Anti-CNTN4 conjugate comprising antibody and use thereof

Abstract

The present invention relates to an antibody-drug conjugate comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof, and a method for producing same. The present invention also relates to a use of the antibody or antigen-binding fragment thereof or the antibody-drug conjugate for preventing, ameliorating, or treating diseases (for example, cancer) associated with CNTN4 function or expression.

Description

Conjugates comprising anti-CNTN4 antibodies and uses thereof

[0001] The present invention relates to a conjugate (e.g., an antibody-drug conjugate) comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof that specifically binds to CNTN4 protein, a pharmaceutical composition comprising the conjugate, and a method for preparing and using the conjugate.

[0002] In addition, the conjugate of the present invention can be used for the prevention, improvement or treatment of diseases related to the function or expression of the CNTN4 protein, such as cancer.

[0003] This invention claims the benefit of Korean Patent Application No. 10-2024-0044949, filed April 2, 2024, Korean Patent Application No. 10-2024-0153281, filed November 1, 2024, and Korean Patent Application No. 10-2025-0042404, filed April 1, 2025, the entire contents of which are incorporated herein by reference.

[0004] The tumor microenvironment is known to influence tumor growth, metastasis, and interactions with the immune system. The tumor microenvironment is a comprehensive concept encompassing not only the constituent cell populations within the tumor, such as vascular cells, stromal cells, and immune cells, but also the surrounding environment (slightly acidified and hypoxic).

[0005] Meanwhile, the interaction between T cells and immune checkpoint proteins is known to cause changes in the tumor microenvironment. Furthermore, immune checkpoint inhibitors, which block the activation of these immune checkpoint proteins, exert their anticancer effects by activating T cells. Therefore, immune checkpoint inhibitors targeting various immune checkpoint proteins are being developed, and in this process, the anti-CTLA-4 monoclonal antibody YERVOY has been developed. ®(Active ingredient: ipilimumab), OPDIVO, a PD-1 monoclonal antibody ® (Active name: nivolumab) are already on the market. However, the use of the above-mentioned commercially available immune checkpoint inhibitors has the disadvantage of showing a strong therapeutic response only in some patients, and the target proteins of the commercially available immune checkpoint inhibitors are not inhibited in all tumors.

[0006] Furthermore, even monoclonal antibodies that bind specifically to cancer cells do not exhibit efficacy in all patients. Therefore, the development of new treatments with improved anticancer effects while maintaining a similar form to monoclonal antibodies is required. One such new treatment is the antibody-drug conjugate (ADC). ADCs bind to antigens specifically expressed on the surface of cancer cells, bind to target cancer cells, and then undergo internalization, entering the cancer cells. After internalization, degradative enzymes present in intracellular lysosomes degrade the bond between the drug and antibody constituting the ADC, allowing the drug to exert an additional cancer cell-killing effect. Therefore, the use of ADCs is expected to have an increased anticancer effect compared to the use of antibodies alone. Therefore, since the FDA approved Mylotag® (Gemcitabine ozogamicin) as the first ADC in 2000, various types of ADCs with different target antigens have been developed, and the development of targeted treatments for various diseases, such as inflammatory diseases, is active in addition to anticancer agents.

[0007] Effective ADC development requires solving technical challenges, including selecting target antigens specifically expressed in cancer cells, screening antibodies with excellent cancer cell internalization ability, and selecting appropriate linkers that are stable in plasma and enable drug release into target cells.

[0008] Meanwhile, CNTN4 (contactin family immunoglobulin member), a GPI (Glycosyl phosphatidylinositol)-anchored membrane protein in the brain, is involved in cell adhesion between T cells and antigen-presenting cells and T cell proliferation. CNTN4 has a low expression in normal cells or immune cells, but is expressed in various tumor cells, and it has been reported that CNTN4 can function as an immune checkpoint protein (Mi Young Cha et al. CNTN4 is a novel immune checkpoint protein that inhibits proliferation of T cells by interacting with APP, Cancer Res (2021) 81 (13_Supplement): 492).

[0009] However, whether CNTN4 is suitable as a target antigen for ADC, an antibody that specifically binds to CNTN4 and has excellent internalization ability, and an ADC manufactured using the same are unknown.

[0010] There is a need to develop novel anticancer therapeutics that can inhibit tumor growth or kill tumors by selectively binding to proteins whose expression is observed in cancer cells and tumor-specifically and releasing therapeutic moieties, such as cytotoxic drugs, through an internalization process that allows them to enter the cell.

[0011] The inventors of the present invention have confirmed that an antibody-drug conjugate prepared by applying a cytotoxic therapeutic moiety and a linking group to an anti-CNTN4 antibody can release the therapeutic moiety within cancer cells in the body, and have completed the present invention.

[0012] [1] In one aspect, the present invention relates to an antibody-drug conjugate represented by general formula I.

[0013] [General Formula I]

[0014] M-[LD]n

[0015] The above M is an anti-CNTN4 antibody or an antigen-binding fragment thereof that binds to the CNTN4 protein, the above L is a linker, the above D is a therapeutic moiety, and the above n is an average binding number per antibody of the LD structure binding to the antibody, and is in the range of 1 to 20.

[0016] [2] In the above [1], the anti-CNTN4 antibody may be an anti-CNTN4 antibody or an antigen-binding fragment thereof comprising a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 2; a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 3; a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 4; a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 6.

[0017] [3] In the above [1], the antibody or antigen-binding fragment thereof may include a heavy chain variable region including an amino acid sequence having 95% or more sequence homology with the amino acid sequence of SEQ ID NO: 7 and a light chain variable region including an amino acid sequence having 95% or more sequence homology with the amino acid sequence of SEQ ID NO: 8.

[0018] [4] In the above [3], the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 8.

[0019] [5] In any one of [1] to [4] above, the antibody or antigen-binding fragment thereof may comprise an amino acid residue engineered for binding to or inhibiting binding of the linker at one or more selected positions.

[0020] [6] In any one of [1] to [5] above, the antibody or antigen-binding fragment thereof may include an amino acid residue engineered to bind or inhibit binding of the linker to the carboxyl terminal of the light or heavy chain.

[0021] [7] In the above [6], the amino acid residue may be selected from a group of peptide moieties including alanine, cysteine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, or arginine.

[0022] [8] In any one of the above [1] to [7], the linker may include a cleavable peptide moiety.

[0023] [9] In the above [8], the cleavable peptide moiety may be cleavable by an enzyme.

[0024]

[0010] In any one of the above [1] to [9], the linking group may include an amino acid unit.

[0025]

[0011] In the above

[0010] , the amino acid unit is valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), alanine-alanine (Ala-Ala), alanine-citrulline (Ala-Cit), citrulline-alanine (Cit-Ala), asparagine-citrulline (Asn-Cit), citrulline-asparagine (Cit-Asn), citrulline-citrulline (Cit-Cit), valine-glutamic acid (Val-Glu), glutamic acid-valine (Glu-Val), serine-citrulline (Ser-Cit), citrulline-serine (Cit-Ser), lysine-citrulline (Lys-Cit), citrulline-lysine (Cit-Lys), aspartic acid-citrulline (Asp-Cit), citrulline-aspartic acid (Cit-Asp), Alanine-valine (Ala-Val), valine-alanine (Val-Ala), phenylalanine-lysine (Phe-Lys), lysine-phenylalanine (Lys-Phe), valine-lysine (Val-Lys), lysine-valine (Lys-Val), alanine-lysine (Ala-Lys), lysine-alanine (Lys-Ala), phenylalanine-citrulline (Phe-Cit), citrulline-phenylalanine (Cit-Phe), leucine-citrulline (Leu-Cit), citrulline-leucine (Cit-Leu), isoleucine-citrulline (Ile-Cit), citrulline-isoleucine (Cit-Ile), phenylalanine-arginine (Phe-Arg), arginine-phenylalanine (Arg-Phe), citrulline-tryptophan (Cit-Trp), It may be selected from the group consisting of tryptophan-citrulline (Trp-Cit), alanine-alanine-aspartic acid (Ala-Ala-Asp), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), and glutamic acid-valine-citrulline (Glu-Val-Cit), and preferably from the group consisting of valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), alanine-valine (Ala-Val), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), and glutamic acid-valine-citrulline (Glu-Val-Cit).

[0026]

[0012] In any one of the above [1] to

[0011] , the linking group may include a direct bond, or a moiety including a succinic acid derivative or a pyrimidine.

[0027]

[0013] In the above

[0012] , the moiety including the succinic acid derivative may be a succinimide moiety, a succinamic acid moiety, or a succinamic ester moiety.

[0028]

[0014] In the above

[0013] , the succinimide moiety may include succinimidocaproyl, succinimidopropanoyl or succinimidobutanoyl.

[0029]

[0015] In any one of the above [1] to

[0014] , the linker may optionally include a polyethylene glycol (PEG) moiety.

[0030]

[0016] In the above

[0015] , the polyethylene glycol (PEG) moiety is -(CH2CH2O) m - may contain a structure represented by, where m may be an integer from 1 to 24.

[0031]

[0017] In any one of the above [1] to

[0016] , the linking group comprises one or more alkylene, alkenylene, alkynylene, cycloalkylene or arylene, and any carbon atom of the alkylene, alkenylene, alkynylene, cycloalkylene or arylene may be optionally replaced with one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom.

[0032]

[0018] In any one of the above [1] to

[0017] , the linking group may include a moiety represented by the following general formula II:

[0033] [General Formula II]

[0034]

[0035] In the above general formula II,

[0036] The above a is an integer from 0 to 24,

[0037] The above b is 0 or 1,

[0038] The above X comprises one or more linked groups selected from the group consisting of alkylene, alkenylene, alkynylene, cycloalkylene and arylene, and the alkylene and cycloalkylene constituting the above X are C 1-30 Alkyl or C 3-30 is unsubstituted or substituted with cycloalkyl, and any carbon atom of said alkylene, alkenylene, alkynylene, cycloalkylene or arylene is optionally replaced by one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom,

[0039] wherein Q is a direct bond or a moiety containing a succinic acid derivative or a pyrimidine;

[0040] The above Q is linked to an antibody.

[0041]

[0019] In the above

[0018] , X is C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 1-30 alkylene-C 3-30 Cycloalkylene, C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene-OC 1-30 Alkylene, C 1-30 Alkylene-OC1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 6-30 Arylene, C 6-30 Arylene-C 1-30 Alkylene, C 1-30 alkylene-C 6-30 Arylene-C 1-30 C selected from the group consisting of alkylene and constituting said X 1-30 Alkylene and C 3-30 Cycloalkylene is C 1-30 Alkyl or C 3-30 Substituted or unsubstituted with cycloalkyl, and the above C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene or C 6-30 Any carbon atom of arylene may be optionally replaced by one or more of a carbonyl, an oxygen atom, or a nitrogen atom.

[0042]

[0020] In any one of [1] to

[0019] above, the linker may optionally include a self-immolative moiety.

[0043]

[0021] In any one of the above [1] to

[0020] , the linking group does not include a self-immolative moiety, and the linking group is a succinimide moiety or a succinamic acid, glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) and -(CH2CH2O) m - (wherein, m is an integer from 1 to 24) may include a polyethylene glycol moiety.

[0044]

[0022] In the above

[0020] , the self-immolative moiety may be selected from the group consisting of p-aminobenzyl (PAB), unsubstituted or substituted p-aminobenzyloxycarbonyl (PABC), aminomethylene, N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC), and N-succinimidyl(4-iodo-acetyl)aminobenzoate (SIAB).

[0045]

[0023] In the above

[0022] , the self-immolative moiety is aminomethylene or unsubstituted or substituted PABC, and optionally, the PABC is -C 1-6 Alkylene-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 It may be substituted with alkyl, and m may be an integer from 1 to 24.

[0046]

[0024] In any one of the above [1] to

[0023] , the linking group may include a moiety represented by the following general formula III:

[0047] [General Formula III]

[0048]

[0049] In the above general formula III,

[0050] The above a is an integer from 0 to 24,

[0051] The above b and c are 0 or 1, respectively,

[0052] The above Lp is a self-immolative moiety, and the self-immolative moiety is -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 A group represented by alkyl or -(CH2CH2O) m -C 1-6 It can be substituted with a group represented by alkyl, and m is an integer from 1 to 24,

[0053] The above La is valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), alanine-alanine (Ala-Ala), alanine-citrulline (Ala-Cit), citrulline-alanine (Cit-Ala), asparagine-citrulline (Asn-Cit), citrulline-asparagine (Cit-Asn), citrulline-citrulline (Cit-Cit), valine-glutamic acid (Val-Glu), glutamic acid-valine (Glu-Val), serine-citrulline (Ser-Cit), citrulline-serine (Cit-Ser), lysine-citrulline (Lys-Cit), citrulline-lysine (Cit-Lys), aspartic acid-citrulline (Asp-Cit), citrulline-aspartic acid (Cit-Asp), alanine-valine (Ala-Val), Valine-Alanine (Val-Ala), Phenylalanine-Lys (Phe-Lys), Lysine-Phenylalanine (Lys-Phe), Valine-Lys (Val-Lys), Lysine-Valine (Lys-Val), Alanine-Lys (Ala-Lys), Lysine-Alanine (Lys-Ala), Phenylalanine-Citrulline (Phe-Cit), Citrulline-Phenylalanine (Cit-Phe), Leucine-Citrulline (Leu-Cit), Citrulline-Leucine (Cit-Leu), Isoleucine-Citrulline (Ile-Cit), Citrulline-Isoleucine (Cit-Ile), Phenylalanine-Arginine (Phe-Arg), Arginine-Phenylalanine (Arg-Phe), Citrulline-Tryptophan (Cit-Trp), An amino acid unit selected from the group consisting of tryptophan-citrulline (Trp-Cit), alanine-alanine-aspartic acid (Ala-Ala-Asp), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), and glutamic acid-valine-citrulline (Glu-Val-Cit).

[0054] The above X is C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene and C 6-30 Containing one or more groups connected to each other selected from the group consisting of arylene, and C 1-30 Alkylene, C 2-30 Alkenylene, C2-30 Alkynylene, C 3-30 Cycloalkylene or C 6-30 Any carbon atom of arylene is optionally replaced by one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom,

[0055] wherein Q is a direct bond, a succinimide moiety, a succinamic acid moiety, a succinamic ester moiety, or a pyrimidine moiety;

[0056] The above Q is linked to an antibody.

[0057]

[0025] In the above

[0024] , the linking group may include a moiety represented by the following general formula III', general formula III'', general formula III'' or general formula III''''.

[0058] [General Formula III']

[0059]

[0060] [General Formula III'']

[0061]

[0062] [General Formula III''']

[0063]

[0064] [General Formula III'''']

[0065]

[0066] In the above general formulas III', III'', III''' and III'''',

[0067] The above X' is hydrogen, C 1-30 Alkyl, C 3-30 Cycloalkyl or C 1-30 Alkyl-C 3-30 It is cycloalkyl,

[0068] The above a, b, c, Lp, La and X are as defined in the general formula III,

[0069] * is linked to an antibody.

[0070]

[0026] In the above

[0024] , the X is C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 1-30 alkylene-C 3-30 Cycloalkylene, C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene-OC 1-30 Alkylene, C 1-30 Alkylene-OC 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 6-30 Arylene, C 6-30 Arylene-C 1-30 Alkylene and C 1-30 alkylene-C 6-30 Arylene-C 1-30 C selected from the group consisting of alkylene and constituting said X 1-30 Alkylene and C 3-30 Cycloalkylene is C 1-30 Alkyl or C 3-30 Substituted or unsubstituted with cycloalkyl, and the above C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene or C 6-30 Any carbon atom of arylene may be optionally replaced by one or more of a carbonyl, an oxygen atom, or a nitrogen atom.

[0071]

[0027] In the above

[0024] , c is 1, Lp is aminomethylene, or -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 PABC which is unsubstituted or substituted with a group represented by alkyl (m is an integer from 1 to 12), and La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

[0072]

[0028] In the above

[0027] , c is 1, and Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 PABC substituted with a group represented by alkyl (m is an integer from 1 to 12), and La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

[0073]

[0029] In the above

[0024] , the Q is a succinimide moiety, the c is 0 or 1, and the Lp is aminomethylene, or -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6PABC which is unsubstituted or substituted with a group represented by alkyl (m is an integer from 1 to 12), and La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

[0074]

[0030] In the above

[0029] , the Q may be a succinimide moiety, the c may be 0, and the La may be glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0075]

[0031] In the above

[0024] , the Q is a succinic acid moiety, the c is 0 or 1, and the Lp is aminomethylene or -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 PABC which is unsubstituted or substituted with a group represented by alkyl (m is an integer from 1 to 12), and La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

[0076]

[0032] In the above

[0024] , the Q is a pyrimidine moiety, the c is 1, and the Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6PABC substituted with a group represented by alkyl (m is an integer from 1 to 12), and La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

[0077]

[0033] In the above

[0024] , the Q is a direct bond, the c is 1, and the Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 PABC substituted with a group represented by alkyl (m is an integer from 1 to 12), and La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

[0078]

[0034] In the above

[0024] , c is 1, and Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 PABC substituted with a group represented by alkyl (m is an integer from 1 to 12), wherein X is C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 alkylene-C 6-30 Arylene, C1-30 alkylene-C 6-30 Arylene-C 1-30 Alkylene, C 6-30 Arylene-C 1-30 Alkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 3-30 Cycloalkylene and C 6-30 C selected from the group consisting of arylene and constituting said X 1-30 Alkylene, C 6-30 Arylene or C 3-30 Any carbon atom of cycloalkylene is optionally replaced with one or more of carbonyl, oxygen, or nitrogen atoms, and La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

[0079]

[0035] In any one of the above [1] to

[0034] , the linking group may include a sugar or a sugar acid, and the sugar or sugar acid may be linked by a bond susceptible to enzymatic cleavage in vivo.

[0080]

[0036] In any one of the above

[0012] to

[0035] , the direct bond, or the moiety comprising a succinic acid derivative or a pyrimidine can be attached to the anti-CNTN4 antibody or antigen-binding fragment thereof via a cysteine ​​residue on the antibody or antigen-binding fragment thereof.

[0081]

[0037] In any one of the above [1] to

[0036] , the linking group is a combination of succinimidocaproyl, valine-citrulline (Val-Cit), and PABC; a combination of succinimidocaproyl, Val-Ala, and PABC; a combination of succinimidocaproyl, Val-Ala, and aminomethylene; a combination of succinimidocaproyl, Gly-Gly-Phe-Gly, and aminomethylene; a combination of succinimidocaproyl, Val-Ala, and PABC comprising a -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituent; a combination of succinimidopropanoyl, polyethylene glycol (PEG), and valine-alanine (Val-Ala); a combination of succinimidopropanoyl, PEG, Val-Ala, and PABC; Combinations of succinimidopropanamide, PEG, carbonyl, and Val-Ala; combinations of succinimidopropanamide, PEG, carbonyl, Val-Ala, and PABC; combinations of succinimidopropanamide, alkyl-carbonyl, and Val-Ala; combinations of succinimidopropanamide, alkyl-carbonyl, Val-Ala, and PABC; combinations of succinimidopropanamide, alkyl-carbonyl, PEG, and Val-Ala; combinations of succinimidopropanamide, alkyl-carbonyl, PEG, Val-Ala, and PABC; combinations of succinimidopropanamide, alkyl-amide, PEG, and Val-Ala; combinations of succinimidopropanamide, alkyl-amide, PEG, Val-Ala, and PABC; combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and PABC; Combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Val-Ala, and PABC; combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and aminomethylene; combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Val-Ala, and aminomethylene; combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and PABC; combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Gly-Gly-Phe-Gly, and PABC;Combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly and aminomethylene; combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Gly-Gly-Phe-Gly and aminomethylene; combinations of succinimidopropanamide, PEG, alkyl-carbonyl and Gly-Gly-Phe-Gly; combinations of succinimidopropanamide, alkyl, PEG, carbonyl and Gly-Gly-Phe-Gly; combinations of succinamic acid, alkyl-carbonyl, Val-Ala and PABC; combinations of succinamic acid, alkyl-carbonyl, Val-Ala and aminomethylene; combinations of succinamic acid, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala and PABC; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala, and aminomethylene; combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, and Val-Ala; combinations of succinic acid, alkyl-carbonyl, Gly-Gly-Phe-Gly, and PABC; combinations of succinic acid, alkyl-carbonyl, Gly-Gly-Phe-Gly, and aminomethylene; combinations of succinic acid, alkyl-carbonyl, and Gly-Gly-Phe-Gly; combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and PABC; combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and aminomethylene; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, and Gly-Gly-Phe-Gly; combinations of succinic acid, alkyl-carbonyl, Val-Ala, and PABC containing -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; combinations of succinic ester, PEG, carbonyl, and Val-Ala; combinations of succinic ester, PEG, carbonyl, Val-Ala, and aminomethylene; combinations of succinic ester, PEG, amide, and Val-Ala; combinations of succinic ester, PEG, amide, Val-Ala, and aminomethylene; combinations of succinic ester, alkyl-carbonyl, and Val-Ala;Combination of succinamic ester, alkyl-carbonyl, Val-Ala and aminomethylene; combination of succinamic ester, alkyl-amide and Val-Ala; combination of succinamic ester, alkyl-amide, Val-Ala and aminomethylene; combination of succinamic ester, alkyl, PEG and Val-Ala; combination of succinamic ester, alkyl, PEG, Val-Ala and aminomethylene; combination of succinamic ester, alkyl-carbonyl, PEG and Val-Ala; combination of succinamic ester, alkyl-carbonyl, PEG, Val-Ala and aminomethylene; combination of succinamic ester, alkyl-amide, PEG and Val-Ala; combination of succinamic ester, alkyl-amide, PEG and Val-Ala; combination of succinamic ester, alkyl-amide, PEG and Val-Ala; combination of succinamic ester, alkyl-amide, PEG, Val-Ala and aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, a carbonyl, and Val-Ala; A combination of a succinamic ester, an alkyl-amide, a PEG, a carbonyl, a Val-Ala, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, and Val-Ala; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Val-Ala, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Val-Ala, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Gly-Gly-Phe-Gly, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Gly-Gly-Phe-Gly, and an aminomethylene; Combinations of PABCs comprising succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents;combinations of PABCs comprising succinic esters, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; combinations of PABCs comprising pyrimidines, alkynyls, carbonyls, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; combinations of PABCs comprising alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; and combinations of PABCs comprising alkylamides, alkyl-cycloalkyl, carbonyls, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents.

[0082]

[0038] In any one of the above [1] to

[0037] , the linking group is a combination of succinimidocaproyl, valine-citrulline (Val-Cit), and PABC; a combination of succinimidocaproyl, Gly-Gly-Phe-Gly, and aminomethylene; a combination of succinimidocaproyl, Val-Ala, and PABC comprising a -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituent; a combination of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and PABC; a combination of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and aminomethylene; a combination of succinimidopropanamide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and aminomethylene; Combinations of succinimidopropanamide, PEG, alkyl-carbonyl, and Gly-Gly-Phe-Gly; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala, and aminomethylene; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and aminomethylene; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, and Gly-Gly-Phe-Gly; Combinations of succinic acid, alkyl-carbonyl, Val-Ala, and PABC containing -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents;combinations of PABCs comprising succinic esters, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; combinations of PABCs comprising pyrimidines, alkynyls, carbonyls, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; combinations of PABCs comprising alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; and combinations of PABCs comprising alkylamides, alkyl-cycloalkyl, carbonyls, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents.

[0083]

[0039] In any one of the above [1] to

[0038] , n may be 1 to 15.

[0084]

[0040] In any one of the above [1] to

[0039] , the therapeutic moiety may be a cytotoxic agent, a prodrug of a cytotoxic agent, an immunostimulant, a protein degrader, a protein kinase inhibitor, or a radioactive isotope-labeled compound.

[0085]

[0041] In the above

[0040] , the cytotoxic agent may be a tubulin inhibitor.

[0086]

[0042] In the above

[0041] , the tubulin inhibitor may be an auristatin or maytansinoid series.

[0087]

[0043] In any one of [1] to

[0039] above, the therapeutic moiety may be a tubulin polymerase inhibitor.

[0088]

[0044] In the above

[0043] , the tubulin polymerase inhibitor may be monomethyloristatin F (MMAF) or monomethyloristatin E (MMAE).

[0089]

[0045] In the above

[0040] , the cytotoxic agent may be a DNA damaging agent.

[0090]

[0046] In the above

[0045] , the DNA damaging agent may be a topoisomerase inhibitor or a DNA alkylating agent.

[0091]

[0047] In any one of the above [1] to

[0039] , the therapeutic moiety may be a topoisomerase inhibitor.

[0092]

[0048] In any one of the above [1] to

[0039] , the therapeutic moiety may be a DNA alkylating agent.

[0093]

[0049] In the above

[0046] or

[0047] , the topoisomerase inhibitor may be a camptothecin analogue, and the camptothecin analogue may be irinotecan, rubitecan, topotecan, zimatecan, pegamotecan, lutotecan, karenitecan, apelletecan, homocamptothecin, diplomotecan, belotecan, 9-aminocamptothecin, exatecan, SN-38, DXd, a DXd derivative, or an exatecan analogue.

[0094]

[0050] In the above

[0049] , the camptothecin analogue may be exatecan, DXd, or a derivative thereof.

[0095]

[0051] In the above

[0046] or

[0048] , the DNA alkylating agent may be a pyrrolobenzodiazepine (PBD) dimer, a pyrrolobenzodiazepine (PBD) dimer analog, calicheamicins, a calicheamicin analog, duocarmycin, a duocarmycin analog, cyclophosphamide, ifosfamide, bendamustine, cisplatin, melphalan, or carboplatin.

[0096]

[0052] In any one of the above [1] to

[0039] , the therapeutic moiety may be monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), exatecan or DXd.

[0097]

[0053] In the above

[0052] , an antibody-drug conjugate represented by one of the following structures, wherein the antibody is an anti-CNTN4 antibody or an antigen-binding fragment thereof, and n is 1 to 20:

[0098] ;

[0099] ;

[0100] ;

[0101] ;

[0102] ;

[0103] ;

[0104] ;

[0105] ;

[0106] ;

[0107] ;

[0108] ;

[0109] ;

[0110] ;

[0111] ;

[0112] ;

[0113] ;

[0114] ;

[0115] ; and

[0116] .

[0117]

[0054] In another aspect, the present invention relates to a pharmaceutical composition for treating a disease associated with the function or expression of CNTN4 protein, comprising an antibody-drug conjugate of any one of [1] to

[0053] ; and a pharmaceutically acceptable carrier.

[0118]

[0055] In the above

[0054] , the disease related to the function or expression of the CNTN4 protein may be a cancer selected from the group consisting of leukemia, lymphoma, myeloma, bone and connective tissue sarcoma, brain cancer, breast cancer, adrenal cancer, thyroid cancer, pancreatic cancer, eye cancer, vaginal cancer, vulvar cancer, uterine cancer, ovarian cancer, esophageal cancer, stomach cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, lung cancer, testicular cancer, prostate cancer, penile cancer, oral cancer, salivary gland cancer, skin cancer, kidney cancer, bladder cancer, head and neck cancer, melanoma, appendix cancer, bronchial cancer, choriocarcinoma, chordoma, ependymoma, gastrointestinal stromal tumor (GIST), neuroendocrine cancer, malignant peripheral nerve sheath tumor, tongue cancer, small intestine cancer, heart cancer, duodenal cancer, parathyroid cancer, and urethral cancer.

[0119]

[0056] In the above

[0054] , the disease related to the function or expression of the CNTN4 protein may be a solid tumor.

[0120]

[0057] In another aspect, the present invention relates to a method for treating a subject having a disease associated with the function or expression of a CNTN4 protein, or suspected of having a disease associated with the function or expression of a CNTN4 protein, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate of any one of [1] to

[0053] or a pharmaceutical composition of any one of

[0054] to

[0056] .

[0121]

[0058] In another aspect, the present invention relates to the use of the antibody-drug conjugate of any one of [1] to

[0053] or the pharmaceutical composition of any one of

[0054] to

[0056] for the manufacture of a medicament for the treatment of a disease associated with the function or expression of CNTN4.

[0122]

[0059] In another aspect, the present invention relates to the use of the antibody-drug conjugate of any one of [1] to

[0053] or the pharmaceutical composition of any one of

[0054] to

[0056] for the treatment of a disease associated with the function or expression of CNTN4.

[0123] An antibody-drug conjugate comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof of the present invention binds to human CNTN4 protein with high affinity and has excellent tumor cell internalization activity and apoptosis activity.

[0124] In addition, the antibody-drug conjugate of the present invention can be usefully used for preventing, improving, or treating diseases (e.g., cancer) related to the function or expression of human CNTN4 protein.

[0125]

[0126] Figure 1 is a drawing showing the SEC-HPLC analysis results of Ab1 antibody, a type of anti-CNTN4 antibody.

[0127] Figure 2 is a drawing showing the SEC-HPLC chromatogram of Ab2 antibody, a type of anti-CNTN4 antibody.

[0128] Figure 3 is a drawing showing the results of SDS-PAGE analysis of Ab1 antibody and Ab2 antibody, which are types of anti-CNTN4 antibodies.

[0129] Figure 4 is a drawing showing the SEC-HPLC chromatogram of Ab3 antibody, a type of anti-CNTN4 antibody.

[0130] Figure 5 is a drawing showing the results of SDS-PAGE analysis of Ab3 antibody, a type of anti-CNTN4 antibody.

[0131] Figure 6 is a diagram showing changes in target binding affinity according to increasing concentrations of Ab1 antibody, a type of anti-CNTN4 antibody, and human IgG4 antibody (hIgG4), an isotype control.

[0132] Figure 7 is a drawing showing the change in MFI (Mean Fluorescence Intensity) according to antibody concentration, which is the result of analyzing the degree of binding of Ab1 antibody, a type of anti-CNTN4 antibody, and human IgG4 antibody (hIgG4), an isotype control, to the CNTN4 protein antigen using FACS.

[0133] Figure 8 is a diagram showing changes in cell fluorescence signals with increasing incubation time when the concentrations (0.5, 1, 2, or 4 μg / ml) of Ab1 antibody, a type of anti-CNTN4 antibody, and human IgG4 antibody (hIgG4), an isotype control, were varied in A549 cells overexpressing human CNTN4 antigen protein.

[0134] Figure 9 is a drawing showing the SEC-HPLC chromatogram of Ab-I, a type of anti-CNTN4 antibody-drug conjugate.

[0135] Figure 10 is a drawing showing the SEC-HPLC chromatogram of Ab-II, a type of anti-CNTN4 antibody-drug conjugate.

[0136] Figure 11 is a drawing showing a HIC-HPLC chromatogram of Ab-I, a type of anti-CNTN4 antibody-drug conjugate.

[0137] Figure 12 is a drawing showing a HIC-HPLC chromatogram of Ab-II, a type of anti-CNTN4 antibody-drug conjugate.

[0138] Figure 13 is a diagram illustrating an SEC-HPLC chromatogram of Ab-III, a type of anti-CNTN4 antibody-drug conjugate.

[0139] Figure 14 is a drawing showing a HIC-HPLC chromatogram of Ab-III, a type of anti-CNTN4 antibody-drug conjugate.

[0140] Figure 15 is a drawing showing the SEC-HPLC chromatogram of Ab-IV, a type of anti-CNTN4 antibody-drug conjugate.

[0141] Figure 16 is a diagram showing an LC-MS chromatogram of Ab-IV, a type of anti-CNTN4 antibody-drug conjugate.

[0142] Figure 17 is a diagram illustrating an SEC-HPLC chromatogram of Ab-V, a type of anti-CNTN4 antibody-drug conjugate.

[0143] Figure 18 is a diagram showing an LC-MS chromatogram of Ab-V, a type of anti-CNTN4 antibody-drug conjugate.

[0144] Figure 19 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-VI, a type of anti-CNTN4 antibody-drug conjugate.

[0145] Figure 20 is a drawing showing a SEC-HPLC 370 nm chromatogram of Ab-VI, a type of anti-CNTN4 antibody-drug conjugate.

[0146] Figure 21 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-VII, a type of anti-CNTN4 antibody-drug conjugate.

[0147] Figure 22 is a drawing showing the SEC-HPLC 370 nm chromatogram of Ab-VII, a type of anti-CNTN4 antibody-drug conjugate.

[0148] Figure 23 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-VIII, a type of anti-CNTN4 antibody-drug conjugate.

[0149] Figure 24 is a drawing showing the SEC-HPLC 370 nm chromatogram of Ab-VIII, a type of anti-CNTN4 antibody-drug conjugate.

[0150] Figure 25 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-IX, a type of anti-CNTN4 antibody-drug conjugate.

[0151] Figure 26 is a drawing showing the SEC-HPLC 370 nm chromatogram of Ab-IX, a type of anti-CNTN4 antibody-drug conjugate.

[0152] Figure 27 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-X, a type of anti-CNTN4 antibody-drug conjugate.

[0153] Figure 28 is a drawing showing the SEC-HPLC 370 nm chromatogram of Ab-X, a type of anti-CNTN4 antibody-drug conjugate.

[0154] Figure 29 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-XI, a type of anti-CNTN4 antibody-drug conjugate.

[0155] Figure 30 is a drawing showing a SEC-HPLC 370 nm chromatogram of Ab-XI, a type of anti-CNTN4 antibody-drug conjugate.

[0156] Figure 31 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-XII, a type of anti-CNTN4 antibody-drug conjugate.

[0157] Figure 32 is a drawing showing the SEC-HPLC 370 nm chromatogram of Ab-XII, a type of anti-CNTN4 antibody-drug conjugate.

[0158] Figure 33 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-XIII, a type of anti-CNTN4 antibody-drug conjugate.

[0159] Figure 34 is a drawing showing a SEC-HPLC 370 nm chromatogram of Ab-XIII, a type of anti-CNTN4 antibody-drug conjugate.

[0160] Figure 35 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-XIV, a type of anti-CNTN4 antibody-drug conjugate.

[0161] Figure 36 is a drawing showing a SEC-HPLC 370 nm chromatogram of Ab-XIV, a type of anti-CNTN4 antibody-drug conjugate.

[0162] Figure 37 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-XV, a type of anti-CNTN4 antibody-drug conjugate.

[0163] Figure 38 is a drawing showing a processing chromatogram of LC / MS to confirm the antibody-drug ratio of Ab-XV, a type of anti-CNTN4 antibody-drug conjugate.

[0164] Figure 39 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-XVI, a type of anti-CNTN4 antibody-drug conjugate.

[0165] Figure 40 is a drawing showing a processing chromatogram of LC / MS to confirm the antibody-drug ratio of Ab-XVI, a type of anti-CNTN4 antibody-drug conjugate.

[0166] Figure 41 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-XVII, a type of anti-CNTN4 antibody-drug conjugate.

[0167] Figure 42 is a drawing showing a processing chromatogram of LC / MS to confirm the antibody-drug ratio of Ab-XVII, a type of anti-CNTN4 antibody-drug conjugate.

[0168] Figure 43 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-XVIII, a type of anti-CNTN4 antibody-drug conjugate.

[0169] Figure 44 is a drawing showing a processing chromatogram of LC / MS to confirm the antibody-drug ratio of Ab-XVIII, a type of anti-CNTN4 antibody-drug conjugate.

[0170] Figure 45 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-XIX, a type of anti-CNTN4 antibody-drug conjugate.

[0171] Figure 46 is a drawing showing a processing chromatogram of LC / MS to confirm the antibody-drug ratio of Ab-XIX, a type of anti-CNTN4 antibody-drug conjugate.

[0172] Figure 47 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-XX, a type of anti-CNTN4 antibody-drug conjugate.

[0173] Figure 48 is a drawing showing a processing chromatogram of LC / MS to confirm the antibody-drug ratio of Ab-XX, a type of anti-CNTN4 antibody-drug conjugate.

[0174] Figure 49 is a drawing showing a SEC-HPLC 280 nm chromatogram of Ab-XXI, a type of anti-CNTN4 antibody-drug conjugate.

[0175] Figure 50 is a drawing showing a processing chromatogram of LC / MS to confirm the antibody-drug ratio of Ab-XXI, a type of anti-CNTN4 antibody-drug conjugate.

[0176] Figure 51 is a diagram showing changes in target binding affinity according to increasing concentration of Ab1 and Ab2 antibodies, which are types of anti-CNTN4 antibodies, human IgG4 antibody (hIgG4) and human IgG1 antibody (hIgG1) as isotype controls, and Ab-I and Ab-II, which are types of anti-CNTN4 antibody-drug conjugates.

[0177] Figure 52 is a drawing showing changes in binding affinity on the cell surface according to increasing treatment concentrations of Ab1 and Ab2 antibodies, which are types of anti-CNTN4 antibodies, human IgG4 antibodies (hIgG4) and human IgG1 antibodies (hIgG1) as isotype controls, and Ab-I and Ab-II, which are types of anti-CNTN4 antibody-drug conjugates.

[0178] Figure 53 is a diagram showing changes in cell fluorescence signals as the incubation time increases when the concentration (0.5, 1, 2, or 4 μg / ml) of Ab-I, a type of anti-CNTN4 antibody-drug conjugate, is varied.

[0179] Figure 54 is a diagram showing cell viability (%) according to anti-CNTN4 antibody-drug conjugate treatment in A549, A549 / CNTN4, HT-1080, HT-1080 / CNTN4, Pan02 / Cntn4, and Hs27 cells.

[0180] Figure 55 is a diagram showing cell viability (%) according to anti-CNTN4 antibody-drug conjugate treatment in HT-1080 and HT-1080 / CNTN4.

[0181] Figure 56 is a diagram showing cell viability (%) according to anti-CNTN4 antibody-drug conjugate treatment in HT-1080 / CNTN4.

[0182] Figure 57 is a diagram showing the area of ​​tumors in the pancreas in the negative control group administered PBS or the group administered 10 mg / kg of Ab-I, an anti-CNTN4 antibody-drug conjugate, in the Pan02 / Cntn4 mouse pancreatic cancer orthotopic transplantation model.

[0183] Figure 58 is a diagram showing the area of ​​intrapancreatic tumors in the negative control group administered PBS, the group administered 10 mg / kg or 20 mg / kg of anti-CNTN4 antibody-drug conjugate Ab-IV, the group administered 10 mg / kg of immunotherapy anti-mouse-PD-1, and the group administered a combination of 10 mg / kg of Ab-IV and 10 mg / kg of anti-mouse-PD-1 in the Pan02 / Cntn4 mouse pancreatic cancer orthotopic transplantation model.

[0184] Figure 59 is a diagram showing the change in tumor volume over time in the negative control group administered PBS, the group administered 20 mg / kg of anti-CNTN4 antibody-drug conjugate Ab-V, and the group administered 7.5 mg / kg of Ab-VI in the HT-1080 / CNTN4 human fibrosarcoma xenograft model.

[0185] Figure 60 is a diagram showing the change in tumor volume by date in the negative control group administered PBS, the group administered 5 mg / kg of anti-CNTN4 antibody-drug conjugate Ab-VII, the group administered 5 mg / kg of Ab-IX, and the group administered 10 mg / kg of Ab-XI in the HT-1080 / CNTN4 human fibrosarcoma xenograft model.

[0186] Figure 61 is a diagram showing the change in tumor volume by date in the negative control group administered PBS, the group administered 5 mg / kg of anti-CNTN4 antibody-drug conjugate Ab-V, the group administered 5 mg / kg of Ab-VII, the group administered 5 mg / kg of Ab-VIII, the group administered 5 mg / kg of Ab-IX, and the group administered 5 mg / kg of Ab-X in the HT-1080 / CNTN4 human fibrosarcoma xenograft model.

[0187] Figure 62 is a diagram showing the change in tumor volume by date in the negative control group administered PBS, the group administered 3 mg / kg of anti-CNTN4 antibody-drug conjugate Ab-V, the group administered 3 mg / kg of Ab-XIII, the group administered 3 mg / kg of Ab-XXI, and the group administered 5 mg / kg of Ab-XXI in the HT-1080 / CNTN4 human fibrosarcoma xenograft model.

[0188]

[0189] definition

[0190] The term "CNTN4" used herein refers to a contactin family immunoglobulin member and a GPI (Glycosyl phosphatidylinositol)-anchored membrane protein. CNTN4 functions as an axon-associated synaptic cell adhesion molecule. CNTN4 was previously known as "BIG-2," and the terms can be used interchangeably.

[0191] The term "antibody" as used herein is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, protein, etc., via at least one antigen recognition site located in its variable region. The term "antibody" is used herein in the broadest sense, and thus broadly includes not only an intact polyclonal antibody or monoclonal antibody, but also dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antigen-binding fragments thereof, antibody fragments, fusion proteins comprising any other modified arrangement of an immunoglobulin molecule comprising an antigen recognition site (e.g., a variable region), synthetic antibodies (e.g., "antibody mimetics"), "FynomAbs," and the like.

[0192] There are five types of antibodies: Immunoglobulin (Ig) M, IgD, IgG, IgA, and IgE, each containing a heavy chain made from heavy chain constant region genes μ, δ, γ, α, and ε. The light and heavy chains of antibodies are divided into a variable region whose amino acid sequence differs from antibody to antibody and a constant region whose amino acid sequence is the same.

[0193] The term “antibody variable region” as used herein refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the complementarity determining regions (CDRs) and the framework regions (FRs).

[0194] The term "CDR," as used herein, refers to the amino acid residues of an antibody variable region that are essential for antigen binding. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR3. CDRs comprise most of the residues responsible for the specific interaction of an antibody (or antigen-binding fragment thereof) with an antigen, and thus contribute to the functional activity of the antibody molecule. They are the primary determinants of antigen specificity.

[0195] The term "multispecific antibody" as used herein is an antibody having binding specificities for at least two different sites.

[0196] The terms "humanized antibody" and "CDR-grafted antibody" as used herein refer to an antibody in which one or more CDR sequences from a non-human species, e.g., another mammalian species, are inserted into a framework sequence from a human immunoglobulin molecule. The framework sequence may further be modified, e.g., by mutation. Human Ig sequences can be found, for example, in the NCBI Database (Entez Gene). By using appropriate sequences, the immunogenicity of the antibody can be reduced, or the avidity, affinity, on-rate, off-rate, affinity, specificity, half-life, or any other suitable characteristic can be reduced, enhanced, or altered.

[0197] The term "chimeric antibody," as used herein, refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. Methods for producing chimeric antibodies are well known in the art. See, for example, Morrison, Science 229:1202 (1985), which is incorporated herein by reference in its entirety.

[0198] The term "human antibody" as used herein refers to an antibody comprising a variable region in which both the framework and CDR regions are derived from human immunoglobulin sequences. The constant region of the antibody is also derived from human immunoglobulin sequences.

[0199] The term “anti-CNTN4 antibody” as used herein includes monovalent antibodies having a single specificity as well as multispecific antibodies having at least two arms, wherein the first arm comprises a first (target) antigen and the second arm comprises a second (target) antigen.

[0200] The term “Fab fragment” as used herein refers to a monovalent fragment consisting of the VL, VH, CL and CH1 domains.

[0201] The term "Fab' fragment" as used herein differs from a Fab fragment in that it has several residues added at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region.

[0202] The term "Fab'-SH" as used herein refers to Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group.

[0203] The term "F(ab')2 antibody fragment" as used herein is produced as a pair of Fab' fragments via hinge cysteines between the Fab' fragments.

[0204] The term "Fv," as used herein, refers to the minimum antibody fragment containing a complete antigen recognition site and antigen binding site. This fragment consists of a dimer of one heavy chain variable region and one light chain variable region, tightly and non-covalently associated. These two regions fold into six hypervariable loops (three loops each from the heavy and light chains), which provide amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable region has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

[0205] The term "single-chain antibody scFv," as used herein, refers to an antibody fragment comprising VH and VL antibody domains linked by a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. Also referred to herein as scFv antibody fragment, antigen-binding fragment scFv, scFv antibody, antibody scFv, or simply scFv.

[0206] The term "diabody" as used herein refers to small antibody fragments prepared by constructing scFv fragments using a short linker (about 5-10 residues) between the VH and VL domains such that intrachain, rather than interchain, pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers composed of two "bridged" scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Similarly, triabodies and tetrabodies comprise three and four polypeptide chains, respectively, and form three and four antigen-binding sites, respectively, which may be the same or different.

[0207] The term "Fynomer" as used herein refers to a non-immunoglobulin-derived binding polypeptide derived from the human Fyn SH3 domain. Fyn SH3-derived polypeptides are well known in the art and are discussed, for example, in Grabulovski et al. (2007) JBC, 282, p. 3196-3204, WO 2008 / 022759, and others. Fynomers can be genetically fused with other molecules (e.g., antibodies) to create "FynomAbs," i.e., forms that can be engineered to have dual specificity.

[0208] The terms "dual-affinity re-targeting (DART)" and "TRIDENT" as used herein refer to those designed to simultaneously bind to two or more targets. DART refers to a covalently linked dual-specific diabody, such as a diabody linked via a C-terminal disulfide bridge, and its specific structure and definition are described in references such as [J. Mol. Biol. (2010) 399, 436-449].

[0209] The term "prevention" as used herein refers to any action that suppresses a disease associated with the function or expression of CNTN4 or delays the onset of a disease associated with the function or expression of CNTN4, and "treatment" as used herein refers to any action that improves or beneficially changes the symptoms of a disease associated with the function or expression of CNTN4.

[0210] The term "subject" as used herein is intended to encompass both humans and non-human animals. Non-human animals include all vertebrates, such as mammals, and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. Mammals, such as non-human primates, sheep, dogs, cats, cows, and horses, are preferred. A preferred subject is a human.

[0211] The term "alkyl" as used herein refers to a fully saturated aliphatic hydrocarbon group, which may be branched or unbranched. The alkyl may be an alkyl having 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 1 to 6 carbon atoms. The above alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc., C 1-30 Linear alkyl or C 3-31 It can be substituted with cycloalkyl.

[0212] The term "alkenyl" as used herein refers to an unsaturated branched or straight-chain hydrocarbon group having one or more carbon-carbon double bonds. Alkenyl may include, but is not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, dodecyl, and the like.

[0213] The term "alkynyl" as used herein refers to an unsaturated branched or straight-chain hydrocarbon group having at least one carbon-carbon triple bond. Alkynyl may include, but is not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, and the like.

[0214] The term "cycloalkyl" as used herein means a fully saturated (no double or triple bonds) cyclic hydrocarbon ring. When composed of two or more rings, the rings may be fused, bridged, or spiro-connected. The cycloalkyl may be a cycloalkyl having 3 to 30, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, or 3 to 7 carbon atoms. The cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like. The cycloalkyl is C 1-30 Linear alkyl or C 3-31 It can be substituted with cycloalkyl.

[0215] The term "aryl" as used herein refers to a polyunsaturated, aromatic hydrocarbon group which may be a single ring or multiple rings, fused or covalently bonded. Aryl includes, but is not limited to, phenyl, naphthyl, and tetrahydronaphthyl.

[0216] The term "heterocyclyl" as used herein refers to a group having a single or multiple ring structure containing one or more heteroatoms (e.g., N, O, P, or S), and may contain one or more double or triple bonds within the ring. When composed of two or more rings, the rings may be fused, bridged, or spiro-linked. The heterocyclyl may include, but is not limited to, heteroaryl, heterocycloalkenyl, or heterocycloalkynyl, or fused ring compounds thereof.

[0217] The terms “alkylene,” “alkenylene,” “alkynylene,” “cycloalkylene,” “arylene,” and “heterocyclylene,” as used herein, mean divalent groups having the same structure as alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heterocyclyl, respectively.

[0218] The term "ester" as used herein refers to a group in which an organic radical is substituted for a hydrogen molecule in an acid.

[0219] The term "prodrug" as used herein refers to a compound that can be converted into a pharmaceutically active compound by enzymatic action or pH conditions, etc.

[0220] The term "direct bond" as used herein means that the relevant substituents or atoms are directly connected. For example, if X in AXB is a direct bond, it means a structure in which A and B are directly connected because X is absent between A and B, i.e., a structure of AB.

[0221] The term "substitution" as used herein means replacing one or more hydrogen atoms within a molecular structure with a substituent, such that a chemically stable compound is obtained from such substitution, without exceeding the valence of the designated atom. For example, "group A is replaced with group B" may mean that a hydrogen atom bonded to a carbon atom or heteroatom forming the backbone of group A is replaced with group B, so that groups A and B form a covalent bond.

[0222] Also, the meaning of the present invention that any carbon atom of a hydrocarbon group such as alkylene, alkenylene, alkynylene, cycloalkylene or arylene can be replaced with a heteroatom or group means that any carbon atom in the chemical structure is replaced with a heteroatom or group so as to be chemically stable by such replacement without exceeding the valence of the atom involved. For example, when any carbon atom of alkylene is replaced with an oxygen atom or an amide, alkylene-O-alkylene and alkylene-amide-alkylene can be formed, respectively, and when any carbon atom of cycloalkylene or arylene is replaced with an oxygen atom, a sulfur atom or a nitrogen atom, an aromatic or non-aromatic heterocyclylene can be formed.

[0223] Anti-CNTN4 antibody or antigen-binding fragment thereof

[0224] In one embodiment of the present invention, the anti-CNTN4 antibody or antigen-binding fragment thereof may be an anti-CNTN4 antibody or antigen-binding fragment thereof comprising a heavy chain (VH) CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light chain (VL) CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0225] VH-CDR1: SFNMF (SEQ ID NO: 1)

[0226] VH-CDR2: EISGGGGSTWYAPAVKG (SEQ ID NO: 2)

[0227] VH-CDR3: SAFTWSYGAADISA (SEQ ID NO: 3)

[0228] VL-CDR1: SGRSGSYG (SEQ ID NO: 4)

[0229] VL-CDR2: DNTNRPS (SEQ ID NO: 5)

[0230] VL-CDR3: GGYWGSTDV (SEQ ID NO: 6)

[0231] In one embodiment of the present invention, the CDR was created according to the Kabat criteria.

[0232] In another embodiment of the present invention, the anti-CNTN4 antibody or antigen-binding fragment thereof may be an anti-CNTN4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 95% sequence homology thereto (preferably, at least 96%, at least 97%, at least 98%, at least 99% sequence homology thereto) and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 95% sequence homology thereto (preferably, at least 96%, at least 97%, at least 98%, at least 99% sequence homology thereto).

[0233] In another embodiment of the present invention, the anti-CNTN4 antibody or antigen-binding fragment thereof may be an anti-CNTN4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 7 and a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 8:

[0234] VH:EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFNMFWVRQAPGKGLEWVAEISGGGGSTWYAPAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAFTWSYGAADISAWGQGTLVTVSS (SEQ ID NO: 7)

[0235] VL:ELTQDPAVSVALGQTVRITCSGRSGSYGWYQQKPGQAPVLVIYDNTNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCGGYWGSTDVFGGGTKLTVL (SEQ ID NO: 8)

[0236] In one embodiment of the present invention, the anti-CNTN4 antibody can be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a multispecific antibody (e.g., a bispecific antibody), a heteroconjugate antibody, a humanized antibody, and any other modified form of an immunoglobulin molecule comprising an antigen recognition site of the required specificity, such as a glycosylation variant of the antibody, an amino acid sequence variant of the antibody, and a covalently modified antibody.

[0237] In another embodiment of the invention, the anti-CNTN4 antibody or antigen-binding fragment thereof of the invention is a multispecific, e.g., bispecific or trispecific, antibody molecule. A multispecific antibody molecule comprises multiple variable regions, each variable region having a binding specificity for a different epitope. In one embodiment, the first variable region of the bispecific antibody molecule has a first binding specificity for a first epitope, e.g., a CNTN4 protein, and the second variable region has a second binding specificity for a second epitope, e.g., a target protein other than a CNTN4 protein.

[0238] In one specific embodiment, the multispecific antibody molecule can bind to a protein that binds to an antigen specifically expressed on cancer cells as compared to normal cells (preferably, an antibody that binds to a tumor-specific antigen). The above tumor-specific antigens are antigens known to be specifically overexpressed in cancer cells, such as ADAM9, ALCAM, ALPP, ANGPT2, AXL, CA9, CD22, CD269, CD27, CD274, CD276, CD30, CD33, CD44, CD46, CD70, CD79b, CDH3, CDH6, CEACAM5, CLDN18, CLDN6, CLDN9, CRIPTO, DLL3, DPEP3, EDNRB, EEF1A2, EGFR, EPCAM, EPHA2, EPHA4, EPHA5, ERBB2, ERBB3, F3, FAP, FGFR2, FGFR3, FN1, FOLH1, FOLR1, FUT3, GPC1, GPNMB, GPR20, GUCY1A2, HAVCR1, IGF1R, ITGB6, KAAG1, These include, but are not limited to, LAMP1, LRRC15, LY6E, LY75, LYPD3, MCAM, MELTF, MET, MSLN, MUC1, MUC16, NCAM1, NECTIN4, NOTCH3, PRL, PTK7, ROR1, ROR2, SDC1, SEZ6, SLC1A5, SLC34A2, SLC39A6, SLC44A4, SLITRK6, ST8SIA1, STEAP1, TFRC, THBS1, TM4SF1, TPBG, VTCN1, or WNT4CD19.

[0239] In one specific embodiment, any combination of the above molecules can be made into a multispecific antibody molecule, e.g., a trispecific antibody comprising a first binding specificity for CNTN4 and second and third binding specificities for two or more tumor-specific antigens. Multispecific antibody molecules of the invention can be made using standard molecular biological techniques known to those skilled in the art, such as recombinant DNA and protein expression techniques.

[0240] In addition, the antigen binding fragment of the anti-CNTN4 antibody is Fab, Fab', Fab'-SH, Fv, single chain antibody scFv, F(ab')2 fragment, VL, VH, diabody, triabody, tetrabody, minibody, IgGdeltaCH2, scFv-Fc, (scFv)2-Fc, Fynomer, dual-affinity re-targeting protein, anticalin, FN3-based monobody, DARPin, Affibody, Affilin, Affimer, Affitin, Alphabody, Avimer, Im7, VLR, VNAR, Trimab, CrossMab, TRIDENT, It can be a nanobody, a binanobody or a di-sdFv.

[0241] In one embodiment of the present invention, the anti-CNTN4 antibody or antigen-binding fragment thereof may specifically bind to a CNTN4 protein, such as a human CNTN4 protein. The human CNTN4 protein may be represented by the amino acid sequence of Q8IWV2-1, Q8IW2-2, Q8IWV2-3, or Q8IWV2-4 provided by Uniprot (https: / www.uniprot.org / uniprotkb / Q8IWV2 / entry#sequences), but is not limited thereto, and includes all proteins that exhibit known CNTN4 functions and are classified as CNTN4.

[0242] In one embodiment of the present invention, the ability of the anti-CNTN4 antibody or antigen-binding fragment thereof to bind to CNTN4 protein can be confirmed using Flow Cytometry, ELISA Assay and / or Biolayer Interference Method.

[0243] In another embodiment of the present invention, the anti-CNTN4 antibody or antigen-binding fragment thereof is present in an amount of 1 x 10 -3 M, 1 x 10 -4 M, 1 x 10 -5 M, 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 The bond dissociation equilibrium constant (K) below M D ) can bind to human CNTN4 protein.

[0244] The term "K" used herein D " refers to the binding equilibrium dissociation constant of a specific antibody-antigen interaction, K D = It is calculated through the calculation formula of Kd / Ka (where Ka is the association rate constant and Kd is the dissociation rate constant), and the above K D has units of M. K for antibodies D The value can be measured using methods widely established in the art. The K of the antibody D Preferred methods for measuring the values ​​are surface plasmon resonance (SPR), preferably using a biosensor system such as the Biacore® system, or bio-layer interferometry (BLI), for example the Octet® system. In one specific example, the K mentioned herein D may be a value obtained through SPR.

[0245] In another embodiment of the present invention, the anti-CNTN4 antibody or antigen-binding fragment thereof has an EC of 150 nM or less, e.g., 130 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM, 1 nM or 0.5 nM or less, as measured by ELISA assay or Flow cytometry. 50 can bind to the CNTN4 protein. The above "EC 50 " is a term related to in vitro or in vivo assays using antibodies, and refers to the concentration of antibody that induces 50% of the maximum response, i.e., a response midway between the maximum response and the baseline.

[0246] In one embodiment of the present invention, the anti-CNTN4 antibody has cancer cell internalization activity. The anti-CNTN4 antibody of the present invention can be internalized into cancer cells expressing CNTN4 to deliver a payload, cytotoxic agent, or therapeutic moiety.

[0247] Preparation of anti-CNTN4 antibody or antigen-binding fragment thereof

[0248] In one aspect of the present invention, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention can be produced by any suitable method known in the art to which the present invention pertains.

[0249] In one embodiment of the present invention, the anti-CNTN4 antibody can be prepared using techniques well known in the art, such as hybridoma, recombinant, phage display, transfection, synthetic techniques or combinations thereof or other techniques readily known in the art (see, e.g., Jayasena, SD, Clin. Chem., 45: 1628-50 (1999) and Fellouse, FA, et al, J. Mol. Biol., 373(4):924-40 (2007)).

[0250] In another embodiment of the present invention, the anti-CNTN4 antibody can be produced using phage display technology. Specifically, the heavy and light chain variable region genes of a human antibody are cloned into a phagemid vector in a form fused to a phage surface protein (pIII), expressed in Escherichia coli, and then infected with M13 helper phage to produce an antibody library in which antibody fragments (scFv or Fab) having various combinations of heavy and light chain variable region sequences are displayed on the surface of the phage. From this library, antibody fragments that bind to a specific antigen are isolated using a panning method, and after characterizing the isolated antibody fragments, they are converted into whole IgG forms and mass-expressed in animal cells, thereby producing specific human monoclonal antibodies. A native antibody library that utilizes antibody genes already present in the human body can be used as the library. A synthetic antibody library that increases diversity by inserting random synthetic sequences into the CDR of an antibody can also be used as the library. The above phagemid vector is a plasmid DNA having a phage origin of replication and typically has an antibiotic resistance gene as a selection marker. In the case of the phagemid vector used for phage display, the gIII gene of the M13 phage or a part thereof is included, and the scFv gene is ligated to the 5' end of the gIII gene and expressed through the transformant. In addition, the helper phage is a phage that provides the genetic information necessary for the phagemid to assemble into a phage particle. Since only gIII or a part of the phage gene is present in the phagemid, the host cell (transformant) transformed with the phagemid is infected with the helper phage to supply the remaining phage genes.A variety of phagemids exist, including M13K07 and VCSM13, and most contain antibiotic resistance genes, such as kanamycin, allowing for the selection of transformants infected with helper phage. Furthermore, phagemids have defective packaging signals, allowing phagemid genes to be selectively assembled into phage particles over helper phage genes.

[0251] In another embodiment of the present invention, the anti-CNTN4 antibody can be produced using hybridoma technology. Specifically, the antibody can be produced, for example, in the form of a monoclonal antibody, by injecting a test subject (e.g., a mouse) with the CNTN4 antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional properties.

[0252] In one embodiment of the present invention, the anti-CNTN4 antibody can be produced in the form of a monoclonal antibody, and the base sequence encoding the monoclonal antibody is immediately isolated and sequenced using a conventional method (for example, using an oligonucleotide probe that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody), and hybridoma cells can be used as a preferred source of the base sequence. At this time, after the base sequence is isolated, it is placed into an expression vector and then transfected into a host cell, for example, E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, thereby obtaining the synthesis of the monoclonal antibody in the recombinant host cell.

[0253] In another aspect of the present invention, the present invention provides an isolated host cell that recombinantly produces the anti-CNTN4 antibody or antigen-binding fragment thereof.

[0254] In one embodiment of the present invention, the anti-CNTN4 antibody or antigen-binding fragment thereof can be expressed by introducing the nucleic acid nucleotide into any recombinant expression system, including bacterial, yeast, insect, or mammalian systems. The recombinant expression system can include the isolated host cell. Bacterial cells include Gram-positive bacteria or Gram-negative bacteria, such as several species of the genus Escherichia, such as E. coli and Pseudomonas. Among fungal cells, yeast cells can be preferably used. Expression in yeast can use yeast strains, such as Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha, among others. Insect cells, such as cells derived from Drosophila and Sf9, can also be used as host cells.

[0255] In another embodiment of the present invention, the expression system can be an isolated mammalian host cell such as, but not limited to, CHO cells, DG44 or DUXB11 cells, NS0 myeloma cells, monkey kidney cell lines (e.g., CVI cells, COS cells, etc.), SP2 cells, human embryonic kidney (HEK) cells, Chinese hamster fibroblasts, human cervical carcinoma cells (e.g., HELA), BHK cells, NSO cells or Bowes melanoma cells, murine fibroblasts (e.g., BALBc / 3T3), murine myeloma cells (P3x63-Ag3.653; NS0; SP2 / O), hamster kidney cells (e.g., HAK), murine L cells (e.g., L-929), human lymphocytes (e.g., RAJI), human kidney cells (e.g., 293 and 293T).

[0256] Since the antibody or antigen-binding fragment thereof of the present invention will ultimately be administered to a human, a fully human expression system may be particularly preferred. In this case, the host cell may be a human cell, such as HeLa, 911, AT1080, A549, 293, or HEK293 cells, and in a specific example, Expi293F cells.

[0257] In another aspect of the present invention, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell under conditions for producing the antibody or an antigen-binding fragment thereof, and isolating the antibody or an antigen-binding fragment thereof from the host cell or culture medium.

[0258] In one embodiment of the present invention, when the recombinant expression vector is introduced into a recombinant expression system, the host cell is cultured so that the antibody or antigen-binding fragment thereof can be sufficiently secreted into the culture medium in which the host cell is grown or the antibody or antigen-binding fragment thereof can be recovered using a standard protein purification method.

[0259] In another embodiment of the present invention, the recombinant expression vector can be transfected into a host cell by standard techniques, including known techniques used for the introduction of exogenous nucleic acid nucleotides into cells, such as electroporation, calcium-phosphate precipitation, and DEAE-dextran transfection.

[0260] Conjugate comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof

[0261] In one aspect of the present invention, the present invention may be in a form in which the anti-CNTN4 antibody or fragment thereof is conjugated to a therapeutic moiety.

[0262] In another aspect of the present invention, the above-described anti-CNTN4 antibody or fragment thereof can be prepared as an antibody-drug conjugate by linking it to a therapeutic moiety via a linker. The types and binding methods of the therapeutic moiety, linker, and conjugate may be the same as those of the therapeutic moiety, linker, and conjugate of the antibody-drug conjugate of the following general formula I, but are not limited thereto.

[0263] In another aspect of the present invention, the present invention may be an antibody-drug conjugate represented by the following general formula I:

[0264] [General Formula I]

[0265] M-[LD] n

[0266] The above M is an anti-CNTN4 antibody or fragment thereof that binds to the CNTN4 protein,

[0267] The above L is a connector,

[0268] wherein D is a therapeutic moiety,

[0269] The above n is the average number of bindings per antibody of the LD structure binding to the antibody, and is in the range of 1 to 20.

[0270] (1) Anti-CNTN4 antibody or fragment thereof

[0271] The anti-CNTN4 antibody or fragment thereof included in the antibody-drug conjugate of the present invention is an anti-CNTN4 antibody or fragment thereof that specifically binds to CNTN4 protein, preferably an anti-CNTN4 antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0272] In one embodiment of the present invention, the anti-CNTN4 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence having at least 95% sequence homology to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having at least 95% sequence homology to the amino acid sequence of SEQ ID NO: 8. Preferably, the anti-CNTN4 antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

[0273] More preferably, the anti-CNTN4 antibody or antigen-binding fragment thereof may be an anti-CNTN4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

[0274] To activate the cysteine ​​residue side chain of the anti-CNTN4 antibody or antigen-binding fragment thereof, the disulfide bonds of the antibody may be partially reduced. A reducing agent may be used to partially reduce the disulfide bonds of the antibody, and (tris(2-carboxyethyl)phosphine)(TCEP) may be used as the reducing agent.

[0275] In one embodiment of the present invention, the anti-CNTN4 antibody or antigen-binding fragment thereof may comprise an amino acid residue engineered to bind or inhibit binding of the linker at one or more selected positions.

[0276] In another embodiment of the present invention, the anti-CNTN4 antibody or antigen-binding fragment thereof may comprise an amino acid residue engineered to bind or inhibit binding of the linker to the carboxyl terminus of the light or heavy chain. The amino acid residue may be selected from the group of peptide moieties comprising alanine, cysteine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, or arginine.

[0277] (2) Connector

[0278] The above linker L is a material capable of linking an antibody and a therapeutic moiety, which is prepared using any known linker or linker technology.

[0279] In one embodiment of the present invention, the linker L may be a cleavable linker, a non-cleavable linker, or a combination thereof.

[0280] In another embodiment of the present invention, the linker L may comprise a cleavable peptide moiety.

[0281] The cleavable peptide moiety may be enzymatically cleavable. The cleavable peptide moiety may be recognized and cleaved by an internalization protein enzyme of the lysosome (e.g., Cathepsin B, beta-glucuronidase).

[0282] In another embodiment of the present invention, the linker L may comprise a natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D-α-amino acid. Preferably, the linker L may comprise an amino acid unit.

[0283] In one embodiment of the present invention, the amino acid unit may include alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, derivatives thereof, or combinations thereof.

[0284] The above amino acid units are valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), alanine-alanine (Ala-Ala), alanine-citrulline (Ala-Cit), citrulline-alanine (Cit-Ala), asparagine-citrulline (Asn-Cit), citrulline-asparagine (Cit-Asn), citrulline-citrulline (Cit-Cit), valine-glutamic acid (Val-Glu), glutamic acid-valine (Glu-Val), serine-citrulline (Ser-Cit), citrulline-serine (Cit-Ser), lysine-citrulline (Lys-Cit), citrulline-lysine (Cit-Lys), aspartic acid-citrulline (Asp-Cit), citrulline-aspartic acid (Cit-Asp), alanine-valine (Ala-Val), Valine-Alanine (Val-Ala), Phenylalanine-Lys (Phe-Lys), Lysine-Phenylalanine (Lys-Phe), Valine-Lys (Val-Lys), Lysine-Valine (Lys-Val), Alanine-Lys (Ala-Lys), Lysine-Alanine (Lys-Ala), Phenylalanine-Citrulline (Phe-Cit), Citrulline-Phenylalanine (Cit-Phe), Leucine-Citrulline (Leu-Cit), Citrulline-Leucine (Cit-Leu), Isoleucine-Citrulline (Ile-Cit), Citrulline-Isoleucine (Cit-Ile), Phenylalanine-Arginine (Phe-Arg), Arginine-Phenylalanine (Arg-Phe), Citrulline-Tryptophan (Cit-Trp), It may be selected from the group consisting of tryptophan-citrulline (Trp-Cit), alanine-alanine-aspartic acid (Ala-Ala-Asp), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), and glutamic acid-valine-citrulline (Glu-Val-Cit), but is not limited thereto.

[0285] Preferably, the amino acid unit may be selected from the group consisting of valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), alanine-valine (Ala-Val), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) and glutamic acid-valine-citrulline (Glu-Val-Cit).

[0286] In one embodiment of the present invention, the linking group L may comprise a direct bond, or a moiety comprising a succinic acid derivative or a pyrimidine. The succinic acid derivative may be succinic acid, succinimide, succinamic acid or a succinamic ester, which are in the form of having an α,β-unsaturated carbonyl group in their molecular structure. The structure in which the antibody and the succinic acid derivative are linked may be formed by a linking reaction between the antibody and a maleic acid derivative such as maleic acid, maleimide, maleamic acid or a maleamic ester.

[0287] In another embodiment of the present invention, the moiety comprising the succinic acid derivative may be a succinimide moiety, a succinamic acid moiety, or a succinamic ester moiety.

[0288] Additionally, the succinimide moiety may include succinimidocaproyl, succinimidoacetyl, succinimidovaleroyl, succinimidopropanoyl or succinimidobutanoyl.

[0289] Specifically, the succinimide moiety may include succinimidocaproyl, succinimidopropanoyl or succinimidobutanoyl.

[0290] The above succinimide moiety is -(Succinimid-3-yl-N)-(CH2) h -C(=O)- (wherein h is an integer of 1 or more, preferably 1 to 30) may include a compound represented by the structure. The -(Succinimid-3-yl-N)- is A structure represented by , which binds to the anti-CNTN4 antibody of the present invention or an antigen-binding fragment thereof at position 3, and can bind to a methylene group in another moiety structure on the nitrogen atom at position 1.

[0291] In another embodiment of the present invention, the linker may optionally include a polyethylene glycol (PEG) moiety. The polyethylene glycol moiety is -(CH2CH2O) m - may include a structure represented by m, wherein m may be an integer from 1 to 24. In addition, m may be an integer from 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10 or 2 to 8, or may be 8.

[0292] In another embodiment of the present invention, the linking group comprises one or more alkylene, alkenylene, alkynylene, cycloalkylene or arylene, and any carbon atom of the alkylene, alkenylene, alkynylene, cycloalkylene or arylene may be optionally replaced with one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom.

[0293] In another embodiment of the present invention, the linking group can be alkylene, alkenylene, alkynylene, alkylene-cycloalkylene, alkylene-cycloalkylene-alkylene, alkylene-amide-alkylene-cycloalkylene, alkylene-amide-cycloalkylene, alkylene-carbonyl-cycloalkylene, alkylene-amide-alkylene-O-alkylene-cycloalkylene, alkylene-amide-alkylene-O-cycloalkylene, alkylene-amide-alkylene-O-alkylene-cycloalkylene, alkylene-amide-alkylene-O-alkylene-cycloalkylene, alkylene-cycloalkylene-alkylene-O-alkylene, alkylene-O-alkylene-cycloalkylene, alkylene-O-alkylene-cycloalkylene, alkylene-O-alkylene-cycloalkylene, alkylene-O-cycloalkylene, alkylene-O-alkylene-O-cycloalkylene. Additionally, any carbon atom of the above alkylene, alkenylene, alkynylene, cycloalkylene or arylene may be optionally replaced with one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom, whereby the linking group may include an aromatic or non-aromatic heterocyclylene.

[0294] In another embodiment of the present invention, the linker may comprise a moiety represented by the following general formula II.

[0295] [General Formula II]

[0296]

[0297] In the above general formula II,

[0298] The above a is an integer from 0 to 24,

[0299] The above b is 0 or 1,

[0300] The above X comprises one or more linked groups selected from the group consisting of alkylene, alkenylene, alkynylene, cycloalkylene and arylene, and the alkylene and cycloalkylene constituting the above X are C 1-30 Alkyl or C 3-30is unsubstituted or substituted with cycloalkyl, and any carbon atom of said alkylene, alkenylene, alkynylene, cycloalkylene or arylene is optionally replaced by one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom,

[0301] wherein Q is a direct bond or a moiety comprising a succinic acid derivative or a pyrimidine;

[0302] The above Q is linked to an antibody.

[0303] Specifically, Q is linked to a cysteine ​​residue in the antibody.

[0304] The above a may be an integer of 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10 or 2 to 8, or may be 8.

[0305] In another embodiment of the present invention, X is C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene and C 6-30 C comprising one or more linked groups selected from the group consisting of arylene, and constituting said X 1-30 Alkylene and C 3-30 Cycloalkylene is C 1-30 Alkyl or C 3-30 Substituted or unsubstituted with cycloalkyl, and the above C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene or C 6-30Any carbon atom of arylene may be optionally replaced by one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom.

[0306] In another embodiment of the present invention, X is C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 1-30 alkylene-C 3-30 Cycloalkylene, C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene-OC 1-30 Alkylene, C 1-30 Alkylene-OC 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 6-30 Arylene, C 6-30 Arylene-C 1-30 Alkylene, C 1-30 alkylene-C 6-30 Arylene-C 1-30 C selected from the group consisting of alkylene and constituting said X 1-30 Alkylene and C 3-30 Cycloalkylene is C 1-30 Alkyl or C 3-30 Substituted or unsubstituted with cycloalkyl, and the above C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene or C 6-30 Any carbon atom of arylene may be optionally replaced by one or more of a carbonyl, an oxygen atom, or a nitrogen atom.

[0307] In another embodiment of the present invention, X is C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 Alkynylene, C 1-20 alkylene-C 3-20 Cycloalkylene, C 1-20 Alkylene-amide-C 1-20 alkylene-C 3-20 Cycloalkylene, C 1-20 alkylene-C 3-20 Cycloalkylene-C 1-20 Alkylene-OC 1-20 Alkylene, C 1-20 Alkylene-OC 1-20 alkylene-C 3-20 C selected from the group consisting of cycloalkylene and constituting said X 1-20 Alkylene and C 3-20 Cycloalkylene is C 1-20 Alkyl or C 3-20 It may be substituted or unsubstituted with cycloalkyl.

[0308] In another embodiment of the present invention, X is C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylene, C 1-10 alkylene-C 3-10 Cycloalkylene, C 1-10 Alkylene-amide-C 1-10 alkylene-C 3-10 Cycloalkylene, C 1-10 alkylene-C 3-10 Cycloalkylene-C 1-10 Alkylene-OC 1-10 Alkylene, C 1-10 Alkylene-OC 1-10 alkylene-C 3-10 C selected from the group consisting of cycloalkylene and constituting said X 1-10 Alkylene and C 3-10 Cycloalkylene is C 1-10 Alkyl or C 3-10 It may be substituted or unsubstituted with cycloalkyl.

[0309] In one embodiment of the present invention, the linker may optionally comprise a self-immolative moiety.

[0310] In another embodiment of the present invention, the linking group does not include a self-immolative moiety, and the linking group is a succinimide moiety or a succinamic acid, glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) and -(CH2CH2O) m - (wherein, m is an integer from 1 to 24) may include a polyethylene glycol moiety represented by, and m is an integer from 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10 or 2 to 8, or may be 8.

[0311] In another embodiment of the present invention, the self-immolative moiety may be selected from the group consisting of p-aminobenzyl (PAB), unsubstituted or substituted p-aminobenzyloxycarbonyl (PABC), aminomethylene, N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC), and N-succinimidyl(4-iodo-acetyl)aminobenzoate (SIAB).

[0312] In another embodiment of the present invention, the self-immolative moiety may be an aminomethylene or an unsubstituted or substituted PABC. Optionally, the PABC is -C 1-6 Alkylene-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6It may be substituted with alkyl, and m is an integer from 1 to 24. In addition, m is an integer from 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, or 2 to 8, or may be 8.

[0313] In one embodiment of the present invention, the linking group may comprise a moiety represented by the following general formula III:

[0314] [General Formula III]

[0315]

[0316] In the above general formula III,

[0317] The above a is an integer from 0 to 24,

[0318] The above b and c are 0 or 1, respectively,

[0319] The above Lp is a self-immolative moiety, and the self-immolative moiety is -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 A group represented by alkyl or -(CH2CH2O) m -C 1-6 It can be substituted with a group represented by alkyl, and m is an integer from 1 to 24,

[0320] The above La is valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), alanine-alanine (Ala-Ala), alanine-citrulline (Ala-Cit), citrulline-alanine (Cit-Ala), asparagine-citrulline (Asn-Cit), citrulline-asparagine (Cit-Asn), citrulline-citrulline (Cit-Cit), valine-glutamic acid (Val-Glu), glutamic acid-valine (Glu-Val), serine-citrulline (Ser-Cit), citrulline-serine (Cit-Ser), lysine-citrulline (Lys-Cit), citrulline-lysine (Cit-Lys), aspartic acid-citrulline (Asp-Cit), citrulline-aspartic acid (Cit-Asp), alanine-valine (Ala-Val), Valine-Alanine (Val-Ala), Phenylalanine-Lys (Phe-Lys), Lysine-Phenylalanine (Lys-Phe), Valine-Lys (Val-Lys), Lysine-Valine (Lys-Val), Alanine-Lys (Ala-Lys), Lysine-Alanine (Lys-Ala), Phenylalanine-Citrulline (Phe-Cit), Citrulline-Phenylalanine (Cit-Phe), Leucine-Citrulline (Leu-Cit), Citrulline-Leucine (Cit-Leu), Isoleucine-Citrulline (Ile-Cit), Citrulline-Isoleucine (Cit-Ile), Phenylalanine-Arginine (Phe-Arg), Arginine-Phenylalanine (Arg-Phe), Citrulline-Tryptophan (Cit-Trp), An amino acid unit selected from the group consisting of tryptophan-citrulline (Trp-Cit), alanine-alanine-aspartic acid (Ala-Ala-Asp), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), and glutamic acid-valine-citrulline (Glu-Val-Cit).

[0321] The above X is C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene and C 6-30 Containing one or more groups connected to each other selected from the group consisting of arylene, and C 1-30 Alkylene, C 2-30 Alkenylene, C2-30 Alkynylene, C 3-30 Cycloalkylene or C 6-30 Any carbon atom of arylene is optionally replaced by one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom,

[0322] wherein Q is a direct bond, a succinimide moiety, a succinamic acid moiety, a succinamic ester moiety, or a pyrimidine moiety;

[0323] The above Q is linked to an antibody.

[0324] Specifically, Q is linked to a cysteine ​​residue in the antibody.

[0325] The above self-immolative moiety is the same as the above-described self-immolative moiety, and the a and m are integers of 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10 or 2 to 8, or may be 8. In addition, the X is unsubstituted C 1-6 Alkyl or C 1-6 Alkyl or C 3-7 C substituted with cycloalkyl 1-6 It may be alkyl. Additionally, the La may be selected from the group consisting of valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), alanine-valine (Ala-Val), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), and glutamic acid-valine-citrulline (Glu-Val-Cit).

[0326] In another embodiment of the present invention, the linking group may comprise a moiety represented by the following general formula III', general formula III'', general formula III'' or general formula III''''.

[0327] [General Formula III']

[0328]

[0329] [General Formula III'']

[0330]

[0331] [General Formula III''']

[0332]

[0333] [General Formula III'''']

[0334]

[0335] In the above general formulas III', III'', III''' and III'''',

[0336] The above X' is hydrogen, C 1-30 Alkyl, C 3-30 Cycloalkyl or C 1-30 Alkyl-C 3-30 It is cycloalkyl,

[0337] The above a, b, c, Lp, La and X are as defined in the general formula III,

[0338] * is linked to an antibody.

[0339] In another embodiment of the present invention, X' is hydrogen, C 1-20 Alkyl, C 3-20 Cycloalkyl and C 1-20 Alkyl-C 3-20 It can be selected from the group consisting of cycloalkyl, and preferably, the X' is hydrogen, C 1-10 Alkyl, C 3-10 Cycloalkyl and C 1-10 Alkyl-C 3-10 It can be selected from the group consisting of cycloalkyl.

[0340] In another embodiment of the present invention, X is C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 1-30 alkylene-C 3-30Cycloalkylene, C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene-OC 1-30 Alkylene, C 1-30 Alkylene-OC 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 6-30 Arylene, C 6-30 Arylene-C 1-30 Alkylene and C 1-30 alkylene-C 6-30 Arylene-C 1-30 C selected from the group consisting of alkylene and constituting said X 1-30 Alkylene and C 3-30 Cycloalkylene is C 1-30 Alkyl or C 3-30 Substituted or unsubstituted with cycloalkyl, and the above C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene or C 6-30 Any carbon atom of arylene may be optionally replaced by one or more of a carbonyl, an oxygen atom, or a nitrogen atom.

[0341] In another embodiment of the present invention, X is C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 Alkynylene, C 3-20 Cycloalkylene and C 6-20 Containing one or more groups connected to each other selected from the group consisting of arylene, and C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 Alkynylene, C3-20 Cycloalkylene or C 6-20 Any carbon atom of arylene may be optionally replaced by one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom.

[0342] In another embodiment of the present invention, X is C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylene, C 3-10 Cycloalkylene and C 6-10 Containing one or more groups connected to each other selected from the group consisting of arylene, and C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylene, C 3-10 Cycloalkylene or C 6-10 Any carbon atom of arylene may be optionally replaced by one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom.

[0343] In another embodiment of the present invention, X is C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene-OC 1-30 Alkylene, C 1-30 Alkylene-OC 1-30 alkylene-C 3-30 C selected from the group consisting of cycloalkylene and constituting said X 1-30 Alkylene and C 3-30 Cycloalkylene is C 1-30 Alkyl or C 3-30It may be substituted or unsubstituted with cycloalkyl. The above C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene or C 6-30 Any carbon atom of arylene may be optionally replaced by one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom.

[0344] In the general formula III, c is 1, Lp is aminomethylene, or -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 PABC which is unsubstituted or substituted with a group represented by alkyl (m is an integer from 1 to 12), and La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val). The m may be an integer from 1 to 8, or from 2 to 8, or may be 8.

[0345] In the above general formula III, c is 1 and Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6PABC substituted with a group represented by alkyl (m is an integer from 1 to 12), wherein La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val). The m may be an integer from 1 to 8, or from 2 to 8, or may be 8.

[0346] In the general formula III, Q is a succinimide moiety, c is 0 or 1, and Lp is aminomethylene or -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 PABC which is unsubstituted or substituted with a group represented by alkyl (m is an integer from 1 to 12), and La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val). The m may be an integer from 1 to 8, or from 2 to 8, or may be 8.

[0347] Additionally, in the general formula III, Q may be a succinimide moiety, c may be 0, and La may be glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0348] In the general formula III, Q is a succinic acid moiety, c is 0 or 1, and Lp is aminomethylene or -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m-C 1-6 PABC which is unsubstituted or substituted with a group represented by alkyl (m is an integer from 1 to 12), and La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val). The m may be an integer from 1 to 8, or from 2 to 8, or may be 8.

[0349] In the general formula III, Q is a pyrimidine moiety, c is 1, and Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 PABC substituted with a group represented by alkyl (m is an integer from 1 to 12), wherein La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val). The m may be an integer from 1 to 8, or from 2 to 8, or may be 8.

[0350] In the general formula III, Q is a direct bond, c is 1, and Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6PABC substituted with a group represented by alkyl (m is an integer from 1 to 12), wherein La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val). The m may be an integer from 1 to 8, or from 2 to 8, or may be 8.

[0351] In the general formula III, c is 1 and Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 PABC substituted with a group represented by alkyl (m is an integer from 1 to 12), wherein X is C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 alkylene-C 6-30 Arylene, C 1-30 alkylene-C 6-30 Arylene-C 1-30 Alkylene, C 6-30 Arylene-C 1-30 Alkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 3-30 Cycloalkylene and C 6-30 C selected from the group consisting of arylene and constituting said X 1-30 Alkylene, C 6-30 Arylene or C 3-30Any carbon atom of cycloalkylene is optionally replaced by one or more of carbonyl, oxygen, or nitrogen atoms, and La may be an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val). The m may be an integer of 1 to 8, or 2 to 8, or may be 8.

[0352] In the above general formula III, X is C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 Alkynylene, C 1-20 alkylene-C 3-20 Cycloalkylene, C 1-20 alkylene-C 3-20 Cycloalkylene-C 1-20 Alkylene, C 1-20 alkylene-C 6-20 Arylene-C 1-20 Alkylene, C 1-20 Alkylene-amide-C 1-20 alkylene-C 3-20 Cycloalkylene, C 1-20 Alkylene-amide-C 1-20 alkylene-C 6-20 Arylene, C 3-20 Cycloalkylene and C 6-20 C selected from the group consisting of arylene and constituting said X 1-20 Alkylene, C 3-20 Cycloalkylene and C 6-20 Arylene is C 1-20 Alkyl or C 3-20 It may be substituted or unsubstituted with cycloalkyl.

[0353] In the above general formula III, X is C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylene, C 1-10 alkylene-C 3-10 Cycloalkylene, C 1-10 alkylene-C3-10 Cycloalkylene-C 1-10 Alkylene, C 1-10 alkylene-C 6-10 Arylene-C 1-10 Alkylene, C 1-10 Alkylene-amide-C 1-10 alkylene-C 3-10 Cycloalkylene, C 1-10 Alkylene-amide-C 1-10 alkylene-C 6-10 Arylene, C 3-10 Cycloalkylene and C 6-10 C selected from the group consisting of arylene and constituting said X 1-10 Alkylene, C 3-10 Cycloalkylene and C 6-10 Arylene is C 1-10 Alkyl or C 3-10 It may be substituted or unsubstituted with cycloalkyl.

[0354] In another embodiment of the present invention, the linking group may comprise a sugar or a sugar acid, and the sugar or sugar acid may be linked by a bond susceptible to enzymatic cleavage in vivo.

[0355] In another embodiment of the present invention, the direct bond, or a moiety comprising a succinic acid derivative or a pyrimidine, can be attached to the anti-CNTN4 antibody or antigen-binding fragment thereof via a cysteine ​​residue on the antibody or antigen-binding fragment thereof.

[0356] In another embodiment of the present invention, the linking group is a combination of succinimidocaproyl, valine-citrulline (Val-Cit), and PABC; a combination of succinimidocaproyl, Val-Ala, and PABC; a combination of succinimidocaproyl, Val-Ala, and aminomethylene; a combination of succinimidocaproyl, Gly-Gly-Phe-Gly, and aminomethylene; a combination of succinimidocaproyl, Val-Ala, and PABC comprising a -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituent; a combination of succinimidopropanoyl, polyethylene glycol (PEG), and valine-alanine (Val-Ala); a combination of succinimidopropanoyl, PEG, Val-Ala, and PABC; a combination of succinimidopropanamide, PEG, carbonyl, and Val-Ala; Combinations of succinimidopropanamide, PEG, carbonyl, Val-Ala, and PABC; combinations of succinimidopropanamide, alkyl-carbonyl, and Val-Ala; combinations of succinimidopropanamide, alkyl-carbonyl, Val-Ala, and PABC; combinations of succinimidopropanamide, alkyl-carbonyl, PEG, and Val-Ala; combinations of succinimidopropanamide, alkyl-carbonyl, PEG, Val-Ala, and PABC; combinations of succinimidopropanamide, alkyl-amide, PEG, and Val-Ala; combinations of succinimidopropanamide, alkyl-amide, PEG, Val-Ala, and PABC; combinations of succinimidopropanamide, alkyl-amide, PEG, Val-Ala, and PABC; combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and PABC; Combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Val-Ala, and PABC; combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and aminomethylene; combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Val-Ala, and aminomethylene; combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and PABC; combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Gly-Gly-Phe-Gly, and PABC;Combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly and aminomethylene; combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Gly-Gly-Phe-Gly and aminomethylene; combinations of succinimidopropanamide, PEG, alkyl-carbonyl and Gly-Gly-Phe-Gly; combinations of succinimidopropanamide, alkyl, PEG, carbonyl and Gly-Gly-Phe-Gly; combinations of succinamic acid, alkyl-carbonyl, Val-Ala and PABC; combinations of succinamic acid, alkyl-carbonyl, Val-Ala and aminomethylene; combinations of succinamic acid, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala and PABC; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala, and aminomethylene; combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, and Val-Ala; combinations of succinic acid, alkyl-carbonyl, Gly-Gly-Phe-Gly, and PABC; combinations of succinic acid, alkyl-carbonyl, Gly-Gly-Phe-Gly, and aminomethylene; combinations of succinic acid, alkyl-carbonyl, and Gly-Gly-Phe-Gly; combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and PABC; combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and aminomethylene; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, and Gly-Gly-Phe-Gly; combinations of succinic acid, alkyl-carbonyl, Val-Ala, and PABC containing -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; combinations of succinic ester, PEG, carbonyl, and Val-Ala; combinations of succinic ester, PEG, carbonyl, Val-Ala, and aminomethylene; combinations of succinic ester, PEG, amide, and Val-Ala; combinations of succinic ester, PEG, amide, Val-Ala, and aminomethylene; combinations of succinic ester, alkyl-carbonyl, and Val-Ala;Combination of succinamic ester, alkyl-carbonyl, Val-Ala and aminomethylene; combination of succinamic ester, alkyl-amide and Val-Ala; combination of succinamic ester, alkyl-amide, Val-Ala and aminomethylene; combination of succinamic ester, alkyl, PEG and Val-Ala; combination of succinamic ester, alkyl, PEG, Val-Ala and aminomethylene; combination of succinamic ester, alkyl-carbonyl, PEG and Val-Ala; combination of succinamic ester, alkyl-carbonyl, PEG, Val-Ala and aminomethylene; combination of succinamic ester, alkyl-amide, PEG and Val-Ala; combination of succinamic ester, alkyl-amide, PEG and Val-Ala; combination of succinamic ester, alkyl-amide, PEG and Val-Ala; combination of succinamic ester, alkyl-amide, PEG, Val-Ala and aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, a carbonyl, and Val-Ala; A combination of a succinamic ester, an alkyl-amide, a PEG, a carbonyl, a Val-Ala, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, and Val-Ala; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Val-Ala, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Val-Ala, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Gly-Gly-Phe-Gly, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Gly-Gly-Phe-Gly, and an aminomethylene; Combinations of PABCs comprising succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents;Combinations of PABCs comprising pyrimidine, alkynyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; combinations of PABCs comprising alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; and combinations of PABCs comprising alkylamide, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents.

[0357] Preferably, the linking group is a combination of succinimidocaproyl, valine-citrulline (Val-Cit) and PABC; a combination of succinimidocaproyl, Gly-Gly-Phe-Gly and aminomethylene; a combination of succinimidocaproyl, Val-Ala and PABC comprising a -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituent; a combination of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala and PABC; a combination of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala and aminomethylene; a combination of succinimidopropanamide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly and aminomethylene; a combination of succinimidopropanamide, PEG, alkyl-carbonyl and Gly-Gly-Phe-Gly; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala, and aminomethylene; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and aminomethylene; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, and Gly-Gly-Phe-Gly; Combinations of succinic acid, alkyl-carbonyl, Val-Ala, and PABC comprising a -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituent; Combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and PABC comprising a -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituent; Combinations of PABCs comprising succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinic acid, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents;Combinations of PABCs comprising pyrimidine, alkynyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; combinations of PABCs comprising alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; and combinations of PABCs comprising alkylamide, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents.

[0358] The above linking group may have a structure in which both terminals of each group included in each combination are connected to each other. For example, among the above combinations, a linking group including a combination of succinic acid, alkyl-carbonyl, Val-Ala, and PABC including a -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituent may have a structure such as succinic acid moiety-alkylene-carbonyl-Val-Ala-PABC(-CH2-N(CH3)-C(=O)-(PEG)8-CH3)-.

[0359] (3) Therapeutic moiety

[0360] The therapeutic moiety has a substituent or structure capable of binding to the linker L, and may be appropriately selected according to the therapeutic or diagnostic use, etc., as long as it does not interfere with the specific binding of the anti-CNTN4 antibody of the present invention to the CNTN4 protein, and there is no limitation on its type.

[0361] In one embodiment of the present invention, the therapeutic moiety included in the antibody-drug conjugate of the present invention may be a substance that increases the in vivo half-life of the antibody, a substance that helps increase the in vivo function / effect of the antibody, a substance that can confirm the presence or absence of the antibody, or a substance that functions as a therapeutic substance, and may be a cytotoxic agent (e.g., a chemotherapeutic agent), a therapeutic protein, a biocompatible polymer, an oligonucleotide, an immunomodulator, an imaging agent, a radionuclide, a substance having antitumor activity, a substance having an effect on blood diseases, a substance having an effect on autoimmune diseases, an anti-inflammatory substance, an antibacterial substance, an antifungal substance, an antiparasitic substance, an antiviral substance, or an anti-anesthetic substance. In addition, the therapeutic moiety may be released into its original structure when the portion bonded to the linking group L is cleaved, thereby exhibiting its original intended effect (e.g., an anticancer effect, an anti-inflammatory effect, etc.).

[0362] In another embodiment of the invention, the therapeutic moiety may be a cytotoxic agent. As the cytotoxic agent, anthracyclines, auristatins, camptothecins or camptothecin analogues (e.g., irinotecan, rubitecan, topotecan, zimatecan, pegamotecan, lutotecan, karenitecan, apelletecan, homocampprothecin, diplomotecan, belotecan, 9-aminocamptothecin, exatecan, SN-38, DXd, DXd derivatives), combretastatins, dolastatins, duocarmycins, enedynes, geldanamycin, indolino-benzodiazepine dimers, maytansine, puromycin, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, tubulisin, hemiasterlin, spliceostatins, fladienolides, calcheamicins, 1-(2chloroethyl)-1,2-dimethanesulfonyl hydrazide, 1,8-dihydroxy-bicyclo[7.3.1]Trideca-4,9-dien-2,6-diyn-13-one, 1-dihydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, actinomycin dactinomycin, amanitin, aminopterin, anguidine, anthramycin, bleomycin, busulfan, butyric acid, carminomycin, carmustine, cemadotin, cisplatin, colchicine, combretastatin, cyclophosphamide, cytarabine, cytochalasin B, daunorubicin, decarbazine, auristatins (e.g., doxorubicin, calicheamicin, dolastatin 10, monomethyloristatin E (MMAE), monomethyloristatin F (MMAF)) series, maytansinoids (e.g., DM1, DM4) series, Duocarmycins (e.g. SJG-136), dibromomannitol, dihydroxy anthracindione, disorazole, echinomycin, eleutherobin, emetine, epothilones, esperamicins, estramustine, ethidium bromide, etoposide, geldanamycin, gramicidin D, glucocorticoids, irinotecan, kinesin spindle protein (KSP) inhibitors, leptomycin, leurosine, lidocaine, lomustine (CCNU), maytansinoids, mechlorethamine, melphalan, mercaptopurine, methopterin, methotrexate, mithramycin, mitomycin, mitoxantrone, N8-acetyl spermidine, podophyllotoxin, procaine, propranolol, Pteridine, pyrrolobenzodiazepines (PBDs), rhizoxin, streptozotocin, talisomycin, tenoposide, tetracaine, thioepa chlorambucil, tomeimycin, vinblastine, vincristine, vindesine, vinnorelbine, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum antineoplastic agents (cisplatin or derivatives thereof) and stereoisomers, isosteres, analogs or derivatives thereof.

[0363] In another embodiment of the invention, the therapeutic moiety may be an immunostimulant, a proteolytic agent, a protein kinase inhibitor, or a radiolabeled compound.

[0364] In another embodiment of the present invention, the therapeutic moiety may be a prodrug of a cytotoxic agent. The prodrug of the cytotoxic agent can be converted into a pharmaceutically active cytotoxic agent by the action of enzymes or pH under physiological conditions in vivo.

[0365] In another embodiment of the present invention, the cytotoxic agent may be a DNA damaging agent that damages DNA or a tubulin inhibitor that inhibits tubulin polymerization, preferably a tubulin inhibitor.

[0366] The DNA damaging agent includes a topoisomerase inhibitor or a DNA alkylating agent, and the tubulin inhibitor includes auristatin or maytansinoid series. The DNA alkylating agent is a drug that damages DNA cross-links, and includes pyrrolobenzodiazepine (PBD) dimer, pyrrolobenzodiazepine (PBD) dimer analogs, calicheamicins, calicheamicin analogs, duocarmycin, duocarmycin analogs, cyclophosphamide, ifosfamide, bendamustine, cisplatin, melphalan, or carboplatin.

[0367] In one embodiment of the present invention, the tubulin inhibitor may be an auristatin or maytansinoid series.

[0368] In another embodiment of the invention, the therapeutic moiety may be a tubulin polymerase inhibitor.

[0369] In another embodiment of the present invention, the tubulin polymerase inhibitor may be monomethyloristatin F (MMAF) or monomethyloristatin E (MMAE).

[0370] In another embodiment of the present invention, the cytotoxic agent may be a DNA damaging agent, and the DNA damaging agent may be a topoisomerase inhibitor or a DNA alkylating agent.

[0371] In another embodiment of the invention, the therapeutic moiety may be a topoisomerase inhibitor. In another embodiment of the invention, the therapeutic moiety may be a DNA alkylating agent. In another embodiment of the invention, the topoisomerase inhibitor may be a camptothecin analog. The camptothecin analog may include irinotecan, rubitecan, topotecan, zimatecan, pegamotecan, lutotecan, karenitecan, apelletecan, homocamptothecin, diplomotecan, belotecan, 9-aminocamptothecin, exatecan, SN-38, DXd, a DXd derivative, or an exatecan analog. Preferably, the camptothecin analog may be exatecan, an exatecan analog, SN-38, DXd, or a DXd derivative. Preferably, the camptothecin analogue may be exatecan or Dxd, or a derivative thereof. For example, the derivative may include those in which any atom of exatecan or Dxd is modified, or any carbon is substituted with any functional group consisting of halogen, alkyl, cycloalkyl, amide, oxygen, nitrogen, etc., or any carbon is modified with O, N, etc.

[0372] In one embodiment of the present invention, the therapeutic moiety may be monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), exatecan, or DXd, or a derivative thereof.

[0373] In one embodiment of the invention, the therapeutic moiety can be a therapeutic protein, including but not limited to a toxin, a hormone, an enzyme, and a growth factor. The above toxin proteins (or polypeptides) include, but are not limited to, diphtheria (e.g., diphtheria A chain), Pseudomonas exotoxins and endotoxins, ricins (e.g., ricin A chain), abrins (e.g., abrin A chain), modeccins (e.g., moscins A chain), alpha-sarcins, Aleurites fordii proteins, dianthin proteins, ribonucleases (RNases), DNase I, Staphylococcus enterotoxin-A, American spore antiviral proteins, gelonin, diphtherin toxins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitors, curcins, crotins, Sapaonaria officinalis inhibitors, mitogellins, restrictocins, phenomycins, enomycins, trichothecenes, inhibitor cystine knot (ICK) peptides (e.g., ceratotoxins) and Conotoxins (e.g., KIIIA or SmIIIa) are included, but are not limited to.

[0374] In another embodiment of the present invention, the therapeutic moiety may be a biocompatible polymer capable of increasing the serum half-life and bioactivity of the antibody-drug conjugate and / or extending the in vivo half-life. The biocompatible polymer may be, but is not limited to, one selected from the group consisting of water-soluble polymers or derivatives thereof and zwitterion-containing biocompatible polymers. An example of the water-soluble polymer is polyethylene glycol (PEG), and an example of the zwitterion-containing biocompatible polymer is a phosphorylcholine-containing polymer.

[0375] In another embodiment of the present invention, the therapeutic moiety may be an immunomodulatory agent. The immunomodulatory agent may be used to modulate the activity of immune cells in the vicinity of the antibody-drug conjugate. The above immunomodulators include gancyclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolgate mofetil, methotrexate, glucocorticoids and their analogs, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18 and IL-21), colony-stimulating factors (e.g., granulocyte-colony stimulating factor Factors that may be used include, but are not limited to, granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF), interferons (e.g., interferon-alpha, -beta, and -gamma), stem cell growth factors designated as "S1 factors," erythropoietin, thrombopoietin, or combinations thereof.

[0376] In another embodiment of the present invention, the therapeutic moiety may further comprise an imaging agent. The imaging agent may be a substance that enables imaging of the antibody-drug conjugate in a subject or a biological sample of the subject. In one embodiment, the imaging agent may be a fluorescent substance. In one embodiment, the imaging agent may comprise fluorescein, rhodamine, lanthanide phosphate and derivatives thereof, or a radioisotope bound to a chelator. The fluorescent material may be selected from the group consisting of, but is not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor™ (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700 or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5-TAMRA), tetramethylrhodamine (TMR) and sulforhodamine (SR) (e.g., SR101). The above chelator may be selected from the group consisting of, but is not limited to, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid (deferoxamine), diethylenetriaminepentaacetic acid (DTPA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA).

[0377] In one embodiment of the present invention, the therapeutic moiety may be or comprise a radionuclide, and may be a radionuclide selected from, but not limited to, 225Ac, 212Bi, 213Bi, 131I, 186Re, 227Th, 222Rn, 223Ra, 224Ra, and 90Y.

[0378] In one embodiment of the present invention, the antibody-drug conjugate may be labeled with a detectable or functional label. The detectable label may be a radioactive label (e.g., an isotope). 2 H, 3 H, 14 C, etc.), fluorescent labels, labels commonly used in MRI-CT imaging techniques, chemical labels such as biotin, etc. The functional labels may include substances designed to target the tumor location and cause tumor tissue destruction, and cytotoxic drugs (e.g., 5-fluorouracil, lysine, etc.) or enzymes (e.g., bacterial carboxypeptidase or nitroductase) may be used as functional labels, but are not limited thereto.

[0379] In another aspect of the present invention, n or DAR of the antibody-drug conjugate of the present invention may be 2 or more.

[0380] In one embodiment of the present invention, the LD (i.e., linker-therapeutic moiety) of the antibody-drug conjugate represented by the general formula I of the present invention can be prepared with one compound selected from the group consisting of compounds 1 to 13:

[0381] [Compound 1]

[0382]

[0383] [Compound 2]

[0384]

[0385] [Compound 3]

[0386]

[0387] [Compound 4]

[0388]

[0389] [Compound 5]

[0390]

[0391] [Compound 6]

[0392]

[0393] [Compound 7]

[0394]

[0395] [Compound 8]

[0396]

[0397] [Compound 9]

[0398]

[0399] [Compound 10]

[0400]

[0401] [Compound 11]

[0402]

[0403] [Compound 12]

[0404]

[0405] [Compound 13]

[0406]

[0407] (4) Combined number

[0408] In the general formula I above, n is a synonym for the so-called DAR (Drug-to-Antibody Ratio), and represents the number of therapeutic moieties (e.g., drugs) bound to one antibody. The n is an important factor affecting the efficacy and safety of the antibody-drug conjugate. The production of the antibody-drug conjugate is carried out by specifying the reaction conditions, such as the amount of raw materials and reagents to be reacted, so that the number of therapeutic moieties bound is a constant number. However, unlike the chemical reaction of small-molecule compounds, the antibody-drug conjugate is usually obtained as a mixture in which different numbers of therapeutic moieties are bound. The number of therapeutic moieties bound to one antibody molecule is specified and expressed as an average value. In the present invention, unless otherwise stated, in the case of an antibody-drug conjugate having a DAR included in an antibody-drug conjugate mixture having different drug binding numbers, the n means an average value.

[0409] In one aspect of the present invention, n or DAR in the antibody-drug conjugate of the present invention can be 1 to 20, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, or 4 to 10. Additionally, the n or DAR may be 1 to 15, 2 to 15, 3 to 15, 4 to 15 or 4 to 10. Preferably, the n or DAR may be 1 to 15.

[0410] In another embodiment of the present invention, the antibody-drug conjugate may be represented by one of the following structures, wherein Antibody is the aforementioned anti-CNTN4 antibody or antigen-binding fragment thereof, and n is 1 to 20:

[0411] ;

[0412] ;

[0413] ;

[0414] ;

[0415] ;

[0416] ;

[0417] ;

[0418] ;

[0419] ;

[0420] ;

[0421] ;

[0422] ;

[0423] ;

[0424] ;

[0425] ;

[0426] ;

[0427] ;

[0428] ; and

[0429] .

[0430] (5) Characteristics of antibody-drug conjugates

[0431] In another embodiment of the present invention, the ability of the antibody-drug conjugate to bind to CNTN4 protein can be confirmed using Flow Cytometry, ELISA Assay and / or Biolayer Interference Method.

[0432] In one embodiment of the present invention, the antibody-drug conjugate has an EC similar to that of the anti-CNTN4 antibody before conjugation. 50 Or it can represent the bond dissociation equilibrium constant (KD).

[0433] In another embodiment of the present invention, the antibody-drug conjugate has an EC of 150 nM or less, e.g., 130 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.6 nM, 0.7 nM, 0.6 nM or 0.5 nM or less, as measured by antibody-drug ELISA assay or Flow cytometry. 50 It can bind to the CNTN4 protein.

[0434] In another embodiment of the present invention, the antibody-drug conjugate is 1 x 10 -3 M, 1 x 10 -4 M, 1 x 10 -5 M, 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 The bond dissociation equilibrium constant (K) below M D ) can bind to human CNTN4 protein.

[0435] In one embodiment of the present invention, the antibody-drug conjugate has cancer cell internalization activity. The antibody-drug conjugate of the present invention can exert cytotoxic or cytostatic effects on CNTN4-expressing cancer cells. In some embodiments, the antibody-drug conjugate is internalized and accumulates in CNTN4-expressing cells, and can exert therapeutic effects such as cytotoxicity, cytostatic activity, or immunosuppression in CNTN4-expressing cells.

[0436] Method for preparing a conjugate comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof

[0437] The antibody or antigen-binding fragment thereof that can be used in the manufacture of the antibody-drug conjugate of the present invention is not particularly limited as long as it is an antibody or fragment thereof that can specifically bind to the CNTN4 protein.

[0438] In another aspect of the present invention, the therapeutic moiety, linker and antibody-drug conjugate can be prepared using known techniques.

[0439] In another embodiment of the present invention, a conjugate can be prepared by linking or conjugating a therapeutic moiety to an anti-CNTN4 antibody or antigen-binding fragment thereof for targeted local delivery of the drug to a disease associated with the function or expression of the CNTN4 protein (e.g., a CNTN4 overexpressing cancer).

[0440] In one embodiment of the present invention, an antibody-drug conjugate can be prepared through the following steps.

[0441] Step 1. Side chain activation of anti-CNTN4 antibodies

[0442] At pH 6.75, 1.64 mM or 0.34 mM TCEP, a reducing agent, is added to the anti-CNTN4 antibody. Subsequently, each antibody and TCEP are reacted for 2 hours at 25°C to reduce some of the disulfide bonds in the antibody.

[0443] In addition, to reduce some of the disulfide bonds of the antibody, 1.02 mM of TCEP is added to the anti-CNTN4 antibody at pH 7.2 to 7.4, and the antibody and TCEP are reacted for 6 hours at 40°C, or 0.38 mM of TCEP is added to the anti-CNTN4 antibody at pH 7.2 to pH 7.5, and the antibody and TCEP are reacted for 2 hours at 25°C, or 1.26 mM of TCEP is added to the anti-CNTN4 antibody at pH 7.2 to pH 7.5, and the antibody and TCEP are reacted for 30 minutes or 2 hours at 25°C.

[0444] Step 2. Preparation of antibody-drug conjugates

[0445] An antibody-drug conjugate is prepared by using an anti-CNTN4 antibody in which a portion of the disulfide bond is reduced in step 1 and a compound corresponding to the LD of the general formula I of the present invention by any one of the following steps 2-1 to 2-5.

[0446] Step 2-1.

[0447] The compound corresponding to the LD of the general formula I of the present invention is dissolved in DMSO. 20 equivalents of the compound dissolved in DMSO are added to the anti-CNTN4 antibody in which some of the disulfide bonds are reduced. The concentration of the antibody during the reaction is adjusted to 10 mg / mL or 10 to 11 mg / mL, and the reaction is performed for 3 hours using a chamber at 25°C, and the reaction substance is titrated to pH 5.0 using acetic acid. After binding the titrated substance to a column packed with cation exchange resin chromatography (CEX) resin, impurities are removed with a 50 mM sodium acetate buffer solution (pH 5.0) containing 50 mM sodium chloride, and the target protein is eluted with a 50 mM sodium acetate buffer solution (pH 5.0) containing 400 mM sodium chloride. The elution fraction ranges from 200 mAU to 500 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate can be formulated in a 50 mM sodium acetate buffer solution (pH 5.0) containing 400 mM sodium chloride, 9.5% trehalose, and 0.05% polysorbate 20.

[0448] Step 2-2.

[0449] The compound corresponding to LD of the general formula I of the present invention is dissolved in DMSO. The compound dissolved in DMSO is added to an antibody in which some of the disulfide bonds are reduced so as to be 20 or 30 equivalents. If 20 equivalents of the compound dissolved in DMSO are added, the concentration of the antibody during the reaction is adjusted to 10 to 12 mg / mL, and the reaction is carried out at 25°C for 3 hours. In addition, if 30 equivalents of the compound dissolved in DMSO are added, the concentration of the antibody during the reaction is adjusted to 6 to 8 mg / mL, and the reaction is carried out at 40°C for 20 hours. The reaction material is passed through a column filled with a desalting resin, and the target protein is eluted with a 50 mM sodium phosphate buffer solution (pH 6.5) containing 400 mM sodium chloride. The range of elution fractions is from 20 mAU to 200 mAU at 280 nm, and the finally produced anti-CNTN4 antibody-drug conjugate can be formulated in a 50 mM sodium phosphate buffer solution (pH 6.5) containing 400 mM sodium chloride, 9.5% trehalose, and 0.05% polysorbate 20.

[0450] Step 2-3.

[0451] The compound corresponding to LD of the general formula I of the present invention is dissolved in DMSO. 20 equivalents of the compound dissolved in DMSO are added to an antibody in which a portion of the disulfide bonds is reduced. The concentration of the antibody during the reaction is adjusted to 2.6 to 2.7 mg / mL, and the reaction is carried out for 3 hours using a chamber at 25°C. The reactant is diluted 7-fold with a 50 mM sodium acetate buffer solution (pH 5.0) and titrated to pH 5.0 with acetic acid. The titrated substance is bound to a column packed with a cation exchange resin chromatography (CEX) resin, and impurities are removed with a 50 mM sodium acetate buffer solution (pH 5.0). Thereafter, the target protein is eluted with a concentration gradient using a 50 mM Tris-acetic acid buffer solution (pH 9.0) containing 1 M sodium chloride, and the eluted fraction is eluted in the range of about 200 to 250 mM sodium chloride. The finally produced anti-CNTN4 antibody-drug conjugate can be formulated with a 50 mM sodium acetate buffer solution (pH 5.4) containing 0.05% polysorbate 20 and 250 mM sodium chloride.

[0452] Step 2-4.

[0453] The compound corresponding to LD of the general formula I of the present invention is dissolved in DMA. The compound dissolved in DMA is added to the antibody in which some of the disulfide bonds are reduced so as to be 20 equivalents. The concentration of the antibody during the reaction is adjusted to 6.6 to 6.8 mg / mL, and the reaction is performed for 3 hours using a chamber at 25℃. The material in which the reaction is completed is passed through a column packed with a desalting resin, and the target protein is eluted with a 20 mM L-histidine buffer solution (pH 6.5). The range of the eluted fraction is from 50 mAU to 600 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate can be formulated with 0.05% polysorbate 20 and 20 mM L-histidine buffer solution (pH 6.5).

[0454] Step 2-5.

[0455] The compound corresponding to LD of the general formula I of the present invention is dissolved in DMA. 20 equivalents of the compound dissolved in DMA are added to the antibody in which some of the disulfide bonds are reduced. The concentration of the antibody during the reaction is adjusted to 2.6 to 2.8 mg / mL, and the reaction is performed for 3 hours using a chamber at 25°C. After each material in which the reaction is completed is passed through a column packed with a desalting resin, the target protein is eluted with a 20 mM L-histidine buffer solution (pH 6.5). The range of the eluted fraction is from 50 mAU to 500 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate, Ab-XIII, can be formulated with 0.05% polysorbate 20 and 20 mM L-histidine buffer solution (pH 6.5).

[0456] Step 3. Opening the conjugate

[0457] To increase the stability of the antibody-drug conjugate obtained in step 2, a step of converting the succinimide moiety of the additionally prepared antibody-drug conjugate to succinamide may be included.

[0458] The prepared antibody-drug conjugate is titrated to pH 9.0 to pH 9.5 using 1 M Tris base, and the reaction is carried out at 25°C for 18 to 20 hours. After the reaction is complete, the reaction can be terminated by titrating again to pH 6.5 to pH 7.0 with 1 M acetic acid.

[0459] In another embodiment of the present invention, purification of the antibody-drug conjugate can be performed using known methods such as batch method, circulating process, flow-through process, separation using hydrophobic resin, etc. for antibody-drug conjugate having an optimal drug to antibody ratio (DAR) of a certain numerical value.

[0460] Uses of anti-CNTN4 antibodies or antigen-binding fragments thereof and anti-CNTN4 antibody-drug conjugates

[0461] The antibodies and / or antibody-drug conjugates of the present invention may be useful for a variety of applications, including, but not limited to, therapeutic treatment methods and / or diagnostic treatment methods.

[0462] In one aspect of the present invention, the antibody or antigen-binding fragment thereof and / or the antibody-drug conjugate can be used to specifically bind to CNTN4 protein and prevent, improve and / or treat diseases associated with the function or expression of CNTN4 protein.

[0463] In another embodiment of the present invention, the disease associated with the function or expression of the CNTN4 protein may be cancer.

[0464] The anti-CNTN4 antibody or antigen-binding fragment thereof or antibody-drug conjugate of the present invention can effectively bind to cancer cells expressing CNTN4 by specifically binding to the CNTN4 protein, thereby inhibiting the growth of cancer cells in vivo, and can be usefully used for preventing, improving, or treating cancer. CNTN4 is known to be associated with tumor migration, and its overexpression is known to occur particularly in gastric cancer, colon cancer, liver cancer, gallbladder cancer, endometrial cancer, bladder cancer, prostate cancer, pancreatic cancer, melanoma, etc. Accordingly, the anti-CNTN4 antibody of the present invention can be used for all cancers in which tumor migration is observed, such as metastatic cancer, and can also be used for specific cancer types in which CNTN4 overexpression is observed.

[0465] Examples of cancers whose growth can be inhibited using the anti-CNTN4 antibody or antigen-binding fragment thereof or antibody-drug conjugate of the present invention include (1) leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia such as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, myelodysplastic syndrome, preleukemia and chronic myelomonocytic leukemia (CMML)); (2) lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma); (3) myeloma; (4) bone and connective tissue sarcomas (e.g., osteosarcoma, osteosarcoma, chondrosarcoma, Ewing sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma, fibrosarcoma, Kaposi sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic cancer, schwannoma, rhabdomyosarcoma, and synovial sarcoma); (5) brain cancers (e.g., glioma, glioblastoma, astrocytic, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pinealoma, pineoblastoma, oligodendroglioma, primary brain lymphoma, pituitary cancer); (6) breast cancer; (7) adrenal cancer; (8) thyroid cancer; (9) pancreatic cancer; (10) ocular cancer (e.g., ocular melanomas, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma); (11) vaginal cancer; (12) vulvar cancer; (13) uterine cancer (e.g., endometrial cancer, squamous cell carcinoma, uterine sarcoma, and adenocarcinoma); (14) ovarian cancer (e.g., ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor); (15) esophageal cancer (e.g., squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma); (16) gastric cancer (e.g., adenocarcinoma, mycosis (polypoid), ulcerative, superficial spreading, diffuse expansile, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma); (17) colorectal cancer (e.g., colon cancer, rectal cancer, and anal cancer); (18) Liver cancer (e.g., hepatocellular carcinoma and hepatoblastoma; (19) Gallbladder cancer; (20) Cholangiocarcinoma;(21) lung cancer (e.g., non-small cell lung cancer, squamous cell carcinoma (epidermoid cystic carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer); (22) testicular cancer (e.g., germ cell tumor, seminoma, anaplastic, classical (typical), spermatocyte, nonseminomatous, embryonal carcinoma, teratoma carcinoma, and choriocarcinoma (yolk sac tumor)); (23) prostate cancer (e.g., adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma); (24) penile cancer; (25) oral cancer (e.g., salivary gland cancer, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, squamous cell carcinoma); (26) salivary gland cancer; (27) skin cancer; (28) kidney cancer (e.g., renal cell carcinoma, adenocarcinoma, renal cell carcinoma, fibrosarcoma, and transitional cell carcinoma); (29) bladder cancer (e.g., transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma); (30) head and neck cancer (including cancer of the mouth, nose, pharynx, larynx, paranasal sinuses, or salivary glands, and head and neck squamous cell carcinoma); (31) melanoma; (32) appendix cancer, bronchial cancer, choriocarcinoma, chordoma, ependymoma, gastrointestinal stromal tumor (GIST), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), malignant peripheral nerve sheath tumor (MPNST); (33) tongue cancer; (34) small intestine cancer; (35) heart cancer; (36) duodenal cancer;(37) parathyroid cancer, and urethral cancer, but are not limited thereto (see Fishman et al., 1985, Medicine, 2d Ed., JB Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America). Meanwhile, the tumor may be a metastatic tumor, an unresectable tumor, or a locally advanced tumor. In one embodiment of the present invention, the disease associated with the function or expression of the CNTN4 protein may be a solid tumor.;

[0466] In another embodiment of the present invention, the disease associated with the function or expression of the CNTN4 protein may be a cancer that is refractory to or resistant to existing immune checkpoint inhibitors (e.g., resistant to a PD-1 pathway inhibitor, a PD-L1 pathway inhibitor, or a CTLA-4 pathway inhibitor).

[0467] In one embodiment of the present invention, the present invention provides a method for preventing, ameliorating or treating a disease associated with the function or expression of CNTN4, such as cancer, comprising administering to a subject a therapeutically effective amount of an anti-CNTN4 antibody-drug conjugate.

[0468] In another embodiment of the present invention, the present invention provides the use of an anti-CNTN4 antibody-drug conjugate for the prevention, amelioration or treatment of a disease associated with the function or expression of CNTN4, such as cancer.

[0469] In another aspect of the present invention, the present invention provides a pharmaceutical composition for treating a disease associated with the function or expression of the CNTN4 protein, comprising an anti-CNTN4 antibody-drug conjugate; and a pharmaceutically acceptable carrier.

[0470] In another embodiment of the present invention, a method of treating a subject having a disease associated with the function or expression of a CNTN4 protein, or suspected of having a disease associated with the function or expression of a CNTN4 protein, is provided, comprising administering to the subject a therapeutically effective amount of an anti-CNTN4 antibody-drug conjugate or a pharmaceutical composition comprising the same.

[0471] In another aspect of the present invention, the present invention relates to the use of an anti-CNTN4 antibody-drug conjugate or a pharmaceutical composition comprising the same for the manufacture of a medicament for the treatment of a disease associated with the function or expression of CNTN4.

[0472] In another aspect of the present invention, the present invention provides the use of an anti-CNTN4 antibody-drug conjugate or a pharmaceutical composition comprising the same for the treatment of a disease associated with the function or expression of CNTN4.

[0473] In another aspect of the present invention, a pharmaceutical composition comprising an anti-CNTN4 antibody-drug conjugate for preventing, improving, or treating a disease associated with the function or expression of CNTN4, such as cancer, may be provided. The anti-CNTN4 antibody-drug conjugate may be included in the pharmaceutical composition in a therapeutically effective amount.

[0474] The pharmaceutical composition may contain inactive ingredients, i.e., pharmaceutically acceptable excipients [see Handbook of Pharmaceutical Excipients, etc.]. The pharmaceutical composition may be prepared by mixing with a physiologically acceptable carrier, excipient, or stabilizer, for example, in the form of a lyophilized powder, slurry, aqueous solution, or suspension.

[0475] Suitable routes of administration for the pharmaceutical composition include parenteral administration, such as intramuscular, intravenous, or subcutaneous administration. Administration of the antibodies used in the pharmaceutical compositions of the present invention or for practicing the methods of the present invention can be accomplished by various conventional methods, such as topical application or intradermal, subcutaneous, intraperitoneal, parenteral, intraarterial, or intravenous injection. In one embodiment, the antibodies of the present invention are administered intravenously or subcutaneously.

[0476] The antibody-drug conjugate of the present invention can be used alone or in combination with other therapeutic agents, or preferably, other anticancer therapies. Other anticancer therapies may include, for example, standard cancer therapies (e.g., chemotherapy, radiation therapy, or surgery); or antibodies conjugated to other anticancer agents, such as cytotoxic agents, cytostatic agents, antihormonal agents, antiangiogenic or antimetabolic agents, targeted anticancer agents, immunostimulants or immunomodulators, immune checkpoint inhibitors, or cytotoxic agents, cytostatic agents, and other toxic agents.

[0477] Preferably, the anti-CNTN4 antibody-drug conjugate of the present invention can be used in combination therapy with other anticancer agents, such as immune checkpoint modulators, chemotherapeutic agents, or radiotherapy. Such chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, kinase inhibitors, spindle toxin plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, antiestrogens and selective estrogen receptor modulators (SERMs), antiprogesterones, estrogen receptor downregulators (ERDs), estrogen receptor antagonists, luteinizing hormone-releasing hormone agonists, antiandrogens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, and antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Specific examples of the chemotherapeutic agent of the present invention include gemcitabine, vinorelbine, etoposide (VP-16), platinum analogs such as cisplatin or carboplatin, taxoids such as paclitaxel, albumin-bound paclitaxel, docetaxel, and the like.

[0478] In another embodiment of the present invention, the anti-CNTN4 antibody-drug conjugate of the present invention may be used in patients who have not responded to other immune checkpoint modulator treatments.

[0479] When the antibody-drug conjugate of the present invention is used in combination with another anticancer agent, they may be administered separately or in the form of a combination product in which multiple active ingredients are present in a single pharmaceutical formulation. When administered as separate formulations, the two formulations may be administered sequentially or simultaneously. In the case of simultaneous administration, they are administered to the subject together. In the case of sequential administration, they may be administered with a short interval of time (e.g., within a period of 12 hours or less, or within a period of 6 hours or less) and may be administered to the subject at intervals of 1, 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks or more.

[0480] The pharmaceutical composition described above may additionally include an anticancer agent. The anticancer agent may be administered simultaneously with the antibody-drug conjugate as a single formulation, or simultaneously or sequentially as separate formulations. The type and method of administration of the anticancer agent are as described above.

[0481] In one embodiment, the present invention provides a method for preventing, ameliorating, or treating cancer, comprising administering to a subject a therapeutically effective amount of an antibody-drug conjugate in combination with an additional anticancer agent. This embodiment includes administering the additional anticancer agent simultaneously in a single composition, as well as administering separate compositions containing each of the additional anticancer agent simultaneously or sequentially to a subject in need thereof. In this case, the route of administration includes parenteral administration, such as intramuscular, intravenous, or subcutaneous administration.

[0482] The additional anticancer agent is preferably an immune checkpoint inhibitor, more preferably an anti-CTLA-4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab, nivolumab), or an anti-PD-L1 antibody (e.g., atezolizumab, avelumab, durvalumab). Other preferred additional anticancer agents include chemotherapeutic agents such as gemcitabine, vinorelbine, etoposide (VP-16), platinum analogs such as cisplatin or carboplatin, taxoids such as paclitaxel, albumin-bound paclitaxel, or docetaxel.

[0483] In another aspect of the present invention, the present invention provides the use of an anti-CNTN4 antibody-drug conjugate for use in combination with an additional anticancer agent for the prevention, amelioration or treatment of cancer.

[0484] In one aspect, the present invention provides a pharmaceutical composition or combination comprising an anti-CNTN4 antibody-drug conjugate and an additional anticancer agent for preventing, ameliorating, or treating cancer. The pharmaceutical composition or combination comprising the additional anticancer agent herein includes cases where the two components are physically present together in the form of a single formulation, as well as cases where they are administered simultaneously or sequentially as separate formulations, wherein the two drugs may be provided separately or together in a single kit. Accordingly, the present invention provides a kit for preventing, ameliorating, or treating cancer, comprising an anti-CNTN4 antibody-drug conjugate and an additional anticancer agent.

[0485] In another aspect of the present invention, the present invention provides the use of the anti-CNTN4 antibody-drug conjugate in the manufacture of a medicament for treating a disease associated with the function or expression of CNTN4.

[0486]

[0487] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0488] Manufacturing Example 1: Manufacturing of CNTN4-specific antibodies through phage display

[0489] Manufacturing Example 1.1. Production and Screening of an scFv Antibody Library Using Phage Display

[0490] mRNA for the heavy and light chain variable regions of antibodies obtained from human blood or bone marrow was amplified by PCR to synthesize cDNA. The synthesized cDNA was cloned into a phagemid vector using restriction enzymes and expressed in E. coli via electroporation. The expressed cDNA was then infected with helper phage to produce a human library in the form of scFv.

[0491] Using the above library as a target, biopanning was used to screen for antibodies that bind with high affinity to the target antigen, CNTN4. Positive clones that bind to human CNTN4 protein were selected, and scFv sequences specific for CNTN4 were selected through sequencing.

[0492] Manufacturing Example 1.2. Preparation of Ab1 and Ab2 antibodies

[0493] Two anti-CNTN4 antibodies (Ab1 antibody, Ab2 antibody) were prepared, each comprising a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 7 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 8. The heavy chain constant region of the Ab1 antibody is an IgG4 heavy chain constant region (SEQ ID NO: 9) into which the S228P mutation has been introduced, the heavy chain constant region of the Ab2 antibody is a human IgG1 heavy chain constant region (SEQ ID NO: 11) into which the LALA mutation has been introduced, and the light chain constant regions of the Ab1 and Ab2 antibodies are proteins represented by the amino acid sequence of SEQ ID NO: 10.

[0494] Amino acid sequence information of sequence numbers 7 to 11 is listed in Table 1.

[0495]

[0496] <Ab1 항체의 제조 및 정제>

[0497] The heavy chain variable region protein represented by the amino acid sequence of SEQ ID NO: 7 was fused with the IgG4 heavy chain constant region protein represented by SEQ ID NO: 9. The light chain variable region protein represented by the amino acid sequence of SEQ ID NO: 8 was fused with the human light chain constant region protein represented by the amino acid sequence of SEQ ID NO: 10.

[0498] Codon optimization was performed on the DNA base sequence encoding the above amino acid sequence through a codon optimization process. The optimized codon sequence was then synthesized and cloned into the pCGS3.2 expression vector, and a single cell line using a CHO cell line was produced using this. This cell line was batch-cultured for 14 days, and then a pure culture solution was obtained using a depth filter. The obtained culture solution was used to perform a purification process in the order of protein A, anion exchange resin, and mixed-mode chromatography, and then the buffer was exchanged through ultrafiltration and a formulation process to complete the production of the final antibody, Ab1.

[0499] <Ab2 항체의 제조 및 정제>

[0500] The heavy chain variable region protein represented by the amino acid sequence of SEQ ID NO: 7 was fused with the heavy chain constant region protein of human IgG1 represented by the amino acid sequence of SEQ ID NO: 11, and the light chain variable region protein represented by the amino acid sequence of SEQ ID NO: 8 was fused with the human light chain constant region protein represented by the amino acid sequence of SEQ ID NO: 10.

[0501] Amino acid sequences of SEQ ID NOs: 7, 8, 10, and 11 were codon-optimized using a DNA base sequence codon optimization process. The optimized codon sequences were then synthesized and cloned into the pcDNA3.4 expression vector. Transient transfection using the vectors was performed in ExpiCHO-S cells, and antibody expression was achieved after culturing for 8 days.

[0502] The antibody was purified using affinity chromatography using protein A. The antibody was bound to an affinity chromatography resin equilibrated with 20 mM histidine buffer (pH 6.0), washed with 20 mM histidine buffer (pH 6.0) containing 500 mM sodium chloride, and the target protein was eluted with 100 mM glycine buffer (pH 3.4) at low pH. The eluted protein was formulated with 20 mM histidine buffer (pH 6.5) containing 100 mM sodium chloride, thereby completing the production of the final antibody, Ab2.

[0503] Manufacturing Example 1.3. Purity Analysis of Ab1 and Ab2 Antibodies

[0504] The absorbance of Ab1 and Ab2 antibodies prepared in Manufacturing Example 1.2 above was measured using Nanodrop, and the concentration was determined by applying the measured absorbance values ​​to the A280 / Extinction coefficient equation. In addition, the purity of the purified protein was confirmed by analysis using SDS-PAGE and SEC-HPLC.

[0505] Specifically, 20 μg of Ab1 and Ab2 antibodies were each loaded onto an SEC-HPLC column, and the analysis was performed under the condition of applying 50 mM sodium phosphate buffer (pH 6.8) containing 150 mM sodium chloride at a flow rate of 1 mL / min, and the purity of the antibodies was confirmed using a 214 nm UV detector. The SEC-HPLC analysis results of Ab1 and Ab2 antibodies are shown in Figs. 1 and 2, respectively, and the SDS-PAGE analysis results of Ab1 and Ab2 antibodies are shown in Fig. 3, and the measured purities of Ab1 and Ab2 antibodies are described in Table 2.

[0506]

[0507] The manufactured Ab1 and Ab2 antibodies were found to have a high purity exceeding 98%.

[0508] Manufacturing Example 1.4. Preparation of Ab3 antibody

[0509] The heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 7 was fused with a human IgG1 heavy chain constant region having an LALAPG mutation introduced as SEQ ID NO: 12, and the light chain variable region protein represented by the amino acid sequence of SEQ ID NO: 8 was fused with a human light chain constant region protein represented by the amino acid sequence of SEQ ID NO: 10. Using the fused amino acid sequence, protein production through transient expression was requested to GenScript, and Ab3 antibody was manufactured.

[0510] The amino acid sequence information of sequence number 12 is listed in Table 3.

[0511]

[0512] Codon optimization was performed on the DNA base sequence encoding the above amino acid sequence, and the corresponding sequence was synthesized and cloned into the pcDNA3.4 expression vector. Antibody expression was performed through transient transfection in ExpiCHO-S cells using the expression vector. Subsequently, the obtained antibody was purified using affinity chromatography using protein A and formulated in a PBS buffer solution to complete the production of Ab3.

[0513] The concentration of the manufactured Ab3 antibody was measured using A280 absorbance, and the protein purity was confirmed by analysis using SDS-PAGE and SEC-HPLC. The results of SEC-HPLC analysis of the Ab3 antibody are shown in Figure 4, and the results of SDS-PAGE analysis are shown in Figure 5, and the measured purity of the Ab3 antibody is described in Table 4.

[0514]

[0515] The manufactured Ab3 antibody was found to have high purity.

[0516] Example 1. CNTN4 protein binding ability of Ab1 antibody

[0517] Example 1.1. CNTN4 protein binding ability of Ab1 antibody (ELISA analysis)

[0518] The ability of the Ab1 antibody manufactured in the above Manufacturing Example 1 to bind to human CNTN4 protein was analyzed using the ELISA (Enzyme-Linked Immunosorbent Assay) method.

[0519] Human CNTN4 protein (Biointron, China), an antigen protein, was diluted with PBS buffer to prepare a concentration of 5 nM. 100 μL of human CNTN4 protein diluted with PBS buffer was added to each well of a 96-well plate (96-well plate, Costar, USA, Cat. No. 9018) and coated overnight at 4°C. The following day, all solutions in the 96-well plate were removed, and blocking buffer (3% BSA in PBS) was added to each well, followed by incubation for 1 hour at room temperature. Ab1 antibody and human IgG4 (hIgG4) antibody samples, which are isotype controls, were serially diluted from 20 nM to 11 points (0.3 pM) at a 3-fold dilution ratio with blocking buffer. After the blocking process, the buffer was removed from each well, and 100 μL of the diluted antibody was added to each well and reacted at room temperature for 1 hour. The wells were washed with PBST (0.1% Tween 20 in PBS), and HRP-conjugated anti-human IgG Fc antibody (ThermoFisher Scientific, USA, Cat. No.: 31423) (1:100,000), a secondary antibody for antibody detection, was treated at room temperature for 1 hour, and then washed again with PBST. For color development, 100 μL of TMB solution (TMB solution, ThermoFisher Scientific, USA, Cat. No.: 34028) was added to each well, reacted at room temperature for 10 minutes, and then the absorbance at 450 nm (OD) was measured. 450 ) was measured, OD according to antibody treatment concentration 450 The values ​​are shown in Figure 6.

[0520] EC to indicate the level of binding affinity of Ab1 antibody to CNTN4 protein 50The concentration (half maximal effective concentration) was calculated, and the EC of Ab1 antibody 50 (nM) was 0.138. Therefore, it was determined that the Ab1 antibody had excellent binding ability to human CNTN4 protein.

[0521] Example 1.2. CNTN4 protein binding ability of Ab1 antibody (FACS test)

[0522] The ability of the Ab1 antibody prepared in the above Manufacturing Example 1 to bind to human CNTN4 protein was analyzed by FACS (Fluorescence Activated Cell Sorting, Flow Cytometry) (FACS CantoTM II, BD, USA) test.

[0523] Transfection was performed with the CNTN4 plasmid vector into HEK293 cells (ThermoFisherScientific, USA), and HEK293 cells expressing human CNTN4 antigen protein were designated HEK293 / CNTN4 cells.

[0524] 1 x 10 suspended in FACS buffer 6 HEK293 / CNTN4 cells (cells / mL) were seeded in 100 μL per 96-well plate.

[0525] In addition, Ab1 antibody and human IgG4 (human IgG4) antibody samples, which are isotype controls, were serially diluted from 10 μg / mL to 14 points (6.3 pg / mL) at a 3-fold dilution ratio in FACS buffer. The diluted antibodies were treated on a 96-well plate seeded with cells and incubated at 4°C for 1 hour. Each well was washed with FACS buffer, and Goat Anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody (Alexa Fluor 647 (ThermoFisher Scientific, USA, Cat. No.: A21445) (1:400)) was added, and the 96-well plate was further incubated, followed by washing with FACS buffer. The degree of antigen-antibody binding was measured as the Mean Fluorescence Intensity (MFI) value using a flow cytometer, and the MFI values ​​according to the antibody treatment concentration are shown in Figure 7.

[0526] EC is used to indicate the degree of binding of Ab1 antibody to antigen protein expressed on the cell surface. 50 The concentration was calculated. The calculated EC 50 The concentrations are described in Table 5.

[0527]

[0528] As confirmed in Fig. 7 and Table 5, Ab1 antibody was confirmed to have excellent binding ability to human CNTN4 protein expressed on the cell surface.

[0529] Example 1.3. CNTN4 protein binding ability of Ab1 antibody (Biolayer interferometry)

[0530] The ability of the Ab1 antibody manufactured in the above Manufacturing Example 1 to bind to human CNTN4 protein was analyzed by bio-layer interferometry (BLI) using an Octet device (Octet® R8, Sartorius).

[0531] A biosensor (Anti-human Fc-Capture 2nd Generation (AHC2) Biosensor, Cat. No. 18-5142, Sartorius) that binds to the human antibody constant region was mounted on an Octet instrument and immersed in an Ab1 antibody solution (1 μg / mL) for 5 minutes to bind the Ab1 antibody. The biosensor with the Ab1 antibody bound was then washed with a PBS buffer solution containing 0.02% Tween 20. The washed biosensor was placed in a buffer solution containing the antigen (human CNTN4 protein), and the antibody-protein binding reaction was monitored for 10 minutes, and the antibody-protein dissociation reaction was then observed for 10 minutes. At this time, the antigen solution was serially diluted to determine the association and dissociation rates according to different concentrations. The association (Ka) and dissociation (Kd) rate constants were determined by fitting them to a 1:1 binding model using curve fitting software, and then the association-dissociation equilibrium constant (K D ) was calculated using the equation KD = Kd / Ka, and the binding dynamics data are shown in Table 6 below.

[0532]

[0533] As confirmed in Table 6, it was confirmed that Ab1 antibody had excellent dynamic binding ability to human CNTN4.

[0534] Example 2. Cellular internalization of Ab1 antibody into cancer cells

[0535] An experiment was conducted to confirm whether the Ab1 antibody manufactured in Manufacturing Example 1 was internalized into cancer cells.

[0536] A549 cells (Korean Cell Line Bank, South Korea), a lung cancer cell line, were transfected with the CNTN4 plasmid vector, and A549 cells overexpressing human CNTN4 antigen protein were named A549 / CNTN4 cells.

[0537] 1 x 10 5 A549 / CNTN4 cells (cells / mL) were seeded in 50 μL per well of a 96-well plate (3595, Corning, USA). The next day, the attachment of cells to each well of the plate was confirmed, and the existing cell culture medium was removed.

[0538] The Ab1 antibody manufactured in Manufacturing Example 1 and the human IgG4 antibody as an isotype control were diluted in cell culture medium (RPMI1640 with L-glutamine (300 mg / L), 25 mM HEPES and 25 mM NaHCO3, 90%; heat-inactivated fetal bovine serum (FBS), 10%) to twice the final concentration of 0.5, 1, 2, and 4 μg / mL, respectively. The diluted antibodies were mixed with IncuCyte® FabFluor-pH Red Antibody labeling reagent (Sartorius, 4722) and reacted at 37°C for 15 minutes. 50 μL was processed per well and placed in the IncuCyte® S3 Live-Cell Analysis System, and images were captured at 1-hour intervals for 24 hours. The image results measured for 24 hours are shown in Fig. 8.

[0539] As confirmed in Figure 8, the Ab1 antibody was internalized into cancer cells with increasing incubation time and concentration, resulting in an increase in cell fluorescence signal. However, no change in cell fluorescence signal was observed when treated with human IgG4 antibody. Therefore, this suggests that the antibody of the present invention can transport cytotoxic drugs or therapeutic moieties into cells through cell internalization. In other words, it was confirmed that the antibody of the present invention can be used in the form of an ADC conjugated to a drug.

[0540] Manufacturing Example 2. Manufacturing of a Linker-Therapeutic Moiety

[0541] A compound to be used as an LD (i.e., linker-therapeutic moiety) of an antibody-drug conjugate represented by the general formula I of the present invention was prepared.

[0542] Manufacturing Example 2.1.

[0543] Preparation of (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (Compound 1)

[0544] [Synthetic formula of compound 1]

[0545]

[0546] Compound 1a:

[0547] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (also called Monomethylauristatin F, MMAF)

[0548] Compound 1b:

[0549] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate

[0550] 1.11 g (1.50 mmol) of compound 1b (AK001RR3, Jiangsu Aikon Biopharmaceutical) was dissolved in 20 mL of N,N-dimethylformamide, and 167 mg (1.50 mmol) of 2-hydroxypyridine N-oxide was added at 20°C and stirred for 5 minutes. 1 g (1.37 mmol) of compound 1a (HY-15579, Haoyuan chemexpress) and 3.68 g (34.3 mmol) of 2,6-lutidine were added to the reaction mixture, and the mixture was stirred at 20°C for 20 hours. After distillation under reduced pressure, the obtained residue was separated using a high-performance liquid chromatography system. The obtained residue was lyophilized to obtain 1.28 g of compound 1 (yield: 70%).

[0551] MS (ESI + ): m / z = 1330.6 [M+H] +

[0552] Manufacturing Example 2.2.

[0553] Preparation of 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (compound 4)

[0554] [Synthetic formula of compound 4]

[0555]

[0556] Compound 4a:

[0557] (1S,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (also called exatecan) methanesulfonic acid

[0558] Compound 4b:

[0559] (9H-fluoren-9-yl)methyl ((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate

[0560] Compound 4c:

[0561] 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0562] Compound 4d:

[0563] 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-aza-31-hentriacontanoic acid

[0564] Compound 4e:

[0565] 2,5-Dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oate

[0566] Step 1) Preparation of compound 4c

[0567] Compound 4a (BD630430, Bide) 315 mg (0.59 mmol) was dissolved in 4 mL of N,N-dimethylformamide. 100 mg (0.74 mmol) of 1-hydroxybenzotriazole and 214.94 μL (1.23 mmol) of N,N-diisopropylethylamine were added. To this solution, 420 mg (0.62 mmol) of compound 4b was added, and the mixture was stirred at 15 °C for 4 hours under a nitrogen atmosphere. 746 mg (8.76 mmol) of piperidine was added to the reaction mixture, and the mixture was stirred at 15 °C for 1 hour under a nitrogen atmosphere. Upon completion of the reaction, the reaction mixture was slowly added to 100 mL of methyl tert-butyl ether, filtered under reduced pressure, and the filtrate was distilled under reduced pressure. The obtained residue was separated using a preparative chromatography system. The obtained residue was freeze-dried to obtain 480 mg of compound 4c (yield: 97%).

[0568] MS (ESI + ): m / z = 755.3 [M+H] + .

[0569] Step 2) Preparation of compound 4e

[0570] Compound 4d (2.0 g, 3.37 mmol) was dissolved in 160 mL of dichloromethane, and 583 mg (5.06 mmol) of N-hydroxysuccinimide and 970 mg (5.06 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, followed by stirring at 25 °C for 2 hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane and washed three times with aqueous hydrochloric acid solution. The organic layer was separated, dried over anhydrous sodium sulfate, filtered under reduced pressure, and distilled under reduced pressure. The obtained residue was dried under reduced pressure to quantitatively obtain 2.39 g of compound 4e.

[0571] 1H NMR (400 MHz, DMSO-d6) δ 8.00 (1H, t), 7.00 (1H, s), 5.75 (1H, s), 3.68-3.74 (2H, m), 3.59 (2, t), 3.46-3.56 (28H, m), 3.35-3.39 (2H, m), 3.14 (2H, q), 2.92 (2H, t), 2.81 (4H, s), 2.33 (2H, t).

[0572] Step 3) Preparation of compound 4

[0573] Compound 4c460 mg (0.61 mmol) prepared in step 1) was dissolved in 4.5 mL of N,N-dimethylformamide, and 212.3 μL (1.22 mmol) of N,N-diisopropylethylamine and 4e420 mg (0.61 mmol) prepared in step 2) were added, followed by stirring at 0 °C for 1 hour. Upon completion of the reaction, the reaction mixture was distilled under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system. The obtained residue was freeze-dried to obtain 204 mg of compound 4 (yield: 24%).

[0574] MS (ESI + ): m / z = 1329.6 [M+H] + .

[0575] Manufacturing Example 2.3.

[0576] N-((S)-1-(((S)-1-((((1S,9S)-1-amino-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide trifluoroacetic acid (compound) 5) Manufacturing

[0577] [Synthetic formula of compound 5]

[0578]

[0579] Compound 5a:

[0580] tert-Butyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0581] Compound 5b:

[0582] (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazaundecan-11-yl acetate

[0583] Compound 5c:

[0584] (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((((1S,9S)-1-((tert-butoxycarbonyl)amino)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxo)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[0585] Compound 5d:

[0586] tert-Butyl ((1S,9S)-9-(((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)methoxy)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0587] Compound 5e:

[0588] tert-Butyl ((1S,9S)-9-(((4S,7S)-39-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-7-isopropyl-4-methyl-3,6,9,37-tetraoxo-12,15,18,21,24,27,30,33-octaoxa-2,5,8,36-tetraazanonatriacontyl)oxy)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0589] Step 1) Preparation of compound 5a

[0590] Compound 4a5 g (9.4 mmol) was dissolved in 50 mL of N,N-dimethylformamide, and 3.28 mL (18.8 mmol) of N,N-diisopropylethylamine and 3.24 mL (14.1 mmol) of di-tert-butyl dicarbonate were added, and the mixture was stirred at 25 °C for 1 hour. Upon completion of the reaction, the reaction mixture was diluted with distilled water and extracted three times with 150 mL of ethyl acetate. The organic layer was separated, washed sequentially with distilled water and a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered under reduced pressure, and distilled under reduced pressure. The obtained residue was separated using column chromatography to obtain compound 5a4.6 g (yield: 91%).

[0591] 1H NMR (400 MHz, DMSO-d6) δ 7.56-7.75 (2H, m), 7.27 (1H, s), 6.51 (1H, s), 5.42 (2H, s), 5.13-5.27 (2H, m), 4.95 (1H, d), 3.17-3.26 (1H, m), 3.00-3.11 (1H, m), 2.31 (3H, s), 2.17-2.26 (1H, m), 1.99-2.08 (1H, m), 1.79-1.92 (2H, m), 1.51 (9H, s), 0.87 (3H, t).

[0592] Step 2) Preparation of compound 5c

[0593] Compound 5a (1.0 g, 1.87 mmol) and compound 5b (1.8 g, 3.73 mmol) prepared in the above step 2) were dissolved in 30 mL of tetrahydrofuran, and 3.73 mL (3.73 mmol) of a 1M potassium tert-butoxide tetrahydrofuran solution was slowly added at 0 °C under nitrogen atmosphere, and the mixture was stirred at 0 °C for 5 minutes. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the filtrate was distilled under reduced pressure. The obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain 145 mg of compound 5c (yield: 8%).

[0594] MS (ESI + ): m / z = 957.2 [M+H] + .

[0595] Step 3) Preparation of compound 5d

[0596] 145 mg (0.15 mmol) of compound 5c prepared in the above step 2) was dissolved in 2 mL of N,N-dimethylformamide, 510 mg (5.86 mmol) of morpholine was added, and the mixture was stirred at 25 °C for 1 hour. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain 95 mg of compound 5d (yield: 78%).

[0597] MS (ESI + ): m / z = 735.2 [M+H] + .

[0598] Step 4) Preparation of compound 5e

[0599] In the above step 3), 95 mg (0.13 mmol) of compound 5d and 84 mg (0.14 mmol) of compound 4d prepared were dissolved in 2 mL of N,N-dimethylformamide, and 43 mg (0.16 mmol) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride was added at 0 °C under nitrogen, and the mixture was stirred at 25 °C for 30 minutes. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain 44 mg of compound 5e (yield: 25%).

[0600] MS (ESI + ): m / z = 1309.3 [M+H] + .

[0601] Step 5) Preparation of compound 5

[0602] Compound 5e39 mg (0.03 mmol) prepared in the above step 4) was dissolved in 4 mL of 1,1,1,3,3,3-hexafluoro-2-propanol, 154 mg (1.35 mmol) of trifluoroacetic acid was added at 0 °C, and the mixture was stirred at 0 °C for 24 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain 9.6 mg of compound 5 (yield: 26%).

[0603] MS (ESI + ): m / z = 1209.3 [M+H] + .

[0604] Manufacturing Example 2.4.

[0605] N-((S)-1-(((1S,9S)-1-amino-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide trifluoroacetic acid (compound 6) manufacturing

[0606] [Synthetic formula of compound 6]

[0607]

[0608] Compound 6a:

[0609] (9H-fluoren-9-yl)methyl (2-((((1S,9S)-1-((tert-butoxycarbonyl)amido)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)amino)-2-oxoethyl)carbamate

[0610] Compound 6b:

[0611] tert-Butyl ((1S,9S)-9-((2-aminoacetamido)methoxy)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0612] Compound 6c:

[0613] (1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oyl)glycylglycyl-L-phenylalanine

[0614] Compound 6d:

[0615] tert-Butyl ((1S,9S)-9-(((S)-7-benzyl-45-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,43-hexaoxo-18,21,24,27,30,33,36,39-octaoxa-2,5,8,11,14,42-hexaazapentatetracontyl)oxy)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0616] Step 1) Preparation of compound 6a

[0617] Except that 1.38 g (3.73 mmol) of (2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate was used instead of compound 5b used in step 2) of the above manufacturing example 2.3, the same process as step 2) of the above manufacturing example 2.3 was performed to obtain 850 mg of compound 6a (yield: 86%).

[0618] MS (ESI + ): m / z = 844.2 [M+H] + .

[0619] Step 2) Preparation of compound 6b

[0620] Compound 6a780 mg (0.794 mmol) prepared in the above step 1) was dissolved in 8 mL of N,N-dimethylformamide, 0.91 mL (10.3 mmol) of morpholine was added, and the mixture was stirred at -10 °C for 16 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain 182 mg of compound 6b (yield: 35%).

[0621] MS (ESI + ): m / z = 622.2 [M+H] + .

[0622] Step 3) Preparation of compound 6c

[0623] 2.02 g (2.87 mmol) of compound 4e prepared in step 2) of the above Preparation Example 2.2 and 841 mg (3.01 mmol) of glycyl-glycyl-L-phenylalanine were dissolved in 24 mL of N,N-dimethylformamide, 1.27 g (9.86 mmol) of N,N-diisopropylethylamine was added, and the mixture was stirred at 20 °C for 2 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using a liquid chromatography system and freeze-dried to obtain 2.16 g of compound 6c (yield: 88%).

[0624] 1H NMR (400 MHz, DMSO-d6) δ 8.16 (1H, t), 8.10 (1H, d), 8.00 (2H, q), 7.20-7.28 (5H, m), 7.00 (2H, s), 4.38-4.43 (1H, m), 3.67-3.72 (2H, m), 3.57-3.62 (4H, m), 3.47-3.52 (28H, m), 3.36-3.38 (4H, m), 3.14 (2H, q), 3.02-3.06 (1H, m), 2.85-2.91 (1H, m), 2.39 (2H, t), 2.32 (2H, t).

[0625] Step 4) Preparation of compound 6d

[0626] 112 mg (0.180 mmol) of compound 6b prepared in step 2) and 154 mg (0.180 mmol) of compound 6c prepared in step 3) were dissolved in 3 mL of N,N-dimethylformamide, and 54.8 mg (0.198 mmol) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl) 4-methoxymorpholinium chloride was added, followed by stirring at 25 °C for 30 minutes. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain 149 mg of compound 6d (yield: 56%).

[0627] MS (ESI+ ): m / z = 536.1, 922.3 [M+H] + .

[0628] Step 5) Preparation of compound 6

[0629] Compound 6d79 mg (0.051 mmol) prepared in the above step 4) was dissolved in 2 mL of 1,1,1,3,3,3-hexafluoro-2-propanol, 39.5 μL (0.531 mmol) of trifluoroacetic acid was added, and the mixture was stirred at -5 °C for 24 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain 38.8 mg of compound 6 (yield: 33%).

[0630] MS (ESI+): m / z = 1357.3 [M+H] + .

[0631] Manufacturing Example 2.5.

[0632] 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-(2-((2-(((S)-1-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12 Preparation of H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (compound 7)

[0633] [Synthetic formula of compound 7]

[0634]

[0635] Compound 7a:

[0636] (9H-fluoren-9-yl)methyl (2-((2-(((S)-1-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate

[0637] Compound 7b:

[0638] (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl)-3-phenylpropanamide trifluoroacetic acid

[0639] Step 1) Preparation of compound 7a

[0640] Compound 4a700 mg (1.32 mmol) was dissolved in 6 mL of N,N-dimethylformamide and 510 mg (3.95 mmol) of N,N-diisopropylethylamine was added at -40 °C. Then, a solution of 735 mg (1.32 mmol) of (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycine and 500 mg (1.32 mmol) of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU) in 1.8 mL of N,N-dimethylformamide was added and stirred for 30 minutes under nitrogen at -40 °C. After the reaction was completed, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse phase column chromatography and freeze-dried to obtain 998 mg of compound 7a (yield: 74%).

[0641] 1H NMR (400 MHz, DMSO-d6) δ 8.42 (1H, d), 8.32 (1H, t), 8.09 (1H, d), 8.00 (1H, t), 7.86 (2H, m), 7.79 (1H, d), 7.69 (2H, d), 7.59 (1H, t), 7.35-7.43 (2H, m), 7.26-7.34 (3H, m), 7.08-7.26 (5H, m), 6.14-6.83 (1H, m), 5.50-5.62 (1H, m), 5.31-5.46 (2H, m), 5.23 (2H, s), 4.40-4.50 (1H, m), 4.12-4.33 (3H, m), 3.67-3.77 (3H, m), 3.53-3.62 (3H, m), 3.10-3.24 (2H, m), 2.95-3.05 (1H, m), 2.70-2.83 (1H, m), 2.40 (3H, s), 2.01-2.26 (2H, m), 1.70-1.93 (2H, m), 0.86 (3H, t);

[0642] MS (ESI+): m / z = 976.1 [M+H] + .

[0643] Step 2) Preparation of compound 7b

[0644] Compound 7a998 mg (1.02 mmol) prepared in the above step 1) was dissolved in 13 mL of N,N-dimethylformamide, 1.5 g (20 mmol) of N-ethylethanamine was added, and the mixture was stirred at 25 °C for 1 hour. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography and freeze-dried to obtain 558 mg of compound 7b (yield: 50%).

[0645] MS (ESI+): m / z = 754.1 [M+H] + .

[0646] Step 3) Preparation of compound 7

[0647] Compound 4d210 mg (0.354 mmol) was dissolved in 13 mL of N,N-dimethylformamide, and 76 mg (0.59 mmol) of N,N-diisopropylethylamine and 135 mg (0.35 mmol) of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU) were added, and the mixture was stirred at -40 °C for 20 minutes. To the reaction mixture, a solution of compound 7b320 mg (0.295 mmol) prepared in step 2) and 38 mg (0.29 mmol) of N,N-diisopropylethylamine in 3 mL of N,N-dimethylformamide was added, and the mixture was stirred at -40 °C for 30 minutes under nitrogen. After the reaction was completed, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain 401.6 mg of compound 7 (yield: 59%).

[0648] MS (ESI+): m / z = 1328.3 [M+H] + .

[0649] Manufacturing Example 2.6.

[0650] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)-2-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (Compound 8) manufacturing

[0651] [Synthetic formula of compound 8]

[0652]

[0653] Compound 8a:

[0654] Methyl 2-(bromomethyl)-4-nitrobenzoate

[0655] Compound 8b:

[0656] Methyl 2-(((tert-butoxycarbonyl)(methyl)amino)methyl)-4-nitrobenzoate

[0657] Compound 8c:

[0658] tert-butyl (5-amino-2-(hydroxymethyl)benzyl)(methyl) carbamate

[0659] Compound 8d:

[0660] Methyl 4-amino-2-(((tert-butoxycarbonyl)(methyl)amino)methyl) benzoate

[0661] Compound 8e:

[0662] (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-valyl-L-alanine

[0663] Compound 8f:

[0664] tert-Butyl (5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate

[0665] Compound 8g:

[0666] tert-Butyl (5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate

[0667] Compound 8h:

[0668] tert-Butyl (5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate

[0669] Compound 8i:

[0670] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)-2-((methylamino)methyl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate trifluoroacetic acid

[0671] Step 1) Preparation of compound 8b

[0672] 3.59 g (27.4 mmol) of tert-butyl N-methylcarbamate was dissolved in 100 mL of N,N-dimethylformamide, and 1.37 g (34.2 mmol) of 60% sodium hydride was added. The mixture was stirred at 0 °C for 30 minutes. Then, a solution of 7.5 g (27.4 mmol) of methyl 2-(bromomethyl)-4-nitrobenzoate (compound 8a) dissolved in 34 mL of N,N-dimethylformamide was added, and the mixture was stirred at 0 °C for 1 hour. When the reaction was complete, the reaction mixture was added to 1 L of a saturated ammonium chloride aqueous solution, and then extracted twice with 1 L of ethyl acetate. The organic layer was separated, washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered under reduced pressure, and distilled under reduced pressure. The obtained residue was separated using reverse phase column chromatography and freeze-dried to obtain compound 8b867 mg (yield: 8.3%).

[0673] 1H NMR (400 MHz, DMSO-d6) δ 8.24 (1H, m), 8.09 (1H, d), 7.96 (1H, s), 4.76 (2H, s), 3.91 (3H, s), 2.85 (3H, s), 1.25-1.54 (9H, m).

[0674] Step 2) Preparation of compound 8c

[0675] Compound 8b867 mg (2.67 mmol) prepared in the above step 1) was dissolved in 4 mL of N,N-dimethylformamide, and 1.2 g (13.4 mmol) of tetrahydroxydiboron and 41.8 mg (0.27 mmol) of 4-(4-pyridyl)pyridine were added, and the mixture was stirred at 15 °C for 5 minutes. When the reaction was complete, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography. The obtained residue was basified to pH 11 by adding a saturated aqueous sodium bicarbonate solution, distilled under reduced pressure, and the obtained residue was extracted three times with 200 mL of ethyl acetate. The organic layer was separated, washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered under reduced pressure, and distilled under reduced pressure to obtain compound 8c722 mg (yield: 90%).

[0676] 1H NMR (400 MHz, DMSO-d6) δ 7.67 (1H, d), 6.39-6.48 (1H, m), 6.28-6.38 (1H, m), 6.00 (2H, s), 4.64 (2H, s), 3.70 (3H, s), 2.81 (3H, s), 1.27-1.48 (9H, m).

[0677] Step 3) Preparation of compound 8d

[0678] Compound 8c672 mg (2.28 mmol) prepared in the above step 2) was dissolved in 15 mL of tetrahydrofuran, and 1.83 mL (4.58 mmol) of a 2.5 M lithium aluminum hydride tetrahydrofuran solution was slowly added thereto under nitrogen at 25 °C for 5 minutes, followed by stirring at 25 °C for 2 hours. Upon completion of the reaction, the reaction mixture was cooled to 0 °C, 10 g of sodium sulfate decahydrate was added, 10 mL of ethyl acetate was added, and the mixture was stirred for 10 minutes. The reaction mixture was filtered and distilled under reduced pressure, and the obtained residue was separated using column chromatography to obtain compound 8d516 mg (yield: 85%).

[0679] 1H NMR (400 MHz, DMSO-d6) δ 6.95 (1H, d), 6.39 (1H, m), 6.32 (1H, s), 4.98 (2H, s), 4.70 (1H, t), 4.35 (2H, s), 4.32 (2H, d), 2.72 (3H, s), 1.35-1.55 (9H, m).

[0680] Step 4) Preparation of compound 8f

[0681] 740 mg (1.94 mmol) of (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-valyl-L-alanine (compound 8e) and 516 mg (1.94 mmol) of compound 8d prepared in step 3) above were dissolved in 20 mL of dichloromethane and 20 mL of methanol, and 528 mg (2.13 mmol) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added, followed by stirring at 25 °C for 16 hours. Upon completion of the reaction, the reaction mixture was distilled under reduced pressure, and the obtained residue was separated using column chromatography to obtain 840 mg (yield: 68%) of compound 8f.

[0682] 1H NMR (400 MHz, DMSO-d6) δ 9.87 (1H, brs), 8.13 (1H, d), 7.79 (1H, d), 7.45-7.55 (1H, m), 7.33 (1H, d), 7.28 (1H, d), 6.99 (2H, s), 5.04 (1H, t), 4.44 (2H, d), 4.33-4.42 (3H, m), 4.13-4.20 (1H, m), 3.36-3.39 (2H, m), 2.75 (3H, s), 2.06-2.21 (2H, m), 1.90-2.00 (1H, m), 1.34-1.51 (13H, m), 1.29 (3H, d), 1.14-1.22 (2H, m), 0.83 (6H, m).

[0683] Step 5) Preparation of compound 8g

[0684] Compound 8f840 mg (1.33 mmol) prepared in the above step 4) and bis(4-nitrophenyl)carbonate 528 mg (1.73 mmol) were dissolved in 5 mL of N,N-dimethylformamide, 345 mg (2.67 mmol) of N,N-diisopropylethylamine was added, and the mixture was stirred at 25 °C for 15 hours. Upon completion of the reaction, the reaction mixture was distilled under reduced pressure, and the obtained residue was diluted with 50 mL of tert-butyl methyl ether, filtered under reduced pressure, and distilled under reduced pressure to obtain compound 8g780 mg (yield: 74%).

[0685] 1H NMR (400 MHz, DMSO-d6) δ 10.04 (1H, brs), 8.27-8.34 (2H, m), 8.18 (1H, d), 7.80 (1H, d), 7.51 -7.64(3H, m), 7.47 (1H, s), 7.39 (1H, d), 7.00 (2H, s), 5.28 (2H, s), 4.51 (2H, s), 3.38 (2H, m), 4.34-4.41 (1H, m), 4.14-4.21 (1H, m), 2.77 (3H, s), 2.08-2.19 (2H, m), 1.91-2.00 (1H, m), 1.35-1.52 (13H, m),1.30 (3H, d), 1.13-1.21 (2H, m), 0.84 (6H, m);

[0686] MS (ESI + ): m / z = 817.2 [M+Na] + .

[0687] Step 6) Preparation of compound 8h

[0688] Compound 8g390 mg (0.49 mmol) prepared in the above step 5) and pyridine 1.2 mL (14.9 mmol) were dissolved in 5 mL of N,N-dimethylformamide, 1-hydroxybenzotriazole 66 mg (0.49 mmol) and compound 4a260 mg (0.49 mmol) were added, and the mixture was stirred at 25 °C for 16 hours. Upon completion of the reaction, the reaction mixture was distilled under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography and freeze-dried to obtain compound 8h104 mg (yield: 63%).

[0689] MS (ESI + ): m / z = 1091.3 [M+H] + .

[0690] Step 7) Preparation of compound 8i

[0691] Compound 8h104 mg (0.06 mmol) prepared in the above step 6) was dissolved in 4 mL of dichloromethane, 1 mL (13.4 mmol) of trifluoroacetic acid was added at 0 °C, and the mixture was stirred at 25 °C for 30 minutes. Upon completion of the reaction, the reaction mixture was distilled under reduced pressure to obtain compound 8i100 mg (yield: 58%).

[0692] MS (ESI + ): m / z = 991.3 [M+H] + .

[0693] Step 8) Preparation of compound 8

[0694] 100 mg (0.053 mmol) of compound 8i prepared in step 7) above and 32 mg (0.078 mmol) of 2,5,8,11,14,17,20,23-octaoxahexacosane-26-acid were dissolved in 1.5 mL of N,N-dimethylformamide, and 27 μL (0.157 mmol) of N,N-diisopropylethylamine and 24 mg (0.063 mmol) of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU) were added at 0 °C, and the mixture was stirred at 25 °C for 16 hours. After the reaction was completed, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain compound 819.6 mg (yield: 26%).

[0695] MS (ESI + ): m / z = 1385.4 [M+H] + .

[0696] Manufacturing Example 2.7. 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)-2-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctaconic acid-28-yl)benzyl Preparation of ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (compound 9)

[0697] [Synthetic formula of compound 9]

[0698]

[0699] Compound 9a: (1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oyl)-L-valyl-L-alanine

[0700] Compound 9b: tert-Butyl (5-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacondanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate

[0701] Compound 9c: tert-Butyl (5-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacondanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate

[0702] Compound 9d: tert-butyl (5-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacondanamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbonyl)oxy)methyl)benzyl)(methyl)carbamate

[0703] Compound 9e: 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacondanamido)-2-((methylamino)methyl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0704] Step 1) Preparation of compound 9a

[0705] Except for using 546 mg (2.90 mmol) of L-valyl-L-alanine instead of glycyl-glycyl-L-phenylalanine in step 3) of the above manufacturing example 2.4, the same process as step 3) of the above manufacturing example 2.4 was performed to obtain 1.1 g of compound 9a (yield: 86.5%).

[0706] 1H NMR (400 MHz, DMSO-d6) δ 12.45 (1H, m), 8.24 (1H, d), 8.01 (1H, t), 7.83 (1H, d), 7.00 (2H, s), 4.22 (2H, m), 3.57 (4H, m), 3.48 (30H, m), 3.14 (2H, m), 2.49 (1H, m), 2.32 (3H, m), 1.90 (1H, m), 1.26 (3H, d), 0.86 (3H, d), 0.82 (3H, d).

[0707] Step 2) Preparation of compound 9b

[0708] Except for using 1 g (1.31 mmol) of compound 9a prepared in step 1) instead of compound 8e used in step 4) of manufacturing example 2.6, the same process as step 4) of manufacturing example 2.6 was performed to obtain 731 mg of compound 9b (yield: 49.6%).

[0709] MS (ESI+ ): m / z = 1011.6 [M+H] + .

[0710] Step 3) Preparation of compound 9c

[0711] 31 mg (0.723 mmol) of compound 9b7 prepared in the above step 2) and 286 mg (0.94 mmol) of bis(4-nitrophenyl)carbonate were dissolved in 10 mL of N,N-dimethylformamide, 187 mg (1.45 mmol) of N,N-diisopropylethylamine was added, and the mixture was stirred at 25 °C for 24 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography and freeze-dried to obtain 10 mg (yield: 41.4%) of compound 9c5.

[0712] MS (ESI + ): m / z = 1176.3 [M+H] + .

[0713] Step 4) Preparation of compound 9d

[0714] Except that 510 mg (0.434 mmol) of compound 9c prepared in step 3) was used instead of compound 8g used in step 6) of manufacturing example 2.6, the same process as step 6) of manufacturing example 2.6 was performed to obtain 251 mg of compound 9d (yield: 51.2%).

[0715] MS (ESI + ): m / z = 1473.4 [M+H] + .

[0716] Step 5) Preparation of compound 9e

[0717] Except for using 230 mg (0.156 mmol) of compound 9d prepared in step 4) instead of compound 8h used in step 7) of manufacturing example 2.6, the same process as step 7) of manufacturing example 2.6 was performed to quantitatively obtain 235 mg of compound 9e.

[0718] MS (ESI + ): m / z = 1372.8 [M+H] + .

[0719] Step 6) Preparation of compound 9

[0720] Except that 235 mg (0.158 mmol) of compound 9e prepared in step 5) was used instead of compound 8i used in step 8) of manufacturing example 2.6, the same process as step 8) of manufacturing example 2.6 was performed to obtain 188 mg of compound 9 (yield: 66.7%).

[0721] MS (ESI + ): m / z = 1767.3 [M+H] + .

[0722] Manufacturing Example 2.8. 4-((S)-2-((S)-2-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)-3-methylbutanamido)propanamido)-2-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)benzyl Preparation of ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (Compound 10)

[0723] [Synthetic formula of compound 10]

[0724]

[0725] Compound 10a: 2,5-dioxopyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylate

[0726] Compound 10b: (4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carbonyl)-L-valyl-L-alanine

[0727] Compound 10c: tert-Butyl (5-((S)-2-((S)-2-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)-3-methylbutanamido)propanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate

[0728] Compound 10d: tert-Butyl (5-((S)-2-((S)-2-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)-3-methylbutanamido)propanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate

[0729] Compound 10e: tert-Butyl (5-((S)-2-((S)-2-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)-3-methylbutanamido)propanamido)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate

[0730] Compound 10f: 4-((S)-2-((S)-2-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)-3-methylbutanamido)propanamido)-2-((methylamino)methyl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate trifluoroacetic acid

[0731] Step 1) Preparation of compound 10b

[0732] 4 g (12.0 mmol) of 2,5-dioxopyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylate (compound 10a) and 2.48 g (13.2 mmol) of L-valyl-L-alanine were dissolved in 40 mL of N,N-dimethylformamide, and 5.41 g (41.9 mmol) of N,N-diisopropylethylamine was added, followed by stirring at 20 °C for 12 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography and lyophilized to obtain 2.2 g of compound 10b (yield: 45.1%).

[0733] 1H NMR (400 MHz, DMSO-d6) δ 7.93 (1H, d), 7.73 (1H, d), 7.01 (2H, s), 4.12 (1H, m), 4.01 (1H, m), 3.24 (2H, d), 2.22 (1H, m), 1.96 (1H, m), 1.70 (2H, m), 1.61 (2H, m), 1.50 (1H, m), 1.28 (2H, m), 1.22 (3H, d), 0.90 (2H, m), 0.83 (3H, d), 0.80 (3H, d).

[0734] Step 2) Preparation of compound 10c

[0735] Except that 600 mg (1.32 mmol) of compound 10b prepared in step 1) was used instead of compound 8e used in step 4) of manufacturing example 2.6, the same process as step 4) of manufacturing example 2.6 was performed to obtain 700 mg of compound 10c (yield: 36.7%).

[0736] 1H NMR (400 MHz, DMSO-d6) δ 9.87 (1H, brs), 8.07 (1H, d), 7.68 (1H, d), 7.49 (1H, brd), 7.33 (1H, d), 7.28 (1H, d), 7.00 (2H, s), 5.02 (1H, t), 4.45-4.39 (4H, m), 4.14 (1H, m), 4.10 (1H, m), 3.22 (2H, d), 2.75 (3H, s), 2.22 (1H, m), 1.98 (1H, m), 1.70-1.59 (4H, m), 1.49 (1H, m), 1.45-1.33 (9H, m), 1.30-1.25 (5H, m), 0.88 (2H, m), 0.85 (3H, d), 0.81 (3H, d).

[0737] Step 3) Preparation of compound 10d

[0738] In the above step 2), 400 mg (0.61 mmol) of compound 10c prepared and 241 mg (0.79 mmol) of bis(4-nitrophenyl)carbonate were dissolved in 4 mL of N,N-dimethylformamide, 158 mg (1.22 mmol) of N,N-diisopropylethylamine was added, and the mixture was stirred at 20 °C for 24 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography to obtain 172 mg of compound 10d (yield: 33.3%).

[0739] 1H NMR (400 MHz, DMSO-d6) δ 10.05 (1H, brs), 8.31 (2H, d), 8.13 (1H, d), 7.69 (1H, d), 7.56 (3H, m), 7.47 (1H, s), 7.39 (1H, d), 7.01 (2H, s), 5.29 (2H, s), 4.51 (2H, s), 4.37 (1H, m), 4.14 (1H, t), 3.23 (2H, d), 2.77 (3H, s), 2.23 (1H, m), 1.95 (1H, m), 1.70-1.59 (4H, m), 1.49 (1H, m), 1.43-1.35 (9H, m), 1.30-1.25 (5H, m), 0.90 (2H, m), 0.87 (3H, d), 0.81 (3H, d).

[0740] Step 4) Preparation of compound 10e

[0741] In the above step 3), 38 mg (0.82 mmol) of compound 10d prepared in step 3) and 1.3 g (16.5 mmol) of pyridine were dissolved in 4 mL of N,N-dimethylformamide, 111 mg (0.82 mmol) of 1-hydroxybenzotriazole and 450 mg (0.55 mmol) of compound 4a were added, and the mixture was stirred at 25 °C for 2 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography and freeze-dried to obtain 250 mg of compound 10e (yield: 38.8%).

[0742] 1H NMR (400 MHz, DMSO-d6) δ 9.97 (1H, brs), 8.10 (1H, d), 8.03 (1H, d), 7.78 (1H, d), 7.68 (1H, d), 7.53 (1H, m), 7.41 (1H, s), 7.36 (1H, d), 7.30 (1H, s), 7.00 (2H, s), 5.43 (2H, s), 5.28 (3H, m), 5.11 (2H, s), 4.46 (2H, s), 4.36 (1H, m), 4.14 (1H, m), 3.23 (3H, m), 3.10 (1H, m), 2.76 (3H, s), 2.37 (3H, s), 2.27-2.11 (3H, m), 1.99-1.79 (3H, m), 1.76-1.58 (4H, m), 1.51 (1H, m), 1.40-1.19 (15H, m), 0.89 (2H, m), 0.88-0.78 (9H, m).

[0743] Step 5) Preparation of compound 10f

[0744] Except that 200 mg (0.162 mmol) of compound 10e prepared in step 4) was used instead of compound 8h used in step 7) of manufacturing example 2.6, the same process as step 7) of manufacturing example 2.6 was performed to obtain 183 mg of compound 10f (yield: 94.6%).

[0745] MS (ESI + ): m / z = 1017.5 [M+H] + .

[0746] Step 6) Preparation of compound 10

[0747] 183 mg (0.162 mmol) of compound 10f prepared in the above step 5) and 100 mg (0.243 mmol) of 2,5,8,11,14,17,20,23-octaoxahexacosane-26-acid were dissolved in 3 mL of N,N-dimethylformamide, and 209 mg (1.62 mmol) of N,N-diisopropylethylamine and 92 mg (0.243 mmol) of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU) were added at 0 °C, and the mixture was stirred at 25 °C for 2 hours. After the reaction was completed, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain 157 mg of compound 10 (yield: 65.4%).

[0748] MS (ESI + ): m / z = 1411.9 [M+H] + .

[0749] Manufacturing Example 2.9.4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ainamido)butanamido)propanamido)-2-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (compound) 11) Manufacturing

[0750] [Synthetic formula of compound 11]

[0751]

[0752] Compound 11a: 6-Nitroisobenzofuran-1(3H)-one

[0753] Compound 11b: 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide

[0754] Compound 11c: (2-((methylamino)methyl)-4-nitrophenyl)methanol

[0755] Compound 11d: N-(2-(hydroxymethyl)-5-nitrobenzyl)-N-methyl-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide

[0756] Compound 11e: N-(5-amino-2-(hydroxymethyl)benzyl)-N-methyl-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide

[0757] Compound 11f: 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-phosphorous acid

[0758] Compound 11g: 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-inoate

[0759] Compound 11h: (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-inoyl)-L-valyl-L-alanine

[0760] Compound 11i: N-(2-(hydroxymethyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)propanamido)benzyl)-N-methyl-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide

[0761] Compound 11j: 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)propanamido)-2-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)benzyl (4-nitrophenyl) carbonate

[0762] Step 1) Preparation of compound 11b

[0763] 20 g (111 mmol) of 6-nitroisobenzofuran-1(3H)-one was dissolved in 160 mL of methanol, and 61.4 mL (122.8 mmol) of a 2M methanol solution of methylamine was added at 25 °C for 10 minutes under nitrogen atmosphere, followed by stirring at 25 °C for 16 hours. Upon completion of the reaction, the resulting solid was filtered under reduced pressure, washed twice with 20 mL of methanol, and dried under reduced pressure to obtain 1.1 g (yield: 90.3%) of compound 11b2.

[0764] 1H NMR (400 MHz, DMSO-d6) δ 8.57 (1H, brm), 8.31 (1H, m), 8.20 (1H, d), 7.86 (1H, d), 5.54 (1H, t), 4.72 (2H, d), 2.78 (3H, d).

[0765] Step 2) Preparation of compound 11c

[0766] A solution of 1.2 g (101 mmol) of compound 11b2 prepared in the above step 1) in 12 mL of tetrahydrofuran and 19.16 g (191.6 mmol) of borane dimethyl were simultaneously injected into a flow reactor (PFA coiled reactor, 3.175(1 / 8'') mm, 60.054 mL, 70 °C) via pumps 1 and 2, respectively. The reaction mixture in the flow reactor was collected after 20 minutes of reaction and was completed at 87.2 minutes. 90 mL of a 4M methanol hydrochloric acid solution was added to the collected reaction mixture at 0 °C and stirred at 65 °C for 8 hours. Upon completion of the reaction, the reaction mixture was cooled to 0 °C, and the resulting solid was filtered under reduced pressure and dried under reduced pressure to obtain 1 g of compound 11c2 (yield: 90.2%).

[0767] 1H NMR (400 MHz, DMSO-d6) δ 9.43 (2H, brm), 8.50 (1H, d), 8.26 (1H, m), 7.75 (1H, d), 4.78 (2H, s), 4.26 (2H, m), 2.62 (3H, s).

[0768] Step 3) Preparation of compound 11d

[0769] 571 mg (2.91 mmol) of compound 11c prepared in the above step 2) and 1 g (2.42 mmol) of 2,5,8,11,14,17,20,23-octaoxahexacosane-26-acid were dissolved in 10 mL of N,N-dimethylformamide, and 940 mg (7.27 mmol) of N,N-diisopropylethylamine and 1.11 g (2.91 mmol) of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU) were added at 0 °C, and the mixture was stirred at 0 °C for 1 hour. After the reaction was completed, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse phase column chromatography to obtain 1.05 g of compound 11d (yield: 73%).

[0770] 1H NMR (400 MHz, DMSO-d6) δ 8.12 (1H, d), 7.86 (1H, s), 7.74 (1H, d), 5.27 (1H, brs), 4.65 (4H, m), 3.72 (2H, s), 3.53 (26H, m), 3.45 (2H, m), 3.26 (3H, s), 2.98 (3H, s), 2.67 (2H, m).

[0771] Step 4) Preparation of compound 11e

[0772] Compound 11d900 mg (1.52 mmol) prepared in the above step 3) was dissolved in 20 mL of N,N-dimethylformamide, and 683 mg (7.62 mmol) of tetrahydroxydiboron and 23.8 mg (0.152 mmol) of 4-(4-pyridyl)pyridine were added, followed by stirring at 15 °C for 5 minutes. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography to obtain compound 11e401 mg (yield: 47%).

[0773] MS (ESI+ ): m / z = 561.5 [M+H] + .

[0774] Step 5) Preparation of compound 11g

[0775] 1 g (3.73 mmol) of compound 11f (BD01178203, Bide) was dissolved in 20 mL of N,N-dimethylformamide. 2.41 g (18.6 mmol) of N,N-diisopropylethylamine and 1.68 g (5.59 mmol) of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) were added, and the mixture was stirred at 25 °C for 1 hour. Upon completion of the reaction, the reaction mixture was separated using reverse-phase column chromatography to obtain 1.05 g (yield: 77%) of compound 11g.

[0776] 1H NMR (400 MHz, DMSO-d6) δ 9.13 (2H, s), 3.41 (3H, s), 2.89 (2H, t), 2.82 (4H, m), 2.69 (2H, t), 1.96 (2H, m).

[0777] Step 6) Preparation of compound 11h

[0778] 1.05 g (2.87 mmol) of compound 11g prepared in the above step 5) and 649 mg (3.45 mmol) of L-valyl-L-alanine were dissolved in 10 mL of N,N-dimethylformamide, and 743 mg (5.75 mmol) of N,N-diisopropylethylamine were added at 0 °C, followed by stirring at 20 °C for 1 hour. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography and freeze-dried to obtain 960 mg (yield: 76%) of compound 11h.

[0779] 1H NMR (400 MHz, DMSO-d6) δ 9.13 (2H, s), 8.05 (1H, d), 7.75 (1H, d), 4.14 (1H, m), 3.91 (1H, m), 3.41 (3H, s), 2.55 (2H, m), 2.37 (2H, m), 2.02 (1H, m), 1.82 (2H, m), 1.20 (3H, d), 0.84 (6H, m).

[0780] Step 7) Preparation of compound 11i

[0781] Except that 731 mg (1.67 mmol) of compound 11h prepared in step 6) was used instead of compound 8e used in step 4) of the above Preparation Example 2.6, and 850 g (1.52 mmol) of compound 11e prepared in step 4) was used instead of compound 8d, the same process as step 4) of the above Preparation Example 2.6 was performed to obtain 451 mg of compound 11i (yield: 30%).

[0782] MS (ESI + ): m / z = 981.1 [M+H] + .

[0783] Step 8) Preparation of compound 11j

[0784] Except that 400 mg (0.41 mmol) of compound 11i prepared in step 7) was used instead of compound 9b used in step 3) of manufacturing example 2.7, the same process as step 3) of manufacturing example 2.7 was performed to obtain 183 mg of compound 11j (yield: 39%).

[0785] MS (ESI + ): m / z = 1146.6 [M+H] + .

[0786] Step 9) Preparation of compound 11

[0787] Compound 11j111 mg (0.21 mmol) prepared in the above step 8) was dissolved in 6 mL of N,N-dimethylformamide, and pyridine 331 mg (4.19 mmol) and N,N-diisopropylethylamine 27 mg (0.21 mmol) were added. To the solution, 28.3 mg (0.21 mmol) of 1-hydroxybenzotriazole and compound 4a160 mg (0.14 mmol) were added, and the mixture was stirred at 25 °C for 2 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain 61.1 mg of compound 11 (yield: 30%).

[0788] MS (ESI + ): m / z = 1442.62 [M+H] + .

[0789] Manufacturing Example 2.10. Preparation of 4-((S)-2-((S)-2-(2-bromoatetamido)-3-methylbutanamido)propanamido)-2-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (Compound 12)

[0790] [Synthetic formula of compound 12]

[0791]

[0792] Compound 12a: (tert-butoxycarbonyl)-L-valyl-L-alanine

[0793] Compound 12b: tert-Butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)-3-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbonate

[0794] Compound 12c: tert-Butyl ((S)-3-methyl-1-(((S)-1-((3-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbonate

[0795] Compound 12d: 4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propanamido)-2-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0796] Compound 12e: 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0797] Step 1) Preparation of compound 12b

[0798] 370 mg (1.28 mmol) of (tert-butoxycarbonyl)-L-valyl-L-alanine (compound 12a) and 600 mg (1.07 mmol) of compound 11e prepared in step 4) of Preparation Example 2.9 were dissolved in 10 mL of dichloromethane and 1 mL of methanol, and 318 mg (1.28 mmol) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added, followed by stirring at 25 °C for 16 hours. Upon completion of the reaction, the reaction mixture was distilled under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography and freeze-dried to obtain 870 mg of compound 12b (yield: 97.8%).

[0799] 1H NMR (400 MHz, DMSO-d6) δ 10.04-9.93 (1H, m), 8.09-8.02 (1H, m), 7.65-7.54 (1H, m), 7.29 (1H, m), 7.17 (1H, m), 6.73-6.53 (1H, m), 4.62 (1H, s), 4.53 (1H, s), 4.45 (2H, m), 3.83 (1H, m), 3.68 (2H, m), 3.62 (2H, m), 3.56-3.40 (28H, m), 3.23 (3H, s), 2.88 (3H, d), 2.67 (1H, m), 1.94 (1H, m), 1.38 (9H, s), 1.29 (3H, m), 0.87-0.80 (6H, m).

[0800] Step 2) Preparation of compound 12c

[0801] Compound 12b8 prepared in step 1) above 70 mg (1.05 mmol) and bis(4-nitrophenyl)carbonate 637 mg (2.09 mmol) were dissolved in 10 mL of N,N-dimethylformamide, 677 mg (5.23 mmol) of N,N-diisopropylethylamine was added, and the mixture was stirred at 25 °C for 16 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography and freeze-dried to obtain compound 12c9 14 mg (yield: 87.6%).

[0802] MS (ESI + ): m / z = 996.7 [M+H] + .

[0803] Step 3) Preparation of compound 12d

[0804] Compound 4a288 mg (0.54 mmol) was dissolved in 10 mL of N,N-dimethylformamide, and pyridine 858 mg (10.8 mmol) and N,N-diisopropylethylamine 70 mg (0.54 mmol) were added. To the solution, 73 mg (0.54 mmol) of 1-hydroxybenzotriazole and 360 mg (0.36 mmol) of compound 12c prepared in step 2) were added, and the mixture was stirred at 25 °C for 2 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography and lyophilized to obtain 460 mg of compound 12d (yield: 98.5%).

[0805] MS (ESI + ): m / z = 1292.8 [M+H] + .

[0806] Step 4) Preparation of compound 12e

[0807] Compound 12d220 mg (0.17 mmol) prepared in the above step 3) was dissolved in 5 mL of dichloromethane, 1 mL of trifluoroacetic acid was added under nitrogen, and the mixture was stirred at 25 °C for 1 hour under nitrogen. When the reaction was complete, the reaction mixture was distilled under reduced pressure. The obtained residue was diluted with 2 mL of purified water and 2 mL of acetonitrile, neutralized to pH 7 by adding a saturated aqueous potassium carbonate solution, separated using reverse-phase column chromatography, and freeze-dried to obtain compound 12e115 mg (yield: 56.7%).

[0808] MS (ESI + ): m / z = 1192.0 [M+H] + .

[0809] Step 5) Preparation of compound 12

[0810] Compound 12e95 mg (0.80 mmol) prepared in the above step 4) and 22 mg (0.16 mmol) of 2-bromoacetic acid were dissolved in 2 mL of dichloromethane, and 20 mg (0.16 mmol) of N,N'-diisopropylcarbodiimide was added, followed by stirring at 25 °C for 2 hours. Upon completion of the reaction, the reaction mixture was distilled under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain compound 1255.6 mg (yield: 53.1%).

[0811] MS (ESI + ): m / z = 1312.50 [M+H] + .

[0812] Manufacturing Example 2.11.4-((S)-2-((S)-2-(4-((2-bromoacetamido)methyl)cyclohexane-1-carboxamido)-3-methylbutanamido)propanamido)-2-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (compound) 13) Manufacturing

[0813] [Synthetic formula of compound 13]

[0814]

[0815] Compound 13a: 4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxylic acid

[0816] Compound 13b: 2,5-dioxopyrrolidin-1-yl 4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxylate

[0817] Compound 13c: (4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carbonyl)-L-valyl-L-alanine

[0818] Compound 13d: tert-Butyl ((4-(((S)-1-(((S)-1-((4-(hydroxymethyl)-3-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)cyclohexyl)methyl)carbamate

[0819] Compound 13e: tert-Butyl ((4-(((S)-3-methyl-1-(((S)-1-((3-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamoyl)cyclohexyl)methyl)carbamate

[0820] Compound 13f: 4-((S)-2-((S)-2-(4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-methylbutanamido)propanamido)-2-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0821] Compound 13g: 4-((S)-2-((S)-2-(4-(aminomethyl)cyclohexane-1-carboxamido)-3-methylbutanamido)propanamido)-2-(27-methyl-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azaoctacosan-28-yl)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0822] Step 1) Preparation of compound 13b

[0823] 3.35 g (29.2 mmol) of 4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxylic acid (compound 13a) and 3.35 g (29.2 mmol) of N-hydroxysuccinimide were dissolved in 400 mL of dichloromethane, and 5.59 g (29.2 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, followed by stirring at 20 °C for 1 hour. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane and washed three times with 0.1 M aqueous hydrochloric acid solution. The organic layer was separated, dried over anhydrous sodium sulfate, filtered under reduced pressure, and distilled under reduced pressure to obtain 6.8 g of compound 13b (yield: 98.7%).

[0824] 1H NMR (400 MHz, DMSO-d6) δ 6.83 (1H, t), 2.80-2.76 (6H, m), 2.66 (1H, m), 2.00 (2H, m), 1.74 (2H, m), 1.43 (2H, m), 1.37 (9H, s), 1.34 (1H, m), 0.99 (2H, m).

[0825] Step 2) Preparation of compound 13c

[0826] Except that 4 g (11.3 mmol) of compound 13b prepared in step 1) was used instead of compound 10a used in step 1) of manufacturing example 2.8, the same process as step 1) of manufacturing example 2.8 was performed to obtain 3.8 g of compound 13c (yield: 76.4%).

[0827] 1H NMR (400 MHz, DMSO-d6) δ 12.43 (1H, m), 8.16 (1H, d), 7.63 (1H, d), 6.78 (1H, t), 4.17 (2H, m), 2.75 (2H, t), 2.20 (1H, m), 1.95 (1H, m), 1.69 (4H, m), 1.37 (9H, s), 1.32-1.24 (6H, m), 0.86-0.80 (8H, m).

[0828] Step 3) Preparation of compound 13d

[0829] 45 mg (0.82 mmol) of compound 13c4 prepared in step 2) and 55 mg (0.63 mmol) of compound 11e3 prepared in step 4) of Preparation Example 2.9 were dissolved in 5 mL of dichloromethane and 0.5 mL of methanol, and 188 mg (0.76 mmol) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added, followed by stirring at 20 °C for 16 hours. Upon completion of the reaction, the reaction mixture was distilled under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography and freeze-dried to obtain 506 mg (yield: 73.7%) of compound 13d.

[0830] 1H NMR (400 MHz, DMSO-d6) δ 9.95-9.84 (1H, m), 8.08 (1H, m), 7.73-7.53 (2H, m), 7.29 (1H, m), 7.20-7.10 (1H, m), 6.78 (1H, t), 4.62 (1H, s), 4.53 (1H, s), 4.45 (2H, d), 4.37 (1H, m), 4.15 (1H, m), 3.68 (2H, m), 3.52-3.42 (30H, m), 3.23 (3H, s), 2.88 (3H, d), 2.76 (2H, m), 2.69 (1H, m), 2.21 (1H, m), 1.95 (1H, m), 1.70 (4H, m), 1.37 (9H, s), 1.32-1.19 (6H, m), 0.83 (8H, m).

[0831] Step 4) Preparation of compound 13e

[0832] Compound 13d500 mg (0.52 mmol) prepared in the above step 3) and bis(4-nitrophenyl)carbonate 204 mg (0.67 mmol) were dissolved in 5 mL of N,N-dimethylformamide, 133 mg (1.03 mmol) of N,N-diisopropylethylamine was added, and the mixture was stirred at 20 °C for 16 hours. Upon completion of the reaction, the reaction mixture was filtered under reduced pressure, and the obtained residue was separated using reverse-phase column chromatography to obtain compound 13e350 mg (yield: 59.8%).

[0833] MS (ESI + ): m / z = 1135.6 [M+H] + .

[0834] Step 5) Preparation of compound 13f

[0835] Except for using 350 mg (0.31 mmol) of compound 13e prepared in step 4) instead of compound 12c used in step 3) of manufacturing example 2.10, the same process as step 3) of manufacturing example 2.10 was performed to obtain 400 mg of compound 13f (yield: 83.9%).

[0836] MS (ESI + ): m / z = 1431.9 [M+H] + .

[0837] Step 6) Preparation of compound 13g

[0838] Compound 13f390 mg (0.27 mmol) prepared in the above step 5) was dissolved in 5 mL of dichloromethane, 1 mL of trifluoroacetic acid was added, and the mixture was stirred at 20 °C for 1 hour. Upon completion of the reaction, the reaction mixture was distilled under reduced pressure to quantitatively obtain compound 13g360 mg.

[0839] MS (ESI + ): m / z = 1331.8 [M+H] + .

[0840] Step 7) Preparation of compound 13

[0841] Compound 13g360 mg (0.25 mmol) prepared in the above step 6) was dissolved in 5 mL of dichloromethane, and 756 mg (7.47 mmol) of triethylamine and 53 mg (0.26 mmol) of 2-bromoacetyl bromide were added at 0 °C, followed by stirring at 20 °C for 1 hour. Upon completion of the reaction, the reaction mixture was distilled under reduced pressure, and the obtained residue was separated using a high-performance liquid chromatography system and freeze-dried to obtain compound 1350 mg (yield: 13.3%).

[0842] MS (ESI + ): m / z = 1451.60 [M+H] + .

[0843] Manufacturing Example 3. Preparation of anti-CNTN4 antibody-drug conjugate

[0844] Manufacturing Example 3.1. Preparation of anti-CNTN4 antibody-drug conjugate (Ab-I, Ab-II)

[0845] Using compound 1 and anti-CNTN4 antibody prepared in Manufacturing Example 2.1 above, an antibody-drug conjugate having the following structure was prepared.

[0846]

[0847] The above n can range from 1 to 20.

[0848] The antibody-drug conjugate using the Ab1 antibody prepared in Manufacturing Example 1 as an anti-CNTN4 antibody was named Ab-I, and the antibody-drug conjugate using the Ab2 antibody prepared in Manufacturing Example 1 as an anti-CNTN4 antibody was named Ab-II.

[0849] [Reduction of disulfide bonds in antibodies for side chain activation]

[0850] At pH 6.75, 1.64 mM TCEP, a reducing agent, was added to each of the Ab1 and Ab2 antibodies. Furthermore, 0.34 mM TCEP, a reducing agent, was added to the Ab2 antibody under the same conditions. Subsequently, each antibody and TCEP were reacted for 2 hours at 25°C to reduce some of the disulfide bonds in the antibodies.

[0851] [Antibody-Drug Conjugate Manufacturing]

[0852] Compound 1 prepared in Manufacturing Example 2.1 was dissolved in DMSO. 20 equivalents of Compound 1 dissolved in DMSO were added to each of Ab1 and Ab2 antibodies, which had partially reduced disulfide bonds. The concentration of the antibody during the reaction was adjusted to 10.0 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C, and the reaction material was titrated to pH 5.0 using acetic acid. After binding the titrated material to a column packed with cation exchange resin chromatography (CEX) resin, impurities were removed with 50 mM sodium acetate buffer (pH 5.0) containing 50 mM sodium chloride, and the target protein was eluted with 50 mM sodium acetate buffer (pH 5.0) containing 400 mM sodium chloride. The range of the eluted fraction was from 200 mAU to 500 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugates, Ab-I and Ab-II, were formulated in 50 mM sodium acetate buffer (pH 5.0) containing 400 mM sodium chloride, 9.5% trehalose, and 0.05% polysorbate 20.

[0853] After loading 20 μg of the manufactured Ab-I and Ab-II onto an SEC-HPLC column, SEC-HPLC was performed under the condition of applying 50 mM sodium phosphate buffer solution (pH 6.8) containing 150 mM sodium chloride at a flow rate of 1 mL / min, and the purity of the manufactured antibody-drug conjugate was confirmed using a 280 nm ultraviolet detector. The SEC-HPLC chromatogram of the antibody-drug conjugate Ab-I is shown in Fig. 9, and the SEC-HPLC chromatogram of the antibody-drug conjugate Ab-II is shown in Fig. 10.

[0854] In addition, after loading 40 μg of each of the prepared Ab-I and Ab-II onto the HIC-HPLC column, the drug-antibody ratio (DAR) analysis was performed under the conditions of a gradient from 100% of 20 mM sodium phosphate buffer solution (pH 6.8) containing 1 M ammonium sulfate to 70% of 20 mM sodium phosphate buffer solution (pH 6.8) and 30% of 20% isopropanol for 25 min at 30°C and a flow rate of 0.6 mL / min. The DAR of the prepared antibody-drug conjugate was confirmed using a 280 nm ultraviolet detector. The HIC-HPLC chromatogram of the antibody-drug conjugate Ab-I is shown in Fig. 11, and the HIC-HPLC chromatogram of the antibody-drug conjugate Ab-II is shown in Fig. 12.

[0855] From the obtained chromatogram, the DAR 0, DAR 2, DAR 4, DAR 6, and DAR 8 peaks were selected, and the DAR was calculated using the area of ​​these peaks.

[0856] The yield, purity and DAR of the manufactured antibody-drug conjugates, Ab-I and Ab-II, are described in Table 7.

[0857]

[0858] Manufacturing Example 3.2. Preparation of anti-CNTN4 antibody-drug conjugate (Ab-III)

[0859] To prepare antibody-drug conjugates, compound 2 was purchased from BLDpharm (#BD317613, vcMMAE).

[0860] (Compound 2:

[0861] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropen-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate)

[0862]

[0863] An antibody-drug conjugate having the following structure was prepared using compound 2 and an anti-CNTN4 antibody.

[0864]

[0865] The above n can range from 1 to 20.

[0866] The antibody-drug conjugate prepared using the Ab1 antibody of the above Preparation Example 1 as an anti-CNTN4 antibody and compound 2 was named Ab-III.

[0867] [Reduction of disulfide bonds in antibodies for side chain activation]

[0868] 1.64 mM TCEP, a reducing agent, was added to the Ab1 antibody at pH 6.75. Subsequently, the antibody and TCEP were reacted for 2 hours at 25°C to reduce some of the disulfide bonds in the antibody.

[0869] [Antibody-Drug Conjugate Manufacturing]

[0870] Compound 2 was dissolved in DMSO. 20 equivalents of compound 2 dissolved in DMSO were added to Ab1 antibody, in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 10.0 to 11.0 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25 °C, and the reaction material was titrated to pH 5.0 using acetic acid. After binding the titrated material to a column packed with cation exchange resin chromatography (CEX) resin, impurities were removed with 50 mM sodium acetate buffer (pH 5.0) containing 50 mM sodium chloride, and the target protein was eluted with 50 mM sodium acetate buffer (pH 5.0) containing 400 mM sodium chloride. The range of the eluted fraction was from 200 mAU to 400 mAU at 280 nm. The final produced anti-CNTN4 antibody-drug conjugate, Ab-III, was formulated in a 50 mM sodium acetate buffer (pH 5.0) containing 400 mM sodium chloride, 9.5% trehalose, and 0.05% polysorbate 20.

[0871] After loading 20 μg of the manufactured Ab-III onto a SEC-HPLC column, SEC-HPLC was performed under the condition of applying 50 mM sodium phosphate buffer solution (pH 6.8) containing 150 mM sodium chloride at a flow rate of 1 mL / min, and the purity of the manufactured antibody-drug conjugate was confirmed using a 280 nm ultraviolet detector. The SEC-HPLC chromatogram of the antibody-drug conjugate Ab-III is shown in Figure 13.

[0872] In addition, after loading 40 μg of the prepared Ab-III onto the HIC-HPLC column, the drug-antibody ratio (DAR) analysis was performed under the conditions of a gradient from 100% of 20 mM sodium phosphate buffer solution (pH 6.8) containing 1 M ammonium sulfate to 70% of 20 mM sodium phosphate buffer solution (pH 6.8) and 30% of 20% isopropanol for 25 min at 30°C and a flow rate of 0.6 mL / min. The DAR of the prepared antibody-drug conjugate was confirmed using a 280 nm ultraviolet detector. The HIC-HPLC chromatogram of the antibody-drug conjugate Ab-III is shown in Fig. 14.

[0873] From the obtained chromatogram, the DAR 0, DAR 2, DAR 4, DAR 6, and DAR 8 peaks were selected, and the DAR was calculated using the area of ​​these peaks.

[0874] The yield, purity and DAR of the manufactured antibody-drug conjugate, Ab-III, are described in Table 8.

[0875]

[0876] Manufacturing Example 3.3. Preparation of anti-CNTN4 antibody-drug conjugate (Ab-IV, Ab-V)

[0877] To prepare the antibody-drug conjugate, compound 3 was purchased from MedChemExpress (#HY-13631E).

[0878] (Compound 3:

[0879] N-((S)-10-Benzyl-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)

[0880]

[0881] An antibody-drug conjugate having the following structure was prepared using compound 3 and anti-CNTN4 antibody.

[0882]

[0883] The above n can range from 1 to 20.

[0884] The antibody-drug conjugate using the Ab1 antibody prepared in Manufacturing Example 1 as an anti-CNTN4 antibody was named Ab-IV, and the antibody-drug conjugate using the Ab2 antibody was named Ab-V.

[0885] [Reduction of disulfide bonds in antibodies for side chain activation]

[0886] 1.64 mM TCEP, a reducing agent, was added to the Ab1 antibody at pH 6.75. Subsequently, the antibody and TCEP were reacted for 2 hours at 25°C to reduce some of the disulfide bonds in the antibody.

[0887] 1.02 mM TCEP, a reducing agent, was added to the Ab2 antibody at pH 7.2 to 7.4. Subsequently, the antibody and TCEP were reacted at 40°C for 6 hours to reduce some of the disulfide bonds in the antibody.

[0888] [Antibody-Drug Conjugate Manufacturing]

[0889] Compound 3 was dissolved in DMSO. 20 equivalents of compound 3 dissolved in DMSO were added to Ab1 antibody, in which some of the disulfide bonds were reduced, and 30 equivalents of compound 3 dissolved in DMSO were added to Ab2 antibody, in which some of the disulfide bonds were reduced. During the reaction, the antibody concentrations were adjusted to 10.0 to 12.0 mg / mL for Ab1 and 6.0 to 8.0 mg / mL for Ab2, and the reaction was carried out using a chamber at 25°C for 3 hours and at 40°C for 20 hours for Ab2. The reaction material was passed through a column packed with a desalting resin, and the target protein was eluted with a 50 mM sodium phosphate buffer solution (pH 6.5) containing 400 mM sodium chloride. The range of elution fractions was from 20 mAU to 200 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugates, Ab-IV and Ab-V, were formulated in 50 mM sodium phosphate buffer (pH 6.5) containing 400 mM sodium chloride, 9.5% trehalose, and 0.05% polysorbate 20. After loading 20 μg of the produced Ab-IV or Ab-V onto a SEC-HPLC column, SEC-HPLC was performed under the condition of applying 50 mM sodium phosphate buffer (pH 6.8) containing 150 mM sodium chloride at a flow rate of 1 mL / min, and the purity of the produced antibody-drug conjugate was confirmed using a 280 nm UV detector. The SEC-HPLC chromatogram of antibody-drug conjugate Ab-IV is shown in Fig. 15, and the SEC-HPLC chromatogram of Ab-V is shown in Fig. 17. The LC-MS chromatogram of antibody-drug conjugate Ab-IV is shown in Fig. 16, and the LC-MS chromatogram of Ab-V is shown in Fig. 18. LC-MS analysis of the prepared Ab-IV and Ab-V was performed at Waters, and drug-antibody ratio (DAR) analysis was performed.The yield, purity and DAR of the manufactured antibody-drug conjugates Ab-IV and Ab-V are described in Table 9.

[0890]

[0891] Manufacturing Example 3.4. Preparation of anti-CNTN4 antibody-drug conjugate (Ab-VI)

[0892] To prepare an antibody-drug conjugate having the following structure, compound 4 prepared in Manufacturing Example 2.2 and anti-CNTN4 antibody were used.

[0893]

[0894] The above n can range from 1 to 20.

[0895] The antibody-drug conjugate using the Ab2 antibody prepared in Manufacturing Example 1 as an anti-CNTN4 antibody was named Ab-VI.

[0896] [Reduction of disulfide bonds in antibodies for side chain activation]

[0897] 0.38 mM TCEP, a reducing agent, was added to the Ab2 antibody at pH 7.2 to pH 7.5. Subsequently, the antibody and TCEP were reacted for 2 hours at 25°C to reduce some of the disulfide bonds in the antibody.

[0898] [Antibody-Drug Conjugate Manufacturing]

[0899] Compound 4 prepared in Manufacturing Example 2.2 was dissolved in DMSO. 20 equivalents of compound 4 dissolved in DMSO were added to Ab2 antibody in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 2.6 to 2.7 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C. The reaction material was diluted 7-fold with 50 mM sodium acetate buffer (pH 5.0) and titrated to pH 5.0 with acetic acid. After binding the titrated material to a column packed with cation exchange resin chromatography (CEX) resin, impurities were removed with 50 mM sodium acetate buffer (pH 5.0), and the target protein was eluted with a concentration gradient using 50 mM Tris-acetic acid buffer (pH 9.0) containing 1 M sodium chloride. The eluted fraction was eluted in the range of approximately 200 to 250 mM sodium chloride. The final produced anti-CNTN4 antibody-drug conjugate, Ab-VI, was formulated in a 50 mM sodium acetate buffer (pH 5.4) containing 0.05% polysorbate 20 and 250 mM sodium chloride.

[0900] After loading the manufactured Ab-VI onto a 20 μg SEC-HPLC column, SEC-HPLC was performed under the condition of applying 200 mM potassium phosphate buffer solution (pH 6.5) containing 5% isopropanol and 250 mM potassium chloride at a flow rate of 1 mL / min. The purity and drug-antibody ratio (DAR) of the manufactured antibody-drug conjugate were confirmed using 280 nm and 370 nm UV detectors. The DAR was calculated using the 370 nm and 280 nm areas of the obtained chromatogram. The DAR calculation formula is as follows.

[0901] R = A370nm / A280nm

[0902] DAR = E Ab,280nm / ((E Drug,370nm / R)-E Drug,280nm )

[0903] (A370nm: Peak area using a 370 nm detector, A280nm: Peak area using a 280 nm detector, E Ab,280nm : Molar extinction coefficient of antibody at 280 nm, E Drug,280nm : Molar extinction coefficient of drug at 280 nm, E Drug,370nm : molar extinction coefficient of drug at 370 nm)

[0904] The SEC-HPLC 280 nm chromatogram of the antibody-drug conjugate Ab-VI is shown in Figure 19, and the SEC-HPLC 370 nm chromatogram is shown in Figure 20. The yield, purity, and DAR of the prepared antibody-drug conjugate Ab-VI are described in Table 10.

[0905]

[0906] Manufacturing Example 3.5. Preparation of anti-CNTN4 antibody-drug conjugates (Ab-VII, Ab-VIII)

[0907] To prepare an antibody-drug conjugate having the following structure, compound 5 prepared in Preparation Example 2.3 and an anti-CNTN4 antibody were used. The antibody-drug conjugate using the Ab2 antibody prepared in Preparation Example 1 and compound 5 prepared in Preparation Example 2.3 as the anti-CNTN4 antibody was designated as Ab-VII, and the antibody-drug conjugate obtained by ring-opening the conjugate of Ab-VII was designated as Ab-VIII.

[0908] Ab-VII

[0909]

[0910] The above n can range from 1 to 20.

[0911] Ab-VIII

[0912]

[0913] The above n can range from 1 to 20.

[0914] [Reduction of disulfide bonds in antibodies for side chain activation]

[0915] 1.26 mM TCEP, a reducing agent, was added to the Ab2 antibody at pH 7.2 to pH 7.5. Subsequently, the antibody and TCEP were reacted at 25°C for 30 minutes to reduce some of the disulfide bonds in the antibody.

[0916] [Antibody-Drug Conjugate Manufacturing]

[0917] Compound 5, prepared in Manufacturing Example 2.3, was dissolved in DMA. 20 equivalents of compound 5 dissolved in DMA were added to Ab2 antibody, in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 6.6 to 6.8 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C. The completed reaction material was passed through a column packed with desalting resin, and the target protein was eluted with 20 mM L-histidine buffer (pH 6.5). The range of the eluted fraction was from 50 mAU to 600 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate, Ab-VII, was formulated with 0.05% polysorbate 20 and 20 mM L-histidine buffer (pH 6.5).

[0918] [Antibody-Drug Conjugate Opening]

[0919] To increase the stability of the prepared Ab-VII, the succinimide moiety of Ab-VII was converted to ring-opened succinamide. The prepared antibody-drug conjugate Ab-VII was titrated to pH 9.0 to pH 9.5 using 1 M Tris base, and the reaction was carried out at 25°C for 18 to 20 hours. After completion of the reaction, the reaction was completed by titrating again to pH 6.5 to pH 7.0 with 1 M acetic acid, and the compound obtained was designated Ab-VIII.

[0920] After loading 20 μg of the manufactured Ab-VII and Ab-VIII onto an SEC-HPLC column, SEC-HPLC was performed under the condition of applying 200 mM potassium phosphate buffer solution (pH 6.5) containing 5% isopropanol and 250 mM potassium chloride at a flow rate of 1 mL / min, and the purity and drug-antibody ratio (DAR) of the manufactured antibody-drug conjugate were confirmed using 280 nm and 370 nm UV detectors. The DAR was calculated using the 370 nm area and the 280 nm area in the obtained chromatogram. The DAR calculation formula is the same as the DAR calculation formula of the above Preparation Example 3.4.

[0921] The SEC-HPLC 280 nm and 370 nm chromatograms of the antibody-drug conjugate Ab-VII are shown in Figures 21 and 22, respectively. The SEC-HPLC 280 nm and 370 nm chromatograms of the antibody-drug conjugate ring-opened Ab-VIII are shown in Figures 23 and 24, respectively. The yields, purities, and DARs of the prepared antibody-drug conjugates Ab-VII and Ab-VIII are described in Table 11.

[0922]

[0923] Manufacturing Example 3.6. Preparation of anti-CNTN4 antibody-drug conjugates (Ab-IX, Ab-X)

[0924] To prepare an antibody-drug conjugate having the following structure, compound 6 prepared in Preparation Example 2.4 and an anti-CNTN4 antibody were used. The antibody-drug conjugate using the Ab2 antibody prepared in Preparation Example 1 and compound 6 prepared in Preparation Example 2.4 as the anti-CNTN4 antibody was named Ab-IX, and the antibody-drug conjugate obtained by ring-opening the conjugate of Ab-IX was named Ab-X.

[0925] Ab-IX

[0926]

[0927] The above n can range from 1 to 20.

[0928] Ab-X

[0929]

[0930] The above n can range from 1 to 20.

[0931] [Reduction of disulfide bonds in antibodies for side chain activation]

[0932] 1.26 mM TCEP, a reducing agent, was added to the Ab2 antibody at pH 7.2 to pH 7.5. Subsequently, the antibody and TCEP were reacted at 25°C for 30 minutes to reduce some of the disulfide bonds in the antibody.

[0933] [Antibody-Drug Conjugate Manufacturing]

[0934] Compound 6 prepared in Manufacturing Example 2.4 was dissolved in DMA. 20 equivalents of compound 6 dissolved in DMA were added to Ab2 antibody, in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 6.6 to 6.8 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C. The completed reaction material was passed through a column packed with desalting resin, and the target protein was eluted with 20 mM L-histidine buffer (pH 6.5). The range of the eluted fraction was from 50 mAU to 600 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate, Ab-IX, was formulated with 0.05% polysorbate 20 and 20 mM L-histidine buffer (pH 6.5).

[0935] [Antibody-Drug Conjugate Opening]

[0936] To increase the stability of the Ab-IX prepared above, the succinimide moiety of Ab-IX was converted to ring-opened succinamide. The prepared antibody-drug conjugate Ab-IX was titrated to pH 9.0 to pH 9.5 using 1 M Tris base, and the reaction was carried out at 25°C for 18 to 20 hours. After completion of the reaction, the reaction was completed by titrating again to pH 6.5 to pH 7.0 with 1 M acetic acid, and the compound obtained was designated Ab-X.

[0937] After loading 20 μg of the manufactured Ab-IX and Ab-X onto an SEC-HPLC column, SEC-HPLC was performed under the condition of applying 200 mM potassium phosphate buffer solution (pH 6.5) containing 5% isopropanol and 250 mM potassium chloride at a flow rate of 1 mL / min, and the purity and drug-antibody ratio (DAR) of the manufactured antibody-drug conjugate were confirmed using 280 nm and 370 nm ultraviolet detectors. The DAR was calculated using the 370 nm area and the 280 nm area in the obtained chromatogram. The DAR calculation formula is the same as that in Preparation Example 3.4 above.

[0938] The SEC-HPLC 280 nm chromatogram and 370 nm chromatogram of the antibody-drug conjugate Ab-IX are shown in FIGS. 25 and 26 , respectively. The SEC-HPLC 280 nm chromatogram and 370 nm chromatogram of the antibody-drug conjugate ring-opened Ab-X are shown in FIGS. 27 and 28 , respectively. The yields, purities, and DAR of the prepared antibody-drug conjugates Ab-IX and Ab-X are described in Table 12.

[0939]

[0940] Manufacturing Example 3.7. Preparation of anti-CNTN4 antibody-drug conjugates (Ab-XI, Ab-XII)

[0941] To prepare an antibody-drug conjugate having the following structure, compound 7 prepared in Preparation Example 2.5 and an anti-CNTN4 antibody were used. The antibody-drug conjugate using the Ab2 antibody prepared in Preparation Example 1 and compound 7 prepared in Preparation Example 2.5 as the anti-CNTN4 antibody was named Ab-XI, and the antibody-drug conjugate obtained by ring-opening the conjugate of Ab-XI was named Ab-XII.

[0942] Ab-XI

[0943]

[0944] The above n can range from 1 to 20.

[0945] Ab-XII

[0946]

[0947] The above n can range from 1 to 20.

[0948] [Reduction of disulfide bonds in antibodies for side chain activation]

[0949] 1.26 mM TCEP, a reducing agent, was added to the Ab2 antibody at pH 7.2 to pH 7.5. Subsequently, the antibody and TCEP were reacted at 25°C for 30 minutes to reduce some of the disulfide bonds in the antibody.

[0950] [Antibody-Drug Conjugate Manufacturing]

[0951] Compound 7 prepared in Manufacturing Example 2.5 was dissolved in DMA. 20 equivalents of compound 7 dissolved in DMA were added to Ab2 antibody, in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 6.6 to 6.8 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C. Each material in which the reaction was completed was passed through a column packed with a desalting resin, and the target protein was eluted with 20 mM L-histidine buffer solution (pH 6.5). The range of the eluted fraction was from 50 mAU to 600 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate, Ab-XI, was formulated with 0.05% polysorbate 20 and 20 mM L-histidine buffer solution (pH 6.5).

[0952] [Antibody-Drug Conjugate Opening]

[0953] To increase the stability of the prepared Ab-XI, the succinimide moiety of Ab-XI was converted to ring-opened succinamide. The prepared antibody-drug conjugate Ab-XI was titrated to pH 9.0 to pH 9.5 using 1 M Tris base, and the reaction was carried out at 25°C for 18 to 20 hours. After completion of the reaction, the reaction was completed by titrating again to pH 6.5 to pH 7.0 with 1 M acetic acid, and the compound obtained was designated Ab-XII.

[0954] After loading 20 μg of the manufactured Ab-XI and Ab-XII onto an SEC-HPLC column, SEC-HPLC was performed under the condition of applying 200 mM potassium phosphate buffer solution (pH 6.5) containing 5% isopropanol and 250 mM potassium chloride at a flow rate of 1 mL / min, and the purity and drug-antibody ratio (DAR) of the manufactured antibody-drug conjugate were confirmed using 280 nm and 370 nm ultraviolet detectors. The DAR was calculated using the 370 nm area and the 280 nm area in the obtained chromatogram. The DAR calculation formula is the same as that in Preparation Example 3.4 above.

[0955] The SEC-HPLC 280 nm chromatogram and 370 nm chromatogram of the antibody-drug conjugate Ab-XI are shown in FIGS. 29 and 30 , respectively. The SEC-HPLC 280 nm chromatogram and 370 nm chromatogram of the antibody-drug conjugate ring-opened Ab-XII are shown in FIGS. 31 and 32 , respectively. The yields, purities, and DAR of the prepared antibody-drug conjugates Ab-XI and Ab-XII are described in Table 13.

[0956]

[0957] Manufacturing Example 3.8. Preparation of anti-CNTN4 antibody-drug conjugates (Ab-XIII, Ab-XIV)

[0958] To prepare an antibody-drug conjugate having the following structure, compound 8 prepared in Preparation Example 2.6 and an anti-CNTN4 antibody were used. The antibody-drug conjugate using the Ab2 antibody prepared in Preparation Example 1 and compound 8 prepared in Preparation Example 2.6 as the anti-CNTN4 antibody was named Ab-XIII, and the antibody-drug conjugate obtained by ring-opening the conjugate of Ab-XIII was named Ab-XIV.

[0959] Ab-XIII

[0960]

[0961] The above n can range from 1 to 20.

[0962] Ab-XIV

[0963]

[0964] The above n can range from 1 to 20.

[0965] [Reduction of disulfide bonds in antibodies for side chain activation]

[0966] 1.26 mM TCEP, a reducing agent, was added to the Ab2 antibody at pH 7.2 to pH 7.5. Subsequently, the antibody and TCEP were reacted for 2 hours at 25°C to reduce some of the disulfide bonds of the antibody.

[0967] [Antibody-Drug Conjugate Manufacturing]

[0968] Compound 8, manufactured in Manufacturing Example 2.6, was dissolved in DMA. 20 equivalents of compound 8 dissolved in DMA were added to Ab2 antibody, in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 2.6 to 2.8 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C. Each material in which the reaction was completed was passed through a column packed with a desalting resin, and the target protein was eluted with 20 mM L-histidine buffer solution (pH 6.5). The range of the eluted fraction was from 50 mAU to 500 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate, Ab-XIII, was formulated with 0.05% polysorbate 20 and 20 mM L-histidine buffer solution (pH 6.5).

[0969] [Antibody-Drug Conjugate Opening]

[0970] To increase the stability of the prepared Ab-XIII, the succinimide moiety of Ab-XIII was converted to ring-opened succinamide. The prepared antibody-drug conjugate Ab-XIII was titrated to pH 9.0 to pH 9.5 using 1 M Tris base, and the reaction was carried out at 25°C for 18 to 20 hours. After completion of the reaction, the reaction was completed by titrating again to pH 6.5 to pH 7.0 with 1 M acetic acid, and the compound obtained was designated Ab-XIV.

[0971] After loading 20 μg of the manufactured Ab-XIII and Ab-XIV onto an SEC-HPLC column, SEC-HPLC was performed under the condition of applying 200 mM potassium phosphate buffer solution (pH 6.5) containing 5% isopropanol and 250 mM potassium chloride at a flow rate of 1 mL / min, and the purity and drug-antibody ratio (DAR) of the manufactured antibody-drug conjugate were confirmed using 280 nm and 370 nm ultraviolet detectors. The DAR was calculated using the 370 nm area and the 280 nm area in the obtained chromatogram. The DAR calculation formula is the same as that in Preparation Example 3.4 above.

[0972] The SEC-HPLC 280 nm chromatogram and 370 nm chromatogram of antibody-drug conjugate Ab-XIII are shown in Figures 33 and 34, respectively. The SEC-HPLC 280 nm chromatogram and 370 nm chromatogram of antibody-drug conjugate ring-opened Ab-XIV are shown in Figures 35 and 36, respectively. The yields, purities and DAR of the prepared antibody-drug conjugates Ab-XIII and Ab-XIV are described in Table 14.

[0973]

[0974] Manufacturing Example 3.9. Preparation of anti-CNTN4 antibody-drug conjugates (Ab-XV, Ab-XVI)

[0975] To prepare an antibody-drug conjugate having the following structure, compound 9 prepared in Preparation Example 2.7 and an anti-CNTN4 antibody were used. The antibody-drug conjugate using the Ab3 antibody prepared in Preparation Example 1 and compound 9 prepared in Preparation Example 2.7 as the anti-CNTN4 antibody was named Ab-XV, and the antibody-drug conjugate obtained by ring-opening the conjugate of Ab-XV was named Ab-XVI.

[0976] Ab-XV

[0977]

[0978] The above n can range from 1 to 20.

[0979] Ab-XVI

[0980]

[0981] The above n can range from 1 to 20.

[0982] [Reduction of disulfide bonds in antibodies for side chain activation]

[0983] 1.26 mM TCEP, a reducing agent, was added to the Ab3 antibody at pH 7.2 to pH 7.5. Subsequently, the antibody and TCEP were reacted at 25°C for 2 hours to reduce some of the disulfide bonds in the antibody.

[0984] [Antibody-Drug Conjugate Manufacturing]

[0985] Compound 9 prepared in Manufacturing Example 2.7 was dissolved in DMA. 13 to 20 equivalents of compound 9 dissolved in DMA were added to Ab3 antibody, in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 7.5 to 8.0 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C. The reaction material was diluted 6-fold using 20 mM L-histidine buffer solution (pH 5.5). After binding the titrated material to a column packed with cation exchange resin chromatography (CEX) resin, impurities were removed with 20 mM L-histidine buffer solution (pH 5.5) containing 50 mM sodium chloride, and the target protein was eluted with 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride. The range of elution fractions is from 200 mAU to 500 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate was formulated in a 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride and 0.05% polysorbate 20.

[0986] [Antibody-Drug Conjugate Opening]

[0987] To increase the stability of the prepared Ab-XV, the succinimide moiety of Ab-XV was converted to ring-opened succinamide. The prepared antibody-drug conjugate Ab-XV was titrated to pH 9.0 to pH 9.5 using 1 M Tris base, and the reaction was carried out at 25°C for 18 to 20 hours. After completion of the reaction, the reaction was completed by titrating again to pH 6.5 to pH 7.0 with 1 M acetic acid, and the compound obtained by completing the reaction was designated Ab-XVI.

[0988] After loading 20 μg of the manufactured Ab-XV and Ab-XVI onto an SEC-HPLC column, SEC-HPLC was performed under the condition of applying 200 mM potassium phosphate buffer solution (pH 6.5) containing 5% isopropanol and 250 mM potassium chloride at a flow rate of 1 mL / min, and the purity of the manufactured antibody-drug conjugate was confirmed using a 280 nm ultraviolet detector.

[0989] In addition, 10 μg of each of the prepared Ab-XV and Ab-XVI were loaded onto the RP-HPLC column, and then the concentration gradient was increased from 75% of a purified water solution containing 0.1% formic acid to 25% of an acetonitrile solution containing 0.1% formic acid to 20% of a purified water solution containing 0.1% formic acid to 80% of an acetonitrile solution containing 0.1% formic acid at a flow rate of 0.25 mL / min for 20 minutes, and then detected by mass spectrometry (LC / MS) to perform drug-antibody ratio (DAR) analysis. Each DAR peak was selected from the obtained chromatogram, and the DAR was calculated using the intensity of the peak.

[0990] The SEC-HPLC 280 nm chromatogram and LC-MS chromatogram of the antibody-drug conjugate Ab-XV are shown in Figures 37 and 38, respectively. The SEC-HPLC 280 nm chromatogram and LC-MS chromatogram of the antibody-drug conjugate ring-opened Ab-XVI are shown in Figures 39 and 40, respectively. The yields, purities, and DAR of the prepared antibody-drug conjugates Ab-XV and Ab-XVI are described in Table 15.

[0991]

[0992] Manufacturing Example 3.10. Preparation of anti-CNTN4 antibody-drug conjugate (Ab-XVII)

[0993] To prepare an antibody-drug conjugate having the following structure, compound 10 prepared in Manufacturing Example 2.8 and an anti-CNTN4 antibody were used. The antibody-drug conjugate using the Ab3 antibody prepared in Manufacturing Example 1 and compound 10 prepared in Manufacturing Example 2.8 as the anti-CNTN4 antibody was named Ab-XVII.

[0994] Ab-XVII

[0995]

[0996] The above n can range from 1 to 20.

[0997] [Reduction of disulfide bonds in antibodies for side chain activation]

[0998] 1.26 mM TCEP, a reducing agent, was added to the Ab3 antibody at pH 7.2 to pH 7.5. Subsequently, the antibody and TCEP were reacted at 25°C for 2 hours to reduce some of the disulfide bonds in the antibody.

[0999] [Antibody-Drug Conjugate Manufacturing]

[1000] Compound 10 prepared in Manufacturing Example 2.8 was dissolved in DMA. 20 equivalents of compound 10 dissolved in DMA were added to Ab3 antibody, in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 6.0 to 8.0 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C. The reaction material was diluted 6-fold using 20 mM L-histidine buffer solution (pH 5.5). After binding the titrated material to a column packed with cation exchange resin chromatography (CEX) resin, impurities were removed with 20 mM L-histidine buffer solution (pH 5.5) containing 50 mM sodium chloride, and the target protein was eluted with 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride. The range of elution fractions is from 200 mAU to 500 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate was formulated in a 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride and 0.05% polysorbate 20.

[1001] After loading the manufactured Ab-XVII onto a 20 μg SEC-HPLC column, SEC-HPLC was performed under the condition of applying 200 mM potassium phosphate buffer solution (pH 6.5) containing 5% isopropanol and 250 mM potassium chloride at a flow rate of 1 mL / min, and the purity of the manufactured antibody-drug conjugate was confirmed using a 280 nm ultraviolet detector.

[1002] In addition, after loading 10 μg of Ab-XII prepared on a RP-HPLC column, a concentration gradient was applied from a 75% ratio of purified water solution containing 0.1% formic acid to a 25% ratio of acetonitrile solution containing 0.1% formic acid to a 20% ratio of purified water solution containing 0.1% formic acid to an 80% ratio of acetonitrile solution containing 0.1% formic acid at a flow rate of 0.25 mL / min for 20 min, and then detected by mass spectrometry (LC / MS) to perform drug-antibody ratio (DAR) analysis. Each DAR peak was selected from the obtained chromatogram, and the DAR was calculated using the intensity of the peak.

[1003] The SEC-HPLC 280 nm chromatogram and LC-MS chromatogram of the antibody-drug conjugate Ab-XVII are shown in Figures 41 and 42, respectively. The yield, purity, and DAR of the prepared antibody-drug conjugate Ab-XVII are described in Table 16.

[1004]

[1005] Manufacturing Example 3.11. Preparation of anti-CNTN4 antibody-drug conjugate (Ab-XVIII)

[1006] To prepare an antibody-drug conjugate having the following structure, compound 11 prepared in Manufacturing Example 2.9 and an anti-CNTN4 antibody were used. The antibody-drug conjugate using the Ab3 antibody prepared in Manufacturing Example 1 and compound 11 prepared in Manufacturing Example 2.9 as the anti-CNTN4 antibody was named Ab-XVIII.

[1007] Ab-XVIII

[1008]

[1009] The above n can range from 1 to 20.

[1010] [Reduction of disulfide bonds in antibodies for side chain activation]

[1011] 1.26 mM TCEP, a reducing agent, was added to the Ab3 antibody at pH 7.2 to pH 7.5. Subsequently, the antibody and TCEP were reacted at 25°C for 2 hours to reduce some of the disulfide bonds in the antibody.

[1012] [Antibody-Drug Conjugate Manufacturing]

[1013] Compound 11 prepared in Manufacturing Example 2.9 was dissolved in DMA. 15 to 20 equivalents of compound 11 dissolved in DMA were added to Ab3 antibody, in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 8.5 to 10.0 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C. The reaction material was diluted 6-fold using 20 mM L-histidine buffer solution (pH 5.5). After binding the titrated material to a column packed with cation exchange resin chromatography (CEX) resin, impurities were removed with 20 mM L-histidine buffer solution (pH 5.5) containing 50 mM sodium chloride, and the target protein was eluted with 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride. The range of elution fractions is from 200 mAU to 500 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate was formulated in a 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride and 0.05% polysorbate 20.

[1014] After loading the manufactured Ab-XVIII onto a 20 μg SEC-HPLC column, SEC-HPLC was performed under the condition of applying 200 mM potassium phosphate buffer solution (pH 6.5) containing 5% isopropanol and 250 mM potassium chloride at a flow rate of 1 mL / min, and the purity of the manufactured antibody-drug conjugate was confirmed using a 280 nm ultraviolet detector.

[1015] In addition, after loading 10 μg of Ab-XII prepared on a RP-HPLC column, a concentration gradient was applied from a 75% ratio of purified water solution containing 0.1% formic acid to a 25% ratio of acetonitrile solution containing 0.1% formic acid to a 20% ratio of purified water solution containing 0.1% formic acid to an 80% ratio of acetonitrile solution containing 0.1% formic acid at a flow rate of 0.25 mL / min for 20 min, and then detected by mass spectrometry (LC / MS) to perform drug-antibody ratio (DAR) analysis. Each DAR peak was selected from the obtained chromatogram, and the DAR was calculated using the intensity of the peak.

[1016] The SEC-HPLC 280 nm chromatogram and LC-MS chromatogram of the antibody-drug conjugate Ab-XVIII are shown in Figures 43 and 44, respectively. The yield, purity, and DAR of the prepared antibody-drug conjugate Ab-XVIII are described in Table 17.

[1017]

[1018] Manufacturing Example 3.12. Preparation of anti-CNTN4 antibody-drug conjugate (Ab-XIX)

[1019] To prepare an antibody-drug conjugate having the following structure, compound 12 prepared in Manufacturing Example 2.10 and an anti-CNTN4 antibody were used. The antibody-drug conjugate using the Ab3 antibody prepared in Manufacturing Example 1 and compound 12 prepared in Manufacturing Example 2.10 as the anti-CNTN4 antibody was named Ab-XIX.

[1020] Ab-XIX

[1021]

[1022] The above n can range from 1 to 20.

[1023] [Reduction of disulfide bonds in antibodies for side chain activation]

[1024] 1.26 mM TCEP, a reducing agent, was added to the Ab3 antibody at pH 7.2 to pH 7.5. Subsequently, the antibody and TCEP were reacted at 25°C for 2 hours to reduce some of the disulfide bonds in the antibody.

[1025] [Antibody-Drug Conjugate Manufacturing]

[1026] Compound 12 prepared in Manufacturing Example 2.10 was dissolved in DMA. 15 to 20 equivalents of compound 12 dissolved in DMA were added to Ab3 antibody, in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 8.0 to 10.0 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C. The reaction material was diluted 6-fold using 20 mM L-histidine buffer solution (pH 5.5). After binding the titrated material to a column packed with cation exchange resin chromatography (CEX) resin, impurities were removed with 20 mM L-histidine buffer solution (pH 5.5) containing 50 mM sodium chloride, and the target protein was eluted with 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride. The range of elution fractions is from 200 mAU to 500 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate was formulated in a 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride and 0.05% polysorbate 20.

[1027] After loading the manufactured Ab-XIX onto a 20 μg SEC-HPLC column, SEC-HPLC was performed under the condition of applying 200 mM potassium phosphate buffer solution (pH 6.5) containing 5% isopropanol and 250 mM potassium chloride at a flow rate of 1 mL / min, and the purity of the manufactured antibody-drug conjugate was confirmed using a 280 nm ultraviolet detector.

[1028] In addition, after loading 10 μg of Ab-XIX prepared on a RP-HPLC column, a concentration gradient was applied from a 75% solution of purified water containing 0.1% formic acid to a 25% solution of acetonitrile containing 0.1% formic acid to a 20% solution of purified water containing 0.1% formic acid to an 80% solution of acetonitrile containing 0.1% formic acid at a flow rate of 0.25 mL / min for 20 minutes, and then detection was performed using a mass spectrometer (LC / MS) to perform drug-antibody ratio (DAR) analysis. Each DAR peak was selected from the obtained chromatogram, and the DAR was calculated using the intensity of the peak.

[1029] The SEC-HPLC 280 nm chromatogram and LC-MS chromatogram of the antibody-drug conjugate Ab-XIX are shown in Figures 45 and 46, respectively. The yield, purity, and DAR of the prepared antibody-drug conjugate Ab-XIX are described in Table 18.

[1030]

[1031] Manufacturing Example 3.13. Preparation of an anti-CNTN4 antibody-drug conjugate (Ab-XX)

[1032] To prepare an antibody-drug conjugate having the following structure, compound 13 prepared in Manufacturing Example 2.11 and an anti-CNTN4 antibody were used. The antibody-drug conjugate using the Ab3 antibody prepared in Manufacturing Example 1 and compound 13 prepared in Manufacturing Example 2.11 as the anti-CNTN4 antibody was named Ab-XX.

[1033] Ab-XX

[1034]

[1035] The above n can range from 1 to 20.

[1036] [Reduction of disulfide bonds in antibodies for side chain activation]

[1037] 1.26 mM TCEP, a reducing agent, was added to the Ab3 antibody at pH 7.2 to pH 7.5. Subsequently, the antibody and TCEP were reacted at 25°C for 2 hours to reduce some of the disulfide bonds in the antibody.

[1038] [Antibody-Drug Conjugate Manufacturing]

[1039] Compound 13 prepared in Manufacturing Example 2.11 was dissolved in DMA. 15 to 20 equivalents of compound 13 dissolved in DMA were added to Ab3 antibody, in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 8.0 to 10.0 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C. The reaction material was diluted 6-fold using 20 mM L-histidine buffer solution (pH 5.5). After binding the titrated material to a column packed with cation exchange resin chromatography (CEX) resin, impurities were removed with 20 mM L-histidine buffer solution (pH 5.5) containing 50 mM sodium chloride, and the target protein was eluted with 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride. The range of elution fractions is from 200 mAU to 500 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate was formulated in a 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride and 0.05% polysorbate 20.

[1040] After loading 20 μg of the manufactured Ab-XX onto a SEC-HPLC column, SEC-HPLC was performed under the condition of applying 200 mM potassium phosphate buffer solution (pH 6.5) containing 5% isopropanol and 250 mM potassium chloride at a flow rate of 1 mL / min, and the purity of the manufactured antibody-drug conjugate was confirmed using a 280 nm ultraviolet detector.

[1041] In addition, after loading 10 μg of Ab-XX prepared on a RP-HPLC column, a concentration gradient was applied from a 75% solution of purified water containing 0.1% formic acid to a 25% solution of acetonitrile containing 0.1% formic acid to a 20% solution of purified water containing 0.1% formic acid to an 80% solution of acetonitrile containing 0.1% formic acid at a flow rate of 0.25 mL / min for 20 minutes, and then detected by mass spectrometry (LC / MS) to perform drug-antibody ratio (DAR) analysis. Each DAR peak was selected from the obtained chromatogram, and the DAR was calculated using the intensity of the peak.

[1042] The SEC-HPLC 280 nm chromatogram and LC-MS chromatogram of the antibody-drug conjugate Ab-XX are shown in Figures 47 and 48, respectively. The yield, purity, and DAR of the prepared antibody-drug conjugate Ab-XX are described in Table 19.

[1043]

[1044] Manufacturing Example 3.14. Preparation of an anti-CNTN4 antibody-drug conjugate (Ab-XXI)

[1045] To prepare an antibody-drug conjugate having the following structure, compound 8 prepared in Preparation Example 2.6 and an anti-CNTN4 antibody were used. The antibody-drug conjugate using the Ab3 antibody prepared in Preparation Example 1 and compound 8 prepared in Preparation Example 2.6 as the anti-CNTN4 antibody was named Ab-XXI.

[1046] Ab-XXI

[1047]

[1048] The above n can range from 1 to 20.

[1049] [Reduction of disulfide bonds in antibodies for side chain activation]

[1050] 1.26 mM TCEP, a reducing agent, was added to the Ab3 antibody at pH 7.2 to pH 7.5. Subsequently, the antibody and TCEP were reacted at 25°C for 2 hours to reduce some of the disulfide bonds in the antibody.

[1051] [Antibody-Drug Conjugate Manufacturing]

[1052] Compound 8 prepared in Manufacturing Example 2.6 was dissolved in DMA. 15 to 20 equivalents of compound 8 dissolved in DMA were added to Ab3 antibody, in which some of the disulfide bonds were reduced. The concentration of the antibody during the reaction was adjusted to 8.0 to 10.0 mg / mL, and the reaction was carried out for 3 hours using a chamber at 25°C. The reaction material was diluted 6-fold using 20 mM L-histidine buffer solution (pH 5.5). After binding the titrated material to a column packed with cation exchange resin chromatography (CEX) resin, impurities were removed with 20 mM L-histidine buffer solution (pH 5.5) containing 50 mM sodium chloride, and the target protein was eluted with 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride. The range of elution fractions was from 600 mAU to 600 mAU at 280 nm. The finally produced anti-CNTN4 antibody-drug conjugate was formulated in a 20 mM L-histidine buffer solution (pH 5.5) containing 400 mM sodium chloride and 0.05% polysorbate 20.

[1053] After loading the manufactured Ab-XXI onto a 20 μg SEC-HPLC column, SEC-HPLC was performed under the condition of applying 200 mM potassium phosphate buffer solution (pH 6.5) containing 5% isopropanol and 250 mM potassium chloride at a flow rate of 1 mL / min, and the purity of the manufactured antibody-drug conjugate was confirmed using a 280 nm ultraviolet detector.

[1054] In addition, 10 μg of Ab-XXI prepared on the RP-HPLC column was loaded, and then a concentration gradient was applied from a 75% solution of purified water containing 0.1% formic acid to a 25% solution of acetonitrile containing 0.1% formic acid to a 20% solution of purified water containing 0.1% formic acid to an 80% solution of acetonitrile containing 0.1% formic acid at a flow rate of 0.25 mL / min for 20 min, followed by detection by mass spectrometry (LC / MS) to perform drug-antibody ratio (DAR) analysis. Each DAR peak was selected from the obtained chromatogram, and the DAR was calculated using the intensity of the peak.

[1055] The SEC-HPLC 280 nm chromatogram and LC-MS chromatogram of the antibody-drug conjugate Ab-XXI are shown in Figures 49 and 50, respectively. The yield, purity, and DAR of the prepared antibody-drug conjugate Ab-XXI are described in Table 20.

[1056]

[1057] Example 3. Binding ability of anti-CNTN4 antibody-drug conjugate to human CNTN4 protein

[1058] Example 3.1. Antigen protein binding capacity of Ab-I and Ab-II (ELISA analysis)

[1059] To confirm the binding ability of the antibody-drug conjugates Ab-I and Ab-II prepared in Manufacturing Example 3.1 above to human CNTN4 protein, an ELISA (Enzyme-Linked Immunosorbent Assay) test was conducted.

[1060] Human CNTN4 protein (Biointron, China), an antigen protein, was diluted in phosphate-buffered saline (PBS) buffer to prepare a 5 nM human CNTN4 protein solution. 100 μL of the prepared human CNTN4 protein solution was added to each well of a 96-well plate (9018, Corning, USA) and coated for 2 hours at room temperature. All solution in the plate was removed, and blocking buffer (3% BSA in PBS) was added to each well and incubated for 1 hour at room temperature.

[1061] Ab1 and Ab2 antibodies prepared in Manufacturing Example 1, Ab-I and Ab-II antibody-drug conjugates prepared in Manufacturing Example 3.1, and human IgG4 and IgG1 antibody samples as isotype controls were sequentially diluted in blocking buffer from 200 nM to 8 points (0.02 pM) at a 10-fold dilution ratio.

[1062] After the blocking process in the 96-well plate, the buffer was removed from each well, and 100 μL of the diluted antibody was added to each well and reacted at 37°C for 1 hour. The wells were washed with PBST (0.1% Tween 20 in PBS), and treated with HRP-conjugated anti-human IgG Fc antibody (109-035-098, Jackson ImmunoResearch Inc., USA) (1:100,000), a secondary antibody for detecting antibodies or antibody-drug conjugates, at room temperature for 1 hour, and then washed again with PBST. For color development, 100 μL of TMB solution (ThermoFisher Scientific, USA, Cat. No.: 34028) was added to each well, reacted at room temperature for 10 minutes, and the absorbance at 450 nm (OD 450 ) were measured. OD according to the concentration of Ab1 and Ab2 antibodies, antibody-drug conjugates Ab-I and Ab-II, and human IgG4 and IgG1 antibodies treated 450The values ​​are shown in Figure 51. In addition, EC was used to compare the binding affinity of Ab1 and Ab2 antibodies and antibody-drug conjugates Ab-I and Ab-II to human CNTN4 protein. 50 The concentration was calculated, and the calculated values ​​are shown in Table 21.

[1063]

[1064] As a result, through ELISA experiments, it was confirmed that the tested antibodies Ab1 and Ab2 and antibody-drug conjugates Ab-I and Ab-II exhibited excellent binding ability to human CNTN4 protein, and that Ab-I had a binding ability similar to that of Ab1 and Ab-II had a binding ability similar to that of Ab2, respectively.

[1065] Example 3.2. CNTN4 protein binding ability of Ab-I and Ab-II antibody-drug conjugates (FACS test)

[1066] The binding ability of antibody-drug conjugates Ab-I and Ab-II prepared in Manufacturing Example 3.1 above to human CNTN4 protein was analyzed by FACS (Fluorescence Activated Cell Sorting, Flow Cytometry) (FACS CantoTM II, BD, USA) test.

[1067] Transfection was performed with the CNTN4 plasmid vector into HEK293 cells (ThermoFisherScientific, USA), and HEK293 cells expressing human CNTN4 antigen protein were designated HEK293 / CNTN4 cells.

[1068] 1 x 10 suspended in FACS buffer 6 HEK293 / CNTN4 cells (cells / mL) were seeded in 100 μL per 96-well plate.

[1069] In addition, Ab1 and Ab2 antibodies prepared in Manufacturing Example 1, Ab-I and Ab-II antibody-drug conjugates prepared in Manufacturing Example 3.1, and human IgG4 and IgG1 antibody samples as isotype controls were serially diluted from 100 nM to 9 points (0.256 pM) at a 5-fold dilution ratio in FACS buffer. The diluted antibodies and antibody-drug conjugates were treated in a 96-well plate seeded with cells and incubated at 4°C for 1 hour. Each well was washed with FACS buffer, and Goat Anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody (Alexa Fluor 647 (ThermoFisher Scientific, USA, Cat. No.: A21445) (1:400)) was added, and the 96-well plate was further incubated, followed by washing with FACS buffer. The degree of binding of antigen-antibody and antigen-antibody-drug conjugates was measured by MFI (Mean Fluorescence Intensity) value using a flow cytometer, and the MFI values ​​according to the concentration of antibody and antibody-drug conjugate treatment are shown in Figure 52.

[1070] EC was used to indicate the degree of binding of Ab1 and Ab2 antibodies and Ab-I and Ab-II antibody-drug conjugates to antigen proteins expressed on the cell surface. 50 The concentration was calculated. The calculated EC 50 The concentrations are described in Table 22.

[1071]

[1072] As confirmed in Figure 52 and Table 22, antibodies Ab1 and Ab2 and antibody-drug conjugates Ab-I and Ab-II were confirmed to have excellent binding capacities to human CNTN4 protein expressed on the cell surface. In addition, Ab1 was confirmed to have binding capacities similar to those of Ab-I and Ab2 to those of Ab-II, respectively.

[1073] Example 4. Cancer cell internalization of anti-CNTN4 antibody-drug conjugate

[1074] 1 x 10 5 A549 / CNTN4 (a CNTN4-overexpressing human lung cancer cell line prepared in Example 2) at 10 cells / mL was seeded into each well of a 96-well plate (3595, Corning, USA) at 50 μL. The day after seeding, it was confirmed that the cells had attached to each well of the 96-well plate, and the existing cell culture medium was removed.

[1075] In addition, the antibody-drug conjugate Ab-I prepared in Manufacturing Example 3.1 was diluted in the culture medium to 2 times the final concentration of 0.5, 1, 2, and 4 μg / mL. The 2-fold diluted antibody-drug conjugate Ab-I was mixed with IncuCyte® FabFluor-pH Red Antibody labeling reagent (Sartorius, 4722) and reacted at 37°C for 15 minutes, and then 50 μL was treated per well. The 96-well plate was placed in the IncuCyte® S3 Live-Cell Analysis System, and images were measured at 1-hour intervals for 24 hours, and the measured results are shown in Figure 53.

[1076] As confirmed in Figure 53, the antibody-drug conjugate Ab-I was internalized into cancer cells with increasing incubation time and concentration, resulting in an increase in fluorescence signal. This indicates that the antibody-drug conjugate of the present invention can transport cytotoxic drugs or therapeutic moieties into cells through cellular internalization, similar to that confirmed for the Ab1 antibody shown to have a cancer cell internalization effect in Example 2.

[1077] Example 5. In vitro anticancer effect of anti-CNTN4 antibody-drug conjugate

[1078] Example 5.1. In vitro anticancer effects of antibody-drug conjugates Ab-I and Ab-II

[1079] It was confirmed whether Ab-I and Ab-II, antibody-drug conjugates manufactured in Manufacturing Example 3.1, inhibit the growth of human cancer cell lines and mouse cancer cell lines.

[1080] [Target cell line]

[1081] Using the human lung cancer cell line A549 (Korean Cell Line Bank, South Korea), CNTN4-overexpressing human lung cancer cells, A549 / CNTN4 cells (prepared in Example 2), were prepared. The human fibrosarcoma cell line HT-1080 cells (Korean Cell Line Bank, South Korea) were transfected with the CNTN4 plasmid vector used in Example 2, and the HT-1080 cells overexpressing the human CNTN4 antigen protein were designated HT-1080 / CNTN4 cells.

[1082] In addition, pancreatic cancer cells overexpressing mouse Cntn4 antigen protein were produced by transfection of mouse pancreatic cancer cells, Pan02 cells (National Cancer Institute, USA), with a mouse CNTN4 (hereinafter referred to as Cntn4) plasmid vector. The produced Pan02 cells overexpressing mouse Cntn4 antigen protein were named Pan02 / Cntn4 cells.

[1083] [Analysis of cancer cell and normal cell growth inhibition]

[1084] A549, A549 / CNTN4, HT-1080, HT-1080 / CNTN4, Pan02 / Cntn4, and human dermal fibroblast Hs27 cells (American Type Culture Collection, USA) were seeded at 5 x 10 in each 96-well plate (96 well plate, 3595, Corning, USA). 3Each cell was seeded individually. The next day, the attachment of cells to the 96-well plate was confirmed, and the existing cell culture medium was removed.

[1085] The antibody-drug conjugates Ab-I and Ab-II prepared in Manufacturing Example 3.1 were sequentially diluted in cell culture medium from 10 μM to 6 points (0.1 nM) at a 10-fold dilution ratio. 200 μL of the diluted antibody-drug conjugate was treated per well. The negative control for Ab-I and Ab-II used an antibody-drug conjugate prepared by replacing the Ab1 antibody in Manufacturing Example 2 with a human IgG4 antibody.

[1086] In addition, the optical density (OD) of the 0% control group (0% cell growth comparison group) on the day of Ab-I or Ab-II treatment was measured using the MTT assay method. After removing the culture medium from the 96-well plate, 100 μL of MTT solution was treated per well and reacted in a CO2 incubator for 3 hours. Afterwards, the MTT solution was removed and 100 μL of MTT solvent (6% SDS in DMSO) was treated per well and reacted in a CO2 incubator for 2 hours, and the absorbance at 590 nm was measured using a microplate reader (Envision2105, PerkinElmer, USA).

[1087] Furthermore, the experimental groups treated with Ab-I and Ab-II and the 100% control group (a group not treated with drugs and a 100% comparison group for cell growth) were cultured in a 37℃ CO2 incubator for 72 hours, and the absorbance of the experimental groups and the 100% control group was measured using the MTT assay method in the same manner as the 0% control group above.

[1088] Cell viability in the experimental group (Ab-I or Ab-II treatment) was calculated using the following formula.

[1089] Cell viability (%) = [(experimental group) - (0% control group)] / [(100% control group) - (0% control group)]

[1090] GI of Ab-I and Ab-II in A549, A549 / CNTN4, HT-1080, HT-1080 / CNTN4, Pan02 / Cntn4, and Hs27 cells 50 (half maximal growth inhibition concentration) values ​​were calculated using GraphPad Prism (GraphPad software Inc., USA) using cell viability.

[1091] The cell viability (%) of Ab-I and Ab-II in A549, A549 / CNTN4, HT-1080, HT-1080 / CNTN4, Pan02 / Cntn4 and Hs27 cells is shown in Figure 54, and GI 50 is described in Table 23.

[1092]

[1093] As confirmed in Figure 54 and Table 23, the antibody-drug conjugates Ab-I and Ab-II of the present invention inhibited the cell growth of A549 / CNTN4, HT-1080 / CNTN4, and Pan02 / Cntn4 in a concentration-dependent manner. On the other hand, Ab-I and Ab-II did not affect the cell growth of normal cells (i.e., human skin fibroblasts, Hs27) rather than cancer cells. Therefore, it was confirmed that the antibody-drug conjugates Ab-I and Ab-II of the present invention have excellent cell growth inhibition ability against cancer cells overexpressing CNTN4.

[1094] Example 5.2. In vitro anticancer effects of anti-CNTN4 antibody-drug conjugates Ab-III, Ab-IV, Ab-V, Ab-VI, Ab-VII, Ab-VIII, Ab-IX, Ab-X, Ab-XI, Ab-XII, Ab-XIII, and Ab-XIV

[1095] It was determined whether the antibody-drug conjugates prepared in Manufacturing Examples 3.2 to 3.8 (i.e., Ab-III, Ab-IV, Ab-V, Ab-VI, Ab-VII, Ab-VIII, Ab-IX, Ab-X, Ab-XI, Ab-XII, Ab-XIII and Ab-XIV) inhibit the growth of human cancer cell lines.

[1096] HT-1080 and HT-1080 / CNTN4 cells were seeded at 5 x 10 in each 96-well plate (96 well plate, 3595, Corning, USA). 3 Each cell was seeded individually. The next day, the attachment of cells to the 96-well plate was confirmed, and the existing cell culture medium was removed.

[1097] The antibody-drug conjugates prepared in Manufacturing Examples 3.2 to 3.8 were sequentially diluted in cell culture medium from 10 μM to 6 points (0.1 nM) at a 10-fold dilution ratio. 200 μL of the diluted antibody-drug conjugate was treated per well. The negative control for Ab-III and the negative control for Ab-IV used antibody-drug conjugates prepared by replacing the Ab1 antibody in Manufacturing Example 2 with a human IgG4 antibody.

[1098] In addition, the optical density (OD) of the 0% control group (0% cell growth comparison group) on the day of antibody-drug conjugate treatment was measured using the MTT assay method. After removing the culture medium from the 96-well plate, 100 μL of MTT solution was treated per well and reacted in a CO2 incubator for 3 hours. Afterwards, the MTT solution was removed and 100 μL of MTT solvent (6% SDS in DMSO) was treated per well and reacted in a CO2 incubator for 2 hours, and the absorbance at 590 nm was measured using a microplate reader (Envision2105, PerkinElmer, USA).

[1099] Furthermore, the experimental group treated with the antibody-drug conjugate and the 100% control group (a group not treated with the drug and a 100% comparison group for cell growth) were cultured in a 37℃ CO2 incubator for 72 hours, and the absorbance of the experimental group and the 100% control group was measured using the MTT assay method in the same manner as the 0% control group.

[1100] Cell viability in the experimental group (antibody-drug conjugate treatment) was calculated using the following formula.

[1101] Cell viability (%) = [(experimental group) - (0% control group)] / [(100% control group) - (0% control group)]

[1102] GI of antibody-drug conjugates in HT-1080 and HT-1080 / CNTN4 cells 50 (half maximal growth inhibition concentration) values ​​were calculated using GraphPad Prism (GraphPad software Inc., USA) using cell viability.

[1103] The cell viability (%) of Ab-III, Ab-IV, Ab-V, Ab-VI, Ab-VII, Ab-VIII, Ab-IX, Ab-X, Ab-XI, Ab-XII, Ab-XIII and Ab-XIV in HT-1080 and HT-1080 / CNTN4 cells is shown in Figure 55, and GI 50 is described in Table 24.

[1104]

[1105] As confirmed in FIG. 55 and Table 24, the antibody-drug conjugates Ab-III, Ab-IV, Ab-V, Ab-VI, Ab-VII, Ab-VIII, Ab-IX, Ab-X, Ab-XI, Ab-XII, Ab-XIII, and Ab-XIV of the present invention inhibited the cell growth of HT-1080 / CNTN4 in a concentration-dependent manner. Therefore, it was confirmed that the antibody-drug conjugates Ab-III, Ab-IV, Ab-V, Ab-VI, Ab-VII, Ab-VIII, Ab-IX, Ab-X, Ab-XI, Ab-XII, Ab-XIII, and Ab-XIV of the present invention have excellent cell growth inhibition ability against cancer cells overexpressing CNTN4.

[1106] Example 5.3. In vitro anticancer effects of anti-CNTN4 antibody-drug conjugates Ab-XXI, Ab-XV, Ab-XVI, Ab-XVII, Ab-XVIII, Ab-XIX, and Ab-XX

[1107] It was determined whether the antibody-drug conjugates prepared in Manufacturing Examples 3.9 to 3.14 (i.e., Ab-XV, Ab-XVI, Ab-XVII, Ab-XVIII, Ab-XVIII, Ab-XIX, and Ab-XX) inhibit the growth of human cancer cell lines.

[1108] HT-1080 / CNTN4 cells were seeded at 5 x 10 in each 96-well plate (96 well plate, 3595, Corning, USA). 3Each cell was seeded individually. The next day, the attachment of cells to the 96-well plate was confirmed, and the existing cell culture medium was removed.

[1109] The antibody-drug conjugates prepared in Manufacturing Examples 3.9 to 3.14 were sequentially diluted in cell culture medium from 10 μM to 6 points (0.1 nM) at a 10-fold dilution ratio. 200 μL of the diluted antibody-drug conjugate was administered to each well.

[1110] In addition, the optical density (OD) of the 0% control group (0% cell growth comparison group) on the day of antibody-drug conjugate treatment was measured using the MTT assay method. After removing the culture medium from the 96-well plate, 100 μL of MTT solution was treated per well and reacted in a CO2 incubator for 3 hours. Afterwards, the MTT solution was removed and 100 μL of MTT solvent (6% SDS in DMSO) was treated per well and reacted in a CO2 incubator for 2 hours, and the absorbance at 590 nm was measured using a microplate reader (Envision2105, PerkinElmer, USA).

[1111] Furthermore, the experimental group treated with the antibody-drug conjugate and the 100% control group (a group not treated with the drug and a 100% comparison group for cell growth) were cultured in a 37℃ CO2 incubator for 72 hours, and the absorbance of the experimental group and the 100% control group was measured using the MTT assay method in the same manner as the 0% control group.

[1112] Cell viability in the experimental group (antibody-drug conjugate treatment) was calculated using the following formula.

[1113] Cell viability (%) = [(experimental group) - (0% control group)] / [(100% control group) - (0% control group)]

[1114] GI of antibody-drug conjugates in HT-1080 / CNTN4 cells 50 (half maximal growth inhibition concentration) values ​​were calculated using GraphPad Prism (GraphPad software Inc., USA) using cell viability.

[1115] The cell viability (%) of Ab-XXI, Ab-XV, Ab-XVI, Ab-XVII, Ab-XVIII, Ab-XIX and Ab-XX in HT-1080 / CNTN4 cells is shown in Figure 56, and GI 50 is described in Table 25.

[1116]

[1117] As confirmed in FIG. 56 and Table 25, the antibody-drug conjugates Ab-XXI, Ab-XV, Ab-XVI, Ab-XVII, Ab-XVIII, Ab-XIX, and Ab-XX of the present invention inhibited the cell growth of HT-1080 / CNTN4 in a concentration-dependent manner. Therefore, it was confirmed that the antibody-drug conjugates Ab-XXI, Ab-XV, Ab-XVI, Ab-XVII, Ab-XVIII, Ab-XIX, and Ab-XX of the present invention have excellent cell growth inhibition ability against cancer cells overexpressing CNTN4.

[1118] Example 6. Tumor growth inhibition efficacy of anti-CNTN4 antibody-drug conjugate (in vivo)

[1119] Example 6.1. Tumor growth inhibitory efficacy of Ab-I (in vivo)

[1120] In order to confirm the in vivo anticancer efficacy of the antibody-drug conjugate Ab-I prepared in Manufacturing Example 3.1, the tumor growth inhibitory efficacy was observed in a syngeneic mouse pancreatic cancer orthotopic transplantation model established by transplanting Pan02 / Cntn4, a Cntn4-overexpressing pancreatic cancer cell line prepared in Example 5, into the pancreas of a mouse.

[1121] 2.5 X 10 into the pancreas of 7-week-old female C57BL / 6 mice 4 A pancreatic cancer mouse model was established by transplanting Pan02 / Cntn4 cancer cells. Five days after Pan02 / Cntn4 cell transplantation, the mice were randomly divided into two groups of eight mice each.

[1122] The above two groups were assigned to a negative control group (PBS administration) and a group administered 10 mg / kg of Ab-I. Ab-I was administered to the mice twice a week (QW) by intravenous tail vein administration (D0 and D7). On Day 11, the mice were euthanized, and the pancreata were removed. The tumor area within the pancreas was measured by hematoxylin and eosin (H&E) staining, and the measured tumor area volume values ​​are described in Table 26. In addition, the tumor area inhibition effect within the pancreas in the group administered Ab-I compared to the negative control group is depicted in Figure 57.

[1123]

[1124] **** ,p< 0.0001versus negative control, Student's t test

[1125] As confirmed in Table 26 and Figure 57 above, the tumor area in the pancreas in the negative control group (PBS) was 70.09 mm 2 was measured. On the other hand, when Ab-I was administered at 10 mg / kg, the tumor area was 5.29 mm 2 As measured by , a statistically significant reduction in tumor area was observed compared to the negative control group (p< 0.0001). Therefore, it was confirmed that Ab-I, the antibody-drug conjugate of the present invention, has an excellent tumor formation inhibitory effect.

[1126] Example 6.2. Tumor growth inhibitory efficacy of Ab-IV (in vivo)

[1127] In order to confirm the in vivo efficacy of the antibody-drug conjugate Ab-IV prepared in Manufacturing Example 3.3 alone and in combination with an immuno-oncology agent, the tumor growth inhibitory efficacy was observed in a syngeneic mouse pancreatic cancer orthotopic transplantation model established by transplanting the Cntn4-overexpressing pancreatic cancer cell line Pan02 / Cntn4 prepared in Example 5 into the pancreas of a mouse. The immuno-oncology agent anti-mouse-PD-1 (Clone: ​​29F.1A12) used to confirm the combination efficacy was purchased from BioXCell, LLC (USA).

[1128] 2.5 X 10 into the pancreas of 7-week-old female C57BL / 6 mice 4 A pancreatic cancer mouse model was established by transplanting Pan02 / Cntn4 cancer cells. Five days after Pan02 / Cntn4 cell transplantation, the mice were randomly divided into five groups of eight mice each, and assigned to a negative control group (PBS administration) and experimental groups (Ab-IV 10 mg / kg administration group, Ab-IV 20 mg / kg administration group, anti-mPD-1 antibody 10 mg / kg administration group, and anti-mPD-1 antibody 10 mg / kg and Ab-IV 10 mg / kg combination administration group). The negative control group was administered 5 mL / kg of phosphate-buffered saline (PBS) intraperitoneally (once every 3 days for a total of 4 times: Days 0, 3, 6, and 9). The experimental group was administered the antibody-drug conjugate Ab-IV prepared in Manufacturing Example 3.3 via the tail vein at a dose of 10 mg / kg and a dose of 10 mL / kg, and at a dose of 20 mg / kg and a dose of 10 mL / kg via the tail vein (once a week for a total of 2 times: Days 0 and 7). The anti-mPD-1 antibody was administered 10 mg / kg and a dose of 5 mL / kg via the peritoneum (once every 3 days for a total of 4 times: Days 0, 3, 6, and 9).

[1129] On Day 11, the mice were euthanized, the pancreas was removed, and the tumor area within the pancreas was measured through H&E (Hematoxylin and eosin) staining. The measured tumor area values ​​are shown in Table 27 and Figure 58.

[1130]

[1131] * ,p<0.05 **** ,p< 0.0001versus negative control, One-way ANOVA

[1132] As confirmed in Table 27 and Figure 58 above, the tumor area in the pancreas in the negative control group (PBS) was 70.09 mm 2 The tumor area was measured as 58.00 mm when Ab-IV was administered at 10 mg / kg and 20 mg / kg, respectively. 2 Wow 32.68 mm 2 A dose-dependent effect was observed, measured as . The tumor area in the anti-mPD-1 antibody administration group was 51.69 mm 2 The tumor area of ​​the Ab-IV and anti-mPD-1 antibody combination group was measured as 21.47 mm 2 Statistically significant tumor area reduction was observed compared to each single administration group (vs. Ab-IV (10 mg / kg) alone: ​​p< 0.0001; vs. anti-mPD-1 antibody alone: ​​p< 0.001). Therefore, Ab-IV, the antibody-drug conjugate of the present invention, exhibits excellent tumor formation inhibition efficacy when administered alone, and it is interpreted that an improved effect can be expected when a higher DAR value is obtained (DAR of Ab-IV: 5.1). In addition, it was confirmed that the efficacy was further increased compared to the individual drugs when administered in combination with an immunotherapy anticancer agent.

[1133] Example 6.3. Tumor growth inhibitory efficacy of Ab-V and Ab-VI (in vivo)

[1134] In order to confirm the in vivo anticancer efficacy of antibody-drug conjugate Ab-V prepared in Manufacturing Example 3.3 and Ab-VI prepared in Manufacturing Example 3.4, HT-1080 / CNTN4, a CNTN4-overexpressing human fibrosarcoma cell line prepared in Example 5, was subcutaneously transplanted into immunodeficient nude mice to establish a human fibrosarcoma xenograft model, and the tumor growth inhibitory efficacy of Ab-V and Ab-VI was observed in the model.

[1135] 2 X 10 subcutaneously into 7-week-old female BALB / c-nu mice 6 A human fibrosarcoma xenograft model was established by transplanting HT-1080 / CNTN4 cancer cells. Eleven days after HT-1080 / CNTN4 cell transplantation, the tumor volume was approximately 40–280 mm. 3 When reached, the average tumor volume of each group was similar (the average tumor volume of each group was approximately 140 mm 3 ) Mice were divided into three groups (8 mice per group) and then assigned to a negative control group and an experimental group, respectively. The negative control group was administered phosphate-buffered saline (PBS) at a liquid volume of 10 mL / kg, the experimental group was administered Ab-V, the antibody drug conjugate prepared in Manufacturing Example 3.3, at a dose of 20 mg / kg and a liquid volume of 10 mL / kg, and Ab-VI, the antibody drug conjugate prepared in Manufacturing Example 3.4, at a dose of 7.5 mg / kg and a liquid volume of 10 mL / kg via the tail vein (twice a week on a total of two occasions; Day 0 and Day 7).

[1136] The tumor volumes of the negative control group and experimental group were measured three times a week, and the tumor growth inhibition (TGI) and tumor regression (TR) efficacy of Ab-V and Ab-VI were calculated using the following formulas.

[1137] Tumor growth inhibition (TGI (%))

[1138] = [1 - (relative tumor volume of the experimental group) / (average relative tumor volume of the negative control group)] X 100

[1139] Tumor regression efficacy (TR (%))

[1140] = [1 - (final tumor volume) / (tumor volume on the day of initiation of administration)] X 100

[1141] Figure 59 shows the change in tumor volume by date in the negative control group and experimental group, and the tumor volume is 2,000 mm 3 When the number of individuals exceeding , tumor growth observation of the corresponding group was terminated. Accordingly, the final observation date for tumor growth of the negative control group was confirmed as Day 18, the Ab-V administration group as Day 84, and the Ab-VI administration group as Day 53, respectively.

[1142] The experimental results showed that Ab-V and Ab-VI showed statistically significant HT-1080 / CNTN4 tumor growth ...

Claims

1. Antibody-drug conjugate represented by general formula I: [General Formula I] M-[L-D] n The above M is an anti-CNTN4 antibody or an antigen-binding fragment thereof that binds to human CNTN4 protein, The above L is a connector, wherein D is a therapeutic moiety, The above n is the average number of bindings per antibody of the LD structure binding to the antibody, and is in the range of 1 to 20.

2. In paragraph 1, An antibody-drug conjugate, wherein the anti-CNTN4 antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

3. In the first paragraph, the anti-CNTN4 antibody or antigen-binding fragment thereof is an antibody-drug conjugate comprising a heavy chain variable region comprising an amino acid sequence having 95% or more sequence homology to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having 95% or more sequence homology to the amino acid sequence of SEQ ID NO: 8, or an antigen-binding fragment thereof.

4. An antibody-drug conjugate according to claim 3, wherein the anti-CNTN4 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

8.

5. An antibody-drug conjugate according to any one of claims 1 to 4, wherein the anti-CNTN4 antibody or antigen-binding fragment thereof comprises an amino acid residue engineered for binding to or inhibiting binding of the linker at one or more selected positions.

6. An antibody-drug conjugate according to any one of claims 1 to 5, wherein the anti-CNTN4 antibody or antigen-binding fragment thereof comprises an amino acid residue engineered for binding to or inhibiting binding of the linker to the carboxyl terminal of the light or heavy chain.

7. An antibody-drug conjugate according to claim 6, wherein the amino acid residue is selected from a group of peptide moieties comprising alanine, cysteine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, or arginine.

8. An antibody-drug conjugate according to any one of claims 1 to 7, wherein the linking group comprises a cleavable peptide moiety.

9. An antibody-drug conjugate according to claim 8, wherein the cleavable peptide moiety is cleavable by an enzyme.

10. An antibody-drug conjugate according to any one of claims 1 to 9, wherein the linking group comprises an amino acid unit.

11. In the 10th paragraph, the amino acid unit is valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), alanine-alanine (Ala-Ala), alanine-citrulline (Ala-Cit), citrulline-alanine (Cit-Ala), asparagine-citrulline (Asn-Cit), citrulline-asparagine (Cit-Asn), citrulline-citrulline (Cit-Cit), valine-glutamic acid (Val-Glu), glutamic acid-valine (Glu-Val), serine-citrulline (Ser-Cit), citrulline-serine (Cit-Ser), lysine-citrulline (Lys-Cit), citrulline-lysine (Cit-Lys), aspartic acid-citrulline (Asp-Cit), citrulline-aspartic acid (Cit-Asp), Alanine-valine (Ala-Val), valine-alanine (Val-Ala), phenylalanine-lysine (Phe-Lys), lysine-phenylalanine (Lys-Phe), valine-lysine (Val-Lys), lysine-valine (Lys-Val), alanine-lysine (Ala-Lys), lysine-alanine (Lys-Ala), phenylalanine-citrulline (Phe-Cit), citrulline-phenylalanine (Cit-Phe), leucine-citrulline (Leu-Cit), citrulline-leucine (Cit-Leu), isoleucine-citrulline (Ile-Cit), citrulline-isoleucine (Cit-Ile), phenylalanine-arginine (Phe-Arg), arginine-phenylalanine (Arg-Phe), citrulline-tryptophan (Cit-Trp), An antibody-drug conjugate selected from the group consisting of tryptophan-citrulline (Trp-Cit), alanine-alanine-aspartic acid (Ala-Ala-Asp), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), and glutamic acid-valine-citrulline (Glu-Val-Cit).

12. An antibody-drug conjugate according to any one of claims 1 to 11, wherein the linking group comprises a direct bond or a moiety comprising a succinic acid derivative or a pyrimidine.

13. An antibody-drug conjugate according to claim 12, wherein the moiety comprising the succinic acid derivative is a succinimide moiety, a succinamic acid moiety, or a succinamic ester moiety.

14. An antibody-drug conjugate according to claim 13, wherein the succinimide moiety comprises succinimidocaproyl, succinimidopropanoyl, or succinimidobutanoyl.

15. An antibody-drug conjugate according to any one of claims 1 to 14, wherein the linking group optionally comprises a polyethylene glycol (PEG) moiety.

16. In the 15th paragraph, the polyethylene glycol moiety is -(CH2CH2O) m An antibody-drug conjugate comprising a structure represented by -, wherein m is an integer from 1 to 24.

17. An antibody-drug conjugate according to any one of claims 1 to 16, wherein the linking group comprises one or more alkylene, alkenylene, alkynylene, cycloalkylene, or arylene, and any carbon atom of the alkylene, alkenylene, alkynylene, cycloalkylene, or arylene is optionally replaced with one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine, or nitrogen atom.

18. An antibody-drug conjugate according to any one of claims 1 to 17, wherein the linking group comprises a moiety represented by the following general formula II: [General Formula II] In the above general formula II, The above a is an integer from 0 to 24, The above b is 0 or 1, The above X comprises one or more linked groups selected from the group consisting of alkylene, alkenylene, alkynylene, cycloalkylene and arylene, and the alkylene and cycloalkylene constituting the above X are C 1-30 Alkyl or C 3-30 is unsubstituted or substituted with cycloalkyl, and any carbon atom of said alkylene, alkenylene, alkynylene, cycloalkylene or arylene is optionally replaced by one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom, wherein Q is a direct bond or a moiety containing a succinic acid derivative or a pyrimidine; The above Q is linked to an antibody.

19. In paragraph 18, X is C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 1-30 alkylene-C 3-30 Cycloalkylene, C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene-OC 1-30 Alkylene, C 1-30 Alkylene-OC 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 6-30 Arylene, C 6-30 Arylene-C 1-30 Alkylene, C 1-30 alkylene-C 6-30 Arylene-C 1-30 C selected from the group consisting of alkylene and constituting said X 1-30 Alkylene and C 3-30 Cycloalkylene is C 1-30 Alkyl or C 3-30 Substituted or unsubstituted with cycloalkyl, and the above C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene or C 6-30 An antibody-drug conjugate, wherein any carbon atom of arylene is optionally replaced with one or more of a carbonyl, an oxygen atom, or a nitrogen atom.

20. An antibody-drug conjugate according to any one of claims 1 to 19, wherein the linking group optionally comprises a self-immolative moiety.

21. In any one of claims 1 to 20, the linking group does not include a self-immolative moiety, and the linking group is a succinimide moiety or a succinamic acid, glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) and -(CH2CH2O) m An antibody-drug conjugate comprising a polyethylene glycol moiety represented by -(wherein, m is an integer from 1 to 24).

22. An antibody-drug conjugate according to claim 20, wherein the self-immolative moiety is selected from the group consisting of p-aminobenzyl (PAB), unsubstituted or substituted p-aminobenzyloxycarbonyl (PABC), aminomethylene, N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC), and N-succinimidyl(4-iodo-acetyl)aminobenzoate (SIAB).

23. In the 22nd paragraph, the self-immolative moiety is aminomethylene or unsubstituted or substituted PABC, and optionally, the PABC is -C 1-6 Alkylene-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 An antibody-drug conjugate which may be substituted with an alkyl group, wherein m is an integer from 1 to 24.

24. An antibody-drug conjugate according to any one of claims 1 to 23, wherein the linking group comprises a moiety represented by the following general formula III: [General Formula III] In the above general formula III, The above a is an integer from 0 to 24, The above b and c are 0 or 1, respectively, The above Lp is a self-immolative moiety, and the self-immolative moiety is -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 A group represented by alkyl or -(CH2CH2O) m -C 1-6 It can be substituted with a group represented by alkyl, and m is an integer from 1 to 24, The above La is valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), alanine-alanine (Ala-Ala), alanine-citrulline (Ala-Cit), citrulline-alanine (Cit-Ala), asparagine-citrulline (Asn-Cit), citrulline-asparagine (Cit-Asn), citrulline-citrulline (Cit-Cit), valine-glutamic acid (Val-Glu), glutamic acid-valine (Glu-Val), serine-citrulline (Ser-Cit), citrulline-serine (Cit-Ser), lysine-citrulline (Lys-Cit), citrulline-lysine (Cit-Lys), aspartic acid-citrulline (Asp-Cit), citrulline-aspartic acid (Cit-Asp), alanine-valine (Ala-Val), Valine-Alanine (Val-Ala), Phenylalanine-Lys (Phe-Lys), Lysine-Phenylalanine (Lys-Phe), Valine-Lys (Val-Lys), Lysine-Valine (Lys-Val), Alanine-Lys (Ala-Lys), Lysine-Alanine (Lys-Ala), Phenylalanine-Citrulline (Phe-Cit), Citrulline-Phenylalanine (Cit-Phe), Leucine-Citrulline (Leu-Cit), Citrulline-Leucine (Cit-Leu), Isoleucine-Citrulline (Ile-Cit), Citrulline-Isoleucine (Cit-Ile), Phenylalanine-Arginine (Phe-Arg), Arginine-Phenylalanine (Arg-Phe), Citrulline-Tryptophan (Cit-Trp), An amino acid unit selected from the group consisting of tryptophan-citrulline (Trp-Cit), alanine-alanine-aspartic acid (Ala-Ala-Asp), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), and glutamic acid-valine-citrulline (Glu-Val-Cit). The above X is C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene and C 6-30 Containing one or more groups connected to each other selected from the group consisting of arylene, and C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene or C 6-30 Any carbon atom of arylene is optionally replaced by one or more of carbonyl, amide, oxygen atom, sulfur atom, -S(O)2-, -NH-, alkylamine or nitrogen atom, wherein Q is a direct bond, a succinimide moiety, a succinamic acid moiety, a succinamic ester moiety, or a pyrimidine moiety; The above Q is linked to an antibody.

25. In the 24th paragraph, the linking group comprises a moiety represented by the following general formula III', general formula III'', general formula III'' or general formula III'''', an antibody-drug conjugate: [General Formula III'] [General Formula III''] [General Formula III'''] [General Formula III''''] In the above general formulas III', III'', III''' and III'''', The above X' is hydrogen, C 1-30 Alkyl, C 3-30 Cycloalkyl or C 1-30 Alkyl-C 3-30 It is cycloalkyl, The above a, b, c, Lp, La and X are as defined in the general formula III, * is linked to an antibody.

26. In paragraph 24, X is C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 1-30 alkylene-C 3-30 Cycloalkylene, C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene-OC 1-30 Alkylene, C 1-30 Alkylene-OC 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 6-30 Arylene, C 6-30 Arylene-C 1-30 Alkylene and C 1-30 alkylene-C 6-30 Arylene-C 1-30 C selected from the group consisting of alkylene and constituting said X 1-30 Alkylene and C 3-30 Cycloalkylene is C 1-30 Alkyl or C 3-30 Substituted or unsubstituted with cycloalkyl, and the above C 1-30 Alkylene, C 2-30 Alkenylene, C 2-30 Alkynylene, C 3-30 Cycloalkylene or C 6-30 An antibody-drug conjugate, wherein any carbon atom of arylene is optionally replaced with one or more of a carbonyl, an oxygen atom, or a nitrogen atom.

27. In paragraph 24, The above c is 1, The above Lp is aminomethylene, or -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 PABC substituted or unsubstituted with a group represented by alkyl (m is an integer from 1 to 12), An antibody-drug conjugate, wherein La is an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

28. In paragraph 27, c is 1, and Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 An antibody-drug conjugate, wherein PABC is substituted with a group represented by alkyl (m is an integer from 1 to 12), and La is an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

29. In paragraph 24, wherein Q is a succinimide moiety, c is 0 or 1, and Lp is aminomethylene, or -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 An antibody-drug conjugate, wherein PABC is unsubstituted or substituted with a group represented by alkyl (m is an integer from 1 to 12), and La is an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

30. An antibody-drug conjugate in claim 29, wherein Q is a succinimide moiety, c is 0, and La is glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

31. In paragraph 24, wherein Q is a succinic acid moiety, c is 0 or 1, and Lp is aminomethylene or -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 An antibody-drug conjugate, wherein PABC is unsubstituted or substituted with a group represented by alkyl (m is an integer from 1 to 12), and La is an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

32. In paragraph 24, the Q is a pyrimidine moiety, the c is 1, and the Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 An antibody-drug conjugate, wherein PABC is substituted with a group represented by alkyl (m is an integer from 1 to 12), and La is an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

33. In paragraph 24, Q is a direct bond, c is 1, and Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 An antibody-drug conjugate, wherein PABC is substituted with a group represented by alkyl (m is an integer from 1 to 12), and La is an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

34. In paragraph 24, c is 1, and Lp is -(CH2CH2O) m -C 1-6 Alkyl (m is an integer from 1 to 12) or -CH2-N(CH3)-C(=O)-(CH2CH2O) m -C 1-6 PABC substituted with a group represented by alkyl (m is an integer from 1 to 12), wherein X is C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 3-30 Cycloalkylene-C 1-30 Alkylene, C 1-30 alkylene-C 6-30 Arylene, C 1-30 alkylene-C 6-30 Arylene-C 1-30 Alkylene, C 6-30 Arylene-C 1-30 Alkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene, C 1-30 Alkylene-amide-C 1-30 alkylene-C 3-30 Cycloalkylene-C 1-30 Alkylene, C 3-30 Cycloalkylene and C 6-30 C selected from the group consisting of arylene and constituting said X 1-30 Alkylene, C 6-30 Arylene or C 3-30 An antibody-drug conjugate, wherein any carbon atom of cycloalkylene is optionally replaced with one or more of carbonyl, oxygen, or nitrogen atoms, and wherein La is an amino acid unit selected from the group consisting of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val-Cit), citrulline-valine (Cit-Val), valine-alanine (Val-Ala), and alanine-valine (Ala-Val).

35. An antibody-drug conjugate according to any one of claims 1 to 34, wherein the linking group comprises a sugar or a sugar acid.

36. An antibody-drug conjugate according to any one of claims 12 to 35, wherein the direct bond, or the moiety comprising a succinic acid derivative or a pyrimidine, is attached to the anti-CNTN4 antibody or antigen-binding fragment thereof via a cysteine ​​residue on the antibody or antigen-binding fragment thereof.

37. In any one of paragraphs 1 to 36, the connector Combinations of succinimidocaproyl, valine-citrulline (Val-Cit), and PABC; combinations of succinimidocaproyl, Val-Ala, and PABC; combinations of succinimidocaproyl, Val-Ala, and aminomethylene; combinations of succinimidocaproyl, Gly-Gly-Phe-Gly, and aminomethylene; combinations of succinimidocaproyl, Val-Ala, and PABC comprising a -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituent; combinations of succinimidopropanoyl, polyethylene glycol (PEG), and valine-alanine (Val-Ala); combinations of succinimidopropanoyl, PEG, Val-Ala, and PABC; combinations of succinimidopropanamide, PEG, carbonyl, and Val-Ala; Combinations of succinimidopropanamide, PEG, carbonyl, Val-Ala, and PABC; combinations of succinimidopropanamide, alkyl-carbonyl, and Val-Ala; combinations of succinimidopropanamide, alkyl-carbonyl, Val-Ala, and PABC; combinations of succinimidopropanamide, alkyl-carbonyl, PEG, and Val-Ala; combinations of succinimidopropanamide, alkyl-carbonyl, PEG, Val-Ala, and PABC; combinations of succinimidopropanamide, alkyl-amide, PEG, and Val-Ala; combinations of succinimidopropanamide, alkyl-amide, PEG, Val-Ala, and PABC; combinations of succinimidopropanamide, alkyl-amide, PEG, Val-Ala, and PABC; combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and PABC; Combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Val-Ala, and PABC; combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and aminomethylene; combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Val-Ala, and aminomethylene; combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and PABC; combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Gly-Gly-Phe-Gly, and PABC;Combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly and aminomethylene; combinations of succinimidopropanamide, alkyl, PEG, carbonyl, Gly-Gly-Phe-Gly and aminomethylene; combinations of succinimidopropanamide, PEG, alkyl-carbonyl and Gly-Gly-Phe-Gly; combinations of succinimidopropanamide, alkyl, PEG, carbonyl and Gly-Gly-Phe-Gly; combinations of succinamic acid, alkyl-carbonyl, Val-Ala and PABC; combinations of succinamic acid, alkyl-carbonyl, Val-Ala and aminomethylene; combinations of succinamic acid, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala and PABC; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala, and aminomethylene; combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, and Val-Ala; combinations of succinic acid, alkyl-carbonyl, Gly-Gly-Phe-Gly, and PABC; combinations of succinic acid, alkyl-carbonyl, Gly-Gly-Phe-Gly, and aminomethylene; combinations of succinic acid, alkyl-carbonyl, and Gly-Gly-Phe-Gly; combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and PABC; combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly, and aminomethylene; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, and Gly-Gly-Phe-Gly; combinations of succinic acid, alkyl-carbonyl, Val-Ala, and PABC containing -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; combinations of succinic ester, PEG, carbonyl, and Val-Ala; combinations of succinic ester, PEG, carbonyl, Val-Ala, and aminomethylene; combinations of succinic ester, PEG, amide, and Val-Ala; combinations of succinic ester, PEG, amide, Val-Ala, and aminomethylene; combinations of succinic ester, alkyl-carbonyl, and Val-Ala;Combination of succinamic ester, alkyl-carbonyl, Val-Ala and aminomethylene; combination of succinamic ester, alkyl-amide and Val-Ala; combination of succinamic ester, alkyl-amide, Val-Ala and aminomethylene; combination of succinamic ester, alkyl, PEG and Val-Ala; combination of succinamic ester, alkyl, PEG, Val-Ala and aminomethylene; combination of succinamic ester, alkyl-carbonyl, PEG and Val-Ala; combination of succinamic ester, alkyl-carbonyl, PEG, Val-Ala and aminomethylene; combination of succinamic ester, alkyl-amide, PEG and Val-Ala; combination of succinamic ester, alkyl-amide, PEG and Val-Ala; combination of succinamic ester, alkyl-amide, PEG and Val-Ala; combination of succinamic ester, alkyl-amide, PEG, Val-Ala and aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, a carbonyl, and Val-Ala; A combination of a succinamic ester, an alkyl-amide, a PEG, a carbonyl, a Val-Ala, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, and Val-Ala; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Val-Ala, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Val-Ala, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Gly-Gly-Phe-Gly, and an aminomethylene; A combination of a succinamic ester, an alkyl-amide, a PEG, an alkyl-carbonyl, a Gly-Gly-Phe-Gly, and an aminomethylene; Combinations of PABCs comprising succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs comprising succinimido, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents;An antibody-drug conjugate comprising at least one selected from the group consisting of combinations of PABCs comprising succinic esters, alkyl-cycloalkyl, carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; combinations of PABCs comprising pyrimidines, alkynyls, carbonyls, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; combinations of PABCs comprising alkyl-carbonyl, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; and combinations of PABCs comprising alkylamides, alkyl-cycloalkyl, carbonyls, Val-Ala, and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents.

38. In any one of paragraphs 1 to 37, the connector Combination of succinimidocaproyl, valine-citrulline (Val-Cit) and PABC; A combination of succinimidocaproyl, Gly-Gly-Phe-Gly, and aminomethylene; A combination of PABC containing succinimidocaproyl, Val-Ala and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combination of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala and PABC; Combination of succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala and aminomethylene; Combinations of succinimidopropanamide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly and aminomethylene; Combination of succinimidopropanamide, PEG, alkyl-carbonyl and Gly-Gly-Phe-Gly; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala and aminomethylene; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Gly-Gly-Phe-Gly and aminomethylene; Combinations of succinic acid, alkyl-amide, PEG, alkyl-carbonyl and Gly-Gly-Phe-Gly; Combinations of PABC containing succinic acid, alkyl-carbonyl, Val-Ala and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combination of PABC containing succinimidopropanamide, PEG, alkyl-carbonyl, Val-Ala and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABC containing succinic acid, alkyl-amide, PEG, alkyl-carbonyl, Val-Ala and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs containing succinimido, alkyl-cycloalkyl, carbonyl, Val-Ala and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABC containing succinic acid, alkyl-cycloalkyl, carbonyl, Val-Ala and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs containing succinamic esters, alkyl-cycloalkyl, carbonyl, Val-Ala and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs containing pyrimidine, alkynyl, carbonyl, Val-Ala and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; Combinations of PABCs containing alkyl-carbonyl, Val-Ala and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents; and An antibody-drug conjugate comprising at least one selected from the group consisting of combinations of PABCs comprising alkylamide, alkyl-cycloalkyl, carbonyl, Val-Ala and -CH2-N(CH3)-C(=O)-(PEG)8-CH3 substituents.

39. An antibody-drug conjugate according to any one of claims 1 to 38, wherein n is 1 to 15.

40. An antibody-drug conjugate according to any one of claims 1 to 39, wherein the therapeutic moiety is a cytotoxic agent, a prodrug of a cytotoxic agent, an immunostimulant, a protein degrader, a protein kinase inhibitor, or a radioactive isotope-labeled compound.

41. An antibody-drug conjugate according to claim 40, wherein the cytotoxic agent is a tubulin inhibitor.

42. An antibody-drug conjugate according to claim 41, wherein the tubulin inhibitor is an auristatin or maytansinoid series.

43. An antibody-drug conjugate according to any one of claims 1 to 39, wherein the therapeutic moiety is a tubulin polymerase inhibitor.

44. An antibody-drug conjugate according to claim 43, wherein the tubulin polymerase inhibitor is monomethyloristatin F (MMAF) or monomethyloristatin E (MMAE).

45. An antibody-drug conjugate according to claim 40, wherein the cytotoxic agent is a DNA damaging agent.

46. ​​An antibody-drug conjugate according to claim 45, wherein the DNA damaging agent is a topoisomerase inhibitor or a DNA alkylating agent.

47. An antibody-drug conjugate according to any one of claims 1 to 39, wherein the therapeutic moiety is a topoisomerase inhibitor.

48. An antibody-drug conjugate according to any one of claims 1 to 39, wherein the therapeutic moiety is a DNA alkylating agent.

49. An antibody-drug conjugate according to claim 46 or 47, wherein the topoisomerase inhibitor is a camptothecin analogue.

50. An antibody-drug conjugate according to claim 49, wherein the camptothecin analogue is exatecan or Dxd, or a derivative thereof.

51. An antibody-drug conjugate according to claim 46 or 48, wherein the DNA alkylating agent is a pyrrolobenzodiazepine (PBD) dimer, a pyrrolobenzodiazepine (PBD) dimer analog, calicheamicins, a calicheamicin analog, duocarmycin, a duocarmycin analog, cyclophosphamide, ifosfamide, bendamustine, cisplatin, melphalan, or carboplatin.

52. In any one of paragraphs 1 to 39, An antibody-drug conjugate wherein the therapeutic moiety is monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), exatecan or Dxd, or a derivative thereof.

53. An antibody-drug conjugate in claim 52, wherein the antibody is an anti-CNTN4 antibody or an antigen-binding fragment thereof, and n is 1 to 20, represented by one of the following structures: ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; and .

54. An antibody-drug conjugate according to any one of claims 1 to 53; and a pharmaceutically acceptable carrier, A pharmaceutical composition for treating a disease associated with the function or expression of CNTN4 protein.

55. A pharmaceutical composition for treating a disease associated with the function or expression of the CNTN4 protein, wherein the disease associated with the function or expression of the CNTN4 protein is selected from the group consisting of leukemia, lymphoma, myeloma, bone and connective tissue sarcoma, brain cancer, breast cancer, adrenal cancer, thyroid cancer, pancreatic cancer, eye cancer, vaginal cancer, vulvar cancer, uterine cancer, ovarian cancer, esophageal cancer, stomach cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, lung cancer, testicular cancer, prostate cancer, penile cancer, oral cancer, salivary gland cancer, skin cancer, kidney cancer, bladder cancer, head and neck cancer, melanoma, appendix cancer, bronchial cancer, choriocarcinoma, chordoma, ependymoma, gastrointestinal stromal tumor (GIST), neuroendocrine cancer, malignant peripheral nerve sheath tumor, tongue cancer, small intestine cancer, heart cancer, duodenal cancer, parathyroid cancer, and urethral cancer.

56. A pharmaceutical composition for treating a disease related to the function or expression of the CNTN4 protein, wherein the disease related to the function or expression of the CNTN4 protein in claim 54 is a solid tumor.

57. A method for treating a subject having a disease associated with the function or expression of a CNTN4 protein, or suspected of having a disease associated with the function or expression of a CNTN4 protein, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate of any one of claims 1 to 53 or a pharmaceutical composition of any one of claims 54 to 56.

58. Use of an antibody-drug conjugate according to any one of claims 1 to 53 or a pharmaceutical composition according to any one of claims 54 to 56 for the manufacture of a medicament for the treatment of a disease associated with the function or expression of CNTN4.

59. Use of an antibody-drug conjugate according to any one of claims 1 to 53 or a pharmaceutical composition according to any one of claims 54 to 56 for the treatment of a disease associated with the function or expression of CNTN4.

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