Antibody-drug conjugate comprising a bispecific antibody specifically binding to CD20 and CD22 and uses thereof

Abstract

The present specification relates to antibody-drug conjugates (ADCs) comprising bispecific antibodies that specifically bind to CD20 and CD22, and compositions and uses of said ADCs for the manufacture of drugs for the treatment and / or therapy of diseases, specifically, hyperproliferative and / or angiogenic diseases such as cancer (e.g., blood cancer).

Description

Technology Field

[0001] Related applications

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2023-0155908 filed on November 10, 2023, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] Cancer refers to a disease caused by an abnormally grown mass resulting from the autonomous overgrowth of body tissues, and is the result of uncontrolled cell growth in various tissues. Early-stage tumors can be removed through surgery and radiation therapy, while metastatic tumors are generally treated using chemotherapy.

[0004] The majority of parenterally administered chemotherapeutic agents can cause unwanted side effects as a result of systemic administration and can even induce severe toxicity. Therefore, the focus of development has been on creating therapies that increase efficacy and / or minimize toxicity and side effects in tumor cells or immediately adjacent tissues, such as by selectively applying these chemotherapeutic agents.

[0005] The present specification provides, in particular, antibody-drug conjugates (ADCs) targeting both CD20 and CD22, active metabolites of such ADCs, methods for preparing such ADCs, and compositions and uses of such ADCs for the prevention or treatment of cancer or proliferative diseases. More specifically, the present invention provides bispecific antibodies that specifically bind to CD20 and CD22 and pharmaceutical compositions comprising such antibodies. In some embodiments, the bispecific anti-CD20xCD22 antibody of the present specification has improved cellular internalization compared to antibodies known in the art (e.g., anti-CD20 antibodies).

[0006] In certain aspects, the present specification provides an antibody-drug conjugate comprising a bispecific antibody that specifically binds to CD20 and CD22. In certain embodiments, the antibody disclosed herein binds to CD20 and CD22 expressed in a tumor and may be used to deliver an activator to said tumor. In certain embodiments, the antibody-drug conjugate disclosed herein has improved stability and / or cellular internalization compared to antibody-drug conjugates known in the art.

[0007] In certain aspects, the present specification provides a conjugate having a structure represented by general formula I or a pharmaceutically acceptable salt thereof:

[0008] [General Formula 1]

[0009] Ab-[L-(B) l ] m

[0010] The above

[0011] Ab is a bispecific antibody comprising a first antigen-binding domain that specifically binds to CD20 and a second antigen-binding domain that specifically binds to CD22, and

[0012] Each L is independently a linker, and

[0013] Each B is independently an activator, and

[0014] l and m are each independently 1 to 20. Brief explanation of the drawing

[0015] Figure 1 illustrates the binding affinities of an exemplary anti-CD20xCD22 bispecific antibody (anti-CD20xCD22 bispecific antibody 1), and exemplary anti-CD20, anti-CD22, and control IgG antibodies to cancer cell lines. Figure 2 shows the internalization rates of an exemplary anti-CD20xCD22 bispecific antibody (anti-CD20xCD22 bispecific antibody 1) and exemplary anti-CD20 and control IgG antibodies in Ramos cells. Figure 3 shows the internalization rates of exemplary anti-CD20xCD22 bispecific ADCs (ADC2, 6 and 7) and an exemplary anti-CD20 ADC (ADC1) in Ramos cells. Figure 4 shows the plasma concentration of ADC2 at the indicated time in rats. Figure 5 shows that in a SU-DHL-8 cell model, ADC2 demonstrated superior tumor growth inhibition compared to ADC1 at the same dose (0.3 mg / kg, QDx1). Figure 6 shows that in a Granta-519 cell model, ADC2 demonstrated significant tumor growth inhibition compared to the control group. Figure 7 shows that in a MINO cell model, ADC2 demonstrated significant tumor growth inhibition compared to the control group. Figure 8 illustrates that ADC2 exhibited dose-dependent tumor growth inhibition in a Ramos cell model. ADC2 demonstrated superior tumor growth inhibition compared to an exemplary anti-CD22 ADC (Inotuzumab Ozogamicin) at the same dose. Figure 9 illustrates that ADC2 exhibited dose-dependent tumor growth inhibition in the WSU-DLCL2 cell model. ADC2 showed significant tumor growth inhibition compared to ADC1 or R-CHOP at the same dose (0.3 mg / kg, QDx1). Specific details for implementing the invention

[0016] Antibody-drug conjugates (ADCs) are a targeted technology that combines a toxin or drug with an antibody that binds to an antigen, releasing the toxin within the cell to kill cancer cells and other targets. These ADCs accurately deliver drugs to target cancer cells while minimizing impact on healthy cells, and are designed to release drugs only under specific conditions. Consequently, they possess superior efficacy compared to antibody therapies themselves and can significantly reduce the risk of side effects compared to conventional anticancer drugs.

[0017] The basic structure of the antibody-drug conjugate is "antibody-linker-small molecule drug (toxin)". In this structure, the linker performs a functional role of connecting the antibody and the drug, but in some cases, it also performs a role of ensuring that the drug is released from the antibody at the appropriate time, for example, after the drug has reached the target cell. That is, the stability of the linker can play a very important role in the efficacy and safety, such as systemic toxicity, of the antibody-drug conjugate (Discovery Medicine 2010, 10(53): 329-39).

[0018] The use of monoclonal antibodies for cancer treatment has achieved significant success. For example, monoclonal antibodies are suitable for target-directed addressing of tumor tissues and tumor cells. Antibody-drug conjugates have become a new and powerful option for the treatment of lymphomas and solid tumors, and immunomodulatory antibodies have also achieved significant success in recent clinical trials. The development of therapeutic antibodies is based on a deep understanding of cancer serology, protein engineering technologies and their mechanisms of action, mechanisms of resistance, and the interactions between the immune system and cancer cells.

[0019] Antigens expressed on the surface of human cancer cells are defined as a broad range of targets that are overexpressed, mutated, and selectively expressed compared to normal tissues. The key challenge is identifying appropriate antigens for antibody-based therapies. These therapies exert their efficacy by mediating changes in antigen or receptor function (i.e., acting as agonists or antagonists), modulating the immune system through Fc and T cell activation, and delivering specific drugs that bind to antibodies targeting specific antigens. Molecular technologies capable of altering antibody pharmacokinetics, action function, size, and immune stimulation are emerging as key elements in the development of novel antibody-based therapies. Evidence from clinical trials of therapeutic antibodies in cancer patients highlights the importance of approaches for selecting optimized antibodies, including the affinity and binding of the target antigen to the antibody, the selection of antibody structure, and therapeutic approaches (signaling blockade or immune function).

[0020] The CD20 antigen (human B-lymphocyte-restricted differentiation antigen, also known as BP35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kDa located on pre-B and mature B lymphocytes. This antigen is also expressed in more than 90% of B-cell non-Hodgkin lymphomas (NHL), but may not be found in hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissues. CD20 has been shown to regulate the early step(s) of the activation process for cell cycle initiation and differentiation, and possibly to act as a calcium ion channel.

[0021] CD22 is a 135 kDa B cell-restricted sialoglycoprotein that is expressed on the surface of B cells only during the maturation phase of differentiation. In humans, the major form of CD22 is CD22 beta, which contains seven immunoglobulin superfamily domains in its extracellular domain. One variant form, CD22 alpha, lacks immunoglobulin superfamily domains 3 and 4. Ligands binding to human CD22 have been suggested to be associated with immunoglobulin superfamily domains 1 and 2. In B cell NHL, CD22 expression ranges from 91% to 99% in aggressive and retardant populations, respectively. CD22 can function as both a component of the B cell activation complex and an adhesion molecule. After binding to a natural ligand or its antibody, CD22 is rapidly internalized, providing potent co-stimulatory signals to primary B cells and pro-apoptotic signals to neonatal B cells.

[0022] While not bound by any specific scientific hypothesis, this specification includes the recognition that compositions containing antibodies, such as antibody-drug conjugates (ADCs) (e.g., bispecific anti-CD20xCD20 antibodies), are useful for the treatment of diseases (e.g., cancer). In certain aspects, this specification provides an antibody-drug conjugate comprising a bispecific antibody that specifically binds to CD20 and CD22. In certain embodiments, said antibody disclosed herein binds to CD20 and CD22 expressed in tumors. In certain embodiments, the antibody-drug conjugate disclosed herein has improved stability and / or cellular internalization compared to antibody-drug conjugates known in the art. While not bound by any specific scientific hypothesis, said antibody-drug conjugate disclosed herein enables the drug and / or toxin to be released easily, specifically, and efficiently within cancer cells to maximize efficacy and to stably reach the target cell.

[0023] In certain aspects, the present specification provides a conjugate having a structure represented by general formula I or a pharmaceutically acceptable salt thereof:

[0024] [General Formula I]

[0025] Ab-[L-(B) l ] m

[0026] The above

[0027] Ab is a bispecific antibody comprising a first antigen-binding domain that specifically binds to CD20 and a second antigen-binding domain that specifically binds to CD22, and

[0028] Each L is independently a linker, and

[0029] Each B is independently an activator, and

[0030] l and m are each independently 1 to 20.

[0031] In some embodiments, L is a cleavable linker.

[0032] In some embodiments, a cleavable linker refers to a linker or linker component that connects two moieties by a covalent bond, but under physiologically relevant conditions, the covalent bond between the moieties is broken down and cleaved. In some embodiments, the cleavable linker is in vivo In this case, it is cleaved more rapidly in the intracellular environment than outside the cell, so that the release of the payload (e.g., activator) occurs preferentially inside the target cell. The cleavage may be enzymatic or non-enzymatic. The cleavage may release the drug from the antibody without degrading the antibody. The cleavage may leave the linker or part of the linker component attached to the payload, or release the payload without any residue of the linker.

[0033] In some embodiments, L is a protease-cleavable linker, an acid-cleavable linker, a disulfide linker, or a glycosidase-cleavable linker. In some embodiments, L is a protease-cleavable linker. In some embodiments, the protease may be one or more selected from cysteine ​​protease, metalloprotease, serine protease, threonine protease, and aspartate protease.

[0034] Cysteine ​​proteases, also known as thiol proteases, are proteases that share a common catalytic mechanism containing nucleophilic cysteine ​​thiol within a catalytic tirad or dyad. Metalloproteases are proteases that contain a metal in their catalytic mechanism. Most metalloproteases require zinc, but some use cobalt. The metal ions are incorporated into the protein through three ligands. Serine proteases are enzymes that cleave peptide bonds within proteins. Serine acts as a nucleophilic amino acid in the active site of the enzyme. Serine proteases are classified into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like. Threonine proteases are a family of proteases containing a threonine (Thr) residue within their active site. In the above family, the prototypical member of the enzyme is the catalytic subunit of the proteasome, but the acyltransferase has also convergently evolved the same active site geometry and mechanism. The aspartate protease is a catalytic type protease enzyme that catalyzes a peptide substrate using an activated water molecule bound to one or more aspartate residues. Generally, two highly conserved aspartates are present in the active site, and it is optimally activated at acidic pH. Almost all known aspartate proteases are inhibited by pepstatin.

[0035] In some embodiments, the protease is cathepsin. In some embodiments, L comprises two or more amino acid residues, for example, residues determining the recognition site of the protease. In some embodiments, L comprises two to four amino acid residues, for example, residues determining the recognition site of the protease. In some embodiments, L comprises valine-alanine (Val-Ala), valine-citrulline (Val-Cit), valine-lysine (Val-Lys), valine-arginine (Val-Arg), phenylalanine-lysine-glycine-proline-leucine-glycine (Phe-Lys-Gly-Pro-Leu-Glu) or alanine-alanine-proline-valine (Ala-Ala-Pro-Val). In some embodiments, L comprises Val-Cit, Val-Ala, or Gly-Gly-Phe-Gly.

[0036] In some embodiments, L is a glycosidase cleavable linker. In some embodiments, the glycosidase is β-glucuronidase or β-galactosidase. In some embodiments, L comprises a β-glucuronosyl moiety or a β-galactosyl moiety.

[0037] In some embodiments, L comprises any one of the following components: 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe), lysine-phenylalanine (Lys-Phe), p-aminobenzyloxycarbonyl (PAB), 4-thio-pentanoate (SPP), 4-thio-butyrate (SPDB), 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC), maleimidoethyl (ME), 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), aryl-thiol (PySS), (4-acetyl)aminobenzoate (SIAB), oxybenzylthio, aminobenzylthio, dioxybenzylthio, diaminobenzylthio, Aminooxybenzylthio, alkoxyamino (AOA), ethyleneoxy (EO), 4-methyl-4-dithio-pentanoic acid (MPDP), triazole, dithio, alkylsulfonyl, alkyl sulfonamide, sulfon-bisamide, phosphodiamide, alkylphosphonamide, phosphinic acid, N-methylphosphonamide, N,N'-dimethylphosphonamide, N,N'-dimethylphosphonadiamide, hydrazine, acetimideamide, oxime, acetylacetohydrazide, aminoethyl-amine, aminoethyl-aminoethyl-amine, and L- or D-, natural or non-natural peptides containing 1 to 20 amino acids.

[0038] In some embodiments, L is covalently bonded to Ab by a thioether, thioester, disulfide, hydrazone, ester, carbamate, carbonate, alkoxy, or amide. In some embodiments, L comprises a peptide further comprising at least one hydrophilic amino acid. In some embodiments, the peptide comprises an amino acid having a side chain having a moiety (e.g., amine, guanidine, or carboxyl moiety) that carries a charge in an aqueous solution of neutral pH. In some embodiments, the peptide comprises an amino acid selected from alanine, histidine, arginine, aspartic acid, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine. In some embodiments, the peptide comprises an amino acid selected from alanine, aspartic acid, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine.

[0039] In some embodiments, L comprises a functional group capable of reacting with the terminal moiety of a specific molecule. Examples of functional groups capable of reacting with terminal amines include N-hydroxysuccinimide esters, p-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters, carboxylic acid chlorides, and carboxylic acid anhydrides. Examples of functional groups capable of reacting with terminal thiols include pyridyl disulfide, nitropyridyl disulfide, maleimide, haloacetate, methylsulfonphenyl oxadiazole (ODA), carboxylic acid chlorides, and carboxylic acid anhydrides. Examples of functional groups capable of reacting with terminal ketones or aldehydes include amines, alkoxyamines, hydrazines, acyloxyamines, and hydrazides. Examples of functional groups capable of reacting with terminal azides include alkynes.

[0040] In some embodiments, L comprises an oxime. In some embodiments, L comprises at least one isoprenyl group. In some embodiments, L comprises at least one succinimide group.

[0041] In some embodiments, L is covalently bonded to Ab by a thioether bond, said thioether bond comprises a sulfur atom of cysteine ​​of Ab. In some embodiments, Ab comprises an amino acid motif at the C-terminus that is recognized by an isoprenoid transferase. In some embodiments, said isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase). In some embodiments, L is covalently bonded to Ab by a thioether bond, said thioether bond comprises a sulfur atom of cysteine ​​of said amino acid motif. In some embodiments, said amino acid motif comprises the sequence CY1Y1X, said amino acid motif comprising: C is cysteine; and each Y1 is independently an aliphatic amino acid; X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine. In some embodiments, each Y1 is independently selected from alanine, isoleucine, leucine, methionine, and valine. In some embodiments, the amino acid motif comprises CVIM (SEQ No. 84) or CVLL (SEQ No. 85). In some embodiments, at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine. In some embodiments, the amino acid motif has the sequence GGGGGGGCVIM (SEQ No. 77).

[0042] In some embodiments, L comprises a self-immolative moiety. Examples of said self-immolative moiety include, but are not limited to, 2-aminoimidazole-5-methanol derivatives, heterocyclic PAB analogs, beta-glucuronide, and aromatic compounds electrically similar to para-aminobenzylcarbamoyl (PAB) groups, such as ortho or para-aminobenzylacetal.

[0043] In some embodiments, the conjugate has a structure represented by General Formula II or is a pharmaceutically acceptable salt:

[0044] [General Formula II]

[0045]

[0046] Above:

[0047] B is the above-mentioned active agent, and

[0048] G indicates a sugar moiety or a glucuronic acid moiety;

[0049] R 1 and R 2 is independently hydrogen, C1-C8 alkyl, or C3-C8 cycloalkyl; R 1 and R 2 They combine to form a (C3-C8) cycloalkyl ring;

[0050] W is - * C(O)-, - * C(O)N(R')-, - * N(R')C(O)-, - * (CH2) t N(R')C(O)-, - * C(O)O-, - * S(O2)N(R')-, - * P(O)(R'')N(R')-, - * S(O)N(R')- or - * P(O2)N(R')-, and the C(O), N, CH2, S, or P labeled with * above are directly bonded to the phenyl ring of the above general formula II, and

[0051] R' and R'' are independently hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 Ariligo;

[0052] Z is independently C 1-8 It is an alkyl, halogen, cyano, or nitro, and;

[0053] n1 and n2 are each independently 1 to 20;

[0054] n3 is 0 to 3;

[0055] Y does not exist, or C1-C 50 Alkylene, C1-C 50 It is alkenylene, 1-50 atomic heterocyclylene, or 1-50 atomic heteroalkylene.

[0056] In some embodiments, Y is C 1-50 Alkylene or C 1-50 It comprises heteroalkylene and further comprises at least one of the following:

[0057] (i) one or more unsaturated bonds;

[0058] (ii) heterocyclylene or heteroarylene (e.g., heteroarylene within the alkylene or heteroalkylene chain);

[0059] (iii) at least one C 1-20 alkyl; and

[0060] (iv) at least one isoprenyl group having a structure represented by general formula III:

[0061] [General Formula III]

[0062] ;

[0063] The above n4 is an integer from 1 to 20.

[0064] In some embodiments, the above R 1 and R 2 Each is independently hydrogen. In some embodiments, n3 is 0.

[0065] In some embodiments, the above G has a structure represented by general formula IV:

[0066] [General Formula IV]

[0067]

[0068] The above

[0069] R3 is hydrogen, alkyl, CH2OR3A or CO2R3B;

[0070] R 3A is a hydrogen or hydroxyl protecting group;

[0071] R 3B is a hydrogen or carboxyl protecting group;

[0072] Each R4 is independently a hydrogen or hydroxyl protecting group.

[0073] In some embodiments, R3 is -COOH. In some embodiments, each R4 is independently hydrogen. In some embodiments, W is -*C(O)NR'-.

[0074] In some specific examples, G is is;

[0075] W is -*C(O)NR'-.

[0076] In some embodiments, Y is covalently bonded to Ab by a thioether, thioester, disulfide, hydrazone, ester, carbamate, carbonate, alkoxy, or amide. In some embodiments, Y comprises a peptide further comprising at least one hydrophilic amino acid. In some embodiments, the peptide comprises an amino acid having a side chain having a moiety (e.g., amine, guanidine, or carboxyl moiety) that carries a charge in an aqueous solution of neutral pH. In some embodiments, the peptide comprises an amino acid selected from alanine, histidine, arginine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine. In some embodiments, the peptide comprises an amino acid selected from aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine.

[0077] In some embodiments, Y includes an oxime. In some embodiments, the oxygen atom of the oxime is located on the side of Y connected to W, and the carbon atom of the oxime is located on the side of Y connected to Ab. In some embodiments, the carbon atom of the oxime is located on the side of Y connected to W, and the oxygen atom of the oxime is located on the side of Y connected to Ab.

[0078] In some embodiments, Y is C containing an oxime 1-50It is a heteroalkylene. In some embodiments, the oxygen atom of the oxime is located on the side of Y connected to W, and the carbon atom of the oxime is located on the side of Y connected to Ab. In some embodiments, the carbon atom of the oxime is located on the side of Y connected to W, and the oxygen atom of the oxime is located on the side of Y connected to Ab. In some embodiments, Y comprises an oxime, and at least one isoprenyl unit covalently bonds the oxime to Ab (e.g., at least one isoprenyl unit directly or indirectly bonds the oxime to Ab). In some embodiments, Y comprises at least one isoprenyl group.

[0079] In some embodiments, Y is covalently bonded to Ab by a thioether bond, said thioether bond comprises a sulfur atom of cysteine ​​of Ab. In some embodiments, said Ab comprises an amino acid motif recognized by an isoprenoid transferase at the C-terminus. In some embodiments, said isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase). In some embodiments, Y is covalently bonded to Ab by a thioether bond, said thioether bond comprises a sulfur atom of cysteine ​​of said amino acid motif. In some embodiments, said amino acid motif comprises the sequence CY1Y1X, said: C is cysteine; each Y1 is independently an aliphatic amino acid; X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine. In some embodiments, each Y1 is independently selected from alanine, isoleucine, leucine, methionine, and valine. In some embodiments, the amino acid motif comprises CVIM (SEQ No. 84) or CVLL (SEQ No. 85). In some embodiments, at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine. In some embodiments, the amino acid motif has the sequence GGGGGGGCVIM (SEQ No. 77).

[0080] In some embodiments, the above Y includes a connecting unit represented by the general formula V or the general formula VI:

[0081] [General Formula V]

[0082] -(CH2) r (V(CH2) p ) q -

[0083] [General Formula VI]

[0084] -(CH2CH2X) w -

[0085] Above,

[0086] V is a single bond, -O-, -S-, -NR 5 -, -C(O)NR 6 -, -NR 7 C(O)-, -NR 8 SO2-, or -SO2NR 9 - and;

[0087] X is -O-, C 1-8 Alkylene, or -NR 5 - and;

[0088] R 5 to R 9 are independently hydrogen, C 1-6 Alkyl, C 1-6 Alkyl C 6-20 Aryl or C 1-6 Alkyl-C 3-20 It is heteroaryl;

[0089] r is an integer from 0 to 10;

[0090] p is an integer from 0 to 10;

[0091] q is an integer from 1 to 20;

[0092] w is an integer from 1 to 20.

[0093] In some embodiments, q is 1 to 20. In some embodiments, r is 1 or 2. In some embodiments, p is 1 or 2. In some embodiments, V is -O-. In some embodiments, q is 1 to 10; r and p are each 1 or 2; and V is -O-. In some embodiments, X is -O-. In some embodiments, X is -O-; and w is 1 to 10.

[0094] In some specific examples, L is It comprises at least one polyethylene glycol monomer represented by . In some embodiments, L is or It includes a polyethylene glycol oligomer represented by, and the n40 is 2 to 12.

[0095] In some embodiments, Y includes an oxime, and at least one polyethylene glycol unit covalently bonds the oxime to W.

[0096] In some embodiments, the above Y further comprises a binding unit formed by a reaction between an alkyne and an azide or by a reaction between an aldehyde or ketone and a hydrazine or hydroxylamine.

[0097] In some embodiments, Y further comprises a combining unit represented by the general formulas VIIa, VIIb, VIIc, VIId, or VIIe:

[0098] [General Formula VIIa]

[0099]

[0100] [General Formula VIIb]

[0101]

[0102] [General Formula VIIc]

[0103]

[0104] [General Formula VIId]

[0105]

[0106] [General Formula VIIe]

[0107]

[0108] The above

[0109] L 1 Each is independently a single bond or C 1-30 It is an alkylene;

[0110] R 11 Silver is hydrogen or C 1-10 It is an alkyl.

[0111] In some embodiments, the above composite is Includes

[0112] In some embodiments, the above composite is Comprising a structure represented by or a pharmaceutically acceptable salt thereof; and the above

[0113] n12 is 0 to 30;

[0114] The above wave bond indicates the connection with Ab.

[0115] In some embodiments, Y is branched. In some embodiments, Y includes the following:

[0116] i) A branching unit covalently bonded to Ab by a primary linker;

[0117] ii) a first B (first B) coupled to the branch unit; and

[0118] iiia) A second branch or the second B coupled to the branch unit

[0119] iiib) A second branch in which an alkyl or heteroalkyl (e.g., polyethylene glycol monomer or polyethylene glycol oligomer) is covalently bonded to the branch unit.

[0120] In some embodiments, Y comprises a second branch that combines the second B to the branch unit through a cleavage group. In some embodiments, Y comprises a second branch in which an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) is covalently bonded to the branch unit.

[0121] In some embodiments, the branch unit is , , , or Having a structure represented as,

[0122] The above

[0123] G 1, G 2 and G 3 Each combines independently , , or And;

[0124] R 30 Silver is hydrogen or C 1-30 It is alkyl;

[0125] R 40 Silver is hydrogen or L 5 -COOR 50 And;

[0126] R 50 Silver is hydrogen or C 1-30 It is alkyl;

[0127] L 2 , L 3 and L 4 are each independently combined or -C n' H 2n' -is;

[0128] n' is an integer from 1 to 10.

[0129] In some embodiments, at least one branched linker is covalently bonded to Ab; and at least two B are covalently bonded to said branched linker.

[0130] In some embodiments, the conjugate comprises 1, 2, 3, or 4 branched linkers, and each branched linker comprises 2 B. In some embodiments, the branched unit comprises a lysine residue.

[0131] In some embodiments, the above composite is Comprising a structure represented by or a pharmaceutically acceptable salt thereof, and the above

[0132] Each B is independently an activator;

[0133] n11, n22, and n33 are each independently 0 to 30;

[0134] AA is a peptide containing at least two amino acid residues;

[0135] The above wave bond indicates the connection with Ab.

[0136] bispecific antibody or its antigen-binding domain

[0137] The present specification provides, in particular, a bispecific antibody comprising a first antigen-binding domain that specifically binds to CD20 and a second antigen-binding domain that specifically binds to CD22, for example, an anti-CD20xCD22 bispecific antibody ("Ab").

[0138] In certain aspects, the present specification provides an antibody-drug conjugate comprising an Ab. In certain aspects, the Ab disclosed herein binds to CD20 and CD22 expressed in a tumor and can be used to deliver a drug to said tumor. In certain embodiments, the antibody-drug conjugate and the Ab disclosed herein have improved stability and / or cellular internalization compared to antibody-drug conjugates or antibodies known in the art.

[0139] As mentioned herein, the bispecific antibody according to the present specification is a polypeptide comprising one or more complementarity determining areas or regions (CDRs).

[0140] In some embodiments, the CDR is included in a "framework" region, and the framework orients the CDR(s) so that the CDR(s) can have appropriate antigen binding characteristics.

[0141] In certain embodiments, the antibody according to the present specification may be composed of a single light chain polypeptide or a single heavy chain polypeptide comprising variable regions as shown in Tables 4 to 9 and 16.

[0142] CDR sequences that may be included in the heavy chain and light chain variable regions of the antibody or its antigen-binding fragment according to one embodiment of the present specification are shown in Tables 4 to 9 and 16.

[0143] The antibody according to the present specification shares specific regions or sequences with other antibodies disclosed herein. In certain embodiments, the constant region of the antibody or its antigen-binding fragment may be shared. In certain embodiments, Fc regions may be shared. In certain embodiments, the frame of the variable region may be shared.

[0144] The heavy chain variable region and light chain variable region according to the present specification may be connected to at least a portion of the human constant region. The selection of the constant region may be determined in part by whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity is required. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not have such cytotoxicity. Additionally, human IgG1 and IgG3 induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region may be lambda or kappa.

[0145] The variable regions of immunoglobulin chains generally have an identical overall structure and contain a relatively conserved framework region (FR) connected by three hypervariable regions referred to as "complementarity determining areas or regions or domains" or complementarity determining regions (CDRs). The CDRs of variable regions derived from each chain containing heavy / light chain pairs are typically aligned by the framework region to form a structure that specifically binds to a particular epitope of the target protein. These elements of naturally occurring light and heavy chain variable regions are typically included in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The positions of the amino acid sequences corresponding to each variable region can be determined by Kabat (Kabat et al., (1983) US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest"), Chothia (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), or methods related to the OPAL library (Hye Young Yang et al., 2009 Mol. Cells 27: 225). When comparing the CDRs determined by each of the above definitions, they may overlap or be subsets in which one includes the other. A person skilled in the art will be able to easily select CDR sequences according to the above definitions given the variable region sequence of the antibody.

[0146] In certain embodiments, the amino acid sequence of the CDR is determined according to the Kabat definition. However, it is well known to those skilled in the art that antibody CDRs can be defined according to various methods in the art, such as the Chothia definition based on the location of the structural loop region (Al-Lazikani, B et al., J Mol Biol 273: 927-48 (1997)) and the Kabat definition based on sequence variability (Kabat et al., "Sequences of proteins of immunological interest", fifth edition, National Institutes of Health, Bethesda, MD. (1991).

[0147] In certain embodiments, amino acid residues of the variable region sequence may be determined using a Combined definition that incorporates both the Kabat definition and the Chothia definition. The Combined definition refers to a combination of the ranges of the Kabat definition and the Chothia definition. Unless otherwise specified, it will be understood by those skilled in the art that the "CDR" and "complementarity determining region" of a given antibody or its region (e.g., variable region) should be understood to include a complementarity region defined according to any embodiment described herein.

[0148] The scope of protection claimed in the claims of this specification is based on a sequence defined according to the Kabat definition, but amino acid sequences defined according to other CDR definitions must also be included in the scope of protection of this specification.

[0149] In certain embodiments, the antibody according to the present specification is a humanized antibody. A humanized antibody refers to any antibody in which the constant region of a non-human antibody is completely replaced by a constant region of the human form, and at least a portion of the variable region of the non-human antibody is completely or partially replaced by a corresponding portion of the human antibody, except for three loops of the amino acid sequence outside each variable region that binds to a target structure. In certain embodiments, the antibody according to the present specification is a human antibody.

[0150] Specific mutations can be introduced into the framework region to enhance antibody stability while maintaining antigen-binding activity. The stabilization of therapeutic antibodies can lead to improved serum half-life, reduced dosage, fewer side effects, extended shelf life, and reduced transportation and storage costs.

[0151] In certain embodiments, this specification discloses one or more amino acid sequences having substantial sequence identity with one or more amino acid sequences disclosed herein. Substantial identity means that the effects disclosed herein are maintained even when sequence variations are present. In certain embodiments, the amino acid sequence has about 90% identity, about 95% identity, or about 99% identity with the heavy chain variable regions shown in Tables 1 and 2. In other embodiments, the amino acid sequence has about 90% identity, about 95% identity, or about 99% identity with the light chain variable regions shown in Tables 1 and 2. For example, in the case of variants exhibiting 90% identity, 95% identity, or 99% identity with the antibody or its antigen-binding fragment sequence according to this specification, any mutation occurs within the framework of the variable region rather than the CDR.

[0152] In a specific embodiment, the nucleic acid encoding the antibody or a fragment thereof according to the present specification is a nucleic acid encoding a full-length antibody comprising the CDRs disclosed in the present specification, a variable region including the CDRs, and a variable region and an invariant region. Once the amino acid sequence is determined, the nucleic acid sequence can be easily determined by considering known reverse transcription programs and codon usage, etc.

[0153] In some embodiments, Ab comprises a first constant region connected to the first antigen-binding domain and a second constant region connected to the second antigen-binding domain, and the first constant region and the second constant region each comprise a light chain constant region CL domain and heavy chain constant regions CH1, CH2 and CH3 domains.

[0154] In some embodiments, Ab is a chimeric antibody, a humanized antibody, or a human antibody.

[0155] In some embodiments, Ab is an IgA, IgG, IgM, IgE, or IgD isotype. In some embodiments, Ab is an IgG isotype (e.g., IgG1, IgG2, IgG3, and IgG4).

[0156] In some embodiments, Ab comprises LALA mutations within the first and / or second heavy chain constant regions. In some embodiments, the LALA mutations comprise L234A and L235A according to the EU numbering convetion.

[0157] Knob-in-Hole

[0158] In some embodiments, the bispecific antibodies of this specification are prepared by the method described in U.S. Patent No. 9,637,557 B2, the contents of which are incorporated herein by reference in whole.

[0159] According to some specific examples, the bispecific antibody may be a heteromultimer.

[0160] As used herein, the term "heteromultimer" refers to a protein multimer composed of multiple types of polypeptides, said polypeptides may be related to one another. More specifically, said heteromultimer is a molecule having at least a first polypeptide and a second polypeptide, said second polypeptide having at least one amino acid residue that is different from the first polypeptide in its amino acid sequence.

[0161] In some embodiments, the heteropolymer may include at least one amino acid substitution (e.g., for forming a knob-in-hole structure) within each domain (e.g., a CH3 domain) within the first constant region and the second constant region.

[0162] In this method, the interaction surface of the two CH3 domains is modified to increase the heterodimerization of all heavy chains containing the two CH3 domains. One of the two CH3 domains (of the two heavy chains) may be a “knob” domain and the other may be a “hole” domain. The introduction of the disulfide bridge stabilizes the heterodimers (Merchant, A..M., et al, Nature Biotech 16 (1998) 677-681; Atwell, S., Ridgway, JB, Wells, JA, Carter, P., J MoI Biol 270 (1997) 26-35) and increases the yield.

[0163] The bispecific antibody of this specification may be characterized in that the CH3 domain of one heavy chain and the CH3 domain of another heavy chain form an interface beyond the original interface between the CH3 domains of the antibody; said interface is modified to facilitate the formation of a bivalent bispecific antibody, and said modification is characterized as follows:

[0164] a) The CH3 domain of one heavy chain (e.g., the first constant region) is modified to form a ridge (knob) within the interface of the CH3 domain of one heavy chain that contacts the initial interface of the CH3 domain of another heavy chain (e.g., the second constant region) in a bispecific antibody, by substituting an amino acid residue with an amino acid residue having a larger side chain volume, said knob may be located in a space within the interface of the CH3 domain of the other heavy chain, and

[0165] b) The CH3 domain of another heavy chain (e.g., the second constant region) is modified so as to create a space (hole) within the interface of the CH3 domain of the other heavy chain that contacts the initial interface of the CH3 domain of one heavy chain (e.g., the first constant region) in the bispecific antibody, by substituting an amino acid residue with an amino acid residue having a smaller side chain volume, said hole may be located at a ridge within the interface of the CH3 domain of one heavy chain.

[0166] Preferably, the amino acid residue having the larger side chain volume is selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0167] Preferably, the amino acid residue having the smaller side chain volume is selected from alanine (A), serine (S), threonine (T), and valine (V).

[0168] In some embodiments, both CH3 domains may be further modified so that cysteine ​​(C) as an amino acid is introduced at corresponding positions within each CH3 domain, thereby forming a disulfide bridge between the two CH3 domains.

[0169] In some preferred embodiments, the bispecific antibody comprises a T336W mutation according to the EU index of Kabat in the CH3 domain of the knob chain (e.g., the CH3 domain of the first constant region or the second constant region) and T366S, L368A, and / or Y407V mutations in the CH3 domain of the hole chain (e.g., the CH3 domain of the first constant region or the second constant region). Additional interchain disulfide bridges between the CH3 domains may also be used, for example, by introducing a Y349C mutation into the CH3 domain of the knob chain and an E356C mutation or an S354C mutation into the CH3 domain of the hole chain (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681). Accordingly, in another preferred embodiment, the bispecific antibody comprises Y349C and T366W mutations in one of the two CH3 domains and E356C, T366S, L368A, and Y407V mutations in the other of the two CH3 domains, or the bispecific antibody comprises Y349C and T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, and Y407V mutations in the other of the two CH3 domains (additional Y349C mutations in one CH3 domain and additional E356C or S354C mutations in the other CH3 domain form interchain disulfide bridges).

[0170] In some embodiments, the bispecific antibody comprises a T366W mutation in the CH3 domain of the knob chain and T366S, L368A, and Y407V mutations in the CH3 domain of the hole chain, additionally comprises R409D; K370E mutations in the CH3 domain of the knob chain and D399K; E357K mutations in the CH3 domain of the hole chain.

[0171] In another preferred embodiment, the bispecific antibody comprises Y349C and T366W mutations in one of two CH3 domains and S354C, T366S, L368A, and Y407V mutations in the other of two CH3 domains, or the bispecific antibody comprises Y349C and T366W mutations in one of two CH3 domains and S354C, T366S, L368A, and Y407V mutations in the other of two CH3 domains, and additionally comprises R409D; K370E mutations in the CH3 domain of the knob chain and D399K; E357K mutations in the CH3 domain of the hole chain.

[0172] Accordingly, in some embodiments, the bispecific antibody of the present specification may have a knob-in-hole structure formed by substituting an amino acid residue in the CH3 domain of the first constant region with an amino acid residue having a larger side chain size and substituting an amino acid residue in the CH3 domain of the second constant region with an amino acid residue having a smaller side chain size; or may have a knob-in-hole structure formed by substituting an amino acid residue in the CH3 domain of the second constant region with an amino acid residue having a larger side chain size and substituting an amino acid residue in the CH3 domain of the first constant region with an amino acid residue having a smaller side chain size.

[0173] In some embodiments, the bispecific antibody of this specification may include T366S, L368A, and Y407V according to the Kabat EU index in the CH3 domain of the first constant region and T366W according to the Kabat EU index in the CH3 domain of the second constant region; or may include T366S, L368A, and Y407V according to the Kabat EU index in the CH3 domain of the second constant region and T366W according to the Kabat EU index in the CH3 domain of the first constant region.

[0174] In some embodiments, at least one amino acid residue in the CH3 domain of the first heavy chain constant region is substituted with an amino acid residue having a larger side chain size, and at least one amino acid residue in the CH3 domain of the second heavy chain constant region is substituted with an amino acid residue having a smaller side chain size; or at least one amino acid residue in the CH3 domain of the second heavy chain constant region is substituted with an amino acid residue having a larger side chain size, and at least one amino acid residue in the CH3 domain of the first heavy chain constant region is substituted with an amino acid residue having a smaller side chain size.

[0175] In some embodiments, the amino acid residue having a large side chain size is selected from arginine, phenylalanine, tyrosine, and tryptophan; and the amino acid residue having a smaller side chain size is selected from alanine, serine, threonine, and valine.

[0176] In some embodiments, the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at positions T366, L368, and Y407 according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions at position T366 according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions at positions T366, L368, and Y407 according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at position T366 according to EU number.

[0177] In some embodiments, the CH3 domain of the first heavy chain invariant region comprises T366S, L368A, and Y407V according to EU number, and the CH3 domain of the second heavy chain invariant region comprises T366W according to EU number; or the CH3 domain of the second heavy chain invariant region comprises T366S, L368A, and Y407V according to EU number, and the CH3 domain of the first heavy chain invariant region comprises T366W according to EU number.

[0178] In some embodiments, the CH3 domain of each of the first and second heavy chain constant regions additionally comprises at least one substitution from an amino acid residue to a cysteine ​​residue.

[0179] In some embodiments, (a) the CH3 domain of the first heavy chain invariant region comprises E356C, T366S, L368A, and Y407V according to EU number, and the CH3 domain of the second heavy chain invariant region comprises Y349C and T366W according to EU number; or the CH3 domain of the second heavy chain invariant region comprises E356C, T366S, L368A, and Y407V according to EU number, and the CH3 domain of the first heavy chain invariant region comprises Y349C and T366W according to EU number; or (b) the CH3 domain of the first heavy chain invariant region comprises S354C, T366S, L368A, and Y407V according to EU number, and the CH3 domain of the second heavy chain invariant region comprises Y349C and T366W according to EU number; The CH3 domain of the second heavy chain invariant region comprises S354C, T366S, L368A, and Y407V according to EU numbers, and the CH3 domain of the first heavy chain invariant region comprises Y349C and T366W according to EU numbers.

[0180] In some embodiments, (a) the CH3 domain of the first heavy chain invariant region comprises E356C, E357K, T366S, L368A, D399K and Y407V according to EU number, and the CH3 domain of the second heavy chain invariant region comprises Y349C, T366W, K370E and R409D according to EU number; or the CH3 domain of the second heavy chain invariant region comprises E356C, E357K, T366S, L368A, D399K and Y407V according to EU number, and the CH3 domain of the first heavy chain invariant region comprises Y349C, T366W, K370E and R409D according to EU number; or (b) the CH3 domain of the first heavy chain invariant region comprises S354C, E357K, T366S, L368A, D399K and Y407V according to EU number, and the CH3 domain of the second heavy chain invariant region comprises Y349C, T366W, K370E and R409D according to EU number; or the CH3 domain of the second heavy chain invariant region comprises S354C, E357K, T366S, L368A, D399K and Y407V according to EU number, and the CH3 domain of the first heavy chain invariant region comprises Y349C, T366W, K370E and R409D according to EU number.

[0181] Pentambody

[0182] In some embodiments, the bispecific antibodies of this specification are produced by the method described in U.S. Patent No. 11,498,977 B2, the contents of which are incorporated herein by reference in whole.

[0183] In some embodiments, the bispecific antibody may include one or more amino acid substitutions within the CH3 domains of the first constant region and the second constant region, respectively. The first constant region and the second constant region may include at least five amino acid substitutions at the following positions according to the Kabat EU index within the CH3 domain:

[0184] 1) positions 366 and 399 within the first invariant region (or the second invariant region) and positions 351, 407, and 409 within the second invariant region (or the first invariant region); or 2) positions 366 and 409 within the first invariant region (or the second invariant region) and positions 351, 399, and 407 within the second invariant region (or the first invariant region).

[0185] Specifically, the bispecific antibody may comprise amino acid substitutions at positions T366 and D399 according to the Kabat EU index within the CH3 domain of the first constant region, and amino acid substitutions at positions L351, Y407, and K409 according to the Kabat EU index within the CH3 domain of the second constant region; or may comprise amino acid substitutions at positions T366 and D399 according to the Kabat EU index within the CH3 domain of the second constant region, and amino acid substitutions at positions L351, Y407, and K409 according to the Kabat EU index within the CH3 domain of the first constant region.

[0186] In some embodiments, the first invariant region and the second invariant region comprise at least one of the following substitutions according to the Kabat EU index in the CH3 domain:

[0187] a) Glycine, tyrosine, valine, proline, aspartic acid, glutamic acid, lysine, or tryptophan at the L351 position of the second invariant region (or the first invariant region);

[0188] b) Leucine, proline, tryptophan, or valine at the T366 position of the first invariant region (or the second invariant region);

[0189] c) Cytene, asparagine, isoleucine, glycine, arginine, threonine, or alanine at the D399 position of the first invariant region and / or the second invariant region;

[0190] d) leucine, alanine, proline, phenylalanine, threonine, or histidine at the Y407 position of the second invariant region (or the first invariant region); and

[0191] e) Cysteine, proline, serine, phenylalanine, valine, glutamic acid, or arginine at the K409 position of the first constant region and / or the second constant region.

[0192] The combination of the above amino acid substitutions is possible within the mentioned substitutions, and specifically, the bispecific antibody may include T366L and D399R according to the Kabat EU index in the CH3 domain of the first constant region and L351E, Y407L, and K409Y according to the Kabat EU index in the CH3 domain of the second constant region; or may include T366L and D399R according to the Kabat EU index in the CH3 domain of the second constant region and L351E, Y407L, and K409Y according to the Kabat EU index in the CH3 domain of the first constant region, but is not limited thereto.

[0193] In some embodiments, (a) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at positions T366 and D399 according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions at positions L351, Y407, and K409 according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions at positions T366 and D399 according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at positions L351, Y407, and K409 according to EU number; or (b) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at positions T366 and K409 according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions at positions L351, D399, and Y407 according to EU number; The CH3 domain of the second heavy chain constant region comprises amino acid substitutions at positions T366 and K409 according to EU numbers, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at positions L351, D399, and Y407 according to EU numbers.

[0194] In some embodiments, the amino acid substituted in T366 is selected from leucine, proline, tryptophan, and valine; the amino acid substituted in D399 is selected from cysteine, asparagine, isoleucine, glycine, arginine, threonine, and alanine; the amino acid substituted in L351 is selected from glycine, tyrosine, valine, proline, aspartic acid, glutamate, lysine, and tryptophan; the amino acid substituted in Y407 is selected from leucine, alanine, proline, phenylalanine, threonine, and histidine; and the amino acid substituted in K409 is selected from cysteine, proline, serine, phenylalanine, valine, glutamate, and arginine.

[0195] In some embodiments, (a) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366L and D399R according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351E, Y407L, and K409V according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366L and D399R according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351E, Y407L, and K409V according to EU number; or (b) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366L and D399C according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351G, Y407L, and K409V according to EU number; The CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366L and D399C according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351E, Y407L, and K409C according to EU number; or (c) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366L and D399C according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351Y, Y407A, and K409P according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366L and D399C according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351Y, Y407A, and K409P according to EU number;or (d) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366P and D399N according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351V, Y407P and K409S according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366P and D399N according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351V, Y407P and K409S according to EU number; or (e) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366W and D399G according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351D, Y407P and K409S according to EU number; The CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366W and D399G according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351D, Y407P, and K409S according to EU number; or (f) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366P and D399I according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351P, Y407F, and K409F according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366P and D399I according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351P, Y407F, and K409F according to EU number;or (g) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366V and D399T according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351K, Y407T and K409Q according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366V and D399T according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351K, Y407T and K409Q according to EU number; or (h) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366L and D399A according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351W, Y407H and K409R according to EU number; The CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366L and D399A according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351W, Y407H, and K409R according to EU number.

[0196] In some embodiments, the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366L and D399R, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351E, Y407L, and K409Y; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366L and D399R, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351E, Y407L, and K409Y.

[0197] CrossMab

[0198] In some embodiments, the bispecific antibodies of this specification are produced by the method described in U.S. Patent No. 8,242,247 B2, the contents of which are incorporated herein by reference in whole.

[0199] In some embodiments, the bispecific antibody comprises a first constant region connected to a first antigen-binding domain; and a second constant region connected to a second antigen-binding domain, wherein the first constant region and the second constant region may each comprise CH1, CH2, and CH3 domains of the light chain constant region and the heavy chain constant region of the antibody.

[0200] The bispecific antibody of the present specification may be improved by substituting a specific domain of a heavy chain and light chain (HC / LC) pair. For example, the light chain constant domain of the first constant region and the CH1 domain may be substituted for each other, and / or the light chain constant domain of the second constant region and the CH1 domain may be substituted for each other.

[0201] In some embodiments, the variable heavy chain domain VH of the first invariant region (or the second invariant region) may be substituted with the variable light chain domain VL.

[0202] In some embodiments, the variable heavy chain domain VH of the first invariant region (or the second invariant region) and the heavy chain invariant domain CH1 may be substituted with a light chain domain.

[0203] The structure described above may provide additional advantages in terms of the preparation and efficacy of the anti-CD20xCD22 bispecific antibody-drug conjugate of this specification.

[0204] In some embodiments, (a) the CL and CH1 domains of the first invariant region are substituted with each other; or the CL and CH1 domains of the second invariant region are substituted with each other; (b) the VH and VL domains of the first antigen-binding domain are substituted with each other; or the VH and VL domains of the second antigen-binding domain are substituted with each other; or (c) the VH and CH1 domains of the first invariant region and the VL and CL domains of the first antigen-binding domain are substituted with each other; or the VH and CH1 domains of the second invariant region and the VL and CL domains of the second antigen-binding domain are substituted with each other.

[0205] In some embodiments, the CL and CH1 domains of the first invariant region are substituted for each other; or the CL and CH1 domains of the second invariant region are substituted for each other.

[0206] S-DUAL TM

[0207] In some embodiments, the bispecific antibodies of this specification are produced by the method described in European Patent Application No. 4286408 A1, the contents of which are incorporated herein by reference in whole.

[0208] Through a unique asymmetrical design, S-DUAL TM It can effectively separate and analyze bispecific antibodies in which impurities of different sizes are generated due to improper binding. In addition, S-DUAL TM It applies knob-in-hole technology to prevent mispairing between the two heavy and light chains. S-DUAL TM It has a unique asymmetric structure that ensures high bonding affinity between chains, and accordingly, can achieve high potency and purity for optimized manufacturability.

[0209] In some embodiments, the bispecific antibody of this specification may have an asymmetric structure between the first polypeptide and the second polypeptide. Specifically, a CH3 dimer may be additionally included between the first antigen-binding domain and the CH1 domain or light chain-constant domain of the first constant region, or a CH3 dimer may be additionally included between the second antigen-binding domain and the CH1 domain or light chain-constant domain of the second constant region. By additionally introducing a CH3 dimer into one of the two Fab regions, the two arms of the Fab region may form an asymmetric shape.

[0210] Accordingly, the bispecific antibody according to some embodiments may additionally include the knopinhole structure between the first antigen-binding domain and the CH1 domain of the first constant region; or may additionally include the knopinhole structure between the second antigen-binding domain and the CH1 domain of the second constant region.

[0211] In some embodiments, the first invariant region and the second invariant region have an asymmetric structure with respect to each other. In some embodiments, a knopp-in-hole structure is additionally included between the first antigen-binding domain and the CH1 domain of the first invariant region, or a knopp-in-hole structure is additionally included between the second antigen-binding domain and the CH1 domain of the second invariant region. In some embodiments, the knopp-in-hole structure may include a dimer of a CH3 domain.

[0212] The structures of the bispecific antibodies described herein may be combined with one another in compatible aspects. For example, in some preferred embodiments, structures prepared by the Knob-and-hole method or the pentambody method are CrossmAb or S-DUAL TMIt may be combined with structures prepared by the method. In some embodiments, the combination of said structures may provide additional advantages in terms of preparation and efficacy of the anti-CD20xCD22 bispecific antibody-drug conjugate of this specification.

[0213] Antibodies and their antigen-binding domains

[0214] In some embodiments, the first antigen-binding domain that specifically binds to CD20 comprises a first heavy chain variable region and a first light chain variable region, and the second antigen-binding domain that specifically binds to CD22 comprises a second heavy chain variable region and a second light chain variable region, and the

[0215] (a) The first heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 6; the first light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 13; the second heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 40, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 42, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 44; and the second light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 47, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 49, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 51;

[0216] (b) The first heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 6; the first light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 13; the second heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 58, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 60, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 62; and the second light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 65, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 67, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 69;

[0217] (c) The first heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 22, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 26; the first light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 29, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 31, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 33; the second heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 40, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 42, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 44; and the second light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 47, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 49, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 51; or

[0218] (d) The first heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 22, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 26, and the first light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 29, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 31, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 33, and the second heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 58, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 60, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 62, and the second light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 65, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 67, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 69.

[0219] In some embodiments, (a) the VH domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 15; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 15; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 15, and the VL domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 16; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 16; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 16, and the VH domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 53; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 53; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 53, and the VL domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 54; An amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 54; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 54,

[0220] (b) The VH domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 15; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 15; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 15, and the VL domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 16; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 16; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 16, and the VH domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 71; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 71; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 71, and the VL domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 72; An amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 72; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 72,

[0221] (c) The VH domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 35; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 35; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 35, and the VL domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 36; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 36; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 36, and the VH domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 53; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 53; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 53, and the VL domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 54; An amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 54; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 54,

[0222] (d) The VH domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 35; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 35; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 35, and the VL domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 36; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 36; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 36, and the VH domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 71; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 71; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 71, and the VL domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 72; It includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 72; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 72.

[0223] In some embodiments, (a) the VH domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 15; the VL domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 16; the VH domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 53; and the VL domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 54;

[0224] (b) The VH domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 15; the VL domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 16; the VH domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 71; and the VL domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 72;

[0225] (c) The VH domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 35; the VL domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 36; the VH domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 53; and the VL domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 54;

[0226] (d) The VH domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 35; the VL domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 36; the VH domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 71; and the VL domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 72.

[0227] In some embodiments, Ab comprises a first heavy chain and a first light chain comprising the first antigen-binding domain that specifically binds to CD20; and a second heavy chain and a second light chain comprising the second antigen-binding domain that specifically binds to CD22, wherein (a) the first heavy chain comprises: an amino acid sequence of SEQ ID NO. 20; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 20; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 20, and the first light chain comprises: an amino acid sequence of SEQ ID NO. 18; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 18; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 18, and the second heavy chain comprises: an amino acid sequence of SEQ ID NO. 55; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 55; or comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 55, wherein the second light chain comprises: the amino acid sequence of SEQ ID NO. 56; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 56; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 56, and

[0228] (b) The first heavy chain comprises: the amino acid sequence of SEQ ID NO. 20; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 20; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 20, and the first light chain comprises: the amino acid sequence of SEQ ID NO. 18; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 18; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 18, and the second heavy chain comprises: the amino acid sequence of SEQ ID NO. 73; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 73; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 73, and the second light chain comprises: the amino acid sequence of SEQ ID NO. 74; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 74; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 74, and (c) the first heavy chain comprises: the amino acid sequence of SEQ ID NO. 37; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 37; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 37, and the first light chain comprises: the amino acid sequence of SEQ ID NO. 38; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 38; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 38, and the second heavy chain comprises: the amino acid sequence of SEQ ID NO. 55; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 55;or comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 55, wherein the second light chain comprises: the amino acid sequence of SEQ ID NO. 56; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 56; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 56; and;

[0229] (d) The first heavy chain comprises: the amino acid sequence of SEQ ID NO. 37; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 37; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 37, and the first light chain comprises: the amino acid sequence of SEQ ID NO. 38; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 38; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 38, and the second heavy chain comprises: the amino acid sequence of SEQ ID NO. 73; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 73; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 73, and the second light chain comprises: the amino acid sequence of SEQ ID NO. 74; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 74; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 74,

[0230] (e) The first heavy chain comprises: the amino acid sequence of SEQ ID NO. 75; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 75; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 75, and the first light chain comprises: the amino acid sequence of SEQ ID NO. 18; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 18; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 18, and the second heavy chain comprises: the amino acid sequence of SEQ ID NO. 76; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 76; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 76, and the second light chain comprises: the amino acid sequence of SEQ ID NO. 56; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 56; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 56, or

[0231] (f) The first heavy chain comprises: the amino acid sequence of SEQ ID NO. 20; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 20; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 20, and the first light chain comprises: the amino acid sequence of SEQ ID NO. 18; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 18; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 18, and the second heavy chain comprises: the amino acid sequence of SEQ ID NO. 81; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 81; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 81, and the second light chain comprises: the amino acid sequence of SEQ ID NO. 82; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 82; Or includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 82.

[0232] In some embodiments, (a) the first heavy chain comprises the amino acid sequence of SEQ ID NO. 20; the first light chain comprises the amino acid sequence of SEQ ID NO. 18; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 55; the second light chain comprises the amino acid sequence of SEQ ID NO. 56; (b) the first heavy chain comprises the amino acid sequence of SEQ ID NO. 20; the first light chain comprises the amino acid sequence of SEQ ID NO. 18; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 73; the second light chain comprises the amino acid sequence of SEQ ID NO. 74; (c) the first heavy chain comprises the amino acid sequence of SEQ ID NO. 37; the first light chain comprises the amino acid sequence of SEQ ID NO. 38; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 55; the second light chain comprises the amino acid sequence of SEQ ID NO. 56; and (d) the first heavy chain comprises the amino acid sequence of SEQ ID NO. 37; The first light chain comprises the amino acid sequence of SEQ ID NO. 38; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 73; the second light chain comprises the amino acid sequence of SEQ ID NO. 74; (e) the first heavy chain comprises the amino acid sequence of SEQ ID NO. 75; the first light chain comprises the amino acid sequence of SEQ ID NO. 18; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 76; the second light chain comprises the amino acid sequence of SEQ ID NO. 56; or (f) the first heavy chain comprises the amino acid sequence of SEQ ID NO. 20; the first light chain comprises the amino acid sequence of SEQ ID NO. 18; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 81; and the second light chain comprises the amino acid sequence of SEQ ID NO. 82.

[0233] In some embodiments, Ab is a full-length antibody. In some embodiments, Ab is an IgG-single chain variable fragment (scFv), a trifunctional antibody (triomab), knobs into holes (KIH)-IgG, kparamda-body (κλ-body), crossmab, ortho-Fab IgG, dual variable domain immunoglobulin (DVD-Ig) or 2-in-1-IgG (dual action antibody) or a combination thereof.

[0234] In some embodiments, Ab is an antigen-binding fragment. In some embodiments, Ab is ScFv2-Fc, a bi-nanobody, a bispecific T cell engager (BiTE), a tandem diabody (tandAb), a dual affinity retargeting (DART) antibody, DART-Fc, scFv-human serum albumin (HAS)-scFv, dock-and-lock (DNL)-Fab3, a minibody, a Fab2 fragment (bispecific), a Fab3 fragment (trispecific), a Bis-scFv fragment (bispecific), an sdAb fragment (VH / VHH), a tetrabody, a tribody, or a diabody, or a combination thereof.

[0235] Antibody production

[0236] The bispecific antibodies of this specification may be produced by any means known in the art, such as any chemical, biological, genetic, or enzymatic technique, either alone or in combination, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of full-length monoclonal antibodies. The recombinant expression may occur in any suitable host cell known in the art, including but not limited to mammalian host cells, bacterial host cells, yeast host cells, and insect host cells, or in a cell-free system (e.g., Sutro’s Xpress CF platform, World Wide Web at sutrobio.com / technology / ).

[0237] If the amino acid sequence of the desired sequence is known, a person skilled in the art can easily produce the antibody or polypeptide mentioned by standard techniques for polypeptide production. For example, they can be synthesized using well-known solid-phase methods, preferably using commercially available peptide synthesis devices (such as those manufactured by Applied Biosystems, Foster City, Calif) and according to the manufacturer's instructions. Alternatively, antibodies and other polypeptides can be synthesized by recombinant DNA techniques as well known in the art. For example, such fragments can be obtained as DNA expression products by inserting a DNA sequence encoding the desired (poly)peptide into an expression vector, introducing such a vector into a suitable eukaryotic or prokaryotic host to express the desired polypeptide, and then isolating it from therein using well-known techniques.

[0238] In particular, the present invention describes a method for producing an antibody or polypeptide, said method comprising: (i) culturing a transformed host cell under conditions suitable for allowing the expression of the antibody or polypeptide described below; and (ii) recovering the expressed antibody or polypeptide.

[0239] Antibodies and other polypeptides are appropriately separated from the culture medium by conventional immunoglobulin purification procedures, such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography (HPLC) may also be used for purification. For example, refer to Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), for example, Chapters 1, 4, 6, 8, 9, and 10, the entire contents of each of which are incorporated herein by reference.

[0240] This specification also provides expression vectors and host cells for the production of any of the bispecific antibodies described herein. Various expression vectors may be used to express polynucleotides encoding said bispecific antibodies. Both virus-based and non-viral expression vectors may be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors, episomal vectors (typically comprising expression cassettes for protein or RNA expression), and human artificial chromosomes (e.g., [Harrington et al., Nat Genet.Useful viral vectors include retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus-based vectors, SV40 vectors, papillomavirus vectors, HBP Epstein-Barr virus vectors, vaccinia virus vectors, and Semlicky Forest Virus (SFV)-based vectors, but are not limited thereto.

[0241] The selection of the expression vector depends on the host cell in which the vector is intended to be expressed. Typically, the expression vector comprises a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding a bispecific antibody or antibody fragment (e.g., antigen-binding fragment). In some embodiments, an inducible promoter is used to prevent the expression of the inserted sequence from occurring except under induction conditions. The inducible promoter includes, for example, an arabinose, lacZ, metallothionein promoter, or a heat shock promoter. The transformed organism culture may be proliferated under non-induction conditions so that the population is not biased toward the coding sequence that is better accepted by the host cell.

[0242] The expression vector may also provide a secretion signal sequence site for forming a fusion protein with a polypeptide encoded by the inserted bispecific antibody or antibody fragment (e.g., antigen-binding fragment) sequence. More commonly, the inserted bispecific antibody or antibody fragment (e.g., antigen-binding fragment) sequence is included in the vector after being linked to the signal sequence. Vectors used to include sequences encoding the light chain of the bispecific antibody or antibody fragment (e.g., antigen-binding fragment) and the heavy chain variable domain sometimes also encode the constant region or a portion thereof. Such vectors cause the variable region to be expressed as a fusion protein with the constant region to induce the production of a complete antibody or a fragment thereof.

[0243] In some embodiments, mammalian host cells are used to express and produce the bispecific antibody or antibody fragment (e.g., antigen-binding fragment) polypeptide described herein. For example, such mammalian host cells may be hybridoma cell lines expressing endogenous immunoglobulin genes (e.g., myeloma hybridoma clones described in the examples) or mammalian cell lines possessing exogenous expression vectors (e.g., SP2 / 0 myeloma cells to be illustrated below). The mammalian host cells include any normal apoptotic or normal or abnormal immortalized animal or human cells. For example, a number of suitable host cell lines capable of secreting complete immunoglobulin have been developed, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. Expression vectors for mammalian host cells may include expression regulatory sequences such as replication origins, promoters, and enhancers, and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. These expression vectors generally contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type-specific, step-specific, and / or controllable or regulated promoters. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline-inducible CMV promoters (e.g., human immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

[0244] The method of introducing an expression vector containing the polynucleotide sequence of interest varies depending on the type of host cell. For example, calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment or electroporation can be used for other host cells.

[0245] In this specification, non-human antibodies may be derived from any antibody-producing animal, for example, a mouse, rat, rabbit, goat, donkey, or non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys) or apes (e.g., chimpanzees). Non-human antibodies may be produced by immunizing animals using methods known in the art.

[0246] Chimeric antibodies (e.g., mouse-human chimeras) can be produced by obtaining nucleic acid sequences encoding VL and VH domains as previously described, inserting them into an expression vector for animal cells having genes encoding human antibody CH and human antibody CL to construct a human chimeric antibody expression vector, and then introducing said expression vector into animal cells to express the encoding sequences. The CH domain of the human chimeric antibody may be any region belonging to human immunoglobulins, such as the IgG class or its subclasses, e.g., IgG1, IgG2, IgG3, and IgG4. Similarly, the CL of the human chimeric antibody may be any region belonging to Ig, such as the kappa class or lambda class. Chimeric and humanized monoclonal antibodies containing both human and non-human parts may be prepared using standard recombinant DNA technology. Such chimeric and humanized monoclonal antibodies may be prepared by recombinant DNA technology known in the art, e.g., Robinson et al. International Patent Publication PCT / US86 / 02269; Akira et al. European Patent Application 184,187; Taniguchi, M. European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT Application WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al.It can be produced using the methods described in (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559; Morrison, SL (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S. Patent No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.

[0247] Additionally, humanized antibodies may be manufactured according to standard protocols such as those disclosed in U.S. Patent No. 5,565,332. In other embodiments, antibody chains or specific binding pair members may be produced by recombination between a vector containing a nucleic acid molecule encoding a fusion of a polypeptide chain of a specific binding pair member and a component of a replicatable general display package, and a vector containing a nucleic acid molecule encoding a second polypeptide chain of a single binding pair member, using techniques known in the art, e.g., as described in U.S. Patents No. 5,565,332, No. 5,871,907, or No. 5,733,743. A humanized antibody can be produced by obtaining a nucleic acid sequence encoding a CDR domain as described above, inserting it into an expression vector for animal cells having a gene encoding (i) a heavy chain constant region identical to the heavy chain constant region of a human antibody and (ii) a light chain constant region identical to the light chain constant region of a human antibody to construct a humanized antibody expression vector, and then introducing the expression vector into animal cells to express the gene.

[0248] The humanized antibody expression vector may be of a type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain exist on separate vectors, or a type in which both genes exist on the same vector (tandem type).

[0249] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, e.g., Riechmann L. et al., 1988; Neuberger M S. et al, 1985). Antibodies may be humanized using various techniques known in the art, such as CDR-grafting (EP 239,400; PCT Publication WO91 / 09967; U.S. Patents No. 5,225,539, 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska M A. et al. (1994)), and chain shuffling (U.S. Patent No. 5,565,332). General recombinant DNA techniques for the production of such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0250] Fully human antibodies can be produced by administering an antigen to a transgenic animal containing a human immunoglobulin locus, or by treating a phage display library expressing a human antibody repertoire with an antigen and then selecting a target antibody. The antibody may be polyclonal or monoclonal, or may be synthesized within a cell host through the expression of recombinant DNA. Monoclonal antibodies (mAbs) may be produced using conventional monoclonal antibody methods, for example, standard somatic hybridization techniques described in the literature (Kohler and Milstein, 1975, Nature 256:495).

[0251] The bispecific or multispecific antibodies described herein can be prepared according to standard procedures. For example, triomas and hybrid hybridomas are two examples of cell lines capable of secreting bispecific or multispecific antibodies. Examples of bispecific and multispecific antibodies produced by hybrid hybridomas or triomas are disclosed in U.S. Patent No. 4,474,893. These antibodies can also be constructed by chemical means (Staerz et al. (1985) Nature 314:628 and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453 and Staerz and Bevan (1986) Immunol. Today 7:241). Alternatively, these antibodies may be generated by constructing a heterohybridoma by fusing hybridomas or other cells that produce different antibodies, and then identifying a clone that produces and co-assembles the desired antibody. They can also be generated by chemical or genetic splicing of complete immunoglobulin chains, such as Fab and Fv sequences, or parts thereof.

[0252] Activator and Payload

[0253] The present specification provides, in particular, an activator and an antibody-drug conjugate comprising said activator. The anti-CD20xCD22 bispecific antibody (“Ab”) described herein may be conjugated to a number of identical or different activators using methods known in the art. The said activator in the present specification should not be interpreted as being limited to chemical therapeutic agents. For example, the activator in the present specification may be a protein, peptide, or polypeptide having the desired biological activity.

[0254] In some embodiments, the active agent is a chemotherapy agent or a toxin. In some embodiments, the active agent is selected from chemotherapy compounds, cytotoxic compounds, immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, antiparasitic agents, and combinations thereof. In some embodiments, the cytotoxic compound is selected from mitotic inhibitors, DNA alkylating agents, topoisomerase inhibitors, and combinations thereof. In some embodiments, the cytotoxic compound is selected from auristatin, maytansinoid, tubulisin, calicheamicin, duocarmycin, pyrrolobenzodiazepine and derivatives thereof, camptothecin and derivatives thereof, and combinations thereof. In some embodiments, the orristatin is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0255] In some embodiments, the activator is a pyrrolobenzodiazepine dimer;

[0256] The N10 position of the pyrrobenzodiazepine dimer is substituted with X, or the N'10 position is substituted with X', said X or X' connects the pyrrobenzodiazepine dimer to the linker, and X and X' each independently -C(O)O- * or -C(O)- * is; * refers to the binding point between the above-mentioned pyrrobenzodiazepine dimer and the above-mentioned linker.

[0257] In some embodiments, the activator is a pyrrolobenzodiazepine dimer, and the pyrrolobenzodiazepine dimer has a structure represented by Formula VIII:

[0258] [Essence VIII]

[0259]

[0260] Here,

[0261] The above wavy line indicates the junction point with the linker, and

[0262] The dotted line optionally indicates a double bond;

[0263] R1 and R1 ' Each independently contains H, OH, =O, =CH2, CN, R m , OR m , =CH-R m' , =C(R m' )2, O-SO2-R m , CO2R m , COR m , selected from halo and dihalo;

[0264] R m' is R m , CO2R m , COR m Selected from , CHO, CO2H and halo;

[0265] R m is substituted or unsubstituted C 1-12 Alkyl, substituted or unsubstituted C 2-12 Alkenyl, substituted or unsubstituted C2-12 alkynyl, substituted or unsubstituted C 5-20 Aryl, substituted or unsubstituted C 3-6 Heteroaryl, substituted or unsubstituted C 3-6 Selected from cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocyclyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl and substituted or unsubstituted 5- to 7-membered heteroaryl, and said C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 5-20 Aril, C 5-20 Heteroaryl, C 3-6 When a cycloalkyl, 3- to 7-membered heterocyclile, 3- to 7-membered heterocycloalkyl, or 5- to 7-membered heteroaryl is substituted, the above C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 5-20 Aril, C 5-20 Heteroaryl, C 3-6 Each hydrogen atom of the cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, or 5- to 7-membered heteroaryl is independently a methoxy, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 5-20 Aril, C 5-20 Heteroaryl, C 3-6 It can be substituted with cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl;

[0266] R2, R3, R5, R2 ' , R3 ' and R5 ' are independently H, R m , OH, OR m , SH, SR m , NH2, NHR m , NR m R m'Selected from , NO2, Me3Sn and Halo;

[0267] R4 and R4 ' are independently H, R m , OH, OR m , SH, SR m , NH2, NHR m , NR m R m' , NO2, Me3Sn, halo, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted C 3-6 Cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, substituted or unsubstituted C 5-12 Aryl, substituted or unsubstituted 5- to 7-membered heteroaryl, -CN, -NCO, -OR n , -OC(O)R n , -OC(O)NR n R n' , -OS(O)R n , -OS(O)2R n , -SR n , -S(O)R n , -S(O)2R n , -S(O)NR n R n' , -S(O)2NR n R n' , -OS(O)NR n R n' , -OS(O)2NR n R n' , -NR n R n' , -NR n C(O)R o , -NR n C(O)OR o , -NR n C(O)NR o R o' , -NR n S(O)R o , -NRn S(O)2R o , -NR n S(O)NR o R o' , -NR n S(O)2NR o R o' , -C(O)R n , -C(O)OR n and -C(O)NR n R n' Selected from, and the above C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 The hydrogen atoms of aryls and 5- to 7-membered heteroaryls are C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 In the case of aryls and 5- to 7-membered heteroaryls, each independently C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 Aryl, 5- to 7-membered heteroaryl, -OR p , -OC(O)R p , -OC(O)NR p R p' , -OS(O)R p , -OS(O)2R p , -SR p , -S(O)R p , -S(O)2R p , -S(O)NR p R p' , -S(O)2NR p R p' , -OS(O)NR p R p', -OS(O)2NR p R p' , -NR p R p' , -NR p C(O)R q , -NR p C(O)OR q , -NR p C(O)NR q H, -NR p S(O)R q , -NR p S(O)2R q , -NR p S(O)NR q H, -NR p S(O)2NR q H, -C(O)R p , -C(O)OR p or -C(O)NR p R p It can be replaced with;

[0268] R n , R n' , R o , R o' , R p , R p' and R q H and C are independently 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkinyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Selected from aryls and 5- to 7-membered heteroaryls;

[0269] X is selected from -C(O)O-, -S(O)O-, -C(O)-, -C(O)NR-, -S(O)2NR-, -P(O)R'NR-, -S(O)NR- and -PO2NR-;

[0270] Xa is combined, substituted, or unsubstituted C 1-6 It is an alkylene, and the above C 1-6 When alkylene is substituted, C 1-8 alkyl or C 3-8 Substituted with cycloalkyl;

[0271] R and R' are each independently H, OH, NH2, ONH2, NHNH2, substituted or unsubstituted C 1-8 Alkyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted C 1-8 Alkoxy, substituted or unsubstituted C 1-8 Alkylthio, substituted or unsubstituted C 3-20 Heteroaryl, substituted or unsubstituted C 5-20 Aryl or mono- or di-C 1-8 It is an alkylamino, and the above C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, C 3-20 Heteroaryl and C 5-20 When aryls are substituted, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halo, C 1-6 Alkyl, C 1-6 Alkoxy and C 6-12 Substituted with a substituent selected from aryls;

[0272] Y and Y' are each independently selected from O, S and N(H);

[0273] R6 is substituted or unsubstituted saturated or unsaturated C 3-12 It is a hydrocarbon chain, said chain may be interrupted by one or more heteroatoms, NMe, or substituted or unsubstituted aromatic rings, said chain or aromatic ring at the positions of one or more hydrogen atoms of said chain or aromatic ring -NH, -NR m , -NHC(O)R m , -NHC(O)CH2-[OCH2CH2] n -R or -[CH2CH2O] n It may be substituted with or unsubstituted with -R, and the above R m and R are each the R defined above. m and equal to R, where n is 1 to 12;

[0274] R7 and R7 'Each is independently H, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted C 3-6 Cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5- to 7-membered heteroaryl, -OR r , -OC(O)R r , -OC(O)NR r R r' , -OS(O)R r , -OS(O)2R r , -SR r , -S(O)R r , -S(O)2R r , -S(O)NR r R r' , -S(O)2NR r R r' , -OS(O)NR r R r' , -OS(O)2NR r R r' , -NR r R r' , -NR r C(O)R s , -NR r C(O)OR s , -NR r C(O)NR s R s' , -NR r S(O)R s , -NR r S(O)2R s , -NR r S(O)NR s R s' , -NR r S(O)2NR s R s , -C(O)R r , -C(O)OR s or -C(O)NR r R r' and, the above C 1-6 Alkyl, C2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 The hydrogen atoms of aryls and 5- to 7-membered heteroaryls are C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 In the case of aryls and 5- to 7-membered heteroaryls, each independently C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl, 5- to 7-membered heteroaryl, -OR t , -OC(O)R t , -OC(O)NR t R t' , -OS(O)R t , -OS(O)2R t , -SR t , -S(O)R t , -S(O)2R t , -S(O)NR t R t' , -S(O)2NR t R t' , -OS(O)NR t R t' , -OS(O)2NR t R t' , -NR t R t' , -NR t C(O)R u , -NR t C(O)OR u , -NR t C(O)NR u R u' , -NR t S(O)R u , -NR t S(O)2R u , -NR t S(O)NR u Ru' , -NR t S(O)2NR u R u' , -C(O)R t , -C(O)OR t or -C(O)NR t R t' It can be replaced with;

[0275] R r , R r' , R s , R s' , R t , R t' , R u and R u' H and C are independently 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkinyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 Selected from aryls and 5- to 7-membered heteroaryls;

[0276] G is a glucuronide group or a galactoside group;

[0277] Each Z is H, C 1-8 Alkyl, Halo, NO2, CN, and Selected from;

[0278] R9, R 10 and R 16 H and C are independently 1-8 Alkyl, C 2-6 Alkenyl, C 1-6 Selected from alkoxy and alkyloxyalkyl;

[0279] n30 is 1 to 3.

[0280] In some embodiments, Y is O. In some embodiments, Y' is O. In some embodiments, the dashed line is R1 and R7 or R1 ' and R7 'Indicates the presence of an intercarbon double bond having. In some embodiments, R1 is a substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted C 5-7 Aryl and substituted or unsubstituted C 3-6 It is selected from heteroaryls. In some embodiments, R2, R3, and R5 are each independently H or OH. In some embodiments, R4 is C 1-6 It is an alkoxy. In some embodiments, R4 is methoxy, ethoxy, or butoxy. In some embodiments, X is selected from -C(0)0-, -C(O)-, and -C(0)NR-. In some embodiments, X is -C(O)NR-. In some embodiments, R6 is a substituted or unsubstituted saturated or unsaturated C 3-8 It is a hydrocarbon chain, wherein one or more carbon atoms of the hydrocarbon chain are substituted with a heteroatom or a substituted or unsubstituted aromatic ring, wherein the heteroatom is O, S or N(H), and the aromatic ring is benzene, pyridine, imidazole, or pyrazole, and wherein the chain or aromatic ring is -NHC(O)CH2-[OCH2CH2] at the position of one or more hydrogen atoms of the chain or aromatic ring. n -R or -[CH2CH2O] n It may be substituted with -R; n is 1 to 6. In some embodiments, n is 1 to 6. In some embodiments, Xa is a bond or C 1-3 It is an alkylene.

[0281] In some embodiments, Z is H, and And, the above R9, R 10 and R 16 H and C are independently 1-3 Alkyl, C 1-3It is selected from alkoxy and alkyloxymethyl. In some embodiments, R9 is methyloxyalkyl. In some embodiments, R 10 is a methyloxyalkyl. In some embodiments, R 16 is methyloxyalkyl. In some embodiments, R9, R 10 or R 16 -(CH2CH2O) m -(CH2) m2 CH3, additionally, m is 1 to 6 and m2 is 0 to 2. In some embodiments, m is 1. In some embodiments, m2 is 0. In some embodiments, R2 is H. In some embodiments, R3 is H. In some embodiments, R7 is H. In some embodiments, R4 is an alkoxy (e.g., methoxy). In some embodiments, R5 is OH. In some embodiments, R1 is =CH2, CH3 or phenyl and is optionally substituted with methoxy. In some embodiments, Y is O.

[0282] In some embodiments, R2' is H. In some embodiments, R3' is H. In some embodiments, R7' is H. In some embodiments, R4' is an alkoxy (e.g., methoxy). In some embodiments, R5' is OH. In some embodiments, R1' is =CH2, CH3, or phenyl and is optionally substituted with methoxy. In some embodiments, Y' is O. In some embodiments, X is -C(O)O-. In some embodiments, Xa is CH2. In some embodiments, G is a glucuronide group. In some embodiments, G is is. In some specific examples, n30 is 1.

[0283] In some specific examples, Z is or is. In some embodiments, R9 is H. In some embodiments, R 16 is an alkyloxyalkyl (e.g., methoxyethyl). In some embodiments, Z is is. In some embodiments, R 10is an alkyl (e.g., methyl). In some embodiments, R6 is C 3-12 It is an alkyl (e.g., pentyl).

[0284] In some embodiments, the pyrrolobenzodiazepine dimer is

[0285] , , , and Selected from,

[0286] The above X'' is -OMe or -NH2.

[0287] In a specific embodiment, the composite is

[0288] ,

[0289] ,

[0290] , , , , , , , , , , and Includes,

[0291] The above

[0292] MMAE is monomethyl orristatin E, and MMAF is monomethyl orristatin F;

[0293] The dotted line above indicates the connection with Ab.

[0294] In a specific embodiment, the composite is

[0295] , , , , , ,

[0296]

[0297] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Includes,

[0298] The above

[0299] MMAE is monomethyl orristatin E, and MMAF is monomethyl orristatin F;

[0300] The dotted line above indicates the connection with Ab.

[0301] In some embodiments, the activator is selected from the following:

[0302] (a) erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, Carboquone, meturedopa, uredopa, ethylenimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, calllystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, Duocarmycin, KW-2189, CB1-TM1, Eleutherobin,Pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novelbichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotoxin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, Calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomysins, actinomycin, anthramycin, azacerine, bleomycins, catcinomycin, carabicin, carninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucineDoxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, Quelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin, uvenimex, zinostatin, zorubicin, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, Dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone, propionate,Epithiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatrexate, democolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate, Hydroxyurea, Lentinan, Ionidainine, Maytansine, Ansamitocins, Mitoguazone, Mitoxantrone, Mopidanmol, Pentostatin, Phenamet, Pirarubicin, Losoxantrone, 2-Ethylhydrazide, Procarbazine, Polysaccharide-K, Razoxane, Rhizoxin, Sizofiran, Spirogermanium, Tenuazonic Acid, Triaziquone, 2,2',2''-trichlorotriethylamine, T-2 toxin, verracurin A,Roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, arabinoside, cyclophosphamide, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, Vinblastine, platinum, etoposide, vincristine, vinorelbine, novelrone, teniposide, edatrexate, daunomycin, aminopterin, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine or pharmaceutically acceptable salts thereof, solvates thereof or acids thereof;,

[0303] (b) Monokine, lymphokine, traditional polypeptide hormone, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormone, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-α, tumor necrosis factor-β, mullerian inhibiting substance, mouse gonadotropin-associated peptide, Inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinductive factor, interferon, interferon-α, interferon-β, interferon-γ, colony stimulating factor (CSF), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin;IL), IL-1, IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, polypeptide factor, LIF, kit ligand, or a combination thereof;

[0304] (c) Diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, α-amatinin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivative, indolinobenzodiazepine, pyridinobenzodiazepine, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulisin, geldanamycin, Maytansinoid, calicheamycin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analog, cryptophycin, camptothecin, rhizoxin, rhizoxin derivatives, CC-1065, analogs or derivatives of CC-1065, duocarmycin, enediyne antibiotic, esperamicin, epotilone, toxoid, or combinations thereof;

[0305] (d) an affinity ligand, said affinity ligand is a substrate, an inhibitor, an activator, a neurotransmitter, a radioisotope, or a combination thereof;

[0306] (e) radioactive label, 32P, 35S, fluorescent dye, electron dense reagent, enzyme, biotin, streptavidin, digoxigenin, hapten, immunogenic protein, nucleic acid molecule with a sequence complementary to a target, or a combination thereof;

[0307] (f) immunomodulatory compound, anticancer agent, antiviral agent, antibacterial agent, antifungal agent, antiparasitic agent or combination thereof;

[0308] (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, LY117018, onapristone, or toremifene;

[0309] (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole or anastrozole;

[0310] (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine;

[0311] (j) aromatase inhibitor;

[0312] (k) protein kinase inhibitor;

[0313] (l) lipid kinase inhibitor;

[0314] (m) antisense oligonucleotide;

[0315] (n) ribozymes;

[0316] (o) vaccine; and

[0317] (p) anti-angiogenic agent.

[0318] Use of anti-human anti-CD20xCD22 bispecific antibody-drug conjugates for pharmaceutical purposes

[0319] In cancer, the expression of CD20 and CD22 is known to be associated with a poor prognosis in certain cancers (e.g., hematological cancers and B-cell malignancies) and to influence cancer metastasis. For anticancer antibody therapy, for example, an anti-CD20xCD22 bispecific antibody can be used in a form linked to various activators via a linker, as described herein, to eliminate cancer cells that overexpress CD20 and CD22. Accordingly, a bispecific antibody that binds to CD20 and CD22 can be used in a form bound to an activator, and thus can be used as a targeted therapy directed toward CD20 and CD22-expressing cells.

[0320] In some aspects, the present specification provides a pharmaceutical composition comprising the conjugate described herein and a pharmaceutically acceptable excipient.

[0321] In some aspects, the present specification provides a pharmaceutical composition for the prevention or treatment of a proliferative disease comprising the conjugate described herein. In some embodiments, the proliferative disease is cancer. In some embodiments, the cancer is selected from liver cancer, thyroid cancer, ovarian cancer, brain cancer, multiple myeloma, colon cancer, head and neck cancer, lymphoma, leukemia, bladder cancer, kidney cancer, gastric cancer, breast cancer, uterine cancer, prostate cancer, pancreatic cancer, lung cancer, sarcoma, neuroendocrine tumor, melanoma, and combinations thereof. In some embodiments, the lymphoma or leukemia is selected from Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), T-cell lymphoma, B-cell lymphoma, natural killer cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, lymphoblastic lymphoma, enteropathic intestinal lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, peripheral T-cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and combinations thereof. In some embodiments, the acute lymphoblastic leukemia is B-cell lymphoblastic leukemia (B-ALL).

[0322] Treatment Method: Pharmaceutical Formulation and Route of Administration

[0323] In some embodiments, the present specification also provides a therapeutic method using an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, said antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided to a patient. said antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof binds to CD20 and CD22 expressed on the surface of cancer cells to inhibit the progression of cancer cells. In some embodiments, said antibody binds to CD20 and CD22 expressed on the surface of cancer cells in a form bound to the activator described herein, thereby specifically delivering said activator bound to said antibody to the cancer cells to induce apoptosis of said cancer cells. In some embodiments, said antibody binds to CD20 and CD22 expressed on the surface of cancer cells in the form of antibodies specific to the same target or different targets, thereby increasing the specificity of multiple antibodies against cancer cells or inducing a link between cancer cells and other types of cells, such as immune cells, to induce apoptosis of said cancer cells.

[0324] In some aspects, the present specification provides a method for the prevention or treatment of a proliferative disease comprising the step of administering the conjugate described herein or a pharmaceutically acceptable salt thereof to an individual in need thereof. In some embodiments, the proliferative disease is cancer. In some embodiments, the cancer is selected from liver cancer, thyroid cancer, ovarian cancer, brain cancer, multiple myeloma, colon cancer, head and neck cancer, lymphoma, leukemia, bladder cancer, kidney cancer, gastric cancer, breast cancer, uterine cancer, prostate cancer, pancreatic cancer, lung cancer, sarcoma, neuroendocrine tumor, melanoma, and combinations thereof. In some embodiments, the lymphoma or leukemia is selected from Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), T-cell lymphoma, B-cell lymphoma, natural killer cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, lymphoblastic lymphoma, enteropathic intestinal lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, peripheral T-cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and combinations thereof. In some embodiments, the acute lymphoblastic leukemia (ALL) is B-cell acute lymphoblastic leukemia (B-ALL).

[0325] Pharmaceutical composition

[0326] The present specification provides, in particular, pharmaceutical compositions comprising an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Additionally, a method for treating a cancer patient by administering, for example, such pharmaceutical compositions is provided. The term "patient" includes a human patient.

[0327] The above pharmaceutical composition may include a pharmaceutically acceptable carrier. The term "carrier" is used to mean including excipients, diluents, or adjuvants. The carrier may be selected from the group consisting of, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, saline, a buffer solution such as PBS, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. The above composition may include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, or a combination thereof.

[0328] The above pharmaceutical composition may be prepared in any formulation according to conventional methods. The composition may be formulated as an oral formulation (e.g., powder, tablet, capsule, syrup, pill, or granule) or a parenteral formulation (e.g., injection). Additionally, the composition may be prepared as a systemic or topical formulation.

[0329] The above pharmaceutical composition may contain an effective amount of the antibody or its antigen-binding fragment, an anticancer agent, or a combination thereof. The term "effective amount" refers to an amount sufficient to produce a preventive or therapeutic effect when administered to an individual requiring prevention or treatment. The effective amount may be appropriately selected by a person skilled in the art depending on the selected cell or individual. It may be determined by factors including the severity of the disease, the patient's age, weight, health and gender, the patient's sensitivity to the drug, the time of administration, the route of administration and elimination rate, the duration of treatment, and drugs used in combination with or simultaneously with the composition used, as well as other factors well known in the medical field.

[0330] The dosage of the above pharmaceutical composition may be, for example, in the range of 10 μg / kg to about 30 mg / kg for adults, optionally 0.1 mg / kg to about 30 mg / kg, or alternatively 0.3 mg / kg to about 20 mg / kg. The above pharmaceutical composition may be administered once a day, multiple times a day, once every 1 to 4 weeks, or once to 12 times a year.

[0331] The present disclosure will be explained in more detail below through examples and experimental examples.

[0332] The following examples and experimental examples are intended to aid in understanding the present disclosure and are not intended to limit the scope of the present disclosure.

[0333] definition

[0334] Unless otherwise defined herein, the scientific and technical terms used in this application have the meanings generally understood by those skilled in the art. Generally, the nomenclature and techniques used in connection with chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are well known and commonly used in the art.

[0335] Unless otherwise specified, the methods and techniques of this specification are generally performed according to ordinary methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For example, “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W.H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W.H. Freeman & Co., N.Y. (1999); See Gilbert et al., “Developmental Biology, 6th ed”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0336] Chemical terms used herein, unless otherwise defined herein, are used according to customary usage in the art, as exemplified in “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0337] All of the foregoing and all other documents, patents, and published patent applications set forth in this application are incorporated herein by reference. In the event of a conflict, this specification, including specific definitions, shall prevail.

[0338] As used herein, the term "agent" is an extract made from biological materials such as compounds (e.g., organic or inorganic compounds, mixtures of compounds), biological macromolecules (nucleic acids, humanized, chimeric, and human antibodies and monoclonal antibodies and antibodies including parts thereof, proteins or parts thereof (e.g., peptides), lipids, carbohydrates), or cells or tissues of bacteria, plants, fungi, or animals (particularly mammals). Agents include, for example, agents whose structures are known and agents whose structures are unknown.

[0339] "Patient," "subject," or "individual" are used interchangeably and refer to human or non-human animals. The terms include mammals such as humans, primates, livestock (including cattle, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0340] "Treating" a condition or patient refers to taking measures to obtain a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes may include, but are not limited to, the alleviation or improvement of one or more symptoms or conditions, a reduction in the extent of the disease, a stabilized state of the disease (i.e., not worsening), prevention of disease spread, delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (partial or complete), and may be detectable or undetectable. "Treatment" may also mean extending survival compared to the expected survival time without treatment.

[0341] The term “preventing” is a term recognized in the art and is well understood in the art when used in relation to conditions such as local recurrence (e.g., pain), diseases such as cancer, syndrome complexes such as heart failure, or any other medical conditions, and involves the administration of a composition that reduces the frequency of symptoms of a medical condition or delays the onset of manifestation in a subject compared to a subject who has not been administered the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a group of patients receiving prophylactic treatment compared to an untreated control group and / or delaying the appearance of detectable cancerous growths in a treated group compared to an untreated control group by, for example, a statistically and / or clinically significant amount.

[0342] The "administration of" or "administering" of a substance, compound, or preparation to a subject may be performed using any of the various methods known to a person skilled in the art. For example, a compound or preparation may be administered intravenously, intra-arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, intraocularly, sublingually, orally (ingested), intranasally (inhaled), intraspinally, and transdermally (absorbed, e.g., through a skin duct). Additionally, the compound or preparation may be appropriately introduced by rechargeable or biodegradable polymer devices or other devices (e.g., patches and pumps) or by formulations that provide an extended, slow, or controlled release of said compound or preparation. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0343] The appropriate method of administering a substance, compound, or preparation to a subject may also depend, for example, on the subject's age and / or physical condition and on the chemical and biological properties of the compound or preparation (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, the compound or preparation is administered to the subject orally, for example, by ingestion. In some embodiments, the orally administered compound or preparation is an extended-release or slow-release formulation, or is administered using a device for slow or extended release.

[0344] As used herein, the term “conjoint administration” refers to any form of administering two or more different therapeutic agents, wherein the second agent is administered while the previously administered therapeutic agent is active in the body (e.g., the two agents are simultaneously effective for the patient, which may include a synergistic effect of the two agents). For example, the different therapeutic compounds may be administered simultaneously or sequentially in the same formulation or in separate formulations. Thus, the subject receiving such treatment may benefit from the combined effect of the different therapeutic agents.

[0345] The "therapeutically effective amount" or "therapeutically effective dose" of a drug or preparation refers to the amount of the drug or preparation that produces the intended therapeutic effect when administered to a subject. A full therapeutic effect does not necessarily occur with a single administration but may occur after a series of administrations. Therefore, a therapeutically effective amount may be administered over one or more doses. The exact effective amount required for a specific subject may vary depending, for example, the subject's size, health status and age, and the characteristics and extent of the condition being treated, such as cancer or MDS. A person skilled in the art can easily determine the effective amount suitable for a specific situation through ordinary experimentation.

[0346] As used herein, the terms “optional” or “optionally” mean that the event or situation described below may or may not occur, and the description includes cases where the event or situation occurs as well as cases where it does not occur. For example, “optionally substituted alkyl” includes cases where the alkyl is substituted as well as cases where it is not substituted.

[0347] The substituents and substitution patterns for the compounds of the present invention can be selected by a person skilled in the art to obtain chemically stable compounds, and it is understood that said compounds can be easily synthesized from starting materials that are readily available through techniques known in the art and methods presented below. If the substituent itself is substituted with one or more groups, it is understood that said plurality of groups may be on the same carbon or different carbons as long as a stable structure is formed.

[0348] As used herein, the term “optionally substituted” indicates that one to six hydrogen radicals in a given structure are substituted with radicals of a specified substituent. The radicals of the substituent include, but are not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -0P(0)(0-alkyl)2 or -CH2-0P(0)(0-alkyl)2. Preferably, “optionally substituted” indicates that one to four hydrogen radicals in a given structure are substituted with the aforementioned substituents. More preferably, one to three hydrogen radicals are substituted with the aforementioned substituents. The above substituent is understood to be capable of further substitution.

[0349] As used herein, the term "conjugates" refers to cell binding agents that are covalently bonded to one or more molecules of a cytotoxic compound. In this regard, a "cell binding agent" is a molecule having an affinity for a biological target, which may be, for example, an antibody, in particular, a monoclonal antibody or an antibody fragment, and the binder functions to guide the biologically active compound to the biological target. In certain embodiments, the conjugate may be designed to target tumor cells through cell surface antigens. The antigen may be a cell surface antigen that is overexpressed or expressed in abnormal cell types. Specifically, the target antigen may be one that is expressed only on proliferating cells (e.g., tumor cells). The target antigen may typically be selected based on different expression between proliferating tissue and normal tissue. In this specification, the antibody is bound to the linker.

[0350] In this specification, a “variant” of a polypeptide, such as, for example, an antigen-binding fragment, protein, or antibody, is a polypeptide in which insertion, deletion, addition, and / or substitution has occurred at one or more amino acid residues compared to another polypeptide sequence, and includes fusion polypeptides. Protein variants also include those modified by proteolytic cleavage, phosphorylation, or other post-translational modifications, but retaining biological activity such as binding and specificity to CD20 and / or CD22 of the antibody disclosed herein. Variants may have about 99% identity, about 98% identity, about 97% identity, about 96% identity, about 95% identity, about 94% identity, about 93% identity, about 92% identity, about 91% identity, about 90% identity, about 89% identity, about 88% identity, about 87% identity, about 86% identity, about 85% identity, about 84% identity, about 83% identity, about 82% identity, about 81% identity, or about 80% identity with respect to the sequence of the antibody or its antigen-binding fragment according to this specification. Percent identity (%) or homology may be calculated through methods known in the art.

[0351] In certain embodiments, the percentage homology or identity may be calculated as 100X[(same position) / min(TGA, TGB)], where TGA and TGB are the sum of the number of residues and internal gap positions of sequences A and B being compared (Russell et al., J. Mol Biol., 244: 332-350 (1994).

[0352] As used herein, the term "derivative" of polypeptide means a polypeptide having chemical modifications at one or more residues through combination with other chemical moieties, distinct from insertions, deletions, additions, or substitutions.

[0353] The term "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences in a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for the optimal alignment of the two sequences. Matched positions are all positions where the same nucleotide or amino acid exists in both the target and reference sequences. Since gaps are not nucleotides or amino acids, gaps present in the target sequence are not counted. Similarly, since nucleotides or amino acids in the target sequence that are not nucleotides or amino acids in the reference sequence are counted, gaps present in the reference sequence are not counted. The percentage of sequence identity is calculated by determining the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and then multiplying the result by 100 to obtain the percentage of sequence identity. The comparison of sequences between two sequences and the determination of percent sequence identity can be performed using readily available software programs. Suitable software programs are available from various sources and can align both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST program family provided by the U.S. government’s National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov on the World Wide Web). bl2seq performs the comparison between two sequences using the BLASTN or BLASTP algorithms.BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. Other suitable programs are part of the bioinformatics program EMBOSS family, such as Needle, Stretcher, Water, or Matcher, which are also available on the World Wide Web at ebi.ac.uk / Tools / psa of the European Bioinformatics Institute (EBI).

[0354] As used herein, "homology" means the percentage of amino acid residues of a candidate sequence that match the amino acid residues of a specific peptide or polypeptide sequence, after aligning sequences with respect to a peptide, polypeptide, or antibody sequence and, if necessary, introducing intervals to achieve maximum percentage sequence identity, and no conservative substitutions are considered as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by various methods within the scope of the art, such as using computer software available to the public, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring alignment, including all algorithms known in the art necessary to achieve maximum alignment over the full length of the sequences being compared.

[0355] The above terms “affinity” or “avidity” refer to the strength of the interaction between an antibody or its antigen-binding fragment and an antigen, which is determined by the characteristics of the antigen, such as size, shape, and / or charge, and the CDR sequence of the antibody or antigen-binding fragment. Methods for determining such affinity are known in the art and may be referenced herein.

[0356] The antibody or its antigen-binding fragment used in the present invention has a dissociation constant (KD) ≤10 -6 In the case of M, it is stated that it "specifically binds" to a target such as an antigen. The dissociation constant (KD) is ≤1×10 -8 In the case of M, the antibody binds to the target with "high affinity."

[0357] The “antigen-binding fragment” of the chain (heavy chain or light chain) of the antibody or immunoglobulin as used herein comprises a portion of the antibody that is missing some amino acids compared to the full chain but is capable of specifically binding to an antigen (e.g., CD22 and / or CD20). The fragment may be considered to have biological activity in that it can specifically bind to a target antigen or compete with other antibodies or their antigen-binding fragments to bind to a specific epitope. In certain embodiments, the fragment comprises one or more CDRs present in the full light chain or heavy chain, and in some embodiments, comprises a short chain, a heavy chain and / or a light chain, or a portion thereof. The biologically active fragment may be prepared by recombinant DNA technology, for example, by enzymatically or chemically cleaving an intact antibody.Immunoglobulin fragments with immunological functions include Fab, Fab', scFab, dsFv, Fv, scFV, scFV-Fc, 124cab and dAb, ScFv2-Fc, bi-nanobody, bispecific T cell engager (BiTE), tandem diabody (tandAb), dual affinity retargeting (DART) antibody, DART-Fc, scFv-human serum albumin (HAS)-scFv, dock-and-lock (DNL)-Fab3, minibody, Fab2 fragment (bispecific), Fab3 fragment (triplespecific), Bis-scFv fragment (bispecific), sdAb fragment (VH / VHH), It may be derived from any mammal, including but not limited to humans, mice, rats, camelid animals, or rabbits, including but not limited to tetrabodies, triabody, or diabody. One or more functional portions of antibodies, such as CDRs described herein, may be linked to a secondary protein or small molecule compound via covalent bonding and may be used as a targeted therapeutic agent for a specific target.

[0358] In this specification, the “Fc” region comprises two heavy chain fragments comprising CH2 and CH3 domains of the antibody. The two heavy chain fragments are connected to each other by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0359] In this specification, the "Fab fragment" consists of one light chain and one heavy chain comprising only a variable region and CH1. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules. In scFab, two Fab molecules are connected by a flexible linker.

[0360] In this specification, the "F(ab')2 fragment" comprises a Fab fragment and additionally a region between the CH1 and CH2 domains of a heavy chain. A disulfide bond can be formed between two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0361] In this specification, as described above, the “F(ab')2 fragment” comprises two heavy chains and two light chains, each comprising a variable region, a CH1 and a portion of an invariant region between the CH1 domain and the CH2 domain, and an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments, and the two Fab' fragments are joined to each other by a disulfide bond between them.

[0362] In this specification, the “Fv region” is a fragment of an antibody that includes a variable region of each heavy chain and light chain, but does not include a constant region. In sdFv, the heavy chain and light chain are connected by disulfide bonds. In scFv, Fv is connected by a flexible linker. In scFv-Fc, Fc is connected to scFv. In minibody, CH3 is connected to scFv. Diabody contains two molecules of scFv.

[0363] In this specification, the "single chain Fv" or "scFv" antibody fragment comprises VH and VL domains of the antibody, said domains existing within a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH domain and the VL domain that enables scFv to form a target structure for antigen binding.

[0364] In this specification, the “short-chain antibody (126cab)” is a single polypeptide chain comprising one constant region of the heavy chain or a constant region of the light chain, wherein the heavy chain and the light chain variable regions are connected by a flexible linker. For short-chain antibodies, reference may be made to U.S. Patent No. 5,260,203, which is incorporated herein by reference.

[0365] In this specification, the “domain antibody (dAb)” is an immunologically functional immunoglobulin fragment comprising only the variable region of the heavy chain or the variable region of the light chain. In certain embodiments, two or more VH regions are covalently linked through a peptide linker to form a bivalent domain antibody. The two VH regions of the bivalent domain antibody may target the same antigen or different antigens.

[0366] In this specification, "complementarity determining region" (CDR; i.e., CDR1, CDR2, and CDR3) refers to amino acid residues of variable regions of an antibody required for antigen binding. Each variable region has three CDR domains generally identified as CDR1, CDR2, and CDR3.

[0367] In this specification, the "framework region" (FR) is a variable region residue excluding the CDR residue. Each variable region has four FRs, generally identified as FR1, FR2, FR3, and FR4.

[0368] In this specification, the "bivalent antigen-binding protein" or "bivalent antibody" comprises two antigen-binding sites. The two antigen-binding sites included in the bivalent antibody may have the same antigen specificity, or the antigen-binding sites may be bispecific antibodies that bind to different antigens.

[0369] In this specification, the term “bispecific antibody” refers to an antibody or an antigen-binding fragment thereof capable of binding to two different epitopes on a single antigen or to two different antigens. The bispecific antibody in this specification may be divalent, trivalent, or tetravalent. As used herein, “-valent,” “-valent(s),” or other grammatical variations thereof refer to the number of antigen-binding sites within an antibody molecule.

[0370] In this specification, the "multispecific antigen-binding protein" or "multispecific antibody" targets two or more antigens or epitopes.

[0371] As used herein, the term “chimeric antibody” means an antibody (immunoglobulin) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence of an antibody of a specific species or belonging to a specific antibody class or subclass, while the remaining chain(s) are identical or homologous to the corresponding sequence of an antibody of a different species or belonging to a different antibody class or subclass, and also includes fragments of such antibodies insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). The chimeric antibody of interest herein comprises a PRIMATIZED antibody in which the antigen-binding region of said antibody is derived, for example, from an antibody prepared by immunizing a macaque monkey with the antigen of interest.

[0372] A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody), and is an antibody in which residues of the CDR of said recipient antibody are substituted with residues of the CDR of a non-human species, such as a mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or ability (donor antibody). In some cases, FR residues of said human immunoglobulin are substituted with corresponding non-human residues. Additionally, the humanized antibody may contain residues not found in said recipient antibody or said donor antibody. Such modifications may be made to further improve antibody performance, such as binding affinity. Generally, the humanized antibody comprises substantially all of at least one, typically two, variable domains, and all or substantially all hypervariable loops in the variable domains correspond to hypervariable loops of non-human immunoglobulin sequences, and all or substantially all FR regions are FR regions of human immunoglobulin sequences, although said FR regions may contain one or more individual FR residue substitutions that enhance antibody performance such as binding affinity, isomerization, and immunogenicity. The number of such amino acid substitutions within the FR is generally six or fewer in the H chain and three or fewer in the L chain. The humanized antibody may optionally further comprise at least a portion of an immunoglobulin constant region (Fc), which is generally the constant region of human immunoglobulin. For further details, see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al.See Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Also see, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patents 6,982,321 and 7,087,409.

[0373] "Human antibody" means an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or produced using any technique for producing a human antibody disclosed herein. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies may be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Additionally, for the production of human monoclonal antibodies, methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) may be used. Also refer to van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be produced by administering an antigen to a transgenic animal modified to produce said antibody in response to antigen stimulation but with inactivated endogenous loci, e.g., an immunized xenomice (e.g., U.S. Patents No. 6,075,181 and No. 6,150,584 regarding XENOMOUSE™ technology). Also, regarding human antibodies produced via, for example, human B-cell hybridoma technology, refer to Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006).

[0374] The "human consensus framework" refers to a framework representing the most commonly occurring amino acid residues in selected sequences among human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is made from subgroups of variable domain sequences. Generally, the subgroups of said sequences are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). For example, in the case of VL, said subgroups may be kappa I, kappa II, kappa III, or kappa IV as described in the literature by Kabat et al., etc. Also, in the case of VH, said subgroups may be subgroup I, subgroup II, or subgroup III as described in the literature by Kabat et al.

[0375] An "affinity-matured" antibody refers to an antibody that contains one or more modifications in one or more CDRs and has an enhanced affinity for the antigen compared to a parent antibody that does not have said modifications. In one embodiment, the affinity-matured antibody has an affinity for the target antigen at the nanomolar or picomolar level. The affinity-matured antibody is prepared by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutation induction of CDRs and / or framework residues is described, for example: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. It is described in 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0376] In this specification, "linker" refers to a compound that covalently binds a cytotoxic compound to an antibody.

[0377] In this specification, "unsubstituted or substituted" refers to a parent group that is not substituted or can be substituted, "substituted" means a parent group having at least one substituent, and "substituent" means a chemical moiety covalently bonded or fused to the parent group.

[0378] In this specification, "halo" means fluorine, chlorine, bromine, iodine, etc.

[0379] As used herein, the term "alkyl" means a saturated aliphatic group, and C1-C 10 straight-chain alkyl groups or C1-C 10 It includes, but is not limited to, branched-chain alkyl groups. Preferably, the "alkyl" group means a C1-C6 straight-chain alkyl group or a C1-C6 branched-chain alkyl group. Most preferably, the "alkyl" group means a C1-C4 straight-chain alkyl group or a C1-C4 branched-chain alkyl group. Examples of "alkyl" groups include methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl. Includes, but is not limited to, etc. The "alkyl" group may be optionally substituted.

[0380] The above term "acyl" is a term recognized in the art and means a group represented by the general formula hydrocarbyl C(O)-, preferably meaning alkyl C(O)-.

[0381] The term "acylamino" above is a term recognized in the art and refers to an amino group substituted with an acyl group, and can be represented, for example, as hydrocarbyl C(O)NH-.

[0382] The above term "acyloxy" is a term recognized in the art and refers to a group represented by the general formula hydrocarbyl C(O)O-, preferably alkyl C(O)O-.

[0383] The term "alkoxy" above refers to an alkyl group to which oxygen is attached. Examples of alkoxy groups include methoxy, ethoxy, propoxy, and tert-butoxy.

[0384] The above term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and can be represented by the general formula alkyl-O-alkyl.

[0385] The term "alkyl" above refers to a saturated aliphatic group and includes straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (aliphatic ring) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In a preferred embodiment, the straight-chain or branched-chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., in the case of a straight chain, C 1-30 , in the case of a branched chain, C 3-30 ), more preferably 20 or fewer.

[0386] Additionally, the term “alkyl” used throughout the specification, examples, and claims is intended to include both unsubstituted alkyl groups and substituted alkyl groups, the latter meaning an alkyl moiety having a substituent that replaces a hydrogen at one or more carbons of a hydrocarbon backbone, and includes haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0387] The above terms "Cx-y" or "Cx-Cy" refer to a group containing x to y carbons within a chain when used with chemical moietyes such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy. COalkyl refers to a hydrogen when the group is at a terminal position, and a bond when it is at an internal position. For example, C 1-6 The alkyl group contains 1 to 6 carbon atoms in the chain.

[0388] As used herein, the term "alkylamino" means an amino group substituted with at least one alkyl group.

[0389] As used herein, the term "alkylthio" refers to a thiol group substituted with an alkyl group and can be represented by the general formula alkylS-(alkylS-).

[0390] The term "amido" as used herein is It signifies energy, and

[0391] The above R 9 and R 10 Each independently represents a hydrogen or hydrocarbyl group, or R 9 and R 10 These form a heterocycle having 4 to 8 atoms in the ring structure to which they are attached.

[0392] The above terms "amine" and "amino" are terms recognized in the art and refer to both unsubstituted amines and substituted amines and their salts, for example, or It refers to a moiety that can be represented as, and the above R 9 , R 10 and R 10 Each independently represents a hydrogen or hydrocarbyl group, or R 9 and R10 They form a heterocycle with 4 to 8 atoms in a ring structure together with the N atoms to which they are attached.

[0393] As used herein, the term "aminoalkyl" means an alkyl group substituted with an amino group.

[0394] As used herein, the term "aralkyl" means an alkyl group substituted with an aryl group.

[0395] As used herein, the term "aryl" comprises a substituted or unsubstituted monocyclic aromatic group in which each ring atom is a carbon. Preferably, the ring is a 5- to 7-membered ring, and more preferably a 6-membered ring. The term "aryl" also comprises a polycyclic ring system having two or more cyclic rings, wherein two or more carbons are common between two adjacent rings, at least one of the rings is aromatic, and, for example, the other cyclic ring may be a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic. The aryl group includes benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0396] The above term "carbamate" is a term recognized in the industry, and or It signifies energy, and

[0397] The above R 9 and R 10 Each independently displays a hydrogen or hydrocarbyl group.

[0398] As used herein, the term "carbocyclylalkyl" means an alkyl group substituted with a carbocyclic group.

[0399] The term "carbocycle" comprises a monocyclic ring of 5 to 7 atoms and a bicyclic ring of 8 to 12 atoms. Each ring of the bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. A carbocycle comprises a bicyclic molecule in which one, two, or three or more atoms are shared between two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with another ring. Each ring of the fused carbocycle may be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings, as long as the valence allows, is included in the definition of carbocyclic. Examples of "carbocyclic" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Examples of fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, and bicyclo[4.2.0]octane.Includes 0]octane), 4,5,6,7-tetrahydro-1H-indene and bicyclo[4.1.0]hept-3-ene. The "carbocycle" may be substituted at one or more positions where it may have a hydrogen atom.

[0400] As used herein, the term "carbocyclylalkyl" means an alkyl group substituted with a carbocyclic group.

[0401] The above term "carbonate" is a term recognized in the art and refers to a group represented by -OCO2-.

[0402] As used herein, the term "carboxy" refers to a group represented by the chemical formula CO2H.

[0403] The term "cycloalkyl" comprises a substituted or unsubstituted non-aromatic monocyclic structure, preferably a 4- to 8-membered ring, and more preferably a 4- to 6-membered ring. The term "cycloalkyl" also comprises a polycyclic ring system having two or more cyclic ring structures in which two adjacent rings share two or more carbons, wherein at least one ring is cycloalkyl and a substituent (e.g., R 100) is bonded to the cycloalkyl ring, and, for example, other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl, and the heteroaryl group includes, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, benzodioxane, tetrahydroquinoline, etc.

[0404] The term "ester" as used herein is -C(O)OR 9 It refers to the energy indicated by , and the above R 9 It indicates a hydrocarbyl group.

[0405] As used herein, the term "ether" refers to a hydrocarbyl group bonded to another hydrocarbyl group via oxygen. Accordingly, the ether substituent of the hydrocarbyl group may be hydrocarbyl-O-. The ether may be symmetric or asymmetric. Examples of ethers include, but are not limited to, heterocycle-O-heterocycles and aryl-O-heterocycles. The ether comprises an "alkoxyalkyl" group that can be represented by the general formula alkyl-O-alkyl.

[0406] The terms "halo" and "halogen" as used herein refer to halogens and include chloro, fluoro, bromo, and iodo.

[0407] As used herein, the terms "hetaralkyl" and "heteroaralkyl" refer to an alkyl group substituted with a hetaryl group.

[0408] The terms "heteroaryl" and "hetaryl" comprise a substituted or unsubstituted aromatic single ring structure, preferably a 5- to 7-membered ring, more preferably a 5- to 6-membered ring, wherein the ring structure comprises at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also comprise a polycyclic ring system having two or more cyclic rings and sharing two or more carbons in two adjacent rings, wherein at least one ring is heteroaromatic, and for example, the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0409] As used herein, the term "heteroatom" refers to an atom of an element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0410] As used herein, the term "heterocyclylalkyl" means an alkyl group substituted with a heterocycl group.

[0411] The terms "heterocyclyl," "heterocycle," and "heterocyclic" refer to a non-aromatic ring structure that is substituted or unsubstituted, preferably a ring of 3 to 10 atoms, more preferably a ring of 3 to 7 atoms, wherein the ring structure comprises at least one heteroatom, preferably 1 to 4 heteroatoms, and more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include a polycyclic ring system having two or more cyclic rings and sharing two or more carbons in two adjacent rings, wherein at least one ring is heterocyclic, and for example, the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic. Heterocycline groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, etc.

[0412] As used herein, the term "hydrocarbyl" refers to a group bonded through a carbon atom that does not have an =O or =S substituent, typically having at least one carbon-hydrogen bond and a skeleton composed mainly of carbon, but optionally may include heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for the purposes of this application, but substituents such as acetyl (where the bonded carbon has an =O substituent) and ethoxy (where bonded through oxygen rather than carbon) do not fall under this category. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0413] As used herein, the term "hydroxyalkyl" means an alkyl group substituted with a hydroxyl group.

[0414] The above term “lower” means comprising a group having 10 or fewer atoms in the substituent when used with chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, preferably 6 or fewer. For example, “lower alkyl” means an alkyl group comprising 10 or fewer carbon atoms, preferably 6 or fewer. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein mean lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, and include whether they exist alone or in combination with other substituents such as hydroxyalkyl and aralkyl (in which case, for example, atoms of the aryl group are not counted when calculating the number of carbon atoms of the alkyl substituent).

[0415] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) and refer to a structure in which two or more atoms are common between two adjacent rings, for example, the rings are "fused rings." Each ring of the polycycle may or may not be substituted. In a specific embodiment, each ring of the polycycle contains 3 to 10 atoms in the ring, preferably 5 to 7.

[0416] The above term "sulfate" is a term recognized in the art and means an ↳OSO3H group or a pharmaceutically acceptable salt thereof.

[0417] The above term "sulfonamido" is a term recognized in the art, and the general formula or It refers to the energy indicated by, and the above R 9 and R 10 It independently displays hydrogen or hydrocarbyl.

[0418] The above term "sulfoxide" is a term recognized in the art and means an S(O)- group.

[0419] The term "sulfonate" above is a term recognized in the art and means an SO3H group or a pharmaceutically acceptable salt thereof.

[0420] The term "sulfone" above is a term recognized in the industry and refers to the -S(O)2- group.

[0421] The term "substituted" refers to a moiety having a substituent that replaces a hydrogen at one or more carbons of the skeleton. "Substitution" or "substituted with" is understood to imply the assumption that said substitution is made according to the allowable valence of the atom being substituted and the substituent, and that said substitution forms a stable compound that does not undergo spontaneous transformations such as rearrangement, cyclization, or elimination. As used herein, the term "substituted" is intended to include all allowable substituents of an organic compound. In a broad sense, said allowable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of the organic compound. There may be one or more of said allowable substituents, and they may be identical or different from one another for a suitable organic compound. For the purposes of the present invention, a heteroatom such as nitrogen may have a hydrogen substituent and / or an acceptable substituent of the organic compound described herein, which satisfies the valence of said heteroatom. The substituent may include any substituent used herein and may include, for example, a halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidin, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclil, aracleyl, or aromatic or heteroaromatic moiety. A person skilled in the art will understand that the above moiety substituted in the hydrocarbon chain may be further substituted itself where appropriate.

[0422] As used herein, the term "thioalkyl" means an alkyl group substituted with a thiol group.

[0423] As used herein, the term "thioester" is -C(O)SR 9 or ?SC(O)R 9 Refers to the energy indicated by,

[0424] The above R 9 represents hydrocarbyl.

[0425] As used herein, the term "thioether" is equivalent to ether, and the oxygen is substituted with sulfur.

[0426] The above term "urea" is a term recognized in the art, and the general formula It may be indicated as, and the above R 9 and R 10 It independently displays hydrogen and hydrocarbyl.

[0427] As used herein, the term "DBCO" refers to an optionally substituted dibenzocyclooctyne moiety, e.g., the following structure:

[0428] .

[0429] As used herein, the term “sugar moiety” refers to a natural or modified sugar that forms part of a larger molecule and is connected to the remainder of the larger molecule, for example, through one of the hydroxyl groups present in said sugar. For example, in some embodiments, said sugar is connected to the remainder of the larger molecule through a hydroxyl group on an anomeric carbon. Examples of sugar moiety include, but are not limited to, glucuronosyl and galactosyl.

[0430] "Glycosidase" is an enzyme that breaks down glycosidic bonds in carbohydrates, glycoproteins, and glycolipids. Examples of glycosidases include β-glucuronidase and β-galactosidase. β-glucuronidase is a type of glucuronidase that catalyzes the hydrolysis of β-D-glucuronic acid or β-glucuronosyl residues. β-galactosidase is a glycoside hydrolase that catalyzes the hydrolysis of terminal non-reducing β-D-galactose or β-galactosyl residues in β-D-galactosides.

[0431] As used herein, the term "modulate" includes not only the inhibition or suppression of a function or activity (such as cell proliferation) but also the enhancement of a function or activity.

[0432] The above term "cleavage group" refers to a chemical moiety that dissociates when exposed to acidic conditions, basic conditions, reducing conditions, oxidizing conditions, stimuli such as light or heat, or enzymes such as esterases.

[0433] Examples

[0434] The invention has been generally described, and the invention may be more easily understood by referring to the following examples included merely for the purpose of describing specific aspects and embodiments of the invention, and said examples are not intended to limit the invention.

[0435] Preparation Example 1: Production of an exemplary CD20-specific antibody

[0436] A first exemplary anti-CD20 antibody CD20_1 (rituximab) that specifically binds to CD20 was produced by the method described in U.S. Patent No. 5,736,137 A and U.S. Patent No. 7,422,739 B2, the entire contents of which are incorporated herein by reference. The amino acid sequence of said antibody CD20_1 is shown in Table 1 below. The amino acid sequence of said CDR was defined according to the Kabat definition.

[0437] [Table 1]

[0438] Amino acid sequence of the exemplary CD20 antibody CD20_1 (rituximab)

[0439]

[0440]

[0441] For the synthesis of the ADC, the exemplary antibody clone CD20_1-CaaX was constructed by introducing the CaaX peptide moiety (GGGGGGGCVIM, SEQ ID NO. 77) into the C-terminus of the light chain of SEQ ID NO. 18 in Table 1, by the method disclosed in International Patent Application No. PCT / IB2012 / 001065. The amino acid sequence of CD20_1-CaaX is shown in Table 2 below.

[0442] [Table 2]

[0443] Amino acid sequence of CD20_1-CaaX

[0444]

[0445] Preparation Example 2: Preparation of an exemplary halfmer of a CD20- or CD22- specific antibody

[0446] Halfmers of six exemplary antibodies that specifically bind to CD20 or CD22 were prepared using the Knob-into-Hole method described in U.S. Patent No. 9,637,557 B2 and the pentambody method described in U.S. Patent No. 11,498,977 B2, the entire contents of which are incorporated herein by reference. A list of the exemplary halfmers is shown in Table 3 below.

[0447] [Table 3]

[0448] Exemplary Halfmer

[0449]

[0450] Amino acid positions according to EU numbering conventions

[0451] The amino acid sequences of Halfmer 1 and Halfmer 2 that specifically bind to CD20 are shown in Tables 4 and 5 below, respectively. The amino acid sequences of the CDR according to the Kabat definition are defined below.

[0452] An exemplary half-antibody was prepared by introducing Hole mutations (T370S, L372A, Y411V of SEQ ID NO. 17 in Table 1) and LALA mutations (L238A / L239A of SEQ ID NO. 17 in Table 1) into the heavy chain constant region of a CD20_1 half-antibody.

[0453] [Table 4]

[0454] Exemplary amino acid sequence of Halfmer 1

[0455]

[0456]

[0457] A second exemplary anti-CD20 antibody CD20_2 (ocaratuzumab) that specifically binds to CD20 was produced by the method described in U.S. Patent No. 8,153,125 B2, the entire contents of which are incorporated herein by reference. The exemplary halfmer 2 was prepared by introducing Hole mutations (T370S, L372A, Y411V according to SEQ ID NO. 37) and LALA mutations (L238A / L239A according to SEQ ID NO. 37) into the heavy chain constant region of the anti-antibody of CD20_2.

[0458] [Table 5]

[0459] Exemplary amino acid sequence of Halfmer 2

[0460]

[0461]

[0462] The amino acid sequences of Halfmer3 and Halfmer4, which specifically bind to CD22, are shown in Tables 6 and 7 below, respectively. The amino acid sequences of the above CDRs according to the Kabat definition are defined below.

[0463] The first exemplary anti-CD22 antibody CD22_1 (inotuzumab) was produced by the method described in U.S. Patent No. 8,747,857 B2, the entire contents of which are incorporated herein by reference. The exemplary halfmer 3 was prepared by introducing a Knob mutation (T370W according to SEQ ID NO. 55) into the heavy chain constant region of the anti-antibody of CD22_1 and an LALA mutation (L238A / L239A according to SEQ ID NO. 55) into the heavy chain constant region of the anti-antibody of CD22_1.

[0464] [Table 6]

[0465] Exemplary amino acid sequence of Halfmer 3

[0466]

[0467]

[0468] A second exemplary anti-CD22 antibody, CD22_2 (eparatuzumab), was produced by the method described in U.S. Patent No. 6,198,287 B2, the entire contents of which are incorporated herein by reference. An exemplary halfmer 4 was prepared by introducing a Knob mutation (T365W according to SEQ ID NO. 73) into the heavy chain constant region of the anti-antibody of CD22_2 and an LALA mutation (L238A / L239A according to SEQ ID NO. 73) into the heavy chain constant region of the anti-antibody of CD22_2.

[0469] [Table 7]

[0470] Exemplary amino acid sequence of Halfmer 4

[0471]

[0472]

[0473] The amino acid sequences of Halfmer 5, which specifically binds to CD20, and Halfmer 6, which specifically binds to CD22, are shown in Tables 8 and 9 below, respectively. The amino acid sequences of the CDR according to the Kabat definition are defined below.

[0474] An exemplary halfmer 5 was prepared by introducing a pentambody substitution (L355E, Y411L, K413V of SEQ ID NO. 17 in Table 1) and an LALA mutation (L238A / L239A of SEQ ID NO. 17 in Table 1) into the heavy chain constant region of the CD20_1 anti-antibody.

[0475] [Table 8]

[0476] Exemplary amino acid sequence of Halfmer 5

[0477]

[0478]

[0479] An exemplary halfmer 6 was prepared by introducing a pentambody substitution (T370L and D403R according to SEQ ID NO. 76) into the heavy chain constant region of the CD22_1 anti-antibody and an LALA mutation (L238A / L239A according to SEQ ID NO. 76) into the heavy chain constant region of the CD22_1 anti-antibody.

[0480] [Table 9]

[0481] Exemplary amino acid sequence of Hafmer 6

[0482]

[0483]

[0484] Using CHO cell-based transient expression, the following halfmers were produced: CD20(Hole) halfmer 1, CD20(Hole) halfmer 2, CD22(Knob) halfmer 3, and CD22(Knob) halfmer 4.

[0485] Using HEK293 cell-based transient expression, the following halfmers were produced:

[0486] CD20 (L351E, Y407L, K409V according to EU numbering convention) Hammer 5, and CD22 (T366L, D399R according to EU numbering convention) Hammer 6.

[0487] Preparation Example 3: Preparation of Exemplary CD20- and CD22- Bispecific Antibodies

[0488] For the synthesis of an ADC, an exemplary hafmer-CaaX structure was prepared by introducing a CaaX peptide moiety (GGGGGGGCVIM, SEQ NO. 77) into the C-terminus of each of the light chains of Hafmer 1 (LC1; SEQ NO. 18) of Table 4, Hafmer 5 (LC1; SEQ NO. 18) of Table 8, Hafmer 2 (LC2; SEQ NO. 38) of Table 5, Hafmer 3 (LC3; SEQ NO. 56) of Table 6, Hafmer 6 (LC3; SEQ NO. 56) of Table 9, and Hafmer 4 (LC4; SEQ NO. 74) of Table 7, by the method disclosed in International Patent Application No. PCT / IB2012 / 001065, and the entire contents of said patent application are incorporated herein by reference.

[0489] Exemplary halfmer-CaaX construct pairs 1 and 3, 1 and 4, 2 and 3, and 2 and 4 were reacted using the Knob-into-Hole method described in U.S. Patent No. 9,637,557 B2 to form a bispecific antibody, which was subsequently used in the synthesis of an ADC.

[0490] Specifically, to assemble bispecific antibodies using the knob-into-hole method, each halfmer-CaaX construct was buffer-exchanged with a buffer containing 50 mM arginine (Sigma-Aldrich, A4474) and 200 mM histidine (Sigma-Aldrich, H6034) using vivaspin 20 (Sartorius, VS2002). The hole halfmer-CaaX and knob halfmer-CaaX pairs were then mixed in a 1:1 ratio to achieve a concentration of 2 mg / mL each. Reduced L-glutathione (Sigma-Aldrich, G4251) was added to a final concentration of 2.6 mM. The reaction was then carried out for 6 hours in a shaking incubator at 36 °C and 160 rpm.

[0491] Exemplary halfmer-CaaX construct pairs 5 and 6 were reacted using the pentambody method described in U.S. Patent No. 11,498,977 B2 to form bispecific antibodies for subsequent ADC synthesis.

[0492] The amino acid sequence of the CD20xCD22 bispecific antibody 1 that specifically binds to CD20 and CD22 is shown in Table 10 below.

[0493] [Table 10]

[0494] Amino acid sequence of exemplary CD20xCD22 bispecific antibody 1

[0495]

[0496] The amino acid sequence of the CD20xCD22 bispecific antibody 2 that specifically binds to CD20 and CD22 is shown in Table 11 below.

[0497] [Table 11]

[0498] Amino acid sequence of an exemplary CD20xCD22 bispecific antibody 2

[0499]

[0500]

[0501] The amino acid sequence of the CD20xCD22 bispecific antibody 3 that specifically binds to CD20 and CD22 is shown in Table 12 below.

[0502] [Table 12]

[0503] Amino acid sequence of an exemplary CD20xCD22 bispecific antibody 3

[0504]

[0505] The amino acid sequence of the CD20xCD22 bispecific antibody 4 that specifically binds to CD20 and CD22 is shown in Table 13 below.

[0506] [Table 13]

[0507] Amino acid sequence of an exemplary CD20xCD22 bispecific antibody 4

[0508]

[0509]

[0510] The amino acid sequence of the CD20xCD22 bispecific antibody 5 that specifically binds to CD20 and CD22 is shown in Table 14 below.

[0511] [Table 14]

[0512] Amino acid sequence of an exemplary CD20xCD22 bispecific antibody 5

[0513]

[0514] Two additional types of bispecific antibodies that specifically bind to CD20 and CD22 are the CrossMab method described in U.S. Patent No. 8,242,247 B2 and the S-DUAL described in EP Patent Application No. 4286408 A1. TM Produced using the methods, the entire contents of which are incorporated herein by reference. CrossMab or S-DUAL TM A list of exemplary bispecific antibodies produced by is shown in Table 15 below.

[0515] [Table 15]

[0516] Exemplary CD20xCD22 bispecific antibody

[0517]

[0518] The amino acid sequences of Hafmer 1, which specifically binds to CD20, and Hafmer 7, which specifically binds to CD22, are shown in Table 16 below. The amino acid sequences of the CDR according to the Kabat definition are defined below. Hafmer 7 was prepared based on Hafmer 3 (Table 6) by swapping the heavy chain CH1 and the light chain CL region of Hafmer 3.

[0519] An exemplary bispecific antibody 6 was prepared using Halfmer 1 (Table 4) as is and Halfmer 7.

[0520] [Table 16]

[0521] Exemplary amino acid sequences of Hafmer 1 and Hafmer 7

[0522]

[0523]

[0524]

[0525] For the synthesis of an ADC, an exemplary halfmer-CaaX structure was prepared by introducing a CaaX peptide moiety (GGGGGGGCVIM, SEQ ID NO. 77) into the C-terminus of the light chains of Halfmer 1 (LC1; SEQ ID NO. 18) of Table 4 and Halfmer 7 (LC7; SEQ ID NO. 82) of Table 16, respectively, by the method disclosed in international patent application PCT / IB2012 / 001065, the entire contents of which are incorporated herein by reference.

[0526] The pair of exemplary halfmer-CaaX constructs 1 and 7 were reacted using the CrossMab method described in U.S. Patent No. 8,242,247 B2 to form a bispecific antibody for subsequent ADC synthesis.

[0527] The amino acid sequence of the CD20xCD22 bispecific antibody 6, which specifically binds to CD20 and CD22, is shown in Table 17 below.

[0528] [Table 17]

[0529] Amino acid sequence of exemplary CD20xCD22 bispecific antibody 6

[0530]

[0531] An exemplary CD20xCD22 dual-specific antibody 7 is the S-DUAL described in EP patent application No. 4286408 A1. TMUsing the method, the exemplary anti-CD20 and anti-CD22 antibodies described herein were prepared.

[0532] Preparation Example 4: Synthesis of the exemplary compound of the present invention

[0533] Preparation of Compound 1

[0534]

[0535] Compound 1 was prepared by the method described in U.S. Patent No. 11,654,197 B2, the entire contents of which are incorporated herein by reference.

[0536] EI-MS m / z: [M+H] + 1698.2, [½M+H] + 849.6.

[0537] Preparation of Compound 2

[0538]

[0539] Compound 2 (MC-Val-Cit-PAB-MMAE) has the above structure and was purchased from MedChemExpress (Catalog No. HY-15575).

[0540] Preparation of Compound 3

[0541]

[0542] Compound 3 (MC-Val-Ala-PBD) has the above structure and was purchased from GLPBIO (Catalog No. GC39403).

[0543] Preparation Example 5: Preparation of an exemplary ADC by site-specific conjugation

[0544] An exemplary ADC was manufactured through the following two steps, and LCB14-0606 was manufactured using the method described in U.S. Patent No. 9,669,107 B2, the contents of which are incorporated herein by reference in whole. The structure of said LCB14-0606 is as follows:

[0545]

[0546] LCB14-0606

[0547] Step 1: Preparation of prenylated antibodies using LCB14-0606

[0548] The exemplary anti-CD20 mono- or anti-CD20xCD22 bispecific antibodies listed in Table 18 below were prepared according to Preparation Examples 1-3. Mixtures containing each antibody for the prenylation reaction were prepared and reacted at 30 °C for 16 hours. The reaction mixture for each antibody contained a total of 24 μM of antibody, 400 nM of FTase (Calbiochem #344145), and 0.14 mM of LCB14-0606 in a buffer solution (50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 10 μM ZnCl2, 0.25 mM DTT). After the reaction was complete, the prenylated antibodies were purified using a G25 Sepharose column (AKTA purifier, GE healthcare) equilibrated with PBS buffer.

[0549] Step 2: Drug-conjugation method

[0550] Oxime binding reaction of ADC (conjugation through oxime bond formation)

[0551] The reaction for forming oxime bonds between each prenylated antibody and linker-drug was carried out by mixing 100 mM sodium acetate buffer (pH 5.2, Sigma-Aldrich, S7899), 10% DMSO (Sigma-Aldrich, D4540), 24 μM of prenylated antibody, and 10 equivalents (240 μM) of linker-drug (Compound 1 of Preparation Example 4). The mixture was slowly stirred at 30°C for 6 hours. After the reaction, excess low molecular weight compounds were removed by a desalting process using a G25 Sepharose column, and the protein fraction was collected and concentrated.

[0552] An exemplary ADC produced by the above two steps is shown in Table 18 below.

[0553] [Table 18]

[0554] Exemplary ADC by site-specific junction

[0555]

[0556] The preparation of ADCs was analyzed using hydrophobic interaction chromatography to measure the drug-antibody ratio and size exclusion chromatography for monomer analysis.

[0557] For ADC1, ADC2, ADC3, ADC4, and ADC7, hydrophobic interaction chromatography was performed using a phenyl column (7.5 mm x 75 mm, 10 μm, Tosoh Bioscience, 7573) and potassium phosphate buffer as the mobile phase. For the analysis, buffer A, consisting of 50 mM potassium phosphate buffer (pH 7.0) containing 0.5 M ammonium sulfate, and buffer B, consisting of 50 mM potassium phosphate buffer (pH 7.0) containing 30% acetonitrile, were used. Initial conditions were stabilized at 90% A / 10% B, a linear gradient was applied from 90% A / 10% B to 10% A / 90% B over 30 minutes, followed by further elution at 10% A / 90% B for 5 minutes. The flow rate and temperature were set to 1.0 mL / min and 25 ℃, respectively, and detection was performed at 254 nm and 280 nm.

[0558] For ADC5 and ADC6, the same phenyl column (7.5 mm x 75 mm, 10 μm, Tosoh Bioscience, 7573) and potassium phosphate buffer were used. Buffer A contained 50 mM potassium phosphate (pH 7.0) containing 1.5 M ammonium sulfate, and Buffer B contained 50 mM potassium phosphate (pH 7.0) containing 30% acetonitrile. Initial conditions were stabilized at 70% A / 30% B, a linear gradient was applied from 70% A / 30% B to 10% A / 90% B over 30 minutes, followed by elution at 10% A / 90% B for 5 minutes. The flow rate and temperature were maintained at 1.0 mL / min and 25°C, respectively, and detection was performed at 254 nm and 280 nm.

[0559] For ADC8, hydrophobic interaction chromatography was performed using a butyl column (4.6 mm x 35 mm, 2.5 μm, Tosoh Bioscience, 14947) and potassium phosphate buffer. Buffer A contained 50 mM potassium phosphate (pH 7.0) containing 1.5 M ammonium sulfate, and Buffer B contained 50 mM potassium phosphate (pH 7.0) containing 30% isopropyl alcohol. Initial conditions were stabilized at 100% A / 0% B, followed by a linear gradient from 100% A / 0% B to 0% A / 100% B over 20 minutes, and elution to 0% A / 100% B for 2 minutes. The flow rate and temperature were set to 1.0 mL / min and 25°C, respectively, and detection was performed at 254 nm and 280 nm.

[0560] For size exclusion chromatography of ADC1, ADC2, ADC3, ADC4, ADC5, ADC6, ADC7, and ADC8, a SEC column (7.8 mm x 30 cm, Tosoh Bioscience, 8541) was used with 0.1 M sodium phosphate and 0.3 M sodium chloride (pH 6.8) containing 10% acetonitrile as the mobile phase. The analysis was performed with a linear gradient of 100% buffer for 30 minutes.

[0561] The results of the above ADC manufacturing analysis are shown in Table 19 below.

[0562] [Table 19]

[0563] Exemplary ADC manufacturing analysis results

[0564]

[0565] Preparation Example 6: Preparation of an exemplary ADC by random splicing

[0566] For the preparation of randomly conjugated ADCs, the CD20xCD22 bispecific antibody 1 was conjugated with compound 3 of Preparation Example 4 or compound 2 of Preparation Example 4.

[0567] For the conjugation of the bispecific antibody to the linker-toxin, 74 μM tris(2-carboxyethyl)phosphine (Sigma-Aldrich, C4706) was added to a 24 μM CD20xCD22 bispecific antibody 1 solution containing 4 mM EDTA (Enzynomics, EBE001-500) at pH 7.4, and the mixture was slowly stirred at 25°C. After a reaction time of 2 hours, 216 μM of compound 3 or compound 2 was added, and the mixture was slowly stirred at 25°C for an additional 2 hours. Subsequently, 1 mM dihydroavietic acid (Sigma-Aldrich, 261556) was added, and the mixture was slowly stirred at 25°C for an additional 2 hours. Excess low molecular weight compounds were removed by a desalting process using a G25 Sepharose column, and the protein fraction was collected and concentrated.

[0568] An exemplary ADC manufactured by random splicing is shown in Table 20 below.

[0569] [Table 20]

[0570] Exemplary ADC by Random Conjugation

[0571]

[0572] The preparation of ADC9 and ADC10 was analyzed using reverse-phase chromatography to measure the average drug-antibody ratio and size exclusion chromatography for monomer analysis.

[0573] Reverse-phase chromatography was performed using a PLRP-S 1000 Å column (2.1 mm x 50 mm, 5 μm, Agilent, PL1912-1502) with a mobile phase containing 0.1% trifluoroacetic acid (Sigma-Aldrich, T6508). Water containing 0.1% trifluoroacetic acid was used as Solution A, and acetonitrile containing 0.1% trifluoroacetic acid was used as Solution B. The system was stabilized with 75% A and 25% B for 3 minutes, followed by the application of a linear gradient of 50% A and 50% B for 28 minutes, an additional linear gradient of 5% A and 95% B for 1 minute, and then elution for an additional 2 minutes. The flow rate and temperature were set to 1.0 mL / min and 80°C, respectively. Detection was performed at 254 nm and 280 nm. Size exclusion chromatography was performed using a SEC column (7.8 mm x 30 cm, Tosoh Bioscience, 8541) and a mobile phase (pH 6.8) containing 0.1 M sodium phosphate and 0.3 M sodium chloride in 10% acetonitrile buffer. The analysis was performed with a linear gradient of 100% buffer for 30 minutes.

[0574] The manufacturing analysis results of ADC9 and ADC10 are shown in Table 21 below.

[0575] [Table 21]

[0576] Exemplary ADC manufacturing analysis results

[0577]

[0578] Experimental Example 1: In vitro evaluation of the binding affinity of CD20xCD22 bispecific antibodies to cancer cells

[0579] The cell binding affinity of the anti-CD20xCD22 bispecific antibody 1 prepared in Preparation Example 3 was evaluated in blood cancer cell lines using flow cytometry (FACS). The cell lines tested included SUP-B15, characterized by low CD20 expression and high CD22 expression; Mino, characterized by low CD22 expression and high CD22 expression; and Granta-519, which expresses CD20 and CD22 at high levels.

[0580] Specifically, the cancer cell line was cultured and divided into two groups: a control IgG antibody treatment group and an anti-CD20xCD22 bispecific antibody 1 treatment group. Subsequently, to minimize non-specific antibody binding, the cells were cultured in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) at 4°C for 1 hour. Then, the control antibody and the anti-CD20xCD22 bispecific antibody 1 were cultured with their respective cell groups at 4°C for 1 hour to allow antibody binding to the CD20 or CD22 proteins on the surface of the cancer cells.

[0581] After incubation, unbound antibodies were removed with PBS containing 2% FBS. Subsequently, the cells were incubated at 4°C for 1 hour with a control IgG antibody and a fluorescently labeled secondary antibody specific to anti-CD20xCD22 bispecific antibody 1. Afterward, unbound secondary antibodies were removed by washing with PBS, and the fluorescence intensity of each cell group was measured using a flow cytometer. The binding affinity of the antibody to the cancer cell line was confirmed by comparing the fluorescence intensity of the anti-CD20xCD22 bispecific antibody 1 group with that of the control IgG group. The results are shown in Table 22 below.

[0582] [Table 22]

[0583] Binding affinity of an exemplary CD20xCD22 bispecific antibody

[0584]

[0585] The experimental results (Fig. 1) indicated that the anti-CD20xCD22 bispecific antibody 1 had a binding affinity 10.4 times higher than that of the IgG control group for SUP-B15 cells with low CD20 expression and high CD22 expression. In the case of Mino cells characterized by low CD22 expression and high CD20 expression, the anti-CD20xCD22 bispecific antibody 1 showed 9.9 times stronger binding compared to the IgG control group. In Granta-519 cells expressing high levels of CD20 and CD22, the binding affinity of the anti-CD20xCD22 bispecific antibody 1 was 11.2 times higher compared to the IgG control group. Taken together, these results confirmed that the anti-CD20xCD22 bispecific antibody 1 can bind not only to cancer cells expressing CD20 or CD22 but also to blood cancer cells co-expressing both CD20 and CD22.

[0586] Experimental Example 2: Exemplary antibody internalization rate in cancer cells In vitro evaluation

[0587] To analyze the internalization rate of the anti-CD20 antibody CD20_1-CaaX structure prepared in Preparation Example 1 and the anti-CD20xCD22 bispecific antibody 1 prepared in Preparation Example 3, cell binding experiments were performed using a flow cytometer (FACS).

[0588] Ramos cells, a B-cell non-Hodgkin lymphoma cell line known for CD20 expression, were used, and human IgG was used as a control antibody.

[0589] To evaluate the internalization rate, the anti-CD20 antibody CD20_1-CaaX construct and the anti-CD20xCD22 bispecific antibody 1 were each cultured with Ramos cells at 4°C for 1 hour. Subsequently, the experimental group was cultured at 37°C for 2 or 4 hours to induce internalization, while the control group was maintained at 4°C. To quantify the antibodies remaining on the cell surface in each group, a phycoerythrin (PE)-conjugated secondary antibody (BD, 555787) specific to human IgG was used.

[0590] The above results (Fig. 2) showed that in Ramos cells, the anti-CD20 antibody construct CD20_1-CaaX had an internalization rate of 42% after 2 hours of culture at 37°C and 44% after 4 hours of culture. On the other hand, the anti-CD20xCD22 bispecific antibody 1 showed an internalization rate of 93% after 2 hours and 95% after 4 hours.

[0591] In summary, the above anti-CD20xCD22 bispecific antibody showed an improvement in internalization rate of over 50% in Ramos cells compared to the anti-CD20 antibody.

[0592] Experimental Example 3: Internalization rates of exemplary bispecific ADCs in cancer cells by platform In vitro evaluation

[0593] To evaluate the internalization rate of ADC1 containing an anti-CD20 antibody, and the internalization rates of ADC2 (Knob-into-Hole), ADC6 (Pentambody), and ADC7 (CrossMab) containing an anti-CD20xCD22 bispecific antibody sharing the same variable region as prepared in Preparation Example 3, cell binding experiments were performed using a flow cytometry analyzer (FACS). Ramos cells, a B-cell non-Hodgkin lymphoma cell line expressing CD20, were used, and human IgG was used as a control antibody.

[0594] To analyze the internalization rate, the anti-CD20 ADC (ADC1) and the anti-CD20xCD22 bispecific ADCs (ADC2, 6, and 7) were first cultured at 4°C for 1 hour, respectively. The control group was maintained at 4°C, and the treatment groups were cultured at 37°C for 1 hour or 3 hours to induce internalization. To quantify the antibodies remaining on the cell surface, a PE-conjugated secondary antibody specific to human IgG (BD, 555787) was used.

[0595] The above results (Figure 3) showed that ADC1 had an internalization rate of 19% after 1 hour and 40% after 3 hours in Ramos cells. On the other hand, ADC2 (Knob-into-Hole platform) showed an internalization rate of 94% after 1 hour of culture and 98% after 3 hours of culture. ADC6 (Pentambody platform) showed an internalization rate of 88% after 1 hour of culture and 85% after 3 hours of culture. ADC7 (CrossMab platform) showed an internalization rate of 87% after 1 hour of culture and 88% after 3 hours of culture.

[0596] In summary, the anti-CD20xCD22 bispecific ADC demonstrated a high internalization rate exceeding 50% compared to the anti-CD20 ADC in Ramos cells, regardless of the platform. These results confirm that the anti-CD20xCD22 bispecific ADC has an improved internalization rate compared to CD20-targeted ADCs, regardless of the platform used.

[0597] Experimental Example 4: Exemplary bispecific ADCs by platform In vitro Cytotoxicity evaluation

[0598] As shown in Table 23 below, the cytotoxic activity of the above ADCs against various cancer cell lines was evaluated. The cancer cell lines used were commercially available human B-cell non-Hodgkin lymphoma cell lines (SU-DHL-8, WSU-DLCL2) and acute lymphoblastic leukemia cell lines (SUP-B15 and Reh). The ADCs used to measure cytotoxic activity were ADC1, ADC2, ADC6, and ADC7 prepared in Preparation Example 3.

[0599] Each cancer cell line was seeded into 96-well plates at a density of 5,000 to 20,000 cells per well and treated with each ADC at a concentration range of 0.128 fM to 50 nM (5-fold serial dilution). The number of surviving cells (cell viability) was quantified using CellTiter-Glo (Promega, G7570) after 96 or 144 hours, and the results are shown in Table 23 below.

[0600] [Table 23]

[0601] 50% cytotoxicity concentration of an exemplary ADC in cancer cell lines

[0602]

[0603] As shown in Table 23, in various cancer cell lines (SU-DHL-8, WSU-DLCL2, SUP-B15, Reh), the pyrrobenzodiazepine-based anti-CD20xCD22 bispecific ADCs (ADC2, ADC6, ADC7) exhibited improved efficacy ranging from 22 to 380 times compared to the pyrrobenzodiazepine-based anti-CD20 ADC (ADC1). The anti-CD20xCD22 bispecific ADCs (ADC2, ADC6, and ADC7) consistently exhibited stronger cytotoxic effects on cancer cell lines compared to the anti-CD20 ADC1.

[0604] Therefore, the above anti-CD20xCD22 ADC effectively exhibited cytotoxicity against cancer cells compared to the above anti-CD20 ADC, regardless of the bispecific antibody platform used.

[0605] Experimental Example 5: Exemplary bispecific ADCs by platform In vitro Cytotoxicity evaluation

[0606] As listed in Table 24 below, the cytotoxic activity of the above ADCs against various cancer cell lines was measured. Commercially available human B-cell non-Hodgkin lymphoma cell lines (WSU-DLCL2, WSU-NHL, Mino, and Granta-519) were used in this analysis. The cytotoxicity of ADC2 and ADC8 prepared in Preparation Example 5 was tested.

[0607] Each cancer cell line was seeded into 96-well plates at a density of 5,000 cells per well and treated with each ADC at concentrations ranging from 0.128 fM to 50 nM (5-fold serial dilution). The number of surviving cells (cell viability) was quantified using CellTiter-Glo (Promega, G7570) 96 hours after treatment.

[0608] [Table 24]

[0609] 50% cytotoxicity concentration of an exemplary ADC in cancer cell lines

[0610]

[0611] As shown in Table 24, the results of the cytotoxicity evaluation of cancer cell lines showed that ADC2 produced in a 2-cell system using the Knob-in-Hole method disclosed herein and ADC8 produced in a 1-cell system using the S-DUAL method disclosed herein exhibited similar efficacy across the tested cell lines (WSU-DLCL2, WSU-NHL, Mino, and Granta-519).

[0612] These results confirm that the CD20xCD22 bispecific ADC exhibits consistent cytotoxicity regardless of the bispecific antibody production method.

[0613] Experimental Example 6: ADC containing monoclonal antibodies and bispecific antibodies In vitro Cytotoxicity Comparison

[0614] The cytotoxicity of the drug (SG2057) and ADC was measured in various cell lines and normal cells.

[0615]

[0616] SG2057 has the above structure and was purchased from MedChemExpress (catalog number HY-101160). The cancer cell lines used were commercially available human B-cell non-Hodgkin lymphoma cell lines (Daudi, Ramos, NAMALWA, WSU-NHL, WSU-DLCL2) and acute lymphoblastic leukemia cell lines (SUP-B15 and Reh). The normal cell lines used were PBMC (STEMCELL; catalog number 70002.2) and HSC (STEMCELL; catalog number 70025.2). The ADCs tested were ADC1 and ADC2 prepared in Preparation Example 5.

[0617] Each cancer cell line was seeded in 96-well plates at a density of 5,000 to 20,000 cells per well and treated with SG2057 in a concentration range of 5.12 fM to 2 nM (5-fold serial dilution) or with each ADC in a concentration range of 0.128 fM to 50 nM (5-fold serial dilution). For normal cell lines, cells were treated with SG2057 in a concentration range of 512 fM to 2 nM (5-fold serial dilution) or with ADCs in a concentration range of 10 pM to 5,000 nM (5-fold serial dilution). The number of surviving cells (cancer cell viability) in cancer cell lines was quantified using CellTiter-Glo (Promega, G7570) after 96 or 144 hours. For normal cell lines, PBMC and HSC were quantified using CellTiter-Glo after 144 hours, and the results are shown in Tables 25 and 26 below.

[0618] [Table 25]

[0619] 50% cytotoxic concentration of an exemplary drug or ADC in cancer cell lines

[0620]

[0621] [Table 26]

[0622] 50% cytotoxic concentration of an exemplary drug or ADC in normal cell lines

[0623]

[0624] As shown in Table 25, the results of the cytotoxicity evaluation in cancer cell lines (Daudi, Ramos, NAMALWA, WSU-NHL, WSU-DLCL2, SUP-B15, Reh) showed that ADC2, a pyrrolobenzodiazepine-based anti-CD20xCD22 bispecific ADC, exhibited 4 to 75 times better efficacy compared to ADC1, an anti-CD20 pyrrolobenzodiazepine-based ADC.

[0625] In normal cell lines PBMC and HSC, as shown in Table 26, ADC2 exhibited 8 to 29 times weaker cytotoxicity, which means that the toxicity to blood cells in which CD20 and CD22 are typically expressed in vivo was reduced.

[0626] These results confirm that the cytotoxicity of ADC2 is selectively enhanced in cancer cells and its impact on normal cells is significantly reduced, supporting the potential for targeted cytotoxicity of ADC2 against cancer cells compared to ADC1.

[0627] Experimental Example 7: An exemplary bispecific ADC comprising different antibody sequences In vitro Cytotoxicity evaluation

[0628] The cytotoxic activity of exemplary ADCs against cancer cell lines was measured to observe potential differences in efficacy according to antibody sequence variations. The cancer cell line used was Ramos, a commercially available human B-cell non-Hodgkin lymphoma cell line. The tested ADCs (ADC2, ADC3, ADC4, and ADC5) were prepared in Preparation Example 5.

[0629] Ramos cells were seeded in 96-well plates at a density of 5,000 cells per well and treated with each ADC in a concentration range of 0.128 fM to 50 nM (5-fold serial dilution). The number of surviving cells was quantified after 96 hours using CellTiter-Glo (Promega, G7570), and the results are shown in Table 27 below.

[0630] [Table 27]

[0631] 50% cytotoxicity concentration of an exemplary ADC in cancer cell lines

[0632]

[0633] As shown in Table 27, the results of the cytotoxicity evaluation on cancer cell lines (Ramos) show that all pyrrobenzodiazepine-based anti-CD20xCD22 bispecific ADCs (ADC2, ADC3, ADC4, and ADC5) exhibited strong efficacy regardless of antibody sequence variation.

[0634] The anti-CD20xCD22 bispecific antibody combination ADC was confirmed to consistently exhibit potent cancer cell-specific cytotoxicity, unaffected by variations in the antibody sequence.

[0635] Experimental Example 8: An exemplary bispecific ADC comprising different linker drug combinations in vitro Cytotoxicity evaluation

[0636] The cytotoxic activity of ADC9 and ADC10 prepared in Preparation Example 5 against cancer cell lines was measured to observe potential differences in efficacy depending on variations in the linker-payload combination.

[0637] The cancer cell line used was Ramos, a commercially available human B-cell non-Hodgkin lymphoma cell line. Ramos cells were seeded into 96-well plates at a density of 5,000 cells per well and treated with each ADC at concentrations ranging from 0.128 fM to 50 nM (5-fold serial dilution). The number of viable cells was quantified after 96 hours using CellTiter-Glo (Promega, G7570), and the results are shown in Table 28 below.

[0638] [Table 28]

[0639] 50% cytotoxicity concentration of an exemplary ADC in cancer cell lines

[0640]

[0641] As shown in Table 28, the anti-CD20xCD22 bispecific ADC (ADC9) conjugated with MC-Val-Ala-PBD (Compound 3) exhibited potent cytotoxicity regardless of the specific linker used, and the efficacy was similar to that of ADC2, ADC3, ADC4, and ADC5. Similarly, the anti-CD20xCD22 bispecific ADC (ADC10) conjugated with MC-Val-Cit-PAB-MMAE (Compound 2) exhibited strong efficacy regardless of the specific payload.

[0642] These results suggest that the efficacy of anti-CD20xCD22 bispecific ADCs is not limited by variations in the linker or payload, and that they consistently exhibit cancer cell-specific cytotoxicity.

[0643] Experimental Example 9: Evaluation of Pharmacokinetics (PK)

[0644] Of the ADC2 prepared in Preparation Example 5 in Sprague Dawley (SD) rats in vivo For evaluation, a single dose of ADC2 of 1.0 mg / kg was administered intravenously to 9-week-old male SD rats (Orientbio, South Korea). Pharmacokinetics were studied after the injection of ADC2 into SD rats. Plasma samples were collected at various time points and frozen for analysis. Plasma concentrations of ADC2 at the indicated time points were measured using the LC-MS / MS analysis method.

[0645] Briefly, 250 μL of acetonitrile (ACN) solution was mixed with 50 μL of sample and 50 μL of plasma containing 10 nM dextromethorphan (internal standard), and the solution was mixed vigorously for 5 minutes using a vortex mixer. The sample was then centrifuged at 14,000 rpm at 4°C for 5 minutes. 100 μL of the supernatant was mixed with 100 μL of mobile phase A, namely 0.1% formic acid in water containing 5% ACN, and thoroughly mixed. The sample was measured for ADC2 using the LC-MS / MS method (Nexera LC40 (SHIMADZU) and QTRAP4500 (SCIEX)).

[0646] The PK profile of ADC2 is shown in Table 29 and Figure 4. ADC2 exhibited a stable pharmacokinetic profile in rats.

[0647] [Table 29]

[0648] Exemplary ADC PK profile

[0649]

[0650] Experimental Example 10: In vivo Efficacy Evaluation (SU-DHL-8)

[0651] After culturing the blood cancer cell line SU-DHL-8, 5,000,000 cells were treated with 50 μL of Matrigel in 50 μL of PBS. TM It was mixed with (Corning) and transplanted into CB17 / SCID mice. Tumor size was 70 to 110 mm 3 When [the time] was reached, an exemplary ADC1 or ADC2 was administered via intravenous injection. The results of comparing the efficacy of the ADC samples are shown in Table 30 and Figure 5.

[0652] [Table 30]

[0653] Exemplary ADC in vivo Anticancer efficacy (SU-DHL-8)

[0654]

[0655] As shown in Figure 5 and Table 30, when the pyrrobenzodiazepine-based anti-CD20xCD22 bispecific ADC2 was administered at 0.3 mg / kg, it showed 45.1% tumor growth inhibition compared to the control group (PBS administration group), whereas the anti-CD20 ADC1 at the same dose achieved 13.9% tumor growth inhibition. Therefore, it was indicated that the tumor growth inhibitory efficacy of ADC2 was improved by more than three times compared to ADC1 at the same dose.

[0656] Experimental Example 11: In vivo Efficacy Evaluation (Granta-519)

[0657] After culturing the blood cancer cell line Granta-519, 5,000,000 cells were treated with 50 μL of Matrigel in 50 μL of PBS. TM It was mixed with (Corning) and transplanted into CB17 / SCID mice. Tumor size was 100 mm 3 When [the time] was reached, exemplary ADC2s were administered intravenously at different doses as shown in Table 31 below. The results of verifying the efficacy of the ADC samples are shown in Table 31 and Figure 6.

[0658] [Table 31]

[0659] Exemplary ADC in vivo Anticancer efficacy (Granta-519)

[0660]

[0661] As shown in Figure 6 and Table 31, complete remission was achieved when the pyrrobenzodiazepine-based anti-CD20xCD22 bispecific ADC2 was administered at a dose of 0.1 mg / kg.

[0662] Experimental Example 12: In vivo Efficacy Evaluation (MINO)

[0663] After culturing the blood cancer cell line MINO, 10,000,000 cells were 100 μL of Matrigel in 100 μL of PBS. TM It was mixed with (Corning) and transplanted into CB17 / SCID mice. The tumor size was 126 mm 3 When [the time] was reached, exemplary ADC2s were administered intravenously at different doses as shown in Table 32 below. The results of verifying the efficacy of the ADC samples are shown in Table 32 and Figure 7.

[0664] [Table 32]

[0665] Exemplary ADC in vivo Anticancer efficacy (MINO)

[0666]

[0667] As shown in Figure 7 and Table 32, complete remission was achieved when the pyrrobenzodiazepine-based anti-CD20xCD22 bispecific ADC2 was administered at a dose of 0.2 mg / kg.

[0668] Experimental Example 13: In vivo Efficacy Evaluation (Ramos)

[0669] After culturing the blood cancer cell line MINO, 5,000,000 cells were 100 μL of Matrigel in 100 μL of PBS TM It was mixed with (Corning) and transplanted into BALB / c mice. The tumor size was 70 mm 3 When reached, an exemplary ADC2 or inotuzumab ozogamicin (Besponsa) as shown in Table 33 below. TM ) was administered via intravenous injection at different doses. The results of verifying the efficacy of the ADC samples are shown in Table 33 and Figure 8.

[0670] [Table 33]

[0671] Exemplary ADC in vivo Anticancer efficacy (Ramos)

[0672]

[0673] As shown in Figure 8 and Table 33, the pyrrobenzodiazepine-based anti-CD20xCD22 bispecific drug conjugate ADC2 exhibited dose-dependent tumor growth inhibition at doses of 0.1, 0.3, and 0.5 mg / kg in a Ramos cell xenograft model. Compared to the commercially available CD22-targeted ADC, inotuzumab ozogamicin, the 0.3 mg / kg ADC2 showed a 49% improvement in tumor growth inhibition. At 0.5 mg / kg, the ADC2 achieved 3.14 times stronger tumor growth inhibition than inotuzumab ozogamicin at 2.5 mg / kg. These results confirm that the ADC2 effectively inhibits tumor growth in a xenograft (CDX) model derived from cell lines resistant to inotuzumab ozogamicin.

[0674] Experimental Example 14: In vivo Efficacy Evaluation (WSU-DLCL2)

[0675] After culturing the blood cancer cell line WSU-DLCL2, 10,000,000 cells were 100 μL of Matrigel in 100 μL of PBS TM It was mixed with (Corning) and transplanted into CB17 SCID mice. The tumor size was 150 mm 3 When [the time] was reached, an exemplary ADC1 or ADC2 was administered via intravenous injection as shown in Table 34 below. In the rituximab-CHOP treatment group, rituximab and cyclophosphamide were administered intravenously, while doxorubicin, vincristine, and prednisone were administered orally. The results of verifying the efficacy of the ADC samples and R-CHOP are shown in Table 34 and Figure 9.

[0676] [Table 34]

[0677] Exemplary ADC in vivo Anticancer efficacy (WS-DLCL2)

[0678]

[0679] As shown in Figure 9 and Table 34, the pyrrobenzodiazepine-based anti-CD20xCD22 bispecific drug conjugate ADC2 exhibited dose-dependent tumor growth inhibition at doses of 0.1, 0.3, and 0.5 mg / kg in a WSU-DLCL2 cell CDX model. At 0.3 mg / kg, ADC2 achieved 51% greater tumor growth inhibition compared to ADC1 at the same dose. These results confirm that the anti-CD20xCD22 ADC provides potent tumor growth inhibition by addressing the low internalization problem associated with CD20-targeted ADCs.

[0680] Additionally, ADC2 demonstrated strong tumor growth inhibition at all tested doses when compared to standard R-CHOP therapy, which is commonly used as a first-line treatment for diffuse large B-cell lymphoma. These results indicate that ADC2 is effective in demonstrating tumor growth inhibition even in CDX models resistant to R-CHOP therapy.

[0681] Inclusion by reference

[0682] All publications and patents mentioned herein are incorporated herein by reference in their entirety, as specifically and individually indicated by reference for each individual publication or patent. In the event of a conflict, this application shall prevail, including any definitions provided herein.

[0683] Equalities

[0684] Although specific embodiments of this specification have been discussed, this specification is illustrative and not limiting. Many variations of this specification will become apparent to those skilled in the art upon reviewing this specification and the following claims. The full scope of this specification should be determined by reference to the full scope of the claims and their equivalents, and to the specification and such variations.

Claims

Claim 1 Compounds having a structure represented by the following general formula I or pharmaceutically acceptable salts thereof: [General Formula 1]Ab-[L-(B) l ] m The above Ab is a bispecific antibody comprising a first antigen-binding domain that specifically binds to CD20 and a second antigen-binding domain that specifically binds to CD22, each L is independently a linker, each B is independently an activator, and l and m are each independently 1 to 20. Claim 2 A composite according to claim 1, wherein L is a cleavable linker. Claim 3 A conjugate according to claim 1 or 2, wherein L is a protease-cleavable linker, an acid-cleavable linker, a disulfide linker, or a glycosidase-cleavable linker. Claim 4 A conjugate according to claim 1 or 2, wherein L is a protease-cleaving linker. Claim 5 In claim 4, the protease is a cathepsin, a conjugate. Claim 6 A conjugate according to claim 4 or 5, wherein L comprises 2 to 4 amino acid residues, for example, amino acid residues determining the recognition site of the protease. Claim 7 A conjugate according to claim 6, wherein L comprises Val-Cit, Val-Ala, or Gly-Gly-Phe-Gly. Claim 8 A conjugate according to claim 1 or 2, wherein L is a glycosidase cleavable linker. Claim 9 In claim 8, the glucosidase is a conjugate that is β-glucuronidase or β-galactosidase. Claim 10 A conjugate according to claim 9, wherein L comprises a β-glucuronosyl or β-galactosyl moiety. Claim 11 A conjugate according to any one of claims 1 to 10, wherein L is covalently bonded to Ab by a thioether, thioester, disulfide, hydrazone, ester, carbamate, carbonate, alkoxy, or amide. Claim 12 A conjugate according to any one of claims 1 to 11, wherein L comprises a peptide further comprising at least one hydrophilic amino acid. Claim 13 A conjugate according to claim 12, wherein the peptide comprises an amino acid having a side chain having a charged moiety (e.g., amine, guanidine, or carboxyl moiety) in an aqueous solution of neutral pH. Claim 14 A conjugate according to any one of claims 1 to 13, wherein L comprises an oxime. Claim 15 A conjugate according to any one of claims 1 to 14, wherein L comprises at least one isoprenyl group. Claim 16 A conjugate according to any one of claims 1 to 15, wherein L comprises at least one succinimide group. Claim 17 A conjugate according to any one of claims 1 to 16, wherein L is covalently bonded to Ab by a thioether bond, and the thioether bond comprises a sulfur atom of cysteine ​​of Ab. Claim 18 A conjugate according to any one of claims 1 to 17, wherein Ab comprises an amino acid motif recognized by isoprenoid transferase at the C-terminus. Claim 19 A conjugate according to claim 18, wherein the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase). Claim 20 A conjugate according to claim 18 or 19, wherein L is covalently bonded to Ab by a thioether bond, and the thioether bond comprises a sulfur atom of cysteine ​​of the amino acid motif. Claim 21 A conjugate according to any one of claims 18 to 20, wherein the amino acid motif comprises a CY1Y1X sequence, wherein:C is cysteine; each Y1 is independently an aliphatic amino acid; and X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine. Claim 22 A conjugate according to claim 21, wherein each of the Y1 is independently selected from alanine, isoleucine, leucine, methionine, and valine. Claim 23 A conjugate according to any one of claims 18 to 22, wherein the amino acid motif comprises CVIM (SEQ No. 84) or CVLL (SEQ No. 85). Claim 24 A conjugate according to any one of claims 18 to 23, wherein at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine. Claim 25 A conjugate according to any one of claims 18 to 24, wherein the amino acid motif has the sequence GGGGGGGCVIM (Sequence No. 77). Claim 26 A conjugate according to any one of claims 1 to 25, wherein L comprises a self-immolative moiety. Claim 27 In any one of claims 1 to 10, the conjugate has a structure represented by General Formula II or is a pharmaceutically acceptable salt thereof, conjugate:[General Formula II] Above: B is an activator, G indicates a sugar moiety or a glucuronic acid moiety; R 1 and R 2 is independently hydrogen, C1-C8 alkyl, or C3-C8 cycloalkyl; R 1 and R 2 ... bonds to form a (C3-C8) cycloalkyl ring; W is - * C(O)-, - * C(O)N(R')-, - * N(R')C(O)-, - * (CH2) t N(R')C(O)-, - * C(O)O-, - * S(O2)N(R')-, - * P(O)(R'')N(R')-, - * S(O)N(R')- or - * P(O2)N(R')-, where C(O), N, CH2, S, or P labeled with * are directly bonded to the phenyl ring of the above general formula II, and R' and R'' are each independently hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 Arylgo; Z are each independently C 1-8 It is alkyl, halogen, cyano, or nitro; n1 and n2 are each independently 1 to 20; n3 is 0 to 3; and Y is absent or C1-C 50 Alkylene, C1-C 50 It is alkenylene, 1-50 atomic heterocyclylene, or 1-50 atomic heteroalkylene. Claim 28 In claim 27, the Y is C 1-50 Alkylene or C 1-50 A conjugate comprising a heteroalkylene and further comprising at least one of the following: (i) one or more unsaturated bonds; (ii) a heterocyclylene or a heteroarylene (e.g., a heteroarylene within the alkylene or heteroalkylene chain); (iii) at least one C 1-20 alkyl; and (iv) at least one isoprenyl group having a structure represented by General Formula III: [General Formula III] ; The above n4 is an integer from 1 to 20. Claim 29 In claim 27 or 28, the R 1 and R 2 A conjugate in which each is independently hydrogen. Claim 30 A composite according to any one of claims 27 to 29, wherein n3 is 0. Claim 31 A conjugate according to any one of claims 27 to 30, wherein G has a structure represented by general formula IV: [General formula IV] The above R3 is hydrogen, alkyl, CH2OR3A or CO2R3B, and R 3A is a hydrogen or hydroxyl protecting group;R 3B is a hydrogen or carboxyl protecting group; each R4 is independently a hydrogen or hydroxyl protecting group. Claim 32 A conjugate according to claim 31, wherein R3 is -COOH. Claim 33 A conjugate according to claim 31 or 32, wherein each R4 is independently hydrogen. Claim 34 In any one of claims 27 to 33, the W is - * C(O)NR'-conjugate. Claim 35 In any one of claims 27 to 34, the G is is, and W is - * C(O)NR'-conjugate. Claim 36 A conjugate according to any one of claims 27 to 35, wherein Y is covalently bonded to Ab by a thioether, thioester, disulfide, hydrazone, ester, carbamate, carbonate, alkoxy, or amide. Claim 37 A conjugate according to claim 36, wherein Y comprises a peptide further comprising at least one hydrophilic amino acid. Claim 38 A conjugate according to claim 37, wherein the peptide comprises an amino acid having a side chain having a charged moiety (e.g., amine, guanidine, or carboxyl moiety) in an aqueous solution of neutral pH. Claim 39 A conjugate according to claim 37 or 38, wherein the peptide comprises an amino acid selected from alanine, histidine, arginine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine. Claim 40 A conjugate according to claim 37 or 38, wherein the peptide comprises an amino acid selected from alanine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine. Claim 41 A conjugate according to any one of claims 27 to 40, wherein Y comprises an oxime. Claim 42 A conjugate according to claim 41, wherein the oxygen atom of the oxime is located on the side of Y connected to W, and the carbon atom of the oxime is located on the side of Y connected to Ab. Claim 43 A conjugate according to claim 41, wherein the carbon atom of the oxime is located on the side of Y connected to W, and the oxygen atom of the oxime is located on the side of Y connected to Ab. Claim 44 In any one of claims 27 to 43, the Y comprises C including an oxime 1-50 Heteroalkylene, conjugate. Claim 45 A conjugate according to claim 44, wherein the oxygen atom of the oxime is located on the side of Y connected to W, and the carbon atom of the oxime is located on the side of Y connected to Ab. Claim 46 A conjugate according to claim 44, wherein the carbon atom of the oxime is located on the side of Y connected to W, and the oxygen atom of the oxime is located on the side of Y connected to Ab. Claim 47 A conjugate according to any one of claims 44 to 46, wherein Y comprises an oxime and at least one isoprenyl unit covalently bonds the oxime to Ab (e.g., at least one isoprenyl unit directly or indirectly bonds the oxime to Ab). Claim 48 A conjugate according to any one of claims 27 to 46, wherein Y comprises at least one isoprenyl group. Claim 49 A conjugate according to any one of claims 27 to 48, wherein Y is covalently bonded to Ab by a thioether bond, and said thioether bond comprises a sulfur atom of cysteine ​​of Ab. Claim 50 A conjugate according to any one of claims 27 to 49, wherein Ab comprises an amino acid motif recognized by isoprenoid transferase at the C-terminus. Claim 51 A conjugate according to claim 50, wherein the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase). Claim 52 A conjugate according to claim 50 or 51, wherein Y is covalently bonded to Ab by a thioether bond, and the thioether bond comprises a sulfur atom of cysteine ​​of the amino acid motif. Claim 53 A conjugate according to any one of claims 50 to 52, wherein the amino acid motif comprises a CY1Y1X sequence, wherein:C is cysteine; each Y1 is independently an aliphatic amino acid; and X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine. Claim 54 A conjugate according to claim 53, wherein each of the Y1 is independently selected from alanine, isoleucine, leucine, methionine, and valine. Claim 55 A conjugate according to any one of claims 50 to 54, wherein the amino acid motif comprises CVIM (SEQ No. 84) or CVLL (SEQ No. 85). Claim 56 A conjugate according to any one of claims 50 to 55, wherein at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine. Claim 57 A conjugate according to any one of claims 50 to 56, wherein the amino acid motif has the sequence GGGGGGGCVIM (Sequence No. 77). Claim 58 A composite according to any one of claims 27 to 57, wherein Y comprises a connecting unit represented by general formula V or general formula VI: [General formula V]-(CH2) r (V(CH2) p ) q -[General Formula VI]-(CH2CH2X) w - Above, V is a single bond, -O-, -S-, - NR 5 -, -C(O)NR 6 -, -NR 7 C(O)-, -NR 8 SO2-, or -SO2NR 9 -is;X is -O-, C 1-8 Alkylene, or -NR 5 - and;R 5 to R 9 are independently hydrogen, C 1-6 Alkyl, C 1-6 Alkyl C 6-20 Aryl or C 1-6 Alkyl-C 3-20 It is heteroaryl; r is an integer from 0 to 10; p is an integer from 0 to 10; q is an integer from 1 to 20; and w is an integer from 1 to 20. Claim 59 A composite according to claim 58, wherein q is 1 to 10. Claim 60 A composite according to claim 58 or 59, wherein r is 1 or 2. Claim 61 A composite according to any one of claims 58 to 60, wherein p is 1 or 2. Claim 62 In any one of claims 58 to 61, the conjugate, wherein V is -O-. Claim 63 A conjugate according to claim 58, wherein:q is 1 to 10;r and p are each 1 or 2; andV is -O-. Claim 64 In any one of claims 58 to 63, the conjugate is -O-. Claim 65 A composite according to any one of claims 58 to 64, wherein X is -O- and w is 1 to 10. Claim 66 In any one of claims 58 to 65, the L is A conjugate comprising at least one polyethylene glycol monomer represented by Claim 67 In claim 66, the L is or A conjugate comprising a polyethylene glycol oligomer represented by, wherein n40 is 2 to 12. Claim 68 A conjugate according to claim 66 or 67, wherein Y comprises an oxime, and at least one polyethylene glycol unit covalently bonds the oxime to W. Claim 69 A conjugate according to any one of claims 27 to 68, wherein Y further comprises a binding unit formed by a reaction between an alkyne and an azide or a reaction between an aldehyde or ketone and a hydrazine or hydroxylamine. Claim 70 A conjugate according to any one of claims 27 to 69, wherein Y further comprises a bonding unit represented by the general formula VIIa, VIIb, VIIc, VIId or VIIe: [General formula VIIa] [General Formula VIIb] [General Formula VIIc] [General Formula VIId] [General Formula VIIe] Above L 1 Each is independently a single bond or C 1-30 It is an alkylene; R 11 Silver is hydrogen or C 1-10 It is an alkyl. Claim 71 In any one of claims 27 to 70, the composite is A conjugate that includes Claim 72 In any one of claims 1 to 71, the composite is A conjugate comprising a structure represented by or a pharmaceutically acceptable salt thereof; wherein n12 is 0 to 30; and wherein the wavy bond indicates a connection with Ab. Claim 73 A composite according to any one of claims 27 to 72, wherein Y is branched. Claim 74 In claim 73, the conjugate comprises: i) a branching unit covalently bonded to Ab by a primary linker; ii) a first B (first B) bonded to said branching unit; and iiia) a second branch in which a second B (second B) bonded to said branching unit or iiib) a second branch in which an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) covalently bonded to said branching unit. Claim 75 A composite according to claim 74, wherein Y comprises a second branch that combines the second B to the branch unit through a cleavage group. Claim 76 A conjugate according to claim 74, wherein Y comprises a second branch covalently bonded to the branch unit, wherein an alkyl or heteroalkyl (e.g., polyethylene glycol monomer or polyethylene glycol oligomer) is covalently bonded to the branch unit. Claim 77 In any one of claims 74 to 76, the branch unit , , , or Having a structure indicated by, the above G 1, G 2 and G 3 Each combines independently , , or Igo;R 30 Silver is hydrogen or C 1-30 It is alkyl and;R 40 Silver is hydrogen or L 5 -COOR 50 Igo;R 50 Silver is hydrogen or C 1-30 alkyl and;L 2 , L 3 and L 4 are each independently combined or -C n' H 2n' -is;n' is a conjugate that is an integer from 1 to 10. Claim 78 A conjugate according to any one of claims 74 to 77, wherein: at least one branched linker is covalently bonded to Ab, and at least two Bs are covalently bonded to said branched linker. Claim 79 A conjugate according to claim 78, wherein the conjugate comprises 1, 2, 3 or 4 branched linkers, each branched linker comprising 2 B. Claim 80 A conjugate according to any one of claims 74 to 79, wherein the branching unit comprises a lysine residue. Claim 81 In any one of claims 74 to 80, the composite is A conjugate comprising a structure represented by or a pharmaceutically acceptable salt thereof, wherein each of B is independently an activator; n11, n22, and n33 are each independently 0 to 30; AA is a peptide comprising at least two amino acid residues; and the wave linkage indicates a connection with Ab. Claim 82 A conjugate according to any one of claims 1 to 81, wherein the Ab comprises a first constant region connected to a first antigen-binding domain and a second constant region connected to a second antigen-binding domain, and the first constant region and the second constant region each comprise a light chain constant region CL domain and a heavy chain constant region CH1, CH2, and CH3 domains. Claim 83 A conjugate according to any one of claims 1 to 82, wherein Ab is a chimeric antibody, a humanized antibody, or a human antibody. Claim 84 A conjugate according to any one of claims 1 to 82, wherein Ab is an IgA, IgG, IgM, IgE, or IgD isotype. Claim 85 In claim 84, the conjugate wherein Ab is an IgG isotype (e.g., IgG1, IgG2, IgG3, and IgG4). Claim 86 A conjugate according to any one of claims 1 to 85, wherein the Ab comprises a LALA mutation within the first and / or second heavy chain constant region. Claim 87 A conjugate according to claim 86, wherein the LALA mutation comprises L234A and L235A according to the EU numbering convetion. Claim 88 A conjugate according to any one of claims 83 to 87, wherein at least one amino acid residue in the CH3 domain of the first heavy chain constant region is substituted with an amino acid residue having a larger side chain size, and at least one amino acid residue in the CH3 domain of the second heavy chain constant region is substituted with an amino acid residue having a smaller side chain size; or at least one amino acid residue in the CH3 domain of the second heavy chain constant region is substituted with an amino acid residue having a larger side chain size, and at least one amino acid residue in the CH3 domain of the first heavy chain constant region is substituted with an amino acid residue having a smaller side chain size. Claim 89 A conjugate according to claim 88, wherein the amino acid residue having a larger side chain size is selected from arginine, phenylalanine, tyrosine, and tryptophan; and the amino acid residue having a smaller side chain size is selected from alanine, serine, threonine, and valine. Claim 90 A conjugate according to claim 88 or 89, wherein the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at positions T366, L368, and Y407 according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions at position T366 according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions at positions T366, L368, and Y407 according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at position T366 according to EU number. Claim 91 A conjugate according to claim 90, wherein the CH3 domain of the first heavy chain invariant region comprises T366S, L368A, and Y407V according to EU number, and the CH3 domain of the second heavy chain invariant region comprises T366W according to EU number; or the CH3 domain of the second heavy chain invariant region comprises T366S, L368A, and Y407V according to EU number, and the CH3 domain of the first heavy chain invariant region comprises T366W according to EU number. Claim 92 A conjugate according to any one of claims 88 to 91, wherein the CH3 domain of each of the first and second heavy chain invariant regions additionally comprises at least one substitution from an amino acid residue to a cysteine ​​residue. Claim 93 In claim 88 or 89, (a) the CH3 domain of the first heavy chain invariant region comprises E356C, T366S, L368A, and Y407V according to EU number, and the CH3 domain of the second heavy chain invariant region comprises Y349C and T366W according to EU number; or the CH3 domain of the second heavy chain invariant region comprises E356C, T366S, L368A, and Y407V according to EU number, and the CH3 domain of the first heavy chain invariant region comprises Y349C and T366W according to EU number; or (b) the CH3 domain of the first heavy chain invariant region comprises S354C, T366S, L368A and Y407V according to EU number, and the CH3 domain of the second heavy chain invariant region comprises Y349C and T366W according to EU number; or the CH3 domain of the second heavy chain invariant region comprises S354C, T366S, L368A and Y407V according to EU number, and the CH3 domain of the first heavy chain invariant region comprises Y349C and T366W according to EU number, a conjugate. Claim 94 In claim 88 or 89, (a) the CH3 domain of the first heavy chain invariant region comprises E356C, E357K, T366S, L368A, D399K and Y407V according to EU number, and the CH3 domain of the second heavy chain invariant region comprises Y349C, T366W, K370E and R409D according to EU number; or the CH3 domain of the second heavy chain invariant region comprises E356C, E357K, T366S, L368A, D399K and Y407V according to EU number, and the CH3 domain of the first heavy chain invariant region comprises Y349C, T366W, K370E and R409D according to EU number; or (b) a conjugate wherein the CH3 domain of the first heavy chain invariant region comprises S354C, E357K, T366S, L368A, D399K and Y407V according to EU number, and the CH3 domain of the second heavy chain invariant region comprises Y349C, T366W, K370E and R409D according to EU number; or the CH3 domain of the second heavy chain invariant region comprises S354C, E357K, T366S, L368A, D399K and Y407V according to EU number, and the CH3 domain of the first heavy chain invariant region comprises Y349C, T366W, K370E and R409D according to EU number. Claim 95 In any one of claims 83 to 87, (a) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at positions T366 and D399 according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions at positions L351, Y407, and K409 according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions at positions T366 and D399 according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at positions L351, Y407, and K409 according to EU number; or (b) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at positions T366 and K409 according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions at positions L351, D399 and Y407 according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions at positions T366 and K409 according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions at positions L351, D399 and Y407 according to EU number, conjugate. Claim 96 A conjugate according to claim 95, wherein the amino acid substituted in T366 is selected from leucine, proline, tryptophan, and valine; the amino acid substituted in D399 is selected from cysteine, asparagine, isoleucine, glycine, arginine, threonine, and alanine; the amino acid substituted in L351 is selected from glycine, tyrosine, valine, proline, aspartic acid, glutamate, lysine, and tryptophan; the amino acid substituted in Y407 is selected from leucine, alanine, proline, phenylalanine, threonine, and histidine; and the amino acid substituted in K409 is selected from cysteine, proline, serine, phenylalanine, valine, glutamate, and arginine. Claim 97 In claim 95, (a) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366L and D399R according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351E, Y407L and K409V according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366L and D399R according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351E, Y407L and K409V according to EU number; or (b) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366L and D399C according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351G, Y407L and K409V according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366L and D399C according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351E, Y407L and K409C according to EU number; or (c) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366L and D399C according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351Y, Y407A and K409P according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366L and D399C according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351Y, Y407A and K409P according to EU number;or (d) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366P and D399N according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351V, Y407P and K409S according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366P and D399N according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351V, Y407P and K409S according to EU number; or (e) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366W and D399G according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351D, Y407P and K409S according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366W and D399G according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351D, Y407P and K409S according to EU number; or (f) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366P and D399I according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351P, Y407F and K409F according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366P and D399I according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351P, Y407F and K409F according to EU number;or (g) the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366V and D399T according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351K, Y407T and K409Q according to EU number; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366V and D399T according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351K, Y407T and K409Q according to EU number; or (h) a conjugate wherein the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366L and D399A according to EU number, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351W, Y407H and K409R according to EU number; or wherein the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366L and D399A according to EU number, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351W, Y407H and K409R according to EU number.; Claim 98 A conjugate according to claim 95, wherein the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of T366L and D399R, and the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of L351E, Y407L and K409Y; or the CH3 domain of the second heavy chain constant region comprises amino acid substitutions of T366L and D399R, and the CH3 domain of the first heavy chain constant region comprises amino acid substitutions of L351E, Y407L and K409Y. Claim 99 A conjugate according to any one of claims 83 to 98, wherein (a) the CL and CH1 domains of the first invariant region are replaced with each other; the CL and CH1 domains of the second invariant region are replaced with each other; or (b) the VH and VL domains of the first antigen-binding domain are replaced with each other; the VH and VL domains of the second antigen-binding domain are replaced with each other; or (c) the first invariant region and the VH and CH1 domains and the VL and CL domains of the first antigen-binding domain are replaced with each other; and the second invariant region and the VH and CH1 domains and the VL and CL domains of the second antigen-binding domain are replaced with each other. Claim 100 A conjugate according to claim 99, wherein the CL and CH1 domains of the first invariant region are substituted for each other; and the CL and CH1 domains of the second invariant region are substituted for each other. Claim 101 A composite according to any one of claims 83 to 98, wherein the first invariant region and the second invariant region have a structure asymmetric to each other. Claim 102 A conjugate according to claim 101, wherein the knob-in-hole structure is additionally included between the first antigen-binding domain and the CH1 domain of the first invariant region; or the knob-in-hole structure is additionally included between the second antigen-binding domain and the CH1 domain of the second invariant region. Claim 103 A conjugate according to claim 102, wherein the knopinhole structure comprises a dimer of a CH3 domain. Claim 104 In any one of claims 1 to 102, the first antigen-binding domain specifically binding to CD20 comprises a first heavy chain variable region and a first light chain variable region, and the second antigen-binding domain specifically binding to CD22 comprises a second heavy chain variable region and a second light chain variable region, wherein (a) the first heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 6, and the first light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 13, and the second heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 40, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 42, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 44 The second light chain variable region comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO. 47, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 49, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 51;(b) The first heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 6, and the first light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 13, and the second heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 58, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 60, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 62, and the second light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 65, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 67, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 69; (c) The first heavy chain variable region comprises SEQ ID NO The first light chain variable region comprises a heavy chain CDR1 having an amino acid sequence of 22, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO. 24, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO. 26; the first light chain variable region comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO. 29, a light chain CDR2 having an amino acid sequence of SEQ ID NO. 31, and a light chain CDR3 having an amino acid sequence of SEQ ID NO. 33; the second heavy chain variable region comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO. 40, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO. 42, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO. 44; and the second light chain variable region comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO. 47, a light chain CDR2 having an amino acid sequence of SEQ ID NO. 49, and a light chain CDR3 having an amino acid sequence of SEQ ID NO. 51;or (d) a conjugate wherein the first heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 22, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 26, the first light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 29, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 31, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 33, the second heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 58, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 60, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 62, and the second light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 65, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 67, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 69.; Claim 105 In claim 104, the (a) VH domain of the first antigen-binding domain comprises: an amino acid sequence of SEQ ID NO. 15; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 15; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 15, and the VL domain of the first antigen-binding domain comprises: an amino acid sequence of SEQ ID NO. 16; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 16; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 16, and the VH domain of the second antigen-binding domain comprises: an amino acid sequence of SEQ ID NO. 53; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 53; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 53, and the VL domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 54; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 54; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 54, and (b) the VH domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 15; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 15; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 15, and the VL domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 16; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 16; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 16, and the VH domain of the second antigen-binding domain is: the amino acid sequence of SEQ ID NO. 71;(c) the amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 71; or the amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 71, wherein the VL domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 72; the amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 72; or the amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 72, and (c) the VH domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 35; the amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 35; or the amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 35, wherein the VL domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 36; the amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 36; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 36, and the VH domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 53; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 53; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 53, and the VL domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 54; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 54; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 54, or (d) the VH domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 35; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 35;or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 35, and the VL domain of the first antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 36; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 36; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 36, and the VH domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 71; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 71; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 71, and the VL domain of the second antigen-binding domain comprises: the amino acid sequence of SEQ ID NO. 72; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 72; or a conjugate comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 72.; Claim 106 In claim 104, the (a) the VH domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 15; the VL domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 16; the VH domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 53; and the VL domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 54; (b) the VH domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 15; the VL domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 16; the VH domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 71; and the VL domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 72; and (c) the VH domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 35; and the VL domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 36 A conjugate comprising a sequence; wherein the VH domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 53; the VL domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 54; or (d) wherein the VH domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 35; the VL domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 36; the VH domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO. 71; and the VL domain of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO.

72. Claim 107 In any one of claims 1 to 87, the Ab comprises: a first heavy chain and a first light chain comprising the first antigen-binding domain that specifically binds to CD20; and a second heavy chain and a second light chain comprising the second antigen-binding domain that specifically binds to CD22, wherein (a) the first heavy chain comprises: an amino acid sequence of SEQ ID NO. 20; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 20; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 20, and the first light chain comprises: an amino acid sequence of SEQ ID NO. 18; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 18; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 18, and the second heavy chain comprises: the amino acid sequence of SEQ ID NO. 55; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 55; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 55, and the second light chain comprises: the amino acid sequence of SEQ ID NO. 56; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 56; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 56, and (b) the first heavy chain comprises: the amino acid sequence of SEQ ID NO. 20; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 20; Or, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 20, wherein the first light chain comprises: the amino acid sequence of SEQ ID NO. 18; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 18;or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 18, and the second heavy chain comprises: the amino acid sequence of SEQ ID NO. 73; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 73; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 73, and the second light chain comprises: the amino acid sequence of SEQ ID NO. 74; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 74; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 74, and (c) the first heavy chain comprises: the amino acid sequence of SEQ ID NO. 37; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 37; or comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 37, wherein the first light chain comprises: the amino acid sequence of SEQ ID NO. 38; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 38; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 38, and the second heavy chain comprises: the amino acid sequence of SEQ ID NO. 55; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 55; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 55, and the second light chain comprises: the amino acid sequence of SEQ ID NO. 56; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 56; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 56, and (d) the first heavy chain comprises: the amino acid sequence of SEQ ID NO. 37; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 37;or comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 37, wherein the first light chain comprises: the amino acid sequence of SEQ ID NO. 38; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 38; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 38, and the second heavy chain comprises: the amino acid sequence of SEQ ID NO. 73; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 73; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 73, and the second light chain comprises: the amino acid sequence of SEQ ID NO. 74; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 74; or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 74, and (e) the first heavy chain comprises: the amino acid sequence of SEQ ID NO. 75; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 75; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 75, and the first light chain comprises: the amino acid sequence of SEQ ID NO. 18; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 18; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 18, and the second heavy chain comprises: the amino acid sequence of SEQ ID NO. 76; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 76; Or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 76, wherein the second light chain comprises: the amino acid sequence of SEQ ID NO. 56; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 56;or comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 56, (f) the first heavy chain comprises: the amino acid sequence of SEQ ID NO. 20; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 20; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 20, and the first light chain comprises: the amino acid sequence of SEQ ID NO. 18; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 18; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 18, and the second heavy chain comprises: the amino acid sequence of SEQ ID NO. 81; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 81; A conjugate comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 81, wherein the second light chain comprises: the amino acid sequence of SEQ ID NO. 82; an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 82; or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 82.; Claim 108 In claim 107, the first heavy chain comprises the amino acid sequence of SEQ ID NO. 20; the first light chain comprises the amino acid sequence of SEQ ID NO. 18; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 55; the second light chain comprises the amino acid sequence of SEQ ID NO. 56; (b) the first heavy chain comprises the amino acid sequence of SEQ ID NO. 20; the first light chain comprises the amino acid sequence of SEQ ID NO. 18; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 73; the second light chain comprises the amino acid sequence of SEQ ID NO. 74; (c) the first heavy chain comprises the amino acid sequence of SEQ ID NO. 37; the first light chain comprises the amino acid sequence of SEQ ID NO. 38; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 55; the second light chain comprises the amino acid sequence of SEQ ID NO. 56; and (d) the first heavy chain comprises the amino acid sequence of SEQ ID NO. 37; and the A conjugate comprising: (e) the first light chain comprises the amino acid sequence of SEQ ID NO. 38; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 73; the second light chain comprises the amino acid sequence of SEQ ID NO. 74; (e) the first heavy chain comprises the amino acid sequence of SEQ ID NO. 75; the first light chain comprises the amino acid sequence of SEQ ID NO. 18; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 76; or the second light chain comprises the amino acid sequence of SEQ ID NO. 56; or (f) the first heavy chain comprises the amino acid sequence of SEQ ID NO. 20; the first light chain comprises the amino acid sequence of SEQ ID NO. 18; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 81; and the second light chain comprises the amino acid sequence of SEQ ID NO.

82. Claim 109 A conjugate according to any one of claims 1 to 108, wherein Ab is a full-length antibody. Claim 110 The conjugate of claim 109, wherein the Ab is an IgG-single chain variable fragment (scFv), a trifunctional antibody (triomab), knobs into holes (KIH)-IgG, kparamda-body (κλ-body), crossmab, ortho-Fab IgG, dual variable domain immunoglobulin (DVD-Ig) or 2-in-1-IgG (dual action antibody) or a combination thereof. Claim 111 A conjugate according to any one of claims 1 to 106, wherein Ab is an antigen-binding fragment. Claim 112 In claim 111, the Ab is a conjugate that is ScFv2-Fc, a bi-nanobody, a bispecific T cell engager (BiTE), a tandem diabody (tandAb), a dual affinity retargeting (DART) antibody, DART-Fc, scFv-human serum albumin (HAS)-scFv, dock-and-lock (DNL)-Fab3, a minibody, a Fab2 fragment (bispecific), a Fab3 fragment (trispecific), a Bis-scFv fragment (bispecific), an sdAb fragment (VH / VHH), a tetrabody, a tribody, or a diabody, or a combination thereof. Claim 113 A conjugate according to any one of claims 1 to 112, wherein the activator is a chemotherapy agent or a toxin. Claim 114 A conjugate according to any one of claims 1 to 112, wherein the activator is selected from chemotherapy compounds, cytotoxic compounds, immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, antiparasitic agents, and combinations thereof. Claim 115 A conjugate according to claim 114, wherein the cytotoxic compound is selected from mitotic inhibitors, DNA alkylating agents, topoisomerase inhibitors, and combinations thereof. Claim 116 A conjugate according to claim 114, wherein the cytotoxic compound is selected from auristatin, maytansinoid, tubulisin, calicheamicin, duocarmycin, pyrrolobenzodiazepine and derivatives thereof, camptothecin and derivatives thereof and combinations thereof. Claim 117 A conjugate according to claim 116, wherein the orristatin is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). Claim 118 In any one of claims 1 to 112, the wherein: the activator is a pyrrobenzodiazepine dimer; the N10 position of the pyrrobenzodiazepine dimer is substituted with X or the N'10 position is substituted with X', said X or X' connects the pyrrobenzodiazepine dimer to the linker, and X and X' each independently -C(O)O- * or -C(O)- * is; * A conjugate, which refers to the bonding point between the above-mentioned pyrrobenzodiazepine dimer and the above-mentioned linker. Claim 119 A conjugate according to any one of claims 1 to 112, wherein the activator is a pyrrobenzodiazepine dimer, and the pyrrobenzodiazepine dimer has a structure represented by Formula VIII: [Formula VIII] Here, the wavy line indicates the junction point with the linker, and the dotted line optionally indicates a double bond; R1 and R1 ' Each independently contains H, OH, =O, =CH2, CN, R m , OR m , =CH-R m' , =C(R m' )2, O-SO2-R m , CO2R m , COR m , selected from halo and dihalo; R m' is R m , CO2R m , COR m Selected from , CHO, CO2H and halo;R m is substituted or unsubstituted C 1-12 Alkyl, substituted or unsubstituted C 2-12 Alkenyl, substituted or unsubstituted C 2-12 alkynyl, substituted or unsubstituted C 5-20 Aryl, substituted or unsubstituted C 3-6 Heteroaryl, substituted or unsubstituted C 3-6 Selected from cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocyclyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl and substituted or unsubstituted 5- to 7-membered heteroaryl, and said C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 5-20 Aril, C 5-20 Heteroaryl, C 3-6 When a cycloalkyl, 3- to 7-membered heterocyclile, 3- to 7-membered heterocycloalkyl, or 5- to 7-membered heteroaryl is substituted, the above C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 5-20 Aril, C 5-20 Heteroaryl, C 3-6 Each hydrogen atom of the cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, or 5- to 7-membered heteroaryl is independently a methoxy, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 5-20 Aril, C 5-20 Heteroaryl, C 3-6 It can be substituted with cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl and 5- to 7-membered heteroaryl; R2, R3, R5, R2 ' , R3 ' and R5 ' are independently H, R m , OH, OR m , SH, SR m , NH2, NHR m , NR m R m' Selected from , NO2, Me3Sn and Halo; R4 and R4 ' are independently H, R m , OH, OR m , SH, SR m , NH2, NHR m , NR m R m' , NO2, Me3Sn, halo, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted C 3-6 Cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, substituted or unsubstituted C 5-12 Aryl, substituted or unsubstituted 5- to 7-membered heteroaryl, -CN, -NCO, -OR n , -OC(O)R n , -OC(O)NR n R n' , -OS(O)R n , -OS(O)2R n , -SR n , -S(O)R n , -S(O)2R n , -S(O)NR n R n' , -S(O)2NR n R n' , -OS(O)NR n R n' , -OS(O)2NR n R n' , -NR n R n' , -NR n C(O)R o , -NR n C(O)OR o , -NR n C(O)NR o R o' , -NR n S(O)R o , -NR n S(O)2R o , -NR n S(O)NR o R o' , -NR n S(O)2NR o R o' , -C(O)R n , -C(O)OR n and -C(O)NR n R n' Selected from, and the above C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 The hydrogen atoms of aryls and 5- to 7-membered heteroaryls are C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 In the case of aryls and 5- to 7-membered heteroaryls, each independently C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 Aryl, 5- to 7-membered heteroaryl, -OR p , -OC(O)R p , -OC(O)NR p R p' , -OS(O)R p , -OS(O)2R p , -SR p , -S(O)R p , -S(O)2R p , -S(O)NR p R p' , -S(O)2NR p R p' , -OS(O)NR p R p' , -OS(O)2NR p R p' , -NR p R p' , -NR p C(O)R q , -NR p C(O)OR q , -NR p C(O)NR q H, -NR p S(O)R q , -NR p S(O)2R q , -NR p S(O)NR q H, -NR p S(O)2NR q H, -C(O)R p , -C(O)OR p or -C(O)NR p R p It can be replaced with; R n , R n' , R o , R o' , R p , R p' and R q H and C are independently 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkinyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Selected from aryls and 5- to 7-membered heteroaryls; X is selected from -C(O)O-, -S(O)O-, -C(O)-, -C(O)NR-, -S(O)2NR-, -P(O)R'NR-, -S(O)NR- and -PO2NR-; Xa is a bonded or substituted or unsubstituted C 1-6 It is an alkylene, and the above C 1-6 When alkylene is substituted, C 1-8 alkyl or C 3-8 Substituted with a cycloalkyl; R and R' are each independently H, OH, NH2, ONH2, NHNH2, substituted or unsubstituted C 1-8 Alkyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted C 1-8 Alkoxy, substituted or unsubstituted C 1-8 Alkylthio, substituted or unsubstituted C 3-20 Heteroaryl, substituted or unsubstituted C 5-20 Aryl or mono- or di-C 1-8 It is an alkylamino, and the above C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, C 3-20 Heteroaryl and C 5-20 When aryls are substituted, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halo, C 1-6 Alkyl, C 1-6 Alkoxy and C 6-12 Substituted with a substituent selected from aryl; Y and Y' are each independently selected from O, S and N(H); and R6 is a substituted or unsubstituted saturated or unsaturated C 3-12 It is a hydrocarbon chain, said chain may be interrupted by one or more heteroatoms, NMe, or substituted or unsubstituted aromatic rings, said chain or aromatic ring at the positions of one or more hydrogen atoms of said chain or aromatic ring -NH, -NR m , -NHC(O)R m , -NHC(O)CH2-[OCH2CH2] n -R or -[CH2CH2O] n It may be substituted with or unsubstituted with -R, and the above R m and R are each the R defined above. m and equal to R, where n is 1 to 12; R7 and R7 ' Each is independently H, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted C 3-6 Cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5- to 7-membered heteroaryl, -OR r , -OC(O)R r , -OC(O)NR r R r' , -OS(O)R r , -OS(O)2R r , -SR r , -S(O)R r , -S(O)2R r , -S(O)NR r R r' , -S(O)2NR r R r' , -OS(O)NR r R r' , -OS(O)2NR r R r' , -NR r R r' , -NR r C(O)R s , -NR r C(O)OR s , -NR r C(O)NR s R s' , -NR r S(O)R s , -NR r S(O)2R s , -NR r S(O)NR s R s' , -NR r S(O)2NR s R s , -C(O)R r , -C(O)OR s or -C(O)NR r R r' and, the above C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 The hydrogen atoms of aryls and 5- to 7-membered heteroaryls are C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 In the case of aryls and 5- to 7-membered heteroaryls, each independently C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl, 5- to 7-membered heteroaryl, -OR t , -OC(O)R t , -OC(O)NR t R t' , -OS(O)R t , -OS(O)2R t , -SR t , -S(O)R t , -S(O)2R t , -S(O)NR t R t' , -S(O)2NR t R t' , -OS(O)NR t R t' , -OS(O)2NR t R t' , -NR t R t' , -NR t C(O)R u , -NR t C(O)OR u , -NR t C(O)NR u R u' , -NR t S(O)R u , -NR t S(O)2R u , -NR t S(O)NR u R u' , -NR t S(O)2NR u R u' , -C(O)R t , -C(O)OR t or -C(O)NR t R t' It can be replaced with;R r , R r' , R s , R s' , R t , R t' , R u and R u' H and C are independently 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkinyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 Selected from aryls and 5- to 7-membered heteroaryls; G is a glucuronide group or a galactoside group; and each Z is H, C 1-8 Alkyl, Halo, NO2, CN, and Selected from; R9, R 10 and R 16 H and C are independently 1-8 Alkyl, C 2-6 Alkenyl, C 1-6 Selected from alkoxy and alkyloxyalkyl; n30 is 1 to 3. Claim 120 A composite according to claim 119, wherein Y is O. Claim 121 A conjugate according to claim 119 or 120, wherein Y' is O. Claim 122 In any one of claims 119 to 121, the dotted line is R1 and R7 or R1 ' and R7 ' A conjugate that indicates the presence of a double bond between carbons having Claim 123 In any one of claims 119 to 122, the R1 is substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted C 5-7 Aryl and substituted or unsubstituted C 3-6 A conjugate selected from heteroaryls. Claim 124 A conjugate according to any one of claims 119 to 123, wherein R2, R3 and R5 are each independently H or OH. Claim 125 In any one of claims 119 to 124, the R4 is C 1-6 Alkoxyne, conjugate. Claim 126 A conjugate according to any one of claims 119 to 124, wherein R4 is methoxy, ethoxy, or butoxy. Claim 127 A conjugate according to any one of claims 119 to 126, wherein X is selected from -C(0)0-, -C(O)- and -C(0)NR-. Claim 128 A conjugate according to any one of claims 119 to 127, wherein X is -C(O)NR-. Claim 129 In any one of claims 119 to 128, the R6 is a substituted or unsubstituted saturated or unsaturated C 3-8 It is a hydrocarbon chain, wherein one or more carbon atoms of the hydrocarbon chain are substituted with a heteroatom or a substituted or unsubstituted aromatic ring, wherein the heteroatom is O, S or N(H), and the aromatic ring is benzene, pyridine, imidazole, or pyrazole, and wherein the chain or aromatic ring is -NHC(O)CH2-[OCH2CH2] at the position of one or more hydrogen atoms of the chain or aromatic ring. n -R or -[CH2CH2O] n A conjugate that can be substituted with -R, where n is 1 to 6. Claim 130 A composite according to any one of claims 119 to 129, wherein n is 1 to 6. Claim 131 In any one of claims 119 to 130, the Xa is a combination or C 1-3 Alkylene, conjugate. Claim 132 In any one of claims 119 to 131, the Z is H, and And, the above R9, R 10 and R 16 H and C are independently 1-3 Alkyl, C 1-3 A conjugate selected from alkoxy and alkyloxymethyl. Claim 133 A conjugate according to claim 132, wherein R9 is a methyloxyalkyl. Claim 134 In claim 132 or claim 133, the R 10 A conjugate that is methyloxyalkyl. Claim 135 In any one of claims 132 to 134, the R 16 A conjugate that is methyloxyalkyl. Claim 136 In any one of claims 132 to 135, the R9, R 10 or R 16 -(CH2CH2O) m -(CH2) m2 CH3, additionally, m is 1 to 6 and m2 is 0 to 2, a composite. Claim 137 A composite of claim 136, wherein m is 1. Claim 138 A composite according to claim 136 or 137, wherein m2 is 0. Claim 139 A composite according to claim 119, wherein R2 is H. Claim 140 A composite according to claim 139, wherein R3 is H. Claim 141 A composite according to claim 139 or 140, wherein R7 is H. Claim 142 A conjugate according to any one of claims 139 to 141, wherein R4 is an alkoxy (e.g., methoxy). Claim 143 A conjugate according to any one of claims 139 to 142, wherein R5 is OH. Claim 144 A conjugate according to any one of claims 139 to 143, wherein R1 is =CH2, CH3 or phenyl and optionally substituted with methoxy. Claim 145 A composite according to any one of claims 139 to 144, wherein Y is O. Claim 146 A composite according to any one of claims 139 to 145, wherein R2' is H. Claim 147 A composite according to any one of claims 139 to 146, wherein R3' is H. Claim 148 A composite according to any one of claims 139 to 147, wherein R7' is H. Claim 149 A conjugate according to any one of claims 139 to 148, wherein R4' is an alkoxy (e.g., methoxy). Claim 150 A conjugate according to any one of claims 139 to 149, wherein R5' is OH. Claim 151 A conjugate according to any one of claims 139 to 150, wherein R1' is =CH2, CH3 or phenyl and optionally substituted with methoxy. Claim 152 A conjugate according to any one of claims 139 to 151, wherein Y' is O. Claim 153 A conjugate according to any one of claims 139 to 152, wherein X is -C(O)O-. Claim 154 A conjugate according to any one of claims 139 to 153, wherein Xa is CH2. Claim 155 A conjugate according to any one of claims 139 to 154, wherein G is a glucuronide group. Claim 156 In claim 155, the G is Phosphorus, conjugate. Claim 157 A composite according to any one of claims 139 to 156, wherein n30 is 1. Claim 158 In any one of claims 139 to 157, the Z is or Phosphorus, conjugate. Claim 159 A composite according to claim 158, wherein R9 is H. Claim 160 In claim 158 or 159, the R 16 A conjugate that is an alkyloxyalkyl (e.g., methoxyethyl). Claim 161 In any one of claims 139 to 157, the Z is Phosphorus, conjugate. Claim 162 In claim 161, the R 10 A conjugate that is an alkyl (e.g., methyl). Claim 163 In claim 161, the R6 is C 3-12 A conjugate that is an alkyl (e.g., pentyl). Claim 164 In claim 118, the pyrrolobenzodiazepine dimer is , , , and A conjugate selected from, wherein X'' is -OMe or -NH2. Claim 165 A pharmaceutical composition comprising a conjugate of any one of claims 1 to 164 and a pharmaceutically acceptable excipient. Claim 166 A pharmaceutical composition for the prevention or treatment of a proliferative disease comprising a conjugate of any one of claims 1 to 164. Claim 167 A pharmaceutical composition according to claim 166, wherein the proliferative disease is cancer. Claim 168 A pharmaceutical composition according to claim 167, wherein the cancer is selected from liver cancer, thyroid cancer, ovarian cancer, brain cancer, multiple myeloma, colon cancer, head and neck cancer, lymphoma, leukemia, bladder cancer, kidney cancer, stomach cancer, breast cancer, uterine cancer, prostate cancer, pancreatic cancer, lung cancer, sarcoma, neuroendocrine tumor, melanoma, and combinations thereof. Claim 169 A pharmaceutical composition according to claim 168, wherein the lymphoma or leukemia is selected from Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), T-cell lymphoma, B-cell lymphoma, natural killer cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, lymphoblastic lymphoma, enteropathic intestinal lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, peripheral T-cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and combinations thereof. Claim 170 A pharmaceutical composition according to claim 169, wherein the acute lymphoblastic leukemia (ALL) is B-cell acute lymphoblastic leukemia (B-ALL). Claim 171 A method for the prevention or treatment of a proliferative disease comprising the step of administering a conjugate of any one of claims 1 to 164 or a pharmaceutically acceptable salt thereof to an individual in need thereof. Claim 172 In claim 171, the method wherein the proliferative disease is cancer. Claim 173 A method according to claim 172, wherein the cancer is selected from liver cancer, thyroid cancer, ovarian cancer, brain cancer, multiple myeloma, colon cancer, head and neck cancer, lymphoma, leukemia, bladder cancer, kidney cancer, stomach cancer, breast cancer, uterine cancer, prostate cancer, pancreatic cancer, lung cancer, sarcoma, neuroendocrine tumor, melanoma, and combinations thereof. Claim 174 The method of claim 173, wherein the lymphoma or leukemia is selected from Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), T-cell lymphoma, B-cell lymphoma, natural killer cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, lymphoblastic lymphoma, enteropathic intestinal lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, peripheral T-cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and combinations thereof. Claim 175 The method of claim 174, wherein the acute lymphoblastic leukemia (ALL) is a B-cell acute lymphoblastic leukemia (B-ALL).

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