule, cachet, vial, powder, granule, lozenge, suppository, replenishing powder, or liquid formulation.

The dosage of the active compound or composition used in the method of treatment according to the present invention generally varies depending on the severity of the disease, the weight of the subject and the potency of the active compound. However, as a rule, a suitable unit dose may be from 0.1 to 1000 mg.

The pharmaceutical composition of the present invention may contain, in addition to the active compound, one or more excipients selected from the group consisting of a filler, a diluent, a binder, a wetting agent, a disintegrant, an excipient, and the like. Depending on the route of administration, the composition may contain from 0.1 to 99% by weight of the active compound.

The anti-claudin 18.2 antibody and the antibody-drug conjugate provided in the present invention have good affinity for cell surface antigens, good endocytosis efficiency and high tumor inhibition efficiency, as well as wider application windows of the drug, and are suitable for clinical use of the drug.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1 shows the results of FACS (fluorescence-activated cell sorting) analysis of binding of humanized antibodies to human claudin 18.2 at the cellular level.

Fig. 2 shows endocytosis of humanized antibodies by NUGC4 cells.

Figures 3A-3C show antibody assays for ADCC (antibody-dependent cellular cytotoxicity) effects in NUGC4 cells with different levels of claudin 18.2 expression. Figure 3A shows antibody assays for ADCC effects in wild-type NUGC4 cells (low claudin 18.2 expression); Figure 3B shows antibody assays for ADCC effects in NUGC4 cells with moderate claudin 18.2 expression; Figure 3C shows antibody assays for ADCC effects in NUGC4 cells with high claudin 18.2 expression.

Fig. 4 shows the results of tumor inhibition by ADC-1 of the present invention.

Fig. 5 shows the results of tumor inhibition by ADC-2 of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

1. Terminology

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used to practice or test the present invention, the preferred methods and materials are described herein. In the description and claims of the present invention, the following terms are used in accordance with the definitions below.

When a trade name is used in this description, it is intended to include the composition of the product under the trade name and the generic drug and active drug components of the product under the trade name.

Unless otherwise specified, the terms used in the description and claims have the following meanings.

The term "drug" refers to a chemical substance that can change or establish the physiology and pathological state of an organism and can be used to prevent, diagnose and treat diseases. Drug includes cytotoxic drug. There is no clear boundary between a drug and a toxic substance. Toxic substance refers to a chemical substance that has a toxic effect on organisms and can cause harm to human health even in small doses. Any drug in large doses can cause toxic reactions. Cytotoxic drug refers to a substance that inhibits or prevents cell functions and/or causes cell death or destruction of cells. Cytotoxic drug can kill tumor cells in general when the concentration is high enough; however, due to the lack of specificity, cytotoxic drug can cause apoptosis of normal cells while killing tumor cells, resulting in serious side effects. Cytotoxic drug includes toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes.

The term "linker unit", "linker" or "linker moiety" refers to a chemical structural fragment or linkage that is linked at one end to a ligand (e.g., an antibody or antigen-binding fragment thereof) and at the other end to a drug, or is linked to other linkers prior to being linked to a drug.

The linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"). The linker may comprise stretcher units, spacer units, and amino acid units, and may be synthesized using methods known in the art, such as those described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates the release of drugs in cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers may be used (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

Abbreviations

Linker components include, but are not limited to:

MC is 6-maleimidocaproyl with the structure:

,

Val-Cit or "vc" is valine-citrulline (a typical dipeptide in a protease-cleavable linker),

Citrulline is 2-amino-5-ureidopentanoic acid,

PAB is p-aminobenzyloxycarbonyl (an example of a "self-cleaving" linker component),

Me-Val-Cit is N-methyl-valine-citrulline (where the linker peptide bond has been modified to prevent its cleavage by cathepsin B),

MC(PEG)6-OH is maleimidocaproyl polyethyleneglycol (attached to the cysteine of the antibody),

SPP is N-succinimidyl 4-(2-pyridylthio)valerate,

SPDP is N-succinimidyl 3-(2-pyridyldithio)propionate,

SMCC is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,

IT is an iminothiolane.

The term "ligand drug conjugate" means that the ligand is linked to the biologically active drug via a linker unit. As used herein, the "ligand drug conjugate" is preferably an antibody drug conjugate (ADC), which means that a monoclonal antibody or antibody fragment is linked to a biologically active toxic drug via a linker unit. The antibody can be conjugated to the drug directly or via a linker. The average number of drug units conjugated to each antibody (the average drug loading or drug loading, which can be expressed in units n) can vary, for example, from about 0 to about 20 drug units; in some embodiments, from 1 to about 10 drug units, and in some embodiments, from 1 to about 8 drug units.

The term "average drug loading" or "drug loading" refers to the average amount of cytotoxic drug loaded per ligand in ligand-drug conjugate molecules, and can also be expressed in terms of the drug to antibody ratio. The drug loading can be from 0 to 12, preferably from 1 to 10 cytotoxic drugs per ligand (Pc). In embodiments of the present invention, the drug loading is expressed in units n, which may also be referred to as the DAR (drug-antibody ratio) value and may be a non-zero integer or decimal number from 0 to 12, preferably an integer or decimal number from 1 to 10, more preferably an integer or decimal number from 2 to 8, and most preferably an integer or decimal number from 3 to 8. Examples are an average of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The average number of drugs per ADC molecule after coupling reactions can be characterized by conventional methods such as UV-Vis spectroscopy, mass spectrometry, ELISA (enzyme-linked immunosorbent assay) assays, and HPLC.

The three-letter and one-letter codes for amino acids used in this specification are disclosed in J. biol. chem, 243, p3558 (1968).

Claudin 18 (CLD18) molecules (Genbank accession no. splice variant 1 (CLD18A1): NP_057453, NM016369, and splice variant 2 (CLD18A2 or claudin 18.2): NM_001002026, NP_001002026) are intrinsic transmembrane proteins found in the tight junctions of epithelia and endothelium. At tight junctions, occludins and claudins are the predominant transmembrane protein components. Due to the strong intercellular adhesion property of claudins, they create a primary barrier that prevents and controls paracellular transport of solutes and limits lateral diffusion of membrane lipids and proteins to maintain cell polarity. The structure of epithelial tissues involves proteins that form tight junctions. It is reported that these proteins can hardly approach antibodies in well-constructed epithelium, but become open to attack in tumor cells.

The term "antibody" refers to an immunoglobulin, which has a tetrapeptide chain structure formed by linking two heavy chains and two light chains by interchain disulfide bonds. Based on differences in the amino acid composition and the order of the constant regions of the heavy chain, immunoglobulins can be divided into five classes, otherwise called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ chain, δ chain, γ chain, α chain, and ε chain, respectively. Ig of one class can be divided into different subclasses based on differences in the amino acid composition of the hinge regions and the number and position of the disulfide bonds of the heavy chains; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ or λ chains based on differences in the constant regions. Each of the five Ig classes can have either a κ chain or a λ chain.

In the heavy and light chains of full-length antibodies, the sequences of about 110 amino acids near the N-terminus vary considerably and are thus called variable regions (Fv regions); the remaining amino acid sequences near the C-terminus are relatively stable and are thus called constant regions. The variable regions include 3 hypervariable regions (HVRs) and 4 framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are thus also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) or heavy chain variable region (HCVR) consists of 3 CDRs and 4 FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDRs of the light chain are designated LCDR1, LCDR2, and LCDR3, and the three CDRs of the heavy chain are designated HCDR1, HCDR2, and HCDR3.

The term "fully humanized antibody", "fully human antibody" or "perfectly human antibody", also known as "fully humanized monoclonal antibody", has both a humanized variable region and a constant region. The development of monoclonal antibodies has four stages, namely, mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully humanized monoclonal antibodies. The main suitable technologies for producing fully human antibodies include: human hybridoma technology, EBV (Epstein-Barr virus)-transformed B-lymphocyte technology, phage display technology, transgenic mouse antibody technology, single B-cell antibody technology and the like.

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to bind an antigen. It has been shown that a fragment of a full-length antibody can be used to perform the antigen-binding function of an antibody. The binding fragment included in the "antigen-binding fragment" is selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V region (dsFv) and antigen-binding fragments of peptides containing CDRs; examples include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, divalent fragments comprising two Fab fragments linked by disulfide bridges at the hinge regions; (iii) Fd fragments consisting of the VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of one arm of an antibody; (v) single domains or dAb fragments (Ward et al., (1989) Nature 341:544-546) consisting of VH domains; and (vi) isolated complementarity determining regions (CDRs) or (vii) combinations of two or more isolated CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker by recombination, allowing it to produce a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody. Such antibody fragments are prepared using conventional techniques known to those skilled in the art and are screened for suitability in the same manner as intact antibodies. Antigen-binding portions may be produced using recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of various isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

In general, Fab is an antibody fragment having a molecular weight of about 50,000 and possessing antigen-binding activity, among fragments obtained by treating an IgG antibody molecule with papain protease (e.g., cleavage of the amino acid residue at position 224 of the H chain), in which a portion at the N-terminal side of the H chain is linked to the L chain by a disulfide bond.

In general, F(ab')2 is an antibody fragment produced by cleavage of the portion below the disulfide bond in the hinge region of IgG by the enzyme pepsin. It has a molecular weight of about 100,000, has antigen-binding activity, and contains two Fab regions linked at the hinge position.

In general, Fab' is an antibody fragment having a molecular weight of about 50,000 and possessing antigen-binding activity, obtained by cleavage of the disulfide bond in the hinge region of F(ab')2 described above.

In addition, Fab' can be produced by inserting DNA encoding a Fab' fragment into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express Fab'.

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or VH) and an antibody light chain variable domain (or VL) linked by a linker. Such scFv molecules have the general structure: _NH2 -VL-linker-VH-COOH or _NH2 -VH-linker-VL-COOH. Suitable linkers in the prior art consist of repeating amino acid sequences GGGGS or variants thereof, for example, 1 to 4 repeating variants (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995) Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999) J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

The term "CDR" refers to one of six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. In general, there are three CDRs (HCDR1, HCDR2, and HCDR3) in each variable region of the heavy chain and three CDRs (LCDR1, LCDR2, and LCDR3) in each variable region of the light chain. The amino acid sequence boundaries of the CDRs may be defined using any of a number of well-known schemes. One of the most common definitions for the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. In this context, Kabat's definition of CDRs refers only to CDR1, CDR2, and CDR3 of the variable domain of the light chain and to CDR2 and CDR3 of the variable domain of the heavy chain. Also included are the Chothia numbering scheme, the "ABM" numbering scheme, the "contact" numbering scheme (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), the ImMunoGenTics (IMGT) numbering scheme (Lefranc M.P., Dev. Comp. Immunol., 27, 55-77(2003)) and others.

The term "antibody scaffold" refers to the portion of a VL or VH variable domain that serves as a framework for the CDRs of the variable domain. This is essentially a variable domain without the CDRs.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody binds. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, volume 66, G.E.Morris, Ed. (1996).

The terms "specific binding," "selective binding," "selective binding to," and "specific binding to" refer to the binding of an antibody to an epitope on a given antigen. Typically, an antibody binds with an affinity (KD) of less than about 10^-7M, for example, less than about 10^-8M, 10^-9M or 10^-10M or less.

The term "KD" refers to the equilibrium dissociation constant for an antibody-antigen interaction. In general, an antibody (or antigen-binding fragment thereof) of the present invention binds to claudin 18.2 (or an epitope thereof) with an equilibrium dissociation constant (KD) of less than about ^10-7 M, such as less than about ^10-8 M or ^10-9 M; for example, the KD value is determined using FACS for the affinity of an antibody of the present invention for cell surface antigens.

The term "nucleic acid molecule" refers to a DNA molecule or an RNA molecule. A nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

"Identity" of an amino acid sequence refers to the percentage of amino acid residues shared by a first sequence and a second sequence, wherein gaps are introduced when necessary to achieve the maximum percentage of sequence identity, and any conservative substitution is not considered part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be achieved by various methods that are within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine parameters suitable for measuring alignment, including any algorithm necessary to achieve maximum alignment of the entire length of the aligned sequences.

The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded loop of DNA into which other DNA segments can be ligated. In another embodiment, the vector is a viral vector in which other DNA segments can be ligated into a viral genome. The vectors disclosed herein are capable of autonomously replicating in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors), or are capable of integrating into the host cell's genome after introduction into the host cell and thereby replicating with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, for example, those described in Chapters 5-8 and 15 of Antibodies: A Laboratory Manual, Cold Spring Harbor Press. Antigen-binding fragments can also be produced using conventional methods. The antibody or antigen-binding fragment described in the present invention is genetically engineered to contain one or more additional human FRs in a CDR of non-human origin. Human FR germline sequences can be obtained from the ImMunoGeneTics(IMGT) website https://imgt.cines.fr or from the Journal of Immunoglobulins, Lefranc, G., the Immunoglobulin FactsBook, Academic Press, 2001ISBN012441351, by alignment with the IMGT human antibody germline variable region gene database and the MOE software.

The term "host cell" refers to the cell into which the expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as Escherichia coli or Salmonella strains; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NS0 cells.

The engineered antibody or antigen-binding fragment of the present invention can be produced and purified by conventional techniques. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vectors can be stably transfected into host cells. As more recommended in the prior art, mammalian expression systems will result in glycosylation of the antibody, in particular at the N-terminal site of the Fc region. Positive clones are expanded in the medium in a bioreactor to produce the antibody. The culture with the secreted antibody can be purified using conventional techniques, for example, using an A or G Sepharose FF column. Non-specifically bound fractions are washed off. Bound antibody is eluted by a pH gradient method, and antibody fragments are detected by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods such as molecular sieves and ion exchange. The resulting product should be immediately frozen, e.g. at minus 70°C, or lyophilized.

The term "peptide" refers to a fragment of the connection between an amino acid and a protein. It is formed by joining two or more amino acid molecules by peptide bonds and is a structural and functional fragment of a protein.

The term "sugar" refers to biomacromolecules consisting of C, H and O elements. They can be divided into monosaccharides, disaccharides, polysaccharides, etc.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a linear or branched group containing from 1 to 20 carbon atoms, preferably an alkyl group containing from 1 to 12 carbon atoms, more preferably an alkyl group containing from 1 to 10 carbon atoms, and most preferably an alkyl group containing from 1 to 6 carbon atoms (containing 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-метилгексил, 2,3-диметилпентил, 2,4-диметилпентил, 2,2-диметилпентил, 3,3-диметилпентил, 2-этилпентил, 3-этилпентил, н-октил, 2,3-диметилгексил, 2,4-диметилгексил, 2,5-диметилгексил, 2,2-диметилгексил, 3,3-диметилгексил, 4,4-диметилгексил, 2-этилгексил, 3-этилгексил, 4-этилгексил, 2-метил-2-этилпентил, 2-метил-3-этилпентил, н-нонил, 2-метил-2-этилгексил, 2-метил-3-этилгексил, 2,2-диэтилпентил, н-децил, 3,3-диэтилгексил, 2,2-diethylhexyl, their various isomers with side chains, etc. More preferred is lower alkyl having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available site on the compound, wherein the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from the group consisting of N, O and S, wherein alkyl is as defined above.

The term "alkylene" refers to a saturated linear or branched aliphatic hydrocarbon group having 2 residues derived from a parent alkane by the removal of two hydrogen atoms from the same or two different carbon atoms. It is a linear or branched group containing from 1 to 20 carbon atoms, preferably alkylene containing from 1 to 12 carbon atoms, more preferably alkylene containing from 1 to 6 carbon atoms (containing 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH ₂ -), 1,1-ethylidene (-CH(CH ₃ )-), 1,2-ethylidene (-CH ₂ CH ₂ )-, 1,1-propylidene (-CH(CH ₂ CH ₃ )-), 1,2-propylidene (-CH ₂ CH(CH ₃ )-), 1,3-propyl den (-CH ₂ CH ₂ CH ₂ -), 1,4-butylidene (-CH ₂ CH ₂ CH ₂ CH ₂ -), 1,5-butylidene (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -), etc. Alkylene may be substituted or unsubstituted. When substituting, the substituent may be substituted at any available site on the compound with one or more substituents, preferably independently optionally selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

The term "alkoxy" refers to the group -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutoxy, cyclopentyloxy and cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent. The cycloalkyl ring contains from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 10 carbon atoms, and most preferably from 3 to 8 carbon atoms (containing 3, 4, 5, 6, 7 or 8 carbon atoms). Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc. Polycyclic cycloalkyl includes spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing from 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen and S(O) _m (where m is an integer of 0, 1 or 2), excluding the cyclic portion -OO-, -OS- or -SS-, and the remaining ring atoms are carbon atoms. It preferably contains from 3 to 12 ring atoms, of which from 1 to 4 are heteroatoms (1, 2, 3 or 4 heteroatoms); more preferably, the cycloalkyl ring contains from 3 to 10 ring atoms (3, 4, 5, 6, 7, 8, 9 or 10 ring atoms). Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclyl includes spiroheterocyclyl, fused heterocyclyl, and bridged heterocyclyl.

The term "spiroheterocyclyl" refers to a 5- to 20-membered polycyclic heterocyclyl group in which the monocyclic rings share one atom (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, and S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon atoms. These rings may contain one or more double bonds, but none of them has a completely conjugated π-electron system. Preferably, spiroheterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered. According to the number of spiro atoms shared between the rings, spiroheterocyclyl may be monospiroheterocyclyl, bispiroheterocyclyl or polyspiroheterocyclyl, preferably monospiroheterocyclyl and bispiroheterocyclyl, and more preferably 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiroheterocyclyl. Non-limiting examples of spiroheterocyclyl include:

And
.

The term "fused heterocyclyl" refers to a 5- to 20-membered polycyclic heterocyclyl in which each ring shares a pair of adjacent atoms with other rings in the system, wherein one or more rings may contain one or more double bonds, but none of them has a completely conjugated π-electron system, wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, or S(O) _m (where m is an integer of 0, 1, or 2), and the remaining ring atoms are carbon atoms. Preferably, the fused heterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered (7-, 8-, 9-, or 10-membered ring). According to the number of rings formed, the fused heterocyclyl may be bicyclic, tricyclic, tetracyclic or polycyclic, preferably bicyclic or tricyclic, and more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclyl. Non-limiting examples of fused heterocyclyl include:

And .

The term "bridged heterocyclyl" refers to a 5- to 14-membered polycyclic heterocyclyl in which any two rings share two carbon atoms that are not directly attached to each other, which rings may contain one or more double bonds but none of which has a completely conjugated π-electron system, wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, and S(O) _m (where m is an integer of 0, 1, or 2), and the remaining ring atoms are carbon atoms. Preferably, the fused heterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered (7-, 8-, 9-, or 10-membered ring). According to the number of rings formed, the bridged heterocyclyl may be bicyclic, tricyclic, tetracyclic or polycyclic, preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclyl include:

And
.

A heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl; non-limiting examples include, but are not limited to:

, etc.

Heterocyclyl may be optionally substituted or unsubstituted, and when substituted, the substituent preferably represents one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

The term "aryl" refers to a 6- to 14-membered, preferably 6- to 10-membered (6-, 7-, 8-, 9-, or 10-membered), carbon monocyclic or fused polycyclic (i.e., rings sharing a pair of adjacent carbon atoms) group having a conjugated π-electron system, such as phenyl and naphthyl, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring; non-limiting examples include, but are not limited to:

And .

Aryl may be substituted or unsubstituted, and when substituted, the substituent preferably represents one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio.

The term "heteroaryl" refers to a heteroaromatic system containing from 1 to 4 heteroatoms (1, 2, 3 or 4 heteroatoms) and from 5 to 14 ring atoms, wherein the heteroatoms are selected from the group consisting of oxygen, sulfur and nitrogen. Heteroaryl is preferably 5-10-membered (5-, 6-, 7-, 8-, 9-, 10-membered heteroaryl), more preferably 5- or 6-membered, such as furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl and tetrazolyl. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is heteroaryl; non-limiting examples include, but are not limited to:

And .

Heteroaryl may be optionally substituted or unsubstituted, and when substituted, the substituent preferably represents one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio.

The term "amino protecting group" refers to a group that can be easily removed and is intended to protect the amino group from being altered when the reaction is carried out elsewhere in the molecule. Non-limiting examples include 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, etc. These groups may be optionally substituted with 1 to 3 substituents (1, 2 or 3 substituents) selected from the group consisting of halogen, alkoxy and nitro. The amino protecting group is preferably 9-fluorenylmethoxycarbonyl.

The term "haloalkyl" refers to an alkyl group in which the hydrogen atoms are replaced by one or more halogens, wherein the alkyl group is as defined above.

The term "deuterated alkyl" refers to an alkyl group in which the hydrogen atoms are replaced by one or more deuterium atoms, wherein the alkyl group is as defined above.

The term "hydroxyalkyl" refers to an alkyl group wherein the hydrogen of the alkyl group is replaced by one or more hydroxy groups, wherein alkyl is as defined above.

The term "hydroxy" refers to the -OH group.

The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

The term "amino" refers to -NH₂.

The term "nitro" refers to -NO₂.

The term "cyano" refers to -CN.

The term "acylamino" refers to -C(O)N(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

The term "optional" or "optionally" means that the event or circumstance subsequently described may, but need not, occur, and that the description includes instances where the event or circumstance does or does not occur. For example, "a heterocyclyl group optionally substituted with alkyl" means that alkyl may, but need not, be present, and that the description includes instances where the heterocyclyl group is or is not substituted with alkyl.

"Substituted" means that one or more, preferably up to 5 and more preferably 1, 2 or 3 hydrogen atoms in the group are independently replaced by a substituent. The substituent is only in its possible chemical position, and those skilled in the art will be able to determine (experimentally or theoretically) whether a substitution is possible or impossible without special efforts. For example, it may be unstable when an amino or hydroxy group having a free hydrogen is bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, and other components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration into the body, which facilitates absorption of the active ingredient, thereby exerting biological activity.

The term "pharmaceutically acceptable salt" refers to a salt of a ligand-drug conjugate of the present invention or a salt of an active compound of the present invention. Such salts are safe and effective when used in subjects and have the desired biological activity. The ligand-antibody drug conjugate of the present invention contains at least one amino group and thus can form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodate, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate and p-toluenesulfonate.

In one embodiment of the present invention, the cytotoxic drug is conjugated to the mercapto group of the antibody via a linker unit.

Loading of the ligand-cytotoxic drug conjugate may be controlled by the following non-limiting methods, including:

(1) control of the molar ratio of linker reagent to monoclonal antibody,

(2) control of reaction time and temperature and

(3) selection of different reagents.

For obtaining conventional pharmaceutical compositions, reference is made to the Chinese Pharmacopoeia.

The term "pharmaceutically acceptable carrier" for the drug of the present invention refers to a system that can change the route of entry of the drug into the body of a subject and the distribution of the drug in the subject, control the release rate of the drug, and deliver the drug to the target organ. The release of the drug carrier and the target system can reduce the degradation and loss of the drug, reduce side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble due to their unique amphiphilic structures to form various forms of aggregates, such as micelles, microemulsions, gels, liquid crystals, and vesicles, as preferred examples. The aggregates have the ability to encapsulate drug molecules and have good membrane permeability, and therefore can be used as excellent drug carriers.

The term "excipient" is an additive other than the active compound to a pharmaceutical composition. It may also be called an adjuvant. For example, binders, fillers, disintegrants, lubricants in tablets; bulking agents in semi-solid ointments and cream preparations; preservatives, antioxidants, flavoring agents, co-solvents, emulsifiers, solubilizers, tonicity adjusting agents, coloring agents and the like in liquid formulations may be called excipients.

The term "diluent", also called filler, is primarily used to increase the weight and volume of a tablet. The addition of a diluent not only ensures a certain volume, but also reduces the dose deviation of the main ingredients, and improves the compressibility of the drug, etc. When a drug in the form of a tablet contains oily components, an absorbent is necessarily added to absorb the oily components so as to maintain a "dry" state and thus facilitate the preparation of a tablet. Examples include starch, lactose, inorganic calcium salts, microcrystalline cellulose, etc.

The pharmaceutical composition may be in the form of a sterile aqueous injection solution. Available and acceptable carriers or solvents include water, Ringer's solution and isotonic sodium chloride solution. The sterile injection preparation may be a sterile oil-in-water microemulsion for injection in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerol and processed to form a microemulsion. The injection or microemulsion can be administered locally into the bloodstream of a subject in large quantities. Alternatively, it may be desirable to administer the solution and microemulsion in a manner that maintains a constant circulating concentration of the compound of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device can be used. An example of such a device is the Deltec CADD-PLUS intravenous injection pump. TM. 5400.

The pharmaceutical composition can be in the form of a sterile aqueous or oily injection suspension for intramuscular and subcutaneous administration. The suspension can be prepared according to the prior art using those suitable dispersing or wetting agents and suspending agents mentioned above. The sterile injection composition can also be a sterile injection or suspension prepared in a parenterally acceptable non-toxic diluent or solvent, for example, a solution prepared in 1,3-butanediol. In addition, a sterile fixed oil can usually be used as a solvent or suspending medium. For this purpose, any mixture of fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can also be used in the preparation of injections.

2. Method of synthesis

For the purposes of synthesis, the following technical synthesis schemes are adopted:

The method for obtaining a compound of the general formula (Pc-L _a -YD) includes the following stages:

subjecting the reduced Pc to a coupling reaction with the general formula (L _a -YD) to obtain a compound of the general formula (Pc-L _a -YD), wherein the reducing agent is preferably TCEP (tris(2-carboxyethyl)phosphine); in particular, the disulfide bonds in the antibody are preferably reduced;

Pc, W, L², L³, R¹, R², R⁵-R⁷, m and n are as defined in the general formula (Pc-L_a-Y-D).

One or more embodiments of the present invention are described in detail in the description above. Although any methods and materials similar or identical to those described herein can be used to make or test the present invention, the preferred methods and materials are described below. Other features, objects and advantages of the present invention will be apparent from the description and claims. In the description and claims, the singular forms "a" and "an" include the plural forms unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. All patents and publications cited in the description are incorporated by reference. The following examples are provided to more fully illustrate the preferred embodiments of the present invention. These examples should not be construed in any way as limiting the scope of the present invention, which is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

I. Obtaining antibodies

Example 1-1. Construction of a cell strain with high expression of claudin 18.2

Lentiviral expression vector plasmids pCDH-hClaudin18.2, lentiviral packaging vector system pVSV-G and pCMV-dR8.91 were transfected into 293T viral packaging cells using Lipofectamine 3000 transfection reagent. The medium supernatant containing viruses was collected, filtered and ultra-high-speed centrifuged. Human gastric signet ring cell carcinoma cell strain NUGC4 was allowed to be infected with concentrated virus, screened with puromycin for two to three weeks and subjected to single-cell FACS sorting.

The expression levels of claudin 18.2 were determined according to the tumor IHC (immunohistochemistry) scores. Cells with claudin 18.2 expression levels similar to those of a tumor with an IHC score of 3 were considered high expressing cells, and cells with claudin 18.2 expression levels similar to those of a tumor with an IHC score of 2 were considered moderate expressing cells. According to the claudin 18.2 expression level on the surface of NUGC4 cells determined by FACS, NUGC4/hClaudin18.2 monoclonal cell strains with high claudin 18.2 expression were selected. The expression level of claudin 18.2 on the surface of wild-type NUGC4 cells was also determined by FACS, and NUGC4 clonal cell strains with moderate claudin 18.2 expression were selected. Wild-type NUGC4 cells were cells with low expression of claudin 18.2.

Selected monoclonal cell strains were expanded and preserved by freezing for subsequent experiments.

Claudin 18.2 sequence in Genbank: NP_001002026: (SEQ ID NO: 1)

MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHA SGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV;

DNA sequence of claudin 18.2: (SEQ ID NO: 2)

1 AGAATTGCGC TGTCCACTTG TCGTGTGGCT CTGTGTCGAC ACTGTGCGCC ACCATGGCCG

61 TGACTGCCTG TCAGGGCTTG GGGTTCGTGG TTTCACTGAT TGGGATTGCG GGCATCATTG

121 CTGCCACCTG CATGGACCAG TGGAGCACCC AAGACTTGTA CAACAACCCC GTAACAGCTG

181 TTTTCAACTA CCAGGGGCTG TGGCGCTCCT GTGTCCGAGA GAGCTCTGGC TTCACCGAGT

241 GCCGGGGCTA CTTCACCCTG CTGGGGCTGC CAGCCATGCT GCAGGCAGTG CGAGCCCTGA

301 TGATCGTAGG CATCGTCCTG GGTGCCATTG GCCTCCTGGT ATCCATCTTT GCCCTGAAAT

361 GCATCCGCAT TGGCAGCATG GAGGACTCTG CCAAAGCCAA CATGACACTG ACCTCCGGGA

421 TCATGTTCAT TGTCTCAGGT CTTTGTGCAA TTGCTGGAGT GTCTGTGTTT GCCAACATGC

481 TGGTGACTAA CTTCTGGATG TCCCAGCTA ACATGTACAC CGGCATGGGT GGGATGGTGC

541 AGACTGTTCA GACCAGGTAC ACATTTGGTG CGGCTCTGTT CGTGGGCTGG GTCGCTGGAG

601 GCCTCACACT AATTGGGGGT GTGATGATGT GCATCGCCTG CCGGGGCCTG GCACCAGAAG

661 AAACCAACTA CAAAGCCGTT TCTTATCATG CCTCAGGCCA CAGTGTTGCC TACAAGCCTG

721 GAGGCTTCAA GGCCAGCACT GGCTTTGGT CCAACACCAA AAACAAGAAG ATATACGATG

781 GAGGTGCCCG CACAGAGGAC GAGGTACAAT CTTATCCTTC CAAGCACGAC TATGTGTAAT

841 GCTCTAAGAC CTCTCAGCAC GGGCGGAAGA AACTCCCGGA GAGCTCACCC AAAAAACAAG

901 GAGATCCCAT CTAGATTTCT TCTTGCTTTT GACTCACAGC TGGAAGTTAG AAAAGCCTCG

961 ATTTCATCTT TGGAGAGGCC AAATGGTCTT AGCCTCAGTC TCTGTCTCTA AATATTCCAC

1021 CATAAAACAG CTGAGTTATT TATGAATTAG AGGCTATAGC TCACATTTTC AATCCTCTAT

1081 TTCTTTTTTT AAATATAACT TTCTACTCTG ATGAGAGAAT GTGGTTTTAA TCTCTCTCTC

1141 ACATTTTGAT GATTTAGACA GACTCCCCCT CTTCCTCCTA GTCAATAAAC CCATTGATGA

1201 TCTATTTCCC AGCTTATCCC CAAGAAAACT TTTGAAAGGA AAGAGTAGAC CCAAAGATGT

1261 TATTTTCTGC TGTTTGAATT TTGTCTCCCCC ACCCCCAACT TGGCTAGTAA TAAACACTTA

1321 CTGAAGAAGA AGCAATAAGA GAAAGATATT TGTAATCTCT CCAGCCCATG ATCTCGGTTT

1381 TCTTACACTG TGATCTTAAA AGTTACCAAA CCAAAGTCAT TTTCAGTTTG AGGCAACCAA

1441 ACCTTTCTAC TGCTGTTGAC ATCTTCTTAT TACAGCAACA CCATTCTAGG AGTTTCCTGA

1501 GCTCTCCACT GGAGTCCTCT TTCTGTCGCG GGTCAGAAAT TGTCCCTAGA TGAATGAGAA

1561 AATTATTTTT TTTAATTTAA GTCCTAAATA TAGTTAAAAT AAATAATGTT TTAGTAAAAT

1621 GATACACTAT CTCTGTGAAA TAGCCTCACC CCTACATGTG GATAGAAGGA AATGAAAAAA

1681 TAATTGCTTT GACATTGTCT ATATGGTACT TTGTAAAGTC ATGCTTAAGT ACAAATTCCA

1741 TGAAAAGCTC ACTGATCCTA ATTCTTTCCC TTTGAGGTCT CTATGGCTCT GATTGTACAT

1801 GATAGTAAGT GTAAGCCATG TAAAAAGTAA ATAATGTCTG GGCACAGTGG CTCACGCCTG

1861 TAATCCTAGC ACTTTGGGAG GCTGAGGAGG AAGGATCACT TGAGCCCAGA AGTTCGAGAC

1921 TAGCCTGGGC AACATGGAGA AGCCCTGTCT CTACAAAATA CAGAGAGAAA AAATCAGCCA

1981 GTCATGGTGG CCTACACCTG TAGTCCCAGC ATTCCGGGAG GCTGAGGTGG GAGGATCACT

2041 TGAGCCCAGG GAGGTTGGGG CTGCAGTGAG CCATGATCAC ACCACTGCAC TCCAGCCAGG

2101 TGACATAGCG AGATCCTGTC TAAAAAAATA AAAAATAAAT AATGGAACAC AGCAAGTCCT

2161 AGGAAGTAGG TTAAAACTAA TTCTTTAAAA AAAAAAAAAA GTTGAGCCTG AATTAAATGT

2221 AATGTTTCCA AGTGACAGGT ATCCACATTT GCATGGTTAC AAGCCACTGC CAGTTAGCAG

2281 TAGCACTTTC CTGGCACTGT GGTCGGTTTT GTTTTGTTTT GCTTTGTTTA GAGACGGGGT

2341 CTCACTTTCC AGGCTGGCCT CAAACTCCTG CACTCAAGCA ATTCTTCTAC CCTGGCCTCC

2401 CAAGTAGCTG GAATTACAGG TGTGCGCCAT CACAACTAGC TGGTGGTCAG TTTTGTTACT

2461 CTGAGAGCTG TTCACTTCTC TGAATTCACC TAGAGTGGTT GGACCATCAG ATGTTTGGGC

2521 AAAACTGAAA GCTCTTTGCA ACCACACACC TTCCCTGAGC TTACATCACT GCCCTTTTGA

2581 GCAGAAAGTC TAAATTCCTT CCAAGACAGT AGAATTCCAT CCCAGTACCA AAGCCAGATA

2641 GGCCCCCTAG GAAACTGAGG TAAGAGCAGT CTCTAAAAAC TACCCACAGC AGCATTGGTG

2701 CAGGGGAACT TGGCCATTAG GTTATTATTT GAGAGGAAAG TCCTCACATC AATAGTACAT

2761 ATGAAAGTGA CCTCCAAGGG GATTGGTGAA TACTCATAAG GATCTTCAGG CTGAACAGAC

2821 TATGTCTGGG GAAAGAACGG ATTATGCCCC ATTAAAATAAC AAGTTGTGTT CAAGAGTCAG

2881 AGCAGTGAGC TCAGAGGCCC TTCTCACTGA GACAGCAACA TTTAAACCAA ACCAGAGGAA

2941 GTATTTGTGG AACTCACTGC CTCAGTTTGG GTAAAGGATG AGCAGACAAG TCAACTAAAG

3001 AAAAAAGAAA AGCAAGGAGG AGGGTTGAGC AATCTAGAGC ATGGAGTTTG TTAAGTGCTC

3061 TCTGGATTTG AGTTGAAGAG CATCCATTTG AGTTGAAGGC CACAGGGCAC AATGAGCTCT

3121 CCCTTCTACC ACCAGAAAGT CCCTGGTCAG GTCTCAGGTA GTGCGGTGTG GCTCAGCTGG

3181 GTTTTTAATT AGCGCATTCT CTATCCAACA TTTAATTGTT TGAAAGCCTC CATATAGTTA

3241 GATTGTGCTT TGTAATTTTG TTGTTGTTGC TCTATCTTAT TGTATATGCA TTGAGTATTA

3301 ACCTGAATGT TTTGTTACTT AAATATTAAA AACACTGTTA TCCTACAGTT.

Examples 1-2. Obtaining a monoclonal antibody to human claudin 18.2

1. Immunization

Monoclonal antibodies to human claudin 18.2 were obtained by immunization of mice. Laboratory white SJL mice, female, 6-8 weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd., Animal Production License No.: SCXK(Beijing)2012-0001). Maintenance environment: SPF class (free from specific pathogenic microflora). Purchased mice were kept in the laboratory environment for 1 week, in a 12/12-hour light/dark cycle, at a temperature of 20-25°C, with humidity of 40-60%. Acclimatized mice were immunized according to the following schedule. Antigens for immunization were huClaudin18.2-HEK293 cells (a strain of HEK-293 cells stably transfected with the human claudin 18.2 plasmid).

Immunization schedule: Before the first cell immunization, each mouse was injected intraperitoneally (IP) with 0.1 ml TiterMax® Gold adjuvant (Sigma Cat No. T2684) and half an hour later, 0.1 ml of cell fluid diluted with saline at a concentration of 1 x 10 ⁸ /ml was injected. Cells were pipetted evenly and then inoculated on days 0, 14, 28, 42 and 56. Blood was collected on days 21, 35, 49 and 63 and the serum antibody titer of mice was determined by ELISA. After 4-5 immunizations, mice with high serum antibody titer reaching a plateau were selected for splenocyte fusion. Mice were immunized with a booster dose of 1× ¹⁰⁷ cells by intraperitoneal injection (IP) 3 days before splenocyte fusion.

2. Fusion of splenocytes

Spleen lymphocytes and myeloma cells, Sp2/0 cells (ATCC® CRL-8287™), were fused following an optimized PEG-mediated fusion procedure to generate hybridoma cells. The resulting hybridoma cells were resuspended in complete medium (IMDM medium containing 20% FBS (fetal bovine serum), 1× HAT (hypoxanthine aminopterin thymidine), and 1× OPI) at a density of 0.5-1×10 ⁶ /mL and seeded in 96-well plates at 100 µL/well. The plate was incubated at 37°C with 5% _CO2 for 3-4 days, supplemented with complete HAT medium at 100 µl/well and incubated for another 3-4 days to generate spot clones. The supernatant was removed and complete HT medium (IMDM medium containing 20% FBS, 1× HT (hypoxanthine-thymidine) and 1× OPI) was added at 200 µl/well. The plate was incubated at 37°C with 5% _CO2 for 3 days, followed by ELISA analysis.

3. Screening of hybridoma cells

Hybridoma culture supernatants were analyzed using a combination ELISA method according to the density at which hybridoma cells were grown. Cells that had good binding ability to huClaudin18.2-HEK293 cells but did not bind to HEK293 were selected, expanded, and frozen. Subcloning was performed 2-3 times to obtain single-cell clones.

For each subcloning of cells, a cell binding assay was also performed. Hybridoma clones were obtained by the above-mentioned screening process, and antibodies were further obtained using a serum-free cell culture method. The antibodies were purified according to the purification example for use in test examples.

Examples 1 - 3. Humanization of mouse antibodies

Strains of monoclonal hybridoma cells mAb1901 and mAb1902 with high in vitro activity were selected. The monoclonal antibody sequences contained in them were cloned with subsequent humanization, recombinant expression and activity assessment.

Cloning of sequences from hybridomas was performed as follows. Hybridoma cells growing in log phase were collected and RNA was extracted using Trizol (Invitrogen, 15596-018) (following the procedures in the kit instructions) and reverse transcribed (PrimeScript™ Reverse Transcriptase, Takara, cat # 2680A). The resulting reverse transcribed cDNA was amplified by PCR (polymerase chain reaction) using the mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then submitted to a sequencing company for sequencing. The amino acid sequences corresponding to the obtained hybridoma cell DNA sequences are listed in SEQ ID NOs: 3-6:

Mouse heavy chain variable region of mAb1901 (SEQ ID NO: 3)

EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISRGSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRNAMDYWGQGTSVTVSS;

Mouse light chain variable region of mAb1901 (SEQ ID NO: 4)

DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTKLELK;

Mouse heavy chain variable region of mAb1902 (SEQ ID NO: 5)

EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDYWGQGTTLTVSS;

Mouse light chain variable region of mAb1902 (SEQ ID NO: 6)

DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTKLEIK.

The above-mentioned variable regions of the mouse heavy chain and light chain were fused with the constant region of the human IgG1 antibody heavy chain and the constant region of the human κ light chain described below, respectively, to form the chimeric antibodies ch1901 and ch1902.

The constant regions were selected from a group consisting of the following sequences:

Human IgG1 antibody heavy chain constant region: (SEQ ID NO: 7)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

Human κ light chain constant region: (SEQ ID NO: 8)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Humanization of mouse monoclonal antibodies was performed as described in many publications in the art. Briefly, human constant domains were used in place of parental (mouse antibodies) constant domains, and human germline antibody sequences were selected based on homology between mouse and human antibodies for CDR grafting. The present invention selects candidate molecules with good activity for humanization, and the results are as follows.

1. CDR of mouse antibodies

The VH/VL CDR amino acid residues in Table 1 were identified using the Kabat numbering system and annotated.

The CDR sequences of mouse antibodies are described in Table 1:

Table 1. CDR sequences of mouse antibodies Antibody mAb1901 HCDR1 DYGIH (SEQ ID NO: 9) HCDR2 YISRGSSTIYYADTVKG (SEQ ID NO: 10) HCDR3 GGYDTRNAMDY (SEQ ID NO: 11) LCDR1 KSSQSLLNSGNQKNYLA (SEQ ID NO: 12) LCDR2 GASTRAS (SEQ ID NO: 13) LCDR3 QNDLYYPLT (SEQ ID NO: 14) Antibody mAb1902 HCDR1 SYWMH (SEQ ID NO: 15) HCDR2 MIHPNSGSTNYNEKFKGR (SEQ ID NO: 16) HCDR3 LKTGNSFDY (SEQ ID NO: 17) LCDR1 KSSQSLLNSGNQKNYLT (SEQ ID NO: 18) LCDR2 WASTES (SEQ ID NO: 19) LCDR3 QNAYTYPFT (SEQ ID NO: 20)

2. Selection of human germline FR region sequences

Based on the typical structure of the obtained murine VH/VLCDR antibody, the variable region sequences of the heavy chain and light chain were compared with the Germine antibody database to obtain a human germline template with high homology. The human germline light chain framework region was derived from the human κ light chain gene.

2.1 Humanization of mAb1901 and Reverse Mutation Design

A suitable germline of human antibody was selected for humanization on murine antibody mAb1901. CDR of murine antibody mAb1901 was grafted into the selected humanization template to replace humanized variable regions, followed by recombination with IgG constant region to form a complete antibody. Meanwhile, backmutations were introduced into the FR region in the V region of the humanized antibody. Illustrative backmutations and their combinations are as follows:

Table 2. Humanized antibodies mAb1901 and reverse mutations* Light chain variable regions of humanized antibodies mAb1901 Heavy chain variable regions of humanized antibodies mAb1901 VL1 No VH1 No VL2 N22S VH2 N82T VL3 N22S, V85I, Y87H VH3 V48I, N82T VH4 I69M, N82T

* All amino acid positions in the table are numbered according to the Kabat numbering scheme; in N82T of the heavy chain variable region, 82 refers to position 82A according to the Kabat numbering scheme.

Table 3. Sequences of the variable region of the light chain and heavy chain of humanized antibodies mAb1901 Variable region name (SEQ ID NO:) Subsequence VL1
(SEQ ID NO: 21) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDLYYPLTFGQGTKLEIK VL2
(SEQ ID NO:22) DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDLYYPLTFGQGTKLEIK VL3
(SEQ ID NO:23) DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFSGSGSGTDFTLTISSLQAEDVAIYHCQNDLYYPLTFGQGTKLEIK VH1
(SEQ ID NO:24) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWVAYISRGSSTIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGYDTRNAMDYWGQGTTVTVSS VH2
(SEQ ID NO:25) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWVAYISRGSSTIYYADTVKGRFTISRDNAKNSLYLQMTSLRAEDTAVYYCARGGYDTRNAMDYWGQGTTVTVSS VH3
(SEQ ID NO:26) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWIAYISRGSSTIYYADTVKGRFTISRDNAKNSLYLQMTSLRAEDTAVYYCARGGYDTRNAMDYWGQGTTVTVSS VH4
(SEQ ID NO:27) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWVAYISRGSSTIYYADTVKGRFTMSRDNAKNSLYLQMTSLRAEDTAVYYCARGGYDTRNAMDYWGQGTTVTVSS

The corresponding heavy chain variable region in the above table was linked to the human IgG1 heavy chain constant region indicated in SEQ ID NO: 7 to form a full-length antibody heavy chain, and the light chain variable region was linked to the human κ light chain constant region indicated in SEQ ID NO: 8 to form a full-length antibody light chain. In other embodiments, the heavy chain variable region and the light chain variable region may also be linked to other heavy chain constant regions and light chain constant regions, respectively, to form a full-length antibody.

2.2 Humanization of mAb1902 and Reverse Mutation Design

A suitable germline of human antibody was selected for humanization on murine antibody mAb1902. CDR of murine antibody mAb1902 was grafted into the selected humanization template to replace the humanized variable regions, followed by recombination with the constant region of IgG to form a complete antibody. Meanwhile, backmutations were introduced into the FR region of the V region of the humanized antibody. Illustrative backmutations and their combinations are as follows:

Table 4. Humanized antibodies mAb1902 and their reverse mutation design* Light chain variable region of humanized antibody mAb1902 Variable region of the heavy chain of humanized antibodies mAb1902 VL11 No VH11 No VL12 M4L VH12 I69L, R71L, T73K VL13 M4L, N22S VH13 M48I, R66K, V67A, I69L, R71L, T73K VH14 R38K, A40R, M48I, R66K, V67A, I69L, R71L, T73K

* All amino acid positions in the table are numbered according to the Kabat numbering scheme.

Table 5. Sequences of the variable region of the light chain and heavy chain of the humanized antibody mAb1902 Variable region name (SEQ ID NO:) Subsequence VL11
(SEQ ID NO:28) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYTYPFTFGQGTKLEIK VL12
(SEQ ID NO: 29) DIVLTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYTYPFTFGQGTKLEIK VL13
(SEQ ID NO: 30) DIVLTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYTYPFTFGQGTKLEIK VH11
(SEQ ID NO: 31) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWMGMIHPNSGSTNYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARLKTGNSFDYWGQGTTVTVSS VH12
(SEQ ID NO: 32) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWMGMIHPNSGSTNYNEKFKGRVTLTLDKSASTAYMELSSLRSEDTAVYYCARLKTGNSFDYWGQGTTVTVSS VH13
(SEQ ID NO: 33) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSASTAYMELSSLRSEDTAVYYCARLKTGNSFDYWGQGTTVTVSS VH14
(SEQ ID NO: 34) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVKQRPGQRLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSASTAYMELSSLRSEDTAVYYCARLKTGNSFDYWGQGTTVTVSS

The corresponding heavy chain variable region in the above table was linked to the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the full-length antibody heavy chain, and the light chain variable region was linked to the human κ light chain constant region shown in SEQ ID NO: 8 to form the full-length antibody light chain.

Chimeric antibody ch1901

Heavy chain ch1901: (SEQ ID NO: 35)

EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISRGSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRNAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

Light chain ch1901: (SEQ ID NO: 36)

DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC;

Chimeric antibody ch1902

Heavy chain ch1902 (SEQ ID NO: 37)

EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

Light chain ch1902 (SEQ ID NO: 38)

DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC.

Table 6 shows the humanized antibodies mAb1901:

Table 6. Humanized antibodies mAb1901 Light and heavy chains H1 H2 H3 H4 L1 h1901-1 h1901-2 h1901-3 h1901-4 L2 h1901-5 h1901-6 h1901-7 h1901-8 L3 h1901-9 h1901-10 h1901-11 h1901-12

Note: In the table, the humanized antibody h1901-1 has a heavy chain H1 and a light chain L1. This is also true for other humanized antibodies. The sequences of the light chain and heavy chain of the full-length antibody of the humanized antibody mAb1901 are shown in Table 7 below:

Table 7. Sequences of the light chain and heavy chain of humanized antibodies mAb1901 Variable region name (SEQ ID NO:) Subsequence L1
(SEQ ID NO: 39) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDLYYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC L2
(SEQ ID NO: 40) DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDLYYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC L3
(SEQ ID NO: 41) DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFSGSGSGTDFTLTISSLQAEDVAIYHCQNDLYYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC H1
(SEQ ID NO: 42) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWVAYISRGSSTIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGYDT RNAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H2
(SEQ ID NO: 43) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWVAYISRGSSTIYYADTVKGRFTISRDNAKNSLYLQMTSLRAEDTAVYYCARGGYDT RNAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H3
(SEQ ID NO: 44) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWIAYISRGSSTIYYADTVKGRFTISRDNAKNSLYLQMTSLRAEDTAVYYCARGGYDT RNAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H4
(SEQ ID NO: 45) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWVAYISRGSSTIYYADTVKGRFTMSRDNAKNSLYLQMTSLRAEDTAVYYCARGGYDT RNAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Table 8 shows the humanized antibodies mAb1902:

Table 8. Humanized antibodies mAb1902 Light and heavy chains H11 H12 H13 H14 L11 h1902-1 h1902-2 h1902-3 h1902-4 L12 h1902-5 h1902-6 h1902-7 h1902-8 L13 h1902-9 h1902-10 h1902-11 h1902-12

Note: In the table, the humanized antibody h1902-1 has a heavy chain H11 and a light chain L11. This also applies to other humanized antibodies.

The light chain and heavy chain sequences of humanized mAb1902 antibodies are shown in Table 9 below:

Table 9. Sequences of the light chain and heavy chain of humanized antibodies mAb1901 Variable region name (SEQ ID NO:) Subsequence L11
(SEQ ID NO: 46) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYTYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC L12
(SEQ ID NO: 47) DIVLTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYTYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC L13
(SEQ ID NO: 48) DIVLTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYTYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC H11
(SEQ ID NO: 49) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWMGMIHPNSGSTNYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARLKTGNSFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H12
(SEQ ID NO: 50) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWMGMIHPNSGSTNYNEKFKGRVTLTLDKSASTAYMELSSLRSEDTAVYYCARLKTGNSFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H13
(SEQ ID NO: 51) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSASTAYMELSSLRSEDTAVYYCARLKTGNSFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H14
(SEQ ID NO: 52) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVKQRPGQRLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSASTAYMELSSLRSEDTAVYYCARLKTGNSFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The positive control antibody of the present invention is IMAB-362 (from WO2016166122)

Heavy chain IMAB-362 (SEQ ID NO: 53):

1 QVQLQQPGAE LVRPGASVKL SCKASGYTFT SYWINWVKQR PGQGLEWIGN

51 IYPSDSYTNY NQKFKDKATL TVDKSSSTAY MQLSSPTSED SAVYYCTRSW

101 RGNSFDYWGQ GTTLTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY

151 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI

201 CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD

251 TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST

301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY

351 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD

401 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK;

Light chain IMAB-362 (SEQ ID NO: 54):

1 DIVMTQSPSS LTVTAGEKVT MSCKSSQSLL NSGNQKNYLT WYQQKPGQPP

51 KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCQNDYSY

101 PFTFGSGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA

151 KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC

201 EVTHQGLSSP VTKSFNRGEC.

The above antibodies were cloned, expressed and purified using conventional gene cloning and recombinant expression techniques.

2. Obtaining connections

The experimental procedures without conditions indicated in the examples of the present invention are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer of the starting materials or commercial products. Reagents without indication of a specific origin are commercially available common reagents.

The structures of the compounds were identified using nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR spectra were measured using a Bruker AVANCE-400 nuclear magnetic resonance instrument, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform ( _CDCl3 ) and deuterated methanol ( _CD3OD ) as solvents and tetramethylsilane (TMS) as an internal standard. Chemical shifts are given in units of ^10-6 (ppm).

MS analysis was performed using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

UHPLC (ultra-performance liquid chromatography) analysis was performed using a Waters Acquity UPLC SQD liquid chromatography-mass spectrometry system.

HPLC (high performance liquid chromatography) analysis was performed using an Agilent 1200DAD high-pressure liquid chromatograph (Sunfire C18 150×4.6 mm chromatography column) and a Waters 2695-2996 high-pressure liquid chromatograph (Gimini C18 150×4.6 mm chromatography column).

UV-HPLC (high performance liquid chromatography using UV) analysis was performed using a Thermo nanodrop2000 ultraviolet spectrophotometer.

The degree of proliferation inhibition and IC ₅₀ values were measured using a PHERA starFS microplate reader (BMG, Germany).

Huanghai HSGF254 or Qingdao GF254 silica gel plates with specifications from 0.15 mm to 0.2 mm were used for thin layer chromatography (TLC) and 0.4 mm to 0.5 mm for TLC separation and purification.

Yantai Yellow Sea 200-300 mesh silica gel is commonly used as a carrier in column chromatography.

The known starting materials of the present invention can be synthesized using or according to methods known in the art, or can be purchased from ABCR GmbH & Co.KG, Acros Organnics, Aldrich Chemical Company, Accela ChemBio Inc, Chembee Chemicals, etc.

In the examples, all reactions were carried out under argon or nitrogen atmosphere unless otherwise stated.

Argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a cylinder containing about 1 liter of argon or nitrogen.

Hydrogen atmosphere means that the reaction flask is connected to a cylinder containing about 1 liter of hydrogen.

Parr 3916EKX hydrogenator, Qinglan QL-500 hydrogenator or HC2-SS hydrogenator were used in pressure hydrogenation reactions.

The hydrogenation reaction typically involves 3 cycles of vacuum and hydrogen purge.

A CEM Discover-S microwave reactor type 908860 was used for microwave-assisted reactions.

In the examples, the solution in the reaction refers to an aqueous solution unless otherwise stated.

In the examples, the reaction temperature is room temperature unless otherwise stated.

Room temperature is the optimum reaction temperature, which ranges from 20 to 30°C.

Preparation of PBS (phosphate buffered saline) buffer at pH 6.5 in examples: 8.5 g KH ₂ PO ₄ , 8.56 g K ₂ HPO ₄ ×3H ₂ O, 5.85 g NaCl and 1.5 g EDTA were added to the flask and the volume was adjusted to 2 L. All additions were dissolved by ultrasound and the solution was mixed well by shaking to obtain the required buffer.

The eluent system for column chromatography and the developing solvent system for thin layer chromatography used for the purification of the compound included: A: dichloromethanol and isopropanol system, B: dichloromethanol and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of the solvents was adjusted according to the polarity of the compound, or a small amount of triethylamine and an acidic or basic reagent was added.

Some of the compounds of the present invention were characterized by Q-TOF LC/MS analysis. The Q-TOF LC/MS utilized an Agilent 6530 accurate mass quadrupole time-of-flight mass spectrometer and an Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm, 2.1×75 mm chromatography column).

See PCT/CN2019/107873 for the Y-D drug portion of the antibody drug conjugates of the present invention, and the synthesis and testing of the corresponding compounds are incorporated herein by reference. Non-limiting examples of the synthesis are incorporated by reference as follows:

Example 1

N-((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)-1-hydroxycyclopropane-1-carboxamide 1

To exatecan mesylate 1b (2.0 mg, 3.76 μmol, prepared as described in patent application "EP0737686A1") was added 1 ml of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 1-hydroxycyclopropylcarboxylic acid 1a (1.4 mg, 3.7 μmol, prepared by the known method "Tetrahedron Letters, 25(12), 1269-72; 1984") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.8 mg, 13.7 μmol). After addition, the reaction mixture was stirred at 0-5°C for 2 h, quenched with 5 mL of water and extracted with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 1 (1.6 mg, yield 82.1%).

MS m/z (ESI): 520.2 [M+1]

¹H NMR (400 MHz, CDCl₃): δ 7.90-7.84 (m, 1H), 7.80-7.68 (m, 1H), 5.80-5.70 (m, 1H), 5.62-5.54 ( m, 2H), 5.44-5.32 (m, 2H), 5.28-5.10 (m, 2H), 3.40-3.15 (m, 3H), 2.44 (s, 3H), 2.23(t, 1H), 2.06-1.75 (m, 2H), 1.68-1.56 (m, 1H), 1.22-1.18 (m, 2H) , 1.04-0.98 (m, 2H), 0.89 (t, 3H).

Example 2

(S)-2-cyclopropyl-N-((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)-2-hydroxyacetamide 2-A

(R)-2-cyclopropyl-N-((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)-2-hydroxyacetamide 2-B

To 1b (4 mg, 7.53 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared as described in patent application "WO2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol). After the addition, the reaction mixture was stirred at 0-5°C for 1 hour. The ice water bath was removed, and the reaction mixture was heated to 30°C, stirred for 2 hours, and concentrated under reduced pressure. The obtained crude compound 2 was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A - water (10 mmol NH ₄ OAc), B - acetonitrile, gradient elution, flow rate: 18 mL/min), and the appropriate fractions were collected and concentrated under reduced pressure to give the title product (2-A: 1.5 mg, 2-B: 1.5 mg).

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

Compound with single 2-B configuration (lower retention time)

UHPLC analysis: retention time: 1.06 min; purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7 µm 2.1 x 50 mm; mobile phase: A - water (5 mmol NH ₄ OAc), B - acetonitrile).

^1H NMR (400 MHz, 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).

Compound with single 2-A configuration (longer retention time)

UHPLC analysis: retention time: 1.10 min; purity: 86% (chromatographic column: ACQUITY UPLC BEHC18 1.7 µm 2.1 x 50 mm; mobile phase: A - water (5 mmol NH ₄ OAc), B - acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.35 (d, 1H), 7.78 (d, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 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 3

(S)-N-((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)-3,3,3-trifluoro-2-hydroxypropionamide 3-A

(R)-N-((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)-3,3,3-fluoro-2-hydroxypropionamide 3-B

To 1b (5.0 mg, 9.41 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, and the mixture was cooled to 0-5 °C in an ice water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction mixture was stirred until it became clear. To the reaction mixture were added sequentially 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, purchased from Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol). After addition, the reaction mixture was stirred at 0-5 °C for 10 min. The ice water bath was removed and the reaction mixture was heated to 30 °C, stirred for 8 h and concentrated under reduced pressure. The obtained crude compound 3 was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A - water (10 mmol NH ₄ OAc), B - acetonitrile, gradient elution, flow rate: 18 mL/min), and the appropriate fractions were collected and concentrated under reduced pressure to give the title product (3-A: 1.5 mg, 3-B: 1.5 mg).

MS m/z (ESI): 561.9 [M+1].

Single configuration connection (lower retention time)

UHPLC analysis: retention time: 1.11 min; purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7 µm 2.1 x 50 mm; mobile phase: A - water (5 mmol NH ₄ OAc), B - acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.94 (d, 1H), 7.80 (d, 1H), 7.32 (s, 1H), 7.20 (d, 1H), 6.53 (s, 1H), 5.61-5.55 (m, 1H), 5.45-5.23 (m, 3H), 5.15-5.06 (m, 1H), 4, 66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H), 2.26-2.20 (m, 1H), 2.16- 2.08 (m, 1H), 2.02-1.94 (m, 1H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

Single configuration connection (longer retention time)

UHPLC analysis: retention time: 1.19 min; purity: 90% (chromatographic column: ACQUITY UPLC BEHC18 1.7 µm 2.1 x 50 mm; mobile phase: A - water (5 mmol NH ₄ OAc), B - acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.97 (d, 1H), 7.80 (d, 1H), 7.31 (s, 1H), 7.16 (d, 1H), 6.53 (s, 1H), 5.63-5.55 (m, 1H), 5.45-5.20 (m, 3H), 5.16-5.07 (m, 1H), 4, 66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H), 2.22-2.14 (m, 1H), 2.04- 1.95 (m, 2H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

Example 4

N-((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)-1-hydroxycyclopentane-1-carboxamide 4

To 1b (3.0 mg, 5.64 μmol) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were added 1-hydroxy-cyclopentanecarboxylic acid 4a (2.2 mg, 16.9 μmol, prepared as described in patent application "WO2013106717") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) sequentially. After the addition, the reaction mixture was stirred at 0-5 °C for 1 h, quenched with 5 mL of water and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 4 (2.5 mg, yield 80.9%).

MS m/z (ESI): 548.0 [M+1].

^1H NMR (400 MHz, CDCl ₃ ): δ 7.73-7.62 (m, 2H), 5.75-5.62 (m, 1H), 5.46-5.32 (m, 2H) , 5.26-5.10 (m, 1H), 3.30-3.10 (m, 1H), 2.43 (s, 3H), 2.28-2.20 (m, 2H), 2 .08-1.84 (m, 8H), 1.69-1.58 (m, 2H), 1.04-1.00 (m, 2H), 0.89 (t, 3H).

Example 5

N-((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)-1-(hydroxymethyl)cyclopropane-1-carboxamide 5

To 1b (2.0 mg, 3.76 μmol) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were added 1-(hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared as described in patent application "WO201396771") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol) sequentially. After the addition, the reaction mixture was stirred at 0-5 °C for 2 h, quenched with 5 mL of water and extracted with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 5 (1.0 mg, yield 50%).

MS m/z (ESI): 533.9 [M+1].

^1H NMR (400 MHz, CDCl ₃ ): δ 8.07 (s, 1H), 7.23-7.18 (m, 2H), 6.71-6.64 (m, 1H), 6.55 -6.51 (m, 1H), 5.36-5.27 (m, 2H), 4.67-4.61 (m, 2H), 3.53-3.48 (m, 1H), 3 .30-3.22 (m, 2H), 3.18-3.13 (m, 1H), 2.71-2.61 (m, 2H), 2.35-2.28 (m, 1H) , 2.04-1.91 (m, 4H), 1.53-1.40 (m, 3H), 0.91-0.75 (m, 4H).

Example 6

N-((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)-1-(hydroxymethyl)cyclobutane-1-carboxamide 6

To 1b (3.0 mg, 5.64 μmol) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were added sequentially 1-(hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol, prepared as described in "Journal of the American Chemical Society, 2014, vol.136, #22, p.8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol). After addition, the reaction mixture was stirred at 0-5°C for 1 h, quenched with 5 mL of water and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 6 (2.1 mg, yield 67.9%).

MS m/z (ESI): 548.0 [M+1].

^1H NMR (400 MHz, DMSO- _d6 ): δ 7.85-7.62 (m, 1H), 6.88 (br, 1H), 5.87-5.48 (m, 2H), 5 .47-5.33 (m, 1H), 5.31-5.06 (m, 1H), 4.25-3.91 (m, 2H), 3.25 (br, 1H), 2.60 -2.32 (m, 3H), 2.23 (t, 1H), 2.15-1.95 (m, 3H), 1.70-1.56 (m, 2H), 1.41-1 .17 (m, 9H), 1.03 (s, 1H), 0.95-0.80 (m, 2H).

Example 7

N-((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)-1-hydroxycyclobutane-1-carboxamide 7

To 1b (3.0 mg, 5.64 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, and the mixture was cooled to 0-5 °C in an ice water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction mixture was stirred until it became clear. To the reaction mixture were added sequentially 1-hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 μmol, purchased from PharmaBlock), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol). After addition, the reaction mixture was stirred at 0-5 °C for 10 min. The ice water bath was removed and the reaction mixture was stirred at room temperature for 2 hours and concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 7 (2.5 mg, yield 83.1%).

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

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.28 (d, 1H), 7.75 (d, 1H), 7.29 (s, 1H), 6.51 (s, 1H), 6.12 (s, 1H), 5.59-5.51 (m, 1H), 5.41 (s, 2H), 5.20-5.01 (m, 2H), 3.27-3, 17 (m, 1H), 3.15-3.05 (m, 1H), 2.71-2.63 (m, 1H), 2.37 (s, 3H), 2.12-2.05 ( m, 1H), 2.03-1.94 (m, 2H), 1.92-1.78 (m, 4H), 1.50-1.42 (m, 1H), 0.90-0.83 (m, 4H).

Example 8

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaicosyl)oxy)-N-((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)cyclopropane-1-carboxamide 8

Stage 1

Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylate 8c

Benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104 mg, 0.54 mmol; prepared as described in patent application "US2005/20645") and 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate 8b (100 mg, 0.27 mmol; prepared as described in patent application "CN105829346A") were added to the reaction flask and 5 mL of tetrahydrofuran was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of potassium tert-butoxide (61 mg, 0.54 mmol). The ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 10 min, followed by the addition of 20 ml of ice water and extraction with ethyl acetate (5 ml × 2) and chloroform (5 ml × 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 3 ml of 1,4-dioxane, followed by the addition of 0.6 ml of water, sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (70 mg, 0.27 mmol). The mixture was stirred at room temperature for 1 h. 20 ml of water was added, followed by extraction with ethyl acetate (8 ml × 3). The organic phases were combined, washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing solvent system B to give the title product 8c (100 mg, yield 73.6%).

MS m/z (ESI): 501.0 [M+1].

Stage 2

1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylic acid 8d

8c (50 mg, 0.10 mmol) was dissolved in 3 ml of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1) and palladium on carbon (25 mg, 10% loading) was added. The system was purged with hydrogen three times and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was filtered through celite and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to give the title product 8d (41 mg, 100% yield).

MS m/z (ESI): 411.0 [M+1].

Stage 3

(9H-fluoren-9-yl)methyl (2-(((1-(((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)aminocarbonyl)cyclopropoxy)methyl)amino)-2-oxoethyl)carbamate 8e

1b (7 mg, 0.013 mmol) was added to the reaction flask and 1 ml of N,N-dimethylformamide was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice-water bath, followed by the addition of a drop of triethylamine, a solution of 8d (7 mg, 0.017 mmol) in 0.5 ml of N,N-dimethylformamide and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (7 mg, 0.026 mmol). The reaction mixture was stirred in an ice bath for 35 min. 10 ml of water was added, followed by extraction with ethyl acetate (5 ml × 3). The organic phase was washed with saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 8e (8.5 mg, yield 78.0%).

MS m/z (ESI): 828.0 [M+1].

Stage 4

1-((2-aminoacetylamino)methoxy)-N-((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)cyclopropane-1-carboxamide 8f

8e (4 mg, 4.84 μmol) was dissolved in 0.2 ml of dichloromethane and 0.1 ml of diethylamine was added. The reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; the procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 8f (2.9 mg), which was used directly in the next step without purification.

MS m/z (ESI): 606.0 [M+1].

Stage 5

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaicosyl)oxy)-N-((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)cyclopropane-1-carboxamide 8

Crude 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide. The system was purged with argon three times and the solution was cooled to 0-5 °C in an ice water bath. A solution of (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrol-1-yl)hexanamido)acetylamino)acetylamino)-3-phenylpropionic acid 8g (2.7 mg, 5.80 μmol, prepared as described in patent application "EP2907824") in 0.3 mL of N,N-dimethylformamide was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol). The reaction mixture was stirred in an ice bath for 30 min. Then the ice bath was removed and the reaction mixture was warmed to room temperature, stirred for 15 min and purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A - water (10 mmol NH ₄ OAc), B - acetonitrile, gradient elution, flow rate: 18 mL/min). The appropriate fractions were collected and concentrated under reduced pressure to give the title product 8 (2 mg, yield 39.0%).

MS m/z (ESI): 1060.0 [M+1].

^1H NMR (400 MHz, DMSO- _d6 ): δ 9.01 (d, 1H), 8.77 (t, 1H), 8.21 (t, 1H), 8.08-7.92 (m , 2H), 7.73 (d, 1H), 7.28 (s, 1H), 7.24-7.07 (m, 4H), 6.98 (s, 1H), 6.50 (s, 1H), 5.61 (q, 1H), 5.40 (s, 2H), 5.32 (t, 1H), 5.12 (q, 2H), 4.62 (t, 1H), 4, 52 (t, 1H), 4.40-4.32 (m, 1H), 3.73-3.47 (m, 8H), 3.16-3.04 (m, 2H), 2.89 (dd, 1H), 2.69-2.55 (m, 2H), 2.37-2.23 (m, 4H) , 2.12-1.93 (m, 4H), 1.90-1.74 (m, 2H), 1.52-1.38 (m, 4H), 1.33-1.11 (m, 5H), 0.91-0.81 (m, 4H).

Example 9

N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((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)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A

N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((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)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

Stage 1

Benzyl 2-cyclopropyl-2-hydroxyacetate 9a

2a (1.3 g, 11.2 mmol; prepared as described in patent application "WO2013/106717") was dissolved in 50 mL of acetonitrile and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol) and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added successively. The reaction mixture was stirred at room temperature for 48 h and filtered through celite, and the filter cake was washed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with a developing solvent system C to give the title product 9a (2 g, yield 86.9%).

Stage 2

Benzyl 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate 9b

9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to the reaction flask and 4 ml of tetrahydrofuran was added. The system was purged with argon three times and the reaction mixture was cooled to 0-5 °C in an ice water bath followed by the addition of potassium tert-butoxide (109 mg, 0.98 mmol). The ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 40 min followed by the addition of 10 ml of ice water and extraction with ethyl acetate (20 ml × 2) and chloroform (10 ml × 5). The organic phases were combined and concentrated. The obtained residue was dissolved in 4 ml of dioxane, and 2 ml of water, sodium bicarbonate (49.2 mg, 0.586 mmol), and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added thereto. The mixture was stirred at room temperature for 2 hours. 20 ml of water was added thereto, followed by extraction with ethyl acetate (10 ml × 3). The organic phase was washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography using a C developing solvent system to give the title product 9b (48 mg, yield 19%).

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

Stage 3

10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid 9c

9b (20 mg, 0.038 mmol) was dissolved in 4.5 ml of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1) and palladium on carbon (12 mg, 10% loading, dry substance) was added. The system was purged with hydrogen three times and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was filtered through celite and the filter cake was washed with ethyl acetate. The filtrate was concentrated to give the crude title product 9c (13 mg), which was used directly in the next step without purification.

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

Stage 4

(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 9d

1b (10 mg, 18.8 μmol) was added to the reaction flask and 1 ml of N,N-dimethylformamide was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of triethylamine, crude 9c (13 mg, 30.6 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol). The reaction mixture was stirred in an ice bath for 40 min. 10 ml of water was added, followed by extraction with ethyl acetate (10 ml × 3). The organic phases were combined, washed with saturated sodium chloride solution (10 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 9d (19 mg, yield 73.6%).

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

Stage 5

2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((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)acetamide 9e

9d (19 mg, 22.6 μmol) was dissolved in 2 ml of dichloromethane and 1 ml of diethylamine was added. The reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. 1 ml of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and left to stand. Then, the supernatant was removed and the solid was saved. The solid residue was concentrated under reduced pressure and dried with an oil pump to give the crude title product 9e (17 mg), which was directly used in the next step without purification.

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

Stage 6

N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((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)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A

N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((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)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

Crude 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide. The system was purged with argon three times and the solution was cooled to 0-5 °C in an ice water bath. A solution of 8g (21.2 mg, 44.8 μmol) in 0.3 mL of N,N-dimethylformamide was added followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol). The reaction mixture was stirred in an ice bath for 10 min. Then the ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 1 h to give compound 9. The reaction mixture was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A - water (10 mmol NH ₄ OAc): B - acetonitrile, gradient elution, flow rate: 18 mL/min). The appropriate fractions were collected and concentrated under reduced pressure to give the title products (9-A: 2.4 mg, 9-B: 1.7 mg).

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

Compound with single 9-A configuration (lower retention time):

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

^1H NMR (400 MHz, DMSO- _d6 ): 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), 7.26 -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, 1H), 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, 1H), 2.80-2.62 (m, 1H), 2.45-2.30 (m, 3H), 2.25-2.15 (m , 2H), 2.04-2.15 (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).

Compound with single configuration 9-B (longer retention time):

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

^1H NMR (400 MHz, DMSO- _d6 ): 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, 3H), 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, 2H), 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, 4H), 0.91-0.79 (m, 3H), 0.53-0.34 (m, 4H).

III. Preparation of ADC conjugates of antibodies to claudin 18.2

Drug loading analysis of ADC stock solution

A. UV-HPLC method

The n DAR value was calculated by UV-HPLC for some examples of ADCs according to the present invention, in particular as follows:

1. Method of determination:

Cuvettes containing sodium succinate buffer solution were placed in the reference well and sample well, and the absorbance of the original solvent was subtracted. Then, a cuvette containing the test solution was placed in the sample well, and the absorbance values at 280 nm and 370 nm were determined.

2. Calculation for results: The loading capacity of the ADC stock solution was determined using ultraviolet spectrophotometry (device: Thermo nanodrop2000 ultraviolet spectrophotometer), based on the principle that the total absorbance of the ADC stock solution at a certain wavelength is the sum of the absorbance values of the drug and the monoclonal antibody at that wavelength, namely:

(1) A _{280 nm =} ε _{mab - 280} bC _mab + ε _{drug - 280} bC _drug

ε _drug-280 : the mean molar attenuation coefficient of the drug at 280 nm is 5100;

C _drug : drug concentration;

ε _mab-280 : the mean molar extinction coefficient of the stock solution of monoclonal antibody at 280 nm is 214600;

C _mab : concentration of the stock solution of monoclonal antibody;

b: The optical path length is 1 cm.

Similarly, the equation for the total absorbance of a sample at 370 nm can be written as:

(2) A _{370 nm =} ε _{mab - 370} bC _mab + ε _{drug - 370} bC _drug

ε _drug-370 : the mean molar attenuation coefficient of the drug at 370 nm is 19000;

C _drug : drug concentration;

ε _mab-370 : the attenuation coefficient of the initial monoclonal antibody solution at 370 nm is 0;

C _mab : concentration of the stock solution of monoclonal antibody;

b: The optical path length is 1 cm.

The drug loading can be calculated using both equation (1) and equation (2) and the attenuation coefficients of the monoclonal antibody and drug at both wavelengths and their concentrations.

Drug loading = C _drug /C _mab .

B. RP-HPLC (reversed-phase high-performance liquid chromatography) method

The DAR value was calculated by RP-HPLC (reversed phase high performance liquid chromatography) for some examples of the ADCs of the present invention, in particular as follows:

1. Method of determination:

Naked antibody (unconjugated antibody) and the test sample ADC (at a concentration of 1 mg/mL) were reconstituted with 4 μL DDT (sigma) in a water bath at 37°C for 1 hour and then transferred to the insert. The analysis was performed on an Agilent 1200 HPLC with Agilent PLRP-S 1000A 8 μm 4.6 × 250 mm selected as the chromatography column, column temperature of 80°C, DAD (Diode Array Detector) detector at a wavelength of 280 nm, flow rate of 1 mL/min and injection volume of 40 μL. Comparison was made between the spectra of the sample and naked antibody to identify the localizations of the light chain and heavy chain, and then integration was performed on the spectrum of the test sample to calculate the n DAR value.

2. Obtaining solutions

1) 0.25 M DTT (dithiothreitol) solution:

Preparation example: 5.78 mg DTT was weighed in 150 µl purified water and completely dissolved to obtain 0.25 M DTT solution, which was then stored at -20°C.

2) Mobile phase A (0.1% TFA (trifluoroacetic acid) in water):

Preparation example: 1000 ml of purified water was measured using a graduated cylinder and 1 ml of TFA (sigma) was added. The solution was mixed well before use and stored at 2-8°C for 14 days.

3) Mobile phase B (0.1% TFA in acetonitrile):

Preparation example: 1000 ml of acetonitrile was measured using a graduated cylinder and 1 ml of TFA was added. The solution was mixed well before use and stored at 2-8°C for 14 days.

3. Data analysis

Comparison was performed between the sample and naked antibody spectra to identify the light chain and heavy chain localizations, and then integration was performed on the test sample spectrum to calculate the DAR(n) value.

The calculation formula looks like this:

Name Number of related drugs LC 0 LC+1 2 HC 0 HC+1 2 HC+2 4 HC+3 6

Total peak area LC = peak area LC + peak area LC+1

Total peak area HC = peak area HC + peak area HC+1 + peak area HC+2 + peak area HC+3

LC DAR = Σ(number of bound drugs × percentage peak area)/total LC peak area

HC DAR = Σ(number of bound drugs × percentage peak area)/total HC peak area

DAR = LC DAR + HC DAR.

Examples of obtaining conjugates of antibodies to claudin 18.2 and a drug

Examples 3-1 and 3-2: ADC-1 and ADC-2

To the PBS buffer containing the h1902-5 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 320.0 mL, 21.62 μmol), an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.03 mL, 110.3 μmol) was added at 37°C. The reaction mixture was shaken in a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C in a water bath.

Compound 9-A (350 mg, 303 μmol) was dissolved in 13.2 mL of acetonitrile and 6.6 mL of DMSO (dimethyl sulfoxide), and the resulting solution was added to the above reaction mixture, which was cooled to 25 °C and then shaken on a shaking water bath at 25 °C for 3 hours before stopping the reaction.

The resulting reaction mixture was purified by ultrafiltration membrane successively with 5 L of PBS buffer (50 mM, pH = 6.5, 4% acetonitrile, 2% DMSO) and 5 L of succinic acid buffer (10 mM, pH = 5.3) to remove small molecules. Sucrose at a concentration of 60 mg/mL and Tween-20 at a concentration of 0.2 mg/mL were added to obtain the final product of example ADC-1 antibody-drug conjugate of the general formula h1902-5-9-A (10 mM succinic acid buffer at pH 5.3; 10 mg/mL, 2.626 g). Yield: 81.81%.

Average value calculated by UV-HPLC: n = 6.8.

Using the methods described above, the example ADC-2 antibody drug conjugate product of general formula h1901-11-9-A can be prepared using compound 9-A and antibody h1901-11 instead of h1902-5, wherein the n DAR value is 7.1.

Example 3-3. ADC-3

To the aqueous PBS buffer of h1901-11 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/ml, 1 ml, 67.5 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 10.1 μl, 101 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 9-A (0.58 mg, 540 nmol) was dissolved in 34 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken on a shaking water bath at 25 °C for 3 hours before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5 containing 0.001 M EDTA) to give the product of example ADC-3 antibody drug conjugate h1901-11-9-A in PBS buffer (0.72 mg/mL, 11.2 mL), which was then stored at 4 °C. Average value calculated by RP-HPLC: n = 2.51.

Example 3-4. ADC-4

To the aqueous PBS buffer of h1901-11 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/ml, 1 ml, 67.5 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 16.9 μl, 169 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 9-A (0.73 mg, 680 nmol) was dissolved in 43 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken on a shaking water bath at 25 °C for 3 hours before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5 containing 0.001 M EDTA) to give the example product ADC-4 antibody drug conjugate h1901-11-9-A in PBS buffer (0.62 mg/mL, 12.5 mL), which was then stored at 4 °C. Average value calculated by RP-HPLC: n = 4.06.

Example 3-5. ADC-5

To the aqueous PBS buffer of h1901-11 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/ml, 1 ml, 67.5 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 35.8 μl, 358 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 9-A (1.09 mg, 1015 nmol) was dissolved in 64 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken on a shaking water bath at 25 °C for 3 hours before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5 containing 0.001 M EDTA) to give the product of example ADC-5 antibody drug conjugate h1901-11-9-A in PBS buffer (0.54 mg/mL, 12.5 mL), which was then stored at 4 °C. Average value calculated by RP-HPLC: n = 6.8.

Example 3-6. ADC-6

To the aqueous PBS buffer of h1902-5 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 1.08 mL, 72.9 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 10.9 μL, 109 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 9-A (0.63 mg, 587 nmol) was dissolved in 40 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken on a shaking water bath at 25 °C for 3 hours before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5 containing 0.001 M EDTA) to give the product of example ADC-6 h1902-5-9-A in PBS buffer (0.7 mg/mL, 13.0 mL), which was then stored at 4 °C. Average value calculated by RP-HPLC: n = 2.69.

Example 3-7. ADC-7

To the aqueous PBS buffer of h1902-5 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 1.08 mL, 72.9 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 18.3 μL, 183 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 9-A (0.79 mg, 736 nmol) was dissolved in 50 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken on a shaking water bath at 25 °C for 3 hours before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5 containing 0.001 M EDTA) to give the product of example ADC-7 h1902-5-9-A in PBS buffer (0.6 mg/mL, 14.0 mL), which was then stored at 4 °C. Average value calculated by RP-HPLC: n = 4.25.

Example 3-8. ADC-8

To the aqueous PBS buffer of h1902-5 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 1.08 mL, 72.9 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 38.7 μL, 387 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 9-A (1.18 mg, 1099 nmol) was dissolved in 70 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken on a shaking water bath at 25 °C for 3 hours before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5 containing 0.001 M EDTA) to give the product of example ADC-8 h1902-5-9-A in PBS buffer (0.56 mg/mL, 14.2 mL), which was then stored at 4 °C. Average value calculated by RP-HPLC: n = 7.01.

Example 3-9. ADC-9

To the histidine-acetate-Tris/EDTA buffer (10 mM histidine-acetate-Tris buffer at pH 7.2, 2.5 mM EDTA buffer; 20.6 g/L, 6.49 L, 0.91 mmol) containing the h1902-5 antibody, the prepared histidine buffer TCEP (10 mM histidine buffer; 1.717 mM, 1.16 L, 1.99 mmol) was added at 12°C. The reaction mixture was stirred in a water bath at 12°C for 2 h, after which the reaction was stopped to obtain a solution of intermediate compound I.

Compound 9-A (4.72 g, 4.39 mmol) was dissolved in 0.38 L of DMSO to obtain a DMSO solution of compound 9-A. 0.38 L of DMSO was added to the above solution of intermediate compound I, and then the above solution of compound 9-A in DMSO was added. The reaction mixture was stirred in a water bath at 12 °C for 1 h, after which the reaction was stopped.

The reaction mixture was purified using a Capto S Impact cationic chromatography column, which was washed with 9 column volumes of 0.05 M acetate buffer containing 10% (v/v) DMSO (pH 5.0) and 6 column volumes of 0.05 M acetate buffer (pH 5.0), followed by elution with 0.05 M acetic acid and 0.30 M sodium chloride buffer (pH 5.5) to remove free toxins and residual solvent from the reaction mixture. The cationic eluate was subjected to 7-fold equal-volume ultrafiltration (30 KD polycellulose membrane was used as the ultrafiltration membrane) at 22 °C to give the product of example ADC-9 h1902-5-9-A. Average value calculated by RP-HPLC: n = 4.1.

The drug loading obtained in this example is a non-limiting example, and one skilled in the art can obtain conjugates with different DAR values (from 1 to 10, preferably from 1 to 8, and more preferably from 2 to 8 and from 2 to 7) by adjusting the reaction conditions and reagents.

Biological assessment

Test Case 1: Binding Analysis by ELISA at Cell Level

A cell-based ELISA assay was used to test the binding properties of antibodies to CL18.2. Stably transfected NUGC4 cells expressing CL18.2 were grown in a 96-well cell plate. When grown at 90% density, the cells were immobilized with 4% paraformaldehyde for 1 h. The plate was washed 3 times with PBST buffer (pH 7.4 PBS containing 0.05% Tween 20) and blocking buffer based on 5% skim milk diluted in PBS (skim milk powder from Brightdairy) was added at 200 μl/well. The plate was incubated in an incubator at 37°C for 2.5 h or left to stand at 4°C overnight (16-18 h) for blocking. After blocking, the blocking buffer was removed. The plate was washed three times with PBST buffer and then the test antibody, which was diluted with sample diluent (pH 7.4 PBS containing 1% milk) to various concentrations, was added at 50 µl/well. The plate was incubated in an incubator at 37°C for 2 hours. After incubation, the plate was washed 5 times with PBST and HRP-labeled goat anti-human secondary antibody (Jackson Immuno Research, 109-035-003), which was diluted with sample diluent, was added at 100 µl/well. The plate was incubated at 37°C for 1 hour. The plate was washed 6 times with PBST and then chromogenic TMB (tetramethylbenzidine) substrate (KPL, 52-00-03) was added at 50 µl/well. The plate was incubated at room temperature for 10-15 min and the reaction was stopped by adding 1 _MH2SO4 at 50 µl/well. The absorbance at 450 nm was read using an MD Versa Max™ _microplate reader and the _EC50 value of binding of anti-CL18.2 antibody to CL18.2 was calculated.

Table 10. Binding activity of hybridoma antibodies Antibody IMAB362 ch1901 ch1902 Emax 1,175 1,399 1,272 EC ₅₀ (nM) 0.108 0.098 0.074

Table 11. Binding activity of humanized antibodies mAb1901 Antibody IMAB362 h1901-2 h1901-3 h1901-4 h1901-6 Emax 1,115 1,039 1,1055 0.986 0.937 EC ₅₀ (nM) 0.086 0.076 0.22 0.201 0.091 Antibody h1901-7 h1901-8 h1901-11 h1901-12 Emax 0.921 1,047 1.44 1.22 EC ₅₀ (nM) 0.166 0.091 0.076 0.116

Table 12. Binding activity of humanized antibodies mAb1902 Antibody IMAB362 h1902-1 h1902-2 h1902-3 h1902-4 h1902-5 Emax 0.88 0.87 0.88 0.84 0.82 0.90 EC ₅₀ (nM) 0.187 0.113 0.107 0.175 0.087 0.098 Antibody h1902-6 h1902-7 h1902-8 h1902-9 h1902-10 Emax 0.78 0.75 0.89 0.75 0.89 EC ₅₀ (nM) 0.141 0.121 0.132 0.137 0.133

Test Case 2: Cellular Binding Analysis of Antibodies

Stably transfected NUGC4 cells expressing claudin 18.2 were suspended in FACS buffer (2% fetal bovine serum (Gibco, 10099141), pH 7.4 PBS (Sigma, P4417-100TAB)) to obtain a suspension of 1× ¹⁰⁶ cells/mL, which was then added to a 96-well round-bottomed plate (Corning, 3795) at 100 μL/well. After centrifugation and removal of the supernatant, the test antibody to claudin 18.2, which was diluted with FACS buffer to different concentrations, was added at 50 μL/well. The plate was incubated in the dark in a refrigerator at 4°C for 1 hour. The plate was washed three times with FACS buffer by centrifugation at 300 g and goat anti-human IgG Alexa Fluor 488 (H+L) (Invitrogen, A-11013) was added at a working concentration. The plate was incubated in the dark in a refrigerator at 4 °C for 40 minutes. The plate was washed three times with FACS buffer by centrifugation at 300 g and tested on a BD FACS CantoII flow cytometer for geometric mean fluorescence intensity. The _EC50 value of binding of the anti-CL18.2 antibody to stably transfected NUGC4 cells expressing CL18.2 was calculated. The results are shown in Fig. 1.

Test Case 3. Antibody Endocytosis Analysis

The test antibody to claudin 18.2 pre-labeled with DyLight 488 NHS Ester (thermofisher, 46403) was added to 1× ¹⁰⁶ /ml stably transfected NUGC4 cells expressing claudin 18.2 at a final concentration of 5 μg/ml. The mixture was incubated in the dark on ice for 1 h and washed three times with pre-chilled FACS buffer (pH 7.4 PBS, 2% fetal bovine serum) by centrifugation. After removal of the supernatant, the residue was added to pre-warmed complete medium, followed by incubation in a cell incubator at 37°C with 5% _CO2 . Cells were removed after 0, 0.5, 1, 2, and 4 h and stored in the dark on ice. After all samples were collected, they were centrifuged at 300 g at low temperature and the supernatants were removed. Elution buffer (pH 1.7, 0.05 M glycine, 0.1 M sodium chloride) was added and then the mixtures were incubated at room temperature for 7 min, washed once with FACS buffer by centrifugation at 300 g and tested on a BD FACS CantoII flow cytometer for geometric mean fluorescence intensity. The endocytosis efficiency of the anti-CL18.2 antibody by stably transfected NUGC4 cells expressing CL18.2 was calculated. The results (see Fig. 2) show that the humanized antibodies have good endocytosis efficiency.

Test Case 4: Flow Cytometry Based Antibody Affinity Analysis

On the day of the experiment, HEK293/hClaudin18.2 cells were collected in a 96-well U-bottom plate at 1-2× ¹⁰⁵ cells/well. Human anti-claudin 18.2 antibody, which was 2× serially diluted (12 concentration points) from a starting concentration of 5 μg/mL, was added and the plate was incubated at 4°C for 1 h. IMAB362 was used as a positive control, and a negative control without antibody was also installed. The antibody was removed by centrifugation and FITC-labeled anti-human IgG Fc antibody was added (200×) at 100 μL/well. The plate was incubated in the dark at 4°C for 30 min and washed twice with PBS + 2% FBS before flow cytometry analysis. The BD FACS CantoII was started and pre-warmed, and then the BD FACSDiva software was launched to start a new experiment. The HEK293/hClaudin18.2 negative control sample was tested and the FSC and SSC voltages were adjusted to the appropriate values and saved. Control sample B and standard curve 1 were tested according to the Quantum™ FITC-5 MESF Kit instructions and the FITC voltage was adjusted to the appropriate value and saved. Samples in a 96-well U-bottom plate were tested at the saved voltage and data were recorded. The experimental data were analyzed using Flowjo software to obtain the geometric (Geo) mean, and the MESF (molecule equivalent soluble fluorochrome)-geometric mean standard curve was fitted according to the instructions of the Quantum™ FITC-5 MESF Kit. The molar concentration of human anti-claudin 18.2 antibody bound to HEK293/hClaudin18.2 cells and the concentration of free antibody were calculated according to the fluorescence concentration value of the FITC-anti-human IgG Fc antibody, and the Bmax and dissociation constant KD of the antibody were calculated using Scatchard plots. The results are presented in Table 13.

Table 13. Affinity of humanized antibodies at the cellular level Antibody IMAB362 h1901-11 h1902-5 KD (nM) 10.2 6.8 1.64

Test Case 5. Evaluation of ADCC Antibody Effect

Different NUGC4 cells (high, moderate and low claudin 18.2 expressing) were split, centrifuged at 1000 rpm, resuspended and counted. Cells were resuspended at a density of 3× ¹⁰⁵ cells/ml in RPMI 1640 without phenol red (Gibco, 11835-030) supplemented with 10% FBS (New Zealand fetal bovine serum ultra-low IgG, Gibco, 1921005PJ). 25 μl of cells were added to each well of a 96-well plate (Corning, 3903) (7500 cells/well). The antibody was diluted in phenol red-free medium to give a 3× dilution of the antibody, which was then added to the cell plate at 25 µl/well. The plate was incubated in a 37°C incubator with 5% _CO2 for 0.5 h.

Effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells) were collected, centrifuged at 1000 rpm, resuspended and counted. Cells were resuspended at a density of 3× ¹⁰⁶ cells/mL in RPMI 1640 without phenol red supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum) and 25 μL of cells (7.5× ¹⁰⁴ cells/well) were added to each well of the plate. The plate was incubated in an incubator at 37°C with 5% _CO2 for 6 h.

75 µl Bright-Glo (Promega, E2610) was added to each well of the plate, and chemical luminescence was determined using a microplate reader (PerkinElmer, VITOR3).

The results (see Table 14 and Figs. 3A-3C) show that both h1901-11 and h1902-5 antibodies exhibited high ADCC activity in NUGC4 cells with low (Fig. 3A), moderate (Fig. 3B), and high (Fig. 3C) claudin 18.2 expression.

Table 14. Effect of ADCC antibodies in NUGC4 cells with different levels of claudin 18.2 expression
IC ₅₀ unit (half-maximal inhibition concentration, ng/ml) Claudin expression level 18.2 h1901-11 h1902-5 IMAB362 Low expression 22.42 35.46 183.4 Moderate expression 15.35 30,00 210.4 High expression 26.17 32.16 132.6

Test Example 6. In vitro inhibition of tumor cell proliferation using compounds

A. Purpose

This experiment is designed to test the inhibitory activity of the pharmaceutical compounds of the present invention on the in vitro proliferation of U87MG cells (glioma cells, Cell Bank, Chinese Academy of Sciences, catalog number TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, catalog number HTB-30). The cells were treated in vitro with the compound at different concentrations. After 6 days of culture, the cell proliferation was tested using CTG reagents (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, catalog number G7573) and the in vitro activity of the compound was evaluated according to the IC ₅₀ value.

B. Method

A method for testing the inhibition of in vitro proliferation of U87MG cells is described below as an example of a method for analyzing the inhibitory activity of the compounds of the present invention on in vitro proliferation of tumor cells. The method is also applicable, but not limited to, to tests for inhibitory activity on in vitro proliferation of other tumor cells.

1. Cell culture: U87MG and SK-BR-3 cells were cultured in EMEM medium (GE, catalog no. SH30024.01) containing 10% FBS and McCoy's 5A medium (Gibco, catalog no. 16600-108) containing 10% FBS, respectively.

2. Cell preparation. U87MG and SK-BR-3 cells growing in log phase were washed once with PBS (phosphate buffer, Shanghai BasalMedia Technologies Co., Ltd.) and then digested with 2-3 ml trypsin (0.25% trypsin-EDTA (1×), Gibico, Life Technologies) for 2-3 min. After the cells were completely digested, 10-15 ml cell culture medium was added to elute the digested cells. The mixtures were centrifuged at 1000 rpm for 5 min, and the supernatants were discarded. The cells were then resuspended in 10-20 ml cell culture medium to obtain single-cell suspensions.

3. Cell culture. Single-cell suspensions of U87MG and SK-BR-3 were mixed well and adjusted with cell culture medium to a cell density of 2.75×10³cells/ ml and 8.25×10³cells/ml, respectively. All adjusted cell suspensions were mixed well and added to 96-well cell culture plates at 180 μl/well. Only 200 μl of medium was added to each of the peripheral wells of the 96-well plates. The plate was incubated in an incubator for 24 hours (37°C, 5% CO₂).

4. Preparation of compounds The compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Scientific Co., Ltd.) to obtain a stock solution at an initial concentration of 10 mM.

Low molecular weight compounds were prepared at an initial concentration of 500 nM as follows.

Different test samples at a concentration of 100 μM (30 μl) were added to the first column of a 96-well U-bottom plate, and 20 μl of DMSO was added to each well of the second column and the eleventh column. The samples in the first column (10 μl) were added to 20 μl of DMSO in the second column, and the mixtures were mixed well. The mixtures (10 μl) were added to the third column and so on until the tenth column. The drugs in the plate (5 μl per well) were transferred to EMEM medium (95 μl), and the mixtures were mixed well for subsequent use.

ADC was prepared at an initial concentration of 10 nM or 500 nM as follows.

Different test samples at 100 nM or 5 μM (100 μl) were added to the first column of a 96-well plate and 100 μl of PBS was added to each well of the second column and the eleventh column. The samples in the first column (50 μl) were added to 100 μl of PBS in the second column and the mixtures were mixed well. The mixtures (50 μl) were added to the third column and so on, in 3-fold dilutions, up to the tenth column.

5. Sample addition: Test samples prepared at different concentrations (20 µl) were added to the culture plate, with two duplicate wells set up for each sample. The plate was incubated in an incubator for 6 days (37°C, 5% CO ₂ ).

6. Color development: The 96-well cell culture plate was removed and 90 μl of CTG solution was added to each well, followed by 10 min incubation at room temperature.

7. Plate reading: The 96-well cell culture plate was removed and tested for chemiluminescence in a microplate reader (BMG labtech, PHERAstar FS).

C. Data Analysis

The data was processed and analyzed using Microsoft Excel and Graphpad Prism 5. The results obtained according to this example are shown in the table below.

Table 15. IC ₅₀ values of low molecular weight fragments according to the present invention in inhibiting in vitro proliferation of SK-BR-3 cells and U87 cells Connection example IC ₅₀ (nM) SK-BR-3 U87 Example 1 0.12 0.23 Example 2
2-B with lower retention time 0.33 0.86 Example 2
2-B with long retention time 8.11 2.31 Example 3
Shorter retention time 0.36 0.83 Example 3
Longer retention time 1.67 2.98 Example 4 1.9 / Example 5 / 4.81 Example 6 / 1.83 Example 7 / 1.95

Conclusion: The low molecular weight fragments of the present invention have significant inhibitory activity against the proliferation of SK-BR-3 cells and U87 cells, and the chiral centers have a certain effect on the inhibitory activity of the compounds.

Test Case 7: Cell Viability Assays for ADC Molecules

CellTiter-Glo luminescence-based cell viability assays were used to test ADC molecules for in vitro killing effects against the human gastric cancer cell strain in this experiment. On day 1, NUGC4 cells with low, moderate, and high claudin 18.2 expression were harvested, adjusted to a density of 2.5 x 10 ⁴ /mL, and added to a 96-well white clear plate at 90 μL/well, with approximately 2500 cells per well. Cells were cultured overnight in an incubator at 37°C with 5% CO ₂ . On day 2, samples were 4x diluted serially from a starting concentration of 5 μM in a 96-well U-bottom plate to yield 9 concentration points, and the diluted samples were added to the plate with the cells at 10 μL/well. Cells were cultured at 37°C in 5% _CO2 for 6 days. On day 8, the cell culture plate was removed and Cell Titer-Glo reagent was added at 50 µl/well. The plate was left at room temperature for 2–3 min and fluorescence readings were taken on a PHERAstar FS plate reader. Data analysis was performed using GraphPad Prism software. See Table 16.

Table 16. In vitro killing effects of the ADC molecules of the present invention on cells ADC NUGC4 cells with low expression of claudin 18.2 NUGC4 cells with moderate expression of claudin 18.2 NUGC4 cells with high expression of claudin 18.2 EC ₅₀ (nM) Emax (%) EC ₅₀ (nM) Emax (%) EC ₅₀ (nM) Emax (%) ADC-1 126.8 66.7 23.6 82.1 1.3 91.5 ADC-2 109.0 69.8 16.8 82.3 1.9 91.1 ADC-3 more than 500 49.0 94 78.7 ADC-4 299 61.03 12 84.97 ADC-5 142 69.91 3.5 95.89 ADC-6 more than 500 45.22 11 78,00 ADC-7 284 61.09 3.9 88.51 ADC-8 154 66.74 1.3 97.15

Biological assessment of in vivo activity

Test Case 8: In vivo Evaluation of ADC Molecules Efficacy

Balb/c nude mice were subcutaneously inoculated in the right flank with human gastric cancer cells, NUGC4 cells (moderately expressing claudin 18.2) (5× ¹⁰⁶ cells in 50% Matrigel/mouse) and divided into a total of 5 groups of 8 on day 0. The average tumor volume was about 84.41 ^mm3 .

Each mouse was injected intraperitoneally with ADC at a concentration of 0.1 ml/10 g body weight on days 0, 4, and 11 for a total of 3 injections.

Each mouse was intraperitoneally injected with ADC at a concentration of 0.1 ml/10 g body weight from the day of grouping at intervals of 5 days, for a total of 4 injections.

Tumor volumes and body weights were measured twice weekly and the results were recorded.

Excel 2003 statistical software was used. Mean values were calculated as mean (avg); SD (standard deviation) values were calculated as STDEV (square root of sample variance); SEM (standard error of the mean) values were calculated as STDEV/SQRT (square root); and P value difference between groups was calculated as TTEST.

Tumor volume (V) was calculated as: V = 1/2×L _long ×L _short ²

Relative Volume (RTV) = VT/V0

Tumor inhibition rate (%) = (CRTV - TRTV)/CRTV (%),

where V0 and VT are the tumor volumes at the beginning of the experiment (the day of the first administration is defined as day 0) and at the time of measurement, respectively; CRTV and TRTV are the relative tumor volumes of the blank control group and the experimental groups, respectively, at the end of the experiment. The results are shown in Table 17 and Figs. 4 and 5.

Table 17. Tumor inhibition results using ADC Group Average tumor volume ( ^mm3 ) Average tumor volume ( ^mm3 ) Relative tumor volume % Tumor inhibition rate D32 D0 SEM D32 SEM D0 SEM Blank control group 83.33 0.82 2067,0 102.24 24.83 1.27 - ADC-2 10 mg/kg (mpk) 83.93 1.65 263.13 44.17 3.11 0.51 87.47%** ADC-2 3 mg/kg 84.35 1.83 328.95 45.04 3.86 0.48 84.45%** ADC-1 10 mg/kg 83.60 1.61 123.80 20.99 1.48 0.25 94.04%** ADC-1 3 mg/kg 86,84 1.91 356.41 55.18 4.06 0.58 83.65%**

vs blank: ** p < 0.01.

--->

SEQUENCE LIST

<110> Jiangsu Hengrui Medicine Co., Ltd.

Shanghai Hengrui Pharmaceutical Co., Ltd.

<120>CLAUDINE ANTIBODY-DRUG CONJUGATE AND ITS

PHARMACEUTICAL USE

<130> 702137CPCT

<140> PCT/CN2020/135680

<141> 2020-12-11

<150>CN201911273041.7

<151> 2019-12-12

<150>CN202011060513.3

<151> 2020-09-30

<160> 55

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Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

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Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

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Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

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tagcctgggc aacatggaga agccctgtct ctacaaaata cagagagaaa aaatcagcca 1980

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50 55 60

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65 70 75 80

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115

<210> 6

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<223> Variable region of the light chain of mouse antibody mAb1902

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50 55 60

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Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Gln Asn

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Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

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100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

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165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

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Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

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Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

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Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

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50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 9

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1901 HCDR1

<400> 9

Asp Tyr Gly Ile His

1 5

<210> 10

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1901 HCDR2

<400> 10

Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys

1 5 10 15

Gly

<210> 11

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1901 HCDR3

<400> 11

Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr

1 5 10

<210> 12

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1901 LCDR1

<400> 12

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 13

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1901 LCDR2

<400> 13

Gly Ala Ser Thr Arg Ala Ser

1 5

<210> 14

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1901 LCDR3

<400> 14

Gln Asn Asp Leu Tyr Tyr Pro Leu Thr

1 5

<210> 15

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1902 HCDR1

<400> 15

Ser Tyr Trp Met His

1 5

<210> 16

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1902 HCDR2

<400> 16

Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly Arg

<210> 17

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1902 HCDR3

<400> 17

Leu Lys Thr Gly Asn Ser Phe Asp Tyr

1 5

<210> 18

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1902 LCDR1

<400> 18

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Th

<210> 19

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1902 LCDR2

<400> 19

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 20

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb1902 LCDR3

<400> 20

Gln Asn Ala Tyr Thr Tyr Pro Phe Thr

1 5

<210> 21

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL1

<400> 21

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 22

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL2

<400> 22

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 23

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL3

<400> 23

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr His Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 24

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH1

<400> 24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 25

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH2

<400> 25

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 26

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH3

<400> 26

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 27

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH4

<400> 27

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 28

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL11

<400> 28

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 29

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL12

<400> 29

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 30

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL13

<400> 30

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 31

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH11

<400> 31

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 32

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH12

<400> 32

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 33

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH13

<400> 33

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 34

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH14

<400> 34

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 35

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> heavy chain ch1901

<400> 35

Glu Val Gln Leu Met Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Glu Met Gly Leu Glu Trp Ile

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 36

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> light chain ch1901

<400> 36

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr His Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 37

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> heavy chain ch1902

<400> 37

Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Pro Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 38

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> light chain ch1902

<400> 38

Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 39

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> L1

<400> 39

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 40

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> L2

<400> 40

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 41

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> L3

<400> 41

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr His Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 42

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> H1

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 43

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> H2

<400> 43

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 44

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> H3

<400> 44

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 45

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> H4

<400> 45

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 46

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> L11

<400> 46

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 47

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> L12

<400> 47

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 48

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> L13

<400> 48

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 49

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> H11

<400> 49

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 50

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> H12

<400> 50

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 51

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> H13

<400> 51

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 52

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> H14

<400> 52

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 53

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> heavy chain IMAB-362

<400> 53

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 54

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light Chain IMAB-362

<400> 54

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 55

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Tetrapeptide linker

<400> 55

Gly Gly Phe Gly

1 4

<---

Claims (96)

1. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof for the treatment of cancer with high expression of claudin 18.2: where Y is selected from the group consisting of -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-, -OCR ¹ R ² -(CR ^a R ^b ) _m -, -O-CR ¹ R ² -, -NH-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)- and -S-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-; R ^a and R ^b are the same or different and are each independently selected from the group consisting of hydrogen, deuterium, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl and heterocyclyl; or R ^a and R ^b together with the carbon atoms to which they are attached form cycloalkyl or heterocyclyl; R ¹ is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; R ² is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; or R ¹ and R ² , together with the carbon atoms to which they are attached, form cycloalkyl or heterocyclyl; or R ^a and R ² together with the carbon atoms to which they are attached form cycloalkyl or heterocyclyl; m is an integer between 0 and 4; n is a decimal or integer number between 2 and 8; where the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, where L ¹ is selected from the group consisting of -(succinimidyl-3-yl-N)-W-C(O)-, -CH ₂ -C(O)-NR ³ -WC(O)- and -C(O)-WC(O)-, wherein W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkyl-C _3-6 cycloalkyl and linear heteroalkyl having from 1 to 8 chain atoms, said heteroalkyl containing from 1 to 3 heteroatoms selected from the group consisting of N, O and S, wherein said C _1-8 alkyl, C _1-8 alkyl-C _3-6 cycloalkyl or linear heteroalkyl having from 1 to 8 chain atoms is independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L ² is selected from the group consisting of -NR ⁴ (CH ₂ CH ₂ O) p ¹ CH ₂ CH ₂ C(O)-, -NR ⁴ (CH ₂ CH ₂ O) p ¹ CH ₂ C(O)-, -S(CH ₂ ) p ¹ C(O)- and a chemical bond, where p ¹ is an integer from 1 to 20; L ³ is a peptide residue consisting of 2-7 amino acids, wherein the amino acids are selected from the group consisting of amino acid residues formed from the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L ⁴ is selected from a group consisting of -NR ⁵ (CR ⁶ R ⁷ ) _t -, -C(O)NR ⁵ -, -C(O)NR ⁵ (CH ₂ ) _t - and a chemical bond, where t is an integer from 1 to 6; R ³ , R ⁴ and R ⁵ are the same or different and are each independently selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl; R ⁶ and R ⁷ are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl; RS is an antibody to claudin 18.2 or an antigen-binding fragment thereof, wherein the antibody to claudin 18.2 or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the sequences set forth in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; or iv) the variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3 having the sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the variable region of the light chain comprises LCDR1, LCDR2 and LCDR3 having the sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively. 2. The conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the antibody to claudin 18.2 is a murine antibody, a chimeric antibody, or a humanized antibody. 3. The conjugate or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the antibody to claudin 18.2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein: (1) the variable region of the heavy chain has the amino acid sequence set forth in SEQ ID NO: 3 or has at least 90% identity thereto, and the variable region of the light chain has the amino acid sequence set forth in SEQ ID NO: 4 or has at least 90% identity thereto; (2) the variable region of the heavy chain has the amino acid sequence set forth in SEQ ID NO: 24 or has at least 90% identity thereto, and the variable region of the light chain has the amino acid sequence set forth in SEQ ID NO: 21 or has at least 90% identity thereto; (3) the variable region of the heavy chain has the amino acid sequence set forth in SEQ ID NO: 5 or has at least 90% identity thereto, and the variable region of the light chain has the amino acid sequence set forth in SEQ ID NO: 6 or has at least 90% identity thereto; or (4) the variable region of the heavy chain has the amino acid sequence set forth in SEQ ID NO: 31 or has at least 90% identity thereto, and the variable region of the light chain has the amino acid sequence set forth in SEQ ID NO: 28 or has at least 90% identity thereto. 4. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein the antibody to claudin 18.2 is a humanized antibody comprising a framework region derived from a human antibody or a framework region variant thereof, wherein said framework region variant has back mutations of up to 10 amino acids in the light chain framework region and/or the heavy chain framework region of a human antibody. 5. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein the framework variant comprises mutations selected from (a) or (b): (a) one or more amino acid backmutations are optionally selected from the group consisting of 22S, 85I and 87H contained in the variable region of the light chain; and/or one or more amino acid backmutations are optionally selected from the group consisting of 48I, 82T and 69M contained in the variable region of the heavy chain; or (b) one or more amino acid backmutations are optionally selected from the group consisting of 4L and 22S contained in the variable region of the light chain; and/or one or more amino acid backmutations are optionally selected from the group consisting of 38K, 40R, 48I, 66K, 67A, 69L, 71L and 73K contained in the variable region of the heavy chain. 6. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein the framework variant comprises mutations selected from the group consisting of: (a-1) back mutations of amino acids 22S, 85I and 87H contained in the variable region of the light chain and back mutations of amino acids 48I and 82T contained in the variable region of the heavy chain; or (b-1) reverse mutations of amino acid 4L contained in the variable region of the light chain. 7. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-6, wherein the antibody to claudin 18.2 or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as shown below: (vii) a heavy chain variable region having the sequence set forth in SEQ ID NO: 3 and a light chain variable region having the sequence set forth in SEQ ID NO: 4; (viii) a heavy chain variable region having the sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27 and a light chain variable region having the sequence set forth in SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23; (ix) a heavy chain variable region having the sequence set forth in SEQ ID NO: 5 and a light chain variable region having the sequence set forth in SEQ ID NO: 6; or (x) a heavy chain variable region having the sequence set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34 and a light chain variable region having the sequence set forth in SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30. 8. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-7, wherein the antibody to claudin 18.2 or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as shown below: (xi) a heavy chain variable region having the sequence set forth in SEQ ID NO: 31 and a light chain variable region having the sequence set forth in SEQ ID NO: 29; or (xii) a heavy chain variable region having the sequence set forth in SEQ ID NO: 26 and a light chain variable region having the sequence set forth in SEQ ID NO: 23. 9. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-8, wherein the antibody to claudin 18.2 or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region of an antibody. 10. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-9, wherein the constant region of the heavy chain is selected from the group consisting of the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their normal variants, and the constant region of the light chain is selected from the group consisting of the constant regions of the κ and λ chain of a human antibody and their normal variants. 11. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-10, wherein the antibody to claudin 18.2 or antigen-binding fragment thereof comprises a heavy chain constant region having the sequence indicated in SEQ ID NO: 7, a light chain constant region having the sequence indicated in SEQ ID NO: 8. 12. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-11, wherein the antibody to claudin 18.2 or antigen-binding fragment thereof comprises: a heavy chain having at least 90% sequence identity to the heavy chain set forth in SEQ ID NO: 35 or SEQ ID NO: 42, and a light chain having at least 90% sequence identity to the light chain set forth in SEQ ID NO: 36 or SEQ ID NO: 39; or a heavy chain having at least 90% sequence identity to the heavy chain set forth in SEQ ID NO: 37 or SEQ ID NO: 49, and a light chain having at least 90% sequence identity to the light chain set forth in SEQ ID NO: 38 or SEQ ID NO: 46. 13. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-12, wherein the antibody to claudin 18.2 or antigen-binding fragment thereof comprises: (c) a heavy chain having the sequence set forth in SEQ ID NO: 35 and a light chain having the sequence set forth in SEQ ID NO: 36; (d) a heavy chain having the sequence set forth in SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45 and a light chain having the sequence set forth in SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO: 41; (e) a heavy chain having the sequence set forth in SEQ ID NO: 37 and a light chain having the sequence set forth in SEQ ID NO: 38; or (f) a heavy chain having the sequence set forth in SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 52 and a light chain having the sequence set forth in SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 48. 14. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-13, wherein the antibody to claudin 18.2 is selected from the group consisting of: h1901-11, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 44 and a light chain set forth in SEQ ID NO: 41; and h1902-5, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 49 and a light chain set forth in SEQ ID NO: 47. 15. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-14, wherein n is a decimal or integer from 3.5 to 4.5. 16. A conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-15, wherein: Y represents -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-; R ^a and R ^b are identical or different and each is independently selected from the group consisting of hydrogen, deuterium, halogen and alkyl; R ¹ is haloalkyl or C _3-6 cycloalkyl; R ² is selected from the group consisting of hydrogen, haloalkyl and C _3-6 cycloalkyl; or R ¹ and R ² together with the carbon atoms to which they are attached form C _3-6 cycloalkyl; m represents 0 or 1. 17. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-16, wherein Y is selected from the group consisting of: where the O-terminus of Y is attached to the linker unit L. 18. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 17, wherein the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, wherein L ¹ is and s ¹ is an integer from 2 to 8; L ² is a chemical bond; L ³ is a tetrapeptide residue; L ⁴ is -NR ⁵ (CR ⁶ R ⁷ ) _t -, where R ⁵ , R ⁶ and R ⁷ are the same or different and each independently is hydrogen or alkyl and t is 1 or 2; where the L ¹ end is attached to Pc and the L ⁴ end is attached to Y. 19. The conjugate or pharmaceutically acceptable salt thereof according to claim 18, wherein L ³ is a tetrapeptide residue of GGFG. 20. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-19, wherein -L- is: 21. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-20, wherein -L-Y- is optionally selected from the group consisting of: 22. A conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-21, which is a conjugate of the general formula (Pc-L _a -YD) or a pharmaceutically acceptable salt thereof: where W, L ² , L ³ , R ⁵ , R ⁶ and R ⁷ are as defined in paragraph 1; Rc, n, R ¹ , R ² and m are as defined in paragraph 1. 23. A conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-22, which is a conjugate of the general formula (Pc-L _b -YD) or a pharmaceutically acceptable salt thereof: where s ¹ is an integer from 2 to 8; Rc, R ¹ , R ² , R ⁵ -R ⁷ , m and n are as defined in paragraph 22. 24. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-23, wherein the conjugate is selected from the group consisting of: where Pc and n are as defined in paragraph 1. 25. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-24, wherein the conjugate is selected from the group consisting of: where n is as defined in paragraph 1, and antibodies h1902-5 and h1901-11 are as defined in paragraph 14. 26. A method for producing a conjugate of the general formula (Pc-L _a -YD) or a pharmaceutically acceptable salt thereof, comprising the following steps: subjecting Pc' and a compound of the general formula (L _a -YD) to a coupling reaction to obtain a compound of the general formula (Pc-L _a -YD); wherein Pc is an antibody to claudin 18.2 or an antigen-binding fragment thereof and Pc' is obtained by reduction of Pc; wherein the reducing agent is TCEP; in particular, the disulfide bonds in the antibody are reduced; W, ^L2 , ^L3 , ^R1 , ^R2 , ^R5 - ^R7 , m and n are as defined in paragraph 22. 27. A pharmaceutical composition for treating cancer with high expression of claudin 18.2, comprising a conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-25 and one or more pharmaceutically acceptable excipients, diluents or carriers. 28. Use of a conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-25 or a pharmaceutical composition according to claim 27 for the preparation of a medicinal product for the treatment of cancer with high expression of claudin 18.2. 29. Use of a conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-25 or a pharmaceutical composition according to claim 27 for the preparation of a medicinal product for the treatment or prevention of a tumor or cancer. 30. The use according to claim 29, wherein the tumor and the cancer are selected from the group consisting of: squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, neuroglioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krukenberg tumor, myeloproliferative tumor, squamous cell carcinoma, Ewing sarcoma, systemic light chain amyloidosis, or Merkel cell carcinoma. 31. The use according to claim 30, wherein the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/histiocyte-rich large B-cell lymphoma and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid cell leukemia.