Reactive Conjugates

Abstract

The present invention relates to compounds (reactive conjugates) for the chemical modification of therapeutic antibodies or proteins that allow the regioselective attachment of a payload to an antibody or antibody fragment in one step, thereby producing a modified antibody or antibody fragment that can be used to diagnose, monitor, image, or treat disease.

Description

[Technical Field]

[0001] The present invention relates to compounds for the chemical modification of therapeutic antibodies (hereinafter sometimes referred to as "reactive conjugates") that allow the regioselective attachment of a payload to an antibody or antibody fragment in one step, thereby producing a modified antibody or antibody fragment that can be used to diagnose, monitor, image, or treat disease. [Background technology]

[0002] Traditional cancer treatments, e.g., chemotherapy, can not only be very harsh (due to the severe side effects caused by their toxicity), but also be highly successful and unsuccessful; treatments that are effective in some patients can be completely ineffective in others. As a result, the development of new, less toxic and / or more effective treatments is constantly needed, as is the ability to monitor the effectiveness of treatment, e.g., to distinguish between "responding" and "non-responding" patients.

[0003] In response to these needs, a new class of therapeutic agents called antibody drug conjugates (ADCs) has emerged. ADCs utilize the targeting power of antibodies, e.g., monoclonal antibodies (mAbs), to deliver a payload, e.g., a cytotoxic agent or labeled agent, directly to cancer cells. Specific targeting of cancer cells maximizes the therapeutic effect of the payload while minimizing toxic effects on healthy cells. Depending on the payload, ADCs can fulfill a variety of roles, e.g., diagnostic, monitoring, and / or therapeutic.

[0004] ADCs can be prepared by a variety of methods. However, most of these methods result in heterogeneous mixtures of chemically distinct ADCs with varying payload (drug)-antibody ratios (DARs) and conjugation sites. This heterogeneity complicates manufacturing, leads to high batch-to-batch variability, and can sometimes result in unpredictable safety and efficacy. As a result, there is growing interest in methods that can result in the preparation of homogeneous mixtures, such as regioselective or site-specific conjugation methods. Such methods could dramatically increase the predictability of DARs and payload (drug) conjugation sites, helping to simplify the development and production of more defined ADC products with more predictable safety or efficacy.

[0005] Several approaches have been developed for the position- and site-specific conjugation of payloads to antibodies. However, known approaches often require modification / engineering of the antibody, for example, by incorporating unnatural amino acids or by modifying carbohydrate moieties. Such modifications can negatively affect the therapeutic efficacy / safety of the corresponding ADC, for example, due to undesirable effects on antibody activity, targeting, metabolism, and / or excretion, as well as immune responses against the antibody. Other approaches involve multiple steps, such as those described in WO2018 / 199337. Such multi-step approaches can be expensive and / or laborious, making them less attractive or even suitable for applications where a rapid and simple antibody modification process is desirable (e.g., for point-of-care diagnostic applications).

[0006] Thus, there remains a need to find alternative methods for position- or site-specific conjugation of payloads to antibodies or antibody fragments, e.g., methods that do not require prior manipulation of the antibody or antibody fragment.Furthermore, there is a need to find methods for preparing antibody-drug conjugates in as few steps as possible, preferably in one step. [Prior art documents] [Patent documents]

[0007]

Patent Document 1

Patent document 2

Non-licensed literature

[0008] [Non-licensed document 1] Mosby's Medical Dictionary, Mosby, Elsevier, 10th Edition (2016) [Non-licensed document 2] Oxford Textbook of Oncology, David J. Kerr, OUP Oxford, 3rd Edition (2016) [Non-licensed document 3] Daiら, Nature Com. 2018, 9, page 857

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-patent document 9

Non-patent document 10

Non-patent document 11

Non-patent document 12

Non-patent document 13

Non-patent document 14

Non-patent document 15

Non-patent document 16

Non-patent document 17

Non-patent document 18

Non-patent document 19

[0009] In view of the above, it is an object of the present invention to provide compounds (reactive conjugates) that allow the site-selective conjugation of a payload to an antibody or antibody fragment in one step, without the need for prior manipulation and / or modification of the antibody or antibody fragment, and a further object to provide kits containing such compounds.

[0010] It is yet another object of the present invention to provide methods for producing modified antibodies or modified antibody fragments (e.g., ADCs) that can be used in methods of diagnosing, monitoring, imaging, or treating disease. [Means for solving the problem]

[0011] The present invention provides compounds that allow for regioselective attachment of a payload to an antibody (e.g., a therapeutic antibody) or antibody fragment, optionally incorporated into an Fc-fusion protein. This regioselective attachment can be achieved in a single step. The resulting modified antibody or modified antibody fragment (e.g., an ADC or antibody-radionuclide conjugate) can be used in methods for diagnosing, monitoring, imaging, or treating disease, particularly cancer.

[0012] The compounds of the present invention (reactive conjugates) are represented by the following formula (1): PYSV (1) (In the formula, P is the payload; Y is a reactive moiety capable of reacting with the side chain of an amino acid, e.g., lysine or cysteine, preferably a moiety capable of reacting with the side chain of lysine; V is a vector capable of interacting with the fragment crystallizable (Fc) region of an antibody or fragment thereof, where the antibody fragment is optionally incorporated into an Fc fusion protein; S is a spacer having a length Z, and Z is of a length such that when vector V interacts with the Fc region of an antibody or antibody fragment thereof, reactive moiety Y can react with a side chain of an amino acid residue on the antibody or antibody fragment. It can be expressed as:

[0013] The present invention also relates to a kit for the regioselective modification of antibodies or antibody fragments, where the antibody fragment is optionally incorporated into an Fc-fusion protein, comprising a compound as previously described, optionally immobilized on a solid matrix (e.g., beads), and a buffer.

[0014] Furthermore, the present invention relates to a method for the regioselective modification of antibodies or antibody fragments, where the antibody fragment is optionally incorporated into an Fc fusion protein, using the compounds described above.

[0015] Furthermore, the present invention relates to modified antibodies or modified antibody fragments (e.g., obtained or obtained by the methods described above), where the antibody fragment is optionally incorporated into an Fc-fusion protein, for use in methods of diagnosing, monitoring, imaging, and / or treating disease, in particular cancer.

[0016] The present invention includes, inter alia, the following embodiments ("items"):

[0017] 1.The following formula (1): PYSV (1) (In the formula, P is the payload; Y is a reactive moiety capable of reacting with the side chain of an amino acid, preferably a moiety capable of reacting with the side chain of lysine; V is a vector capable of interacting with the fragment crystallizable (Fc) region of an antibody or fragment thereof, where the antibody fragment is optionally incorporated into an Fc fusion protein; S is a spacer having a length Z, and Z is of a length such that when vector V interacts with the Fc region of an antibody or antibody fragment thereof, reactive moiety Y can react with a side chain of an amino acid residue on the antibody or antibody fragment. A compound represented by:

[0018] 2. The payload is (i) Below: a labeling moiety that may comprise a radionuclide, preferably a chelating agent such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexyldiethylenetriaminepentaacetic acid (CH-X-DTPA), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) or desferrioxamine (DFO), where the chelating agent optionally chelates the radionuclide; · Chromophores; fluorophores, such as fluorescein or rhodamine; and · 125 I, 123 I, 131 I, 18 F, 11 C. 15 O. 18 Labeling moieties containing radionuclides such as F, e.g. 125 I, 123 I or 131 Moieties derived from 4-hydroxyphenylpropionic acid containing radionuclides such as I A portion selected from: (ii) a moiety selected from a moiety comprising a conjugation group comprising an optionally substituted conjugated diene; an optionally substituted tetrazine; an optionally substituted alkyne or azide; an optionally substituted dibenzocyclooctyne (DBCO); an optionally substituted trans-cyclooctene (TCO), an optionally substituted bicyclo[6.1.0]nonyne (BCN); an optionally substituted aldehyde; an optionally substituted ketone; and an optionally substituted hydrazine; (iii) Below: · DNA alkylating agents, e.g., antineoplastic agents such as duocarmycins; Topoisomerase inhibitors, for example, doxorubicin; RNA polymerase II inhibitors, for example, alpha-amanitin; DNA cleaving agents, e.g., calicheamicin; antimitotic or microtubule-disrupting agents, such as taxanes, auristatins or maytansinoids; ·Antimetabolites; Kinase inhibitors such as ipatasertib; Immunomodulators; A portion selected from anti-infective agents A moiety derived from a drug selected from and radioisotopes and / or pharmaceutically acceptable salts thereof. Item 1. The compound according to item 1, comprising:

[0019] 3. The payload is optionally a chelating agent that chelates a radionuclide, preferably DTPA, CH-X-DTPA, DFO, 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-tetraacetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1-glutamic acid-4,7,10-triacetic acid (DOTAGA), 2,2'-(1,4,7-triazacyclononane-1,4-diyl)diacetic acid, Acetic acid (NO2A), DOTA, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminediacetic acid, triethylenetetraminehexaacetic acid (TTHA), 1,4,8,11-tetraazacyclotetradecane (CYCLAM), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,8,11-tetraazabicyclo[6.6.2]Hexadecane-4,11-diacetic acid (CB-TE2A), 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetamide (DO3AM), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), 1,5,9-triazacyclododecane (TACD), (3a1s,5a1s)-dodecahydro-3a,5a,8a,10a-tetraazapyrene (cis-glyoxal-cyclam), 1,4,7-triazacyclononane (TACN), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), Azacyclododecane (cyclene), tri(hydroxypyridinone) (THP), 3-(((4,7-bis((hydroxy(hydroxymethyl)phosphoryl)methyl)-1,4,7-triazonan-1-yl)methyl)(hydroxy)phosphoryl)propanoic acid (NOPO), PCTA, 2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetic acid (TRITA), 2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetic acid 2,2',2''-(1,4,7,10-tetraazacyclotridecane-1,4,7-triyl)triacetamide (TRITAM), 2,2',2''-(1,4,7,10-tetraazacyclotridecane-1,4,7-triyl)triacetamide (TRITRAM), trans-N-dimethyl-cyclam, 2,2',2''-(1,4,7-triazacyclononane-1,4,7-triyl)triacetamide (NOTAM), oxocyclam, dioxocyclam, 1,7-dioxa-4,10-diazacyclododecane, bridged cyclam (CB-cyclam), triazacyclononanephosphinate (TRAP), 3. The compound according to item 1 or 2, which is a chelating agent which is a moiety derived from dipyridoxyl diphosphate (DPDP), meso-tetra-(4-sulfanotophenyl)porphine (TPPS4), ethylenebishydroxyphenylglycine (EHPG), hexamethylenediaminetetraacetic acid, dimethylphosphinomethane (DMPE), methylenediphosphate, dimercaptosuccinic acid (DMPA), or a derivative thereof; more preferably a moiety derived from DTPA, DOTA, DFO, NOTA, PCTA, CH-X-DTPA, NODAGA, or DOTAGA.

[0020] 4. Radionuclides are 124 I, 131 I, 86 Y, 90 Y, 177 Lu, 111 In, 188 Re, 55 Co, 64 Cu, 67 Cu, 68 Ga, 89 Zr, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 72 As, 211 At, 225 Ac, 223 Ra, 97 Ru, 149 Tb, 152 Tb, 161 Tb, 99m Tc, 226 Th, 227 Th, 201 Tl, 89 Sr, 44 / 43 Sc, 47 Sc, 153 Sm, 133 Xe and Al 18 From F, preferably 89 Zr, 111 In, 64 Cu, 177 Lu, 68 Ga, 99m Tc, 203 Pb, 72 As, 55 Co, 97 Ru, 201 Tl, 152 Tb, 133 Xe, 86 Y, and Al 18 From F, more preferably 89 Zr, 111 In, 64 Cu, 177 Lu, 68 Ga, and 99m Tc, in particular 111 4. The compound according to item 2 or 3, wherein In

[0021] 5. The compound according to item 1 or 2, wherein the payload is a moiety derived from exatecan, PNU-159682, amanitin, duocarmycin, auristatin, maytansine, tublysin, calicheamicin, SN-38, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, a pyrrolobenzodiazepine, a pyrrole-based kinesin spindle protein (KSP) inhibitor, an indolino-benzodiazepine dimer, or a radioisotope and / or a pharmaceutically acceptable salt thereof.

[0022] 6.P is expressed by the following formula (2): P 1 -L-- *' (2) (In the formula, P 1 is a payload defined in any of items 2-5; L is a linker, preferably a linker comprising one or more atoms selected from carbon, nitrogen, oxygen, and sulfur, which is optionally cleavable; *' indicates a covalent bond to a reactive moiety (Y) 6. The compound according to any one of items 1 to 5, represented by:

[0023] 7. The linker (a1) an alkylene group having 1 to 12 carbon atoms, preferably an alkylene group having 2 to 6 carbon atoms, such as a propylene group; (b1) a polyalkylene oxide group having 2 or 3 carbon atoms and having 1 to 36 repeating units; preferably a group of the formula -NH-(CH2CH2O) n1 a group represented by -CH2CH2- (wherein n1 is an integer of 0 to 35, for example, 1 to 20); (c1) a peptide group having 2 to 12 amino acids Item 7. The compound according to item 6, selected from:

[0024] 8. The reactive moiety has the following formula (3a): ** --(F1-RC-F2)-- *(3a) (In the formula, R C is a reactive center, preferably an electrophilic reactive center, more preferably a group selected from C=O and C=S; F1 is a single covalent bond, an atom, or a group of atoms; preferably an atom selected from O and S, or a group of atoms comprising one or more atoms selected from C, N, O, and S; more preferably an atom selected from O and S; F2 represents an atom or a group of atoms; preferably an atom selected from O and S, or a group of atoms containing one or more atoms selected from C, N, O and S; more preferably an atom selected from O and S; * indicates a covalent bond to the spacer (S); ** indicates covalent attachment to the payload (P) 8. The compound according to any one of items 1 to 7, represented by:

[0025] 9. The reactive moiety is selected from the group consisting of the following formulas (4a) to (4m):

[0026] [ka]

[0027] (In the formula, * indicates a covalent bond to the spacer (S), ** indicates covalent attachment to the payload (P) The compound according to item 8, which is represented by one of the following:

[0028] 10. The reactive moiety has the following formula (3b): ** --(F1-RC-F2)-(M)-- * (3b) (In the formula, R C is a reactive center, preferably an electrophilic reactive center, more preferably a group selected from C=O and C=S; F1 is a single covalent bond, an atom, or a group of atoms; preferably an atom selected from O and S, or a group of atoms comprising one or more atoms selected from C, N, O, and S; more preferably an atom selected from O and S; F2 represents an atom or a group of atoms; preferably an atom selected from O and S, or a group of atoms containing one or more atoms selected from C, N, O and S; more preferably an atom selected from O and S; M is a group capable of modulating the electron density and stability of F2, preferably a group capable of withdrawing electrons; * indicates a covalent bond to the spacer (S); ** indicates covalent attachment to the payload (P) 8. The compound according to any one of items 1 to 7, represented by:

[0029] 11. The group capable of modulating the electron density and stability of F2 is represented by the following formula (3c): ***' --M'-BC-- * (3c) (In the formula, M' is an aryl group having 6, 10, or 14 ring members and 1, 2, or 3 fused rings, respectively, which may be substituted with one or more substituents, or a heteroaryl group having 5 to 20 ring members, 1, 2, or 3 fused rings, and 1 to 4 heteroatoms independently selected from N, O, and S; preferably a phenyl group, a naphthyl group, a pyridyl group, a quinolinyl group, an isoquinolinyl group, or a benzotriazolyl group, which may be substituted with one or more substituents, and each substituent is preferably -F, -Br, -Cl, -I, -NO2, -CN, -C 1~6 Alkyl, -C 1~6 Alkoxy, -C 1~6 Amides, for example, selected from -C(=O)NH2 and combinations thereof, such as -CCl3, -CF3 or -CH2NO2; B is a single covalent bond, O, S, NR' (wherein R' is a hydrogen atom, OH, an alkyl or cycloalkyl group, C2~6 Alkenylene, C 2~6 Alkynylene, general formula: -(CH2) n1 -(H 1 ) x1 -(CH2) n2 -(H 2 ) x2 -(CH2) n3 -(H 3 ) x3 -(CH2) n4 - (3c') (In the formula, n1, n2, n3, and n4 each represent an integer independently selected from 0 to 10 such that n1+n2+n3+n4 is 10 or less; x1, x2, and x3 are each independently selected from 0 and 1; H 1 , H 2 and H 3 are each an atom independently selected from N, O and S, However, if x1+x2=2, then n2≧1; if x2+x3=2, then n3≧0; if x1+x3=2, then n2≧1 or n3≧1; if x1+x2+x3 is 3, then n2≧1 and n3≧1. represents a group having the formula: or any combination thereof; preferably a single covalent bond, NH or C 1~10 an alkylene group; more preferably a single covalent bond; C is C=O, C=S, C(=NR'') (wherein R'' represents a hydrogen atom, OH, an alkyl or cycloalkyl group, S=O, or S(=O)2; preferably C=O); * indicates a covalent bond to the spacer (S); ***' indicates a covalent bond to F2) Item 11. The compound according to item 10, represented by

[0030] 12. The moiety (F1-RC-F2) is one of the following formulae (4a') to (4m'), and / or M is independently one of the following formulae (5a) to (5j'):

[0031] [ka]

[0032] [ka]

[0033] [ka]

[0034] (In the formula, * indicates a covalent bond to the spacer (S), ** indicates a covalent bond to the payload (P), *** indicates a covalent bond to M, ***' indicates a covalent bond to F2).

[0035] 13. The reactive moiety is selected from the group consisting of the following formulae (6a) to (6l'):

[0036] [ka]

[0037] [ka]

[0038] [ka]

[0039] (In the formula, * indicates a covalent bond to the spacer (S), ** indicates covalent attachment to the payload (P) The compound according to any one of items 10 to 12, represented by one of:

[0040] 14. The spacer has a length of 10 to 35 Å; preferably, a group having 12 to 120 atoms, for example, 16 to 80 atoms, selected from carbon, nitrogen, oxygen, and sulfur in the main chain; more preferably, (a2) a polyalkylene oxide group having 6 to 36 repeating units, for example, 8 to 24 repeating units; preferably, a polyalkylene oxide group represented by the following formula (7): -X 1 -(CH2CH2O) n2 -CH2CH2-X 2 - (7) (In the formula, X 1 is NH, O or S; preferably NH; X 2 If is covalently linked to the vector, X 2 is NH or C=O, preferably C=O; n2 is an integer of 4 to 28, for example, 6 to 20, for example, 10. A group represented by: (b2) A peptide group having 6 to 25 amino acids in the main chain, for example, 9 amino acids in the main chain, each of which is preferably selected from Pro, Gly, Ala, Asn, Asp, Thr, Glu, Gln, and Ser; more preferably, Pro, Gly, or Ser. 14. The compound according to any one of items 1 to 13, wherein

[0041] 15. The compound according to any of items 1 to 13, wherein the spacer comprises a polyethylene oxide group having 4 to 36 repeating units, preferably 6 to 28 repeating units, more preferably 7 to 24 repeating units.

[0042] 16. The vector is a peptide comprising a sequence of 11 to 17 amino acids, for example, 13 to 17 amino acids, preferably represented by the following formulas (8a) and (8b):

[0043] [ka]

[0044] (In the formula, Bxx, Cxx, Dxx, Exx, and Fxx each independently represent an amino acid; Axx is an amino acid, a dicarboxylic acid, or a group represented by the following formula (9a): ---Axx1-Axx2-Axx3--- (9a) (In formula (9a), Axx1 represents a single covalent bond or an amino acid such as Arg; Axx2 represents an amino acid such as Gly or Cys; Axx3 represents an amino acid such as Asp or Asn) represents a peptide moiety represented by: Gxx is an amino acid or a group represented by the following formula (9b): ---Gxx1-Gxx2-Gxx3--- (9b) (In formula 9(b), Gxx1 represents an amino acid such as Thr; Gxx2 represents an amino acid such as Tyr or Cys; Gxx3 represents a single covalent bond or an amino acid such as His) represents a peptide moiety represented by: The side chain of Axx2 may be covalently linked to the side of Gxx2 to form a ring; When Axx2 is Cys and Gxx2 is Cys, preferably the side chains of Axx2 and GXX2 are linked together to form a group of the formula -(SX 4 -S)- group (wherein X 4 represents a single covalent bond or a divalent group containing one or more atoms selected from carbon, nitrogen and oxygen, such as a divalent maleimide group, a divalent acetone group or a divalent arylene group, preferably a single covalent bond); Hxx represents a single covalent bond or a trifunctional amino acid such as a diamino-carboxylic acid; The Z1 is a group covalently attached to the C-terminus of Gxx when Hxx is a single covalent bond selected from -N(H)(R), where R represents a hydrogen atom, an alkyl group or a cycloalkyl group, and a moiety derived from a compound containing a conjugation group selected from biotin, DBCO, TCO, BCN, alkyne, azide, bromoacetamide, maleimide, and thiol; a group covalently attached to the C-terminus of Hxx when Hxx is a trifunctional amino acid and Y' is attached to the side chain of Hxx, preferably N(H)(R), where R represents a hydrogen atom, an alkyl group or a cycloalkyl group when Z1 is covalently attached to the C-terminus of Hxx; or When Hxx is a trifunctional amino acid and Y' is bonded to the C-terminus of Hxx, it represents a hydrogen atom bonded to the side chain of Hxx; The Z2 is a group covalently attached to the N-terminus of Axx when Hxx is a single covalent bond selected from a hydrogen atom, a carbonyl-containing group such as an acetyl group, and a group containing a conjugation moiety such as biotin; a group covalently attached to the N-terminus of Hxx when Hxx is a trifunctional amino acid and Y' is attached to the side chain of Hxx selected from a hydrogen atom and a carbonyl-containing group such as an acetyl group; or When Hxx is a trifunctional amino acid and Y' is bonded to the N-terminus of Hxx, it represents a hydrogen atom bonded to the side chain of Hxx; Y' is present only if Hxx is a trifunctional amino acid, which is When Z1 binds to the C-terminus of Hxx, or when Z2 binds to the N-terminus of Hxx, the side chain of Hxx, When Z1 binds to the side chain of Hxx, the C-terminus of Hxx, or When Z2 binds to the side chain of Hxx, the N-terminus of Hxx represents a moiety covalently attached to; Y' is preferably derived from a compound containing a conjugation group selected from biotin, DBCO, TCO, BCN, alkyne, azide, bromoacetamide, maleimide, and thiol; X 3represents a single covalent bond or a divalent group containing one or more atoms selected from carbon, nitrogen, and oxygen, such as a divalent maleimide group, a divalent acetone group, or a divalent arylene group, preferably a single covalent bond; **** indicates a covalent bond to the spacer (S) 16. The compound according to any one of items 1 to 15, which is a peptide represented by one of the following:

[0045] 17. At least one of Axx, Bxx, Cxx, Dxx, Exx, Fxx, Gxx, and Hxx is defined as follows: Axx represents an amino acid selected from Ala, 2,3-diamino-propionic acid (Dap), Asp, Glu, 2-aminosuberic acid, α-aminobutyric acid, Asn, and Gln, a dicarboxylic acid selected from succinic acid, glutaric acid, and adipic acid; preferably Ala, Asp, or Asn; more preferably Asp; or a peptide moiety of formula (9a) (wherein Axx1 is a single covalent bond, Axx2 is Cys, and Axx3 is Asp); Bxx represents an amino acid selected from Trp, Phe, Tyr, phenylglycine (Phg), 3-benzothiopen-2-yl-L-alanine, 3-naphthalen-2-yl-L-alanine, 3-biphenyl-4-yl-L-alanine, and 3-naphthalen-1-yl-L-alanine; preferably Trp; Cxx represents an amino acid selected from His, Ala, 3-pyridin-2-yl-L-alanine, meta-tyrosine (mTyr) and Phe; preferably His, Ala or mTyr; more preferably His; Dxx represents an amino acid selected from Ala, Abu, Gly, Leu, Ile, Val, Met, cyclohexylalanine (Cha), Phe, Thr, Cys, Tyr, and norleucine (Nle); preferably, Ala, Nle, or Leu; more preferably, Leu; Exx represents an amino acid selected from Ala, Gly, Asn, Ser, Abu, and Asp; preferably, Ala or Gly; more preferably, Gly; Fxx represents an amino acid selected from Ala, Glu, Asp, Gln, His, Arg, Ser, and Asn; preferably, Asp or Glu; more preferably, Glu; Gxx represents an amino acid selected from Thr, Ser, Ala, Asn, Val, 2-amino-butyric acid (Abu), Ile, Met, Leu, Pro, Gln, and Cys; preferably, Thr or Ser; more preferably, Thr; or a peptide moiety of formula (9b) wherein Gxx1 is Thr, Gxx2 is Cys, and Gxx3 is a single covalent bond; and Hxx represents an amino acid selected from Dap, Dab, Lys, Orn and homo-lysine (homo-Lys), preferably an amino acid selected from Dap, Dab, Lys, Orn and homo-Lys; The compound according to item 16.

[0046] 18. A vector is represented by the following formulas (8a') to (8d'):

[0047] [ka]

[0048] (In the formula, Z1, Z2, X 3 , X 4 and **** is as defined in item 16) a peptide represented by one of The compound according to any one of items 1 to 17, which is preferably a peptide represented by formula (8a') or (8b').

[0049] 19. The following:

[0050] [ka]

[0051] [ka]

[0052] (wherein P is as defined in any one of items 1 to 5, and Y' is as defined in item 16) 19. The compound according to any one of items 1 to 18, selected from:

[0053] 20. The following:

[0054] [ka]

[0055] [ka]

[0056] [ka]

[0057] [ka]

[0058] [ka]

[0059] 20. The compound according to any one of items 1 to 19, selected from:

[0060] 21. A kit for site-specific modification of an antibody or a fragment thereof, wherein the antibody fragment is optionally incorporated into an Fc fusion protein, the kit comprising a compound according to any one of items 1 to 20 and a buffering agent, wherein the buffering agent has a pH of 5.5 to 11, more preferably 7.5 to 9.5.

[0061] 22. A kit for site-selective modification of an antibody or a fragment thereof according to item 21, wherein the compound is immobilized on a solid matrix, for example, a bead.

[0062] 23. A method for site-selective modification of an antibody or a fragment thereof, comprising the step of reacting an antibody or antibody fragment, wherein the antibody fragment is optionally incorporated into an Fc fusion protein, with a compound according to any one of items 1 to 20.

[0063] twenty four. the antibody is a monoclonal antibody, preferably adalimumab, aducanumab, alemtuzumab, altumomab pentetate, atezolizumab, anetumab, avelumab, bapineuzumab, basiliximab, bectumomab, bermekimab, besilesomab, bevacizumab, bezlotoxumab, brentuximab, brentuximab vedotin, brodalumab, blinatumomab, catumaxomab, cemiplimab, cetuximab, simpanemab, clivatuzumab, clivatuzumab tetraxetan, crenezumab Tetraxetan, daclizumab, daratumumab, denosumab, dinutuximab, durvalumab, edrecolomab, elotuzumab, emapalumab, enfortumab, enfortumab vedotin, epratuzumab, epratuzumab-SN38, etaracizumab, gemtuzumab, gemtuzumab ozogamicin, girentuximab, goslanemab, ibritumomab, inebilizumab, infliximab, inotuzumab, inotuzumab ozogamicin, ipilimumab, isatuximab, ixekizumab, J591 PSMA antibody, labetuzumab, lecanemab, mogamulizumab, necitumumab, nimotuzumab, natalizumab, nivolumab, ocrelizumab, ofatumumab, olaratumab, oregovomab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, polatuzumab vedotin, prasinezumab, racotumomab, ramucirumab, rituximab, siltuximab, sacituzumab, sacituzumab govitecan, semolinemab, siltuximab, solanezumab mab, tacatuzumab, tetrotumumab, tirabonemab, tocilizumab, tositumomab, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, TS23, ustekinumab, vedolizumab, votumumab, zagotenemab, zalutumumab, zanolimumab, fragments and derivatives thereof; more preferably, an antibody selected from the group consisting of atezolizumab, durvalumab, pembrolizumab, rituximab and trastuzumab; or the antibody fragment is incorporated into an Fc fusion protein, preferably selected from belatacept, aflibercept, ziv-aflibercept, dulaglutide, rilonacept, romiplostim, abatacept, and alefacept; Item 24. The method according to item 23.

[0064] 25. A modified antibody or modified antibody fragment obtained by reacting an antibody or antibody fragment, wherein the antibody fragment is optionally incorporated into an Fc fusion protein, with a compound according to any one of items 1 to 20, preferably having the same definition as in item 24.

[0065] 26. A modified antibody or modified antibody fragment as defined in item 25 for use in a method for diagnosing, monitoring, imaging or treating a disease, comprising administering the modified antibody or modified antibody fragment to a subject.

[0066] 27. A method for diagnosing, monitoring, imaging, or treating a disease, comprising administering the modified antibody or modified antibody fragment according to item 25 to a subject in need thereof.

[0067] 28. The modified antibody or modified antibody fragment for use according to item 26, or the method according to item 27, wherein the disease is a neurological disease, a cardiovascular disease, an autoimmune disease or cancer.

[0068] 29. The disease or treatment thereof is Alzheimer's disease, amyotrophic lateral sclerosis, cerebral arteriosclerosis, encephalopathy, Huntington's disease, multiple sclerosis, Parkinson's disease, progressive multifocal leukoencephalopathy, systemic lupus erythematosus, systemic sclerosis, angina pectoris including unstable angina, aortic aneurysm, atherosclerosis, heart transplant, cardiac toxicity diagnosis, coronary artery bypass graft, heart failure including systolic dysfunction resulting in atrial fibrillation, hypercholesterolemia, ischemia, myocardial infarction, thromboembolism, thrombosis, ankylosing spondylitis, autoimmune diseases 29. The modified antibody or modified antibody fragment for use according to item 26 or 28, or the method according to item 27 or 28, wherein the antibody or antibody fragment is selected from the group consisting of inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease (IGD), inflammatory bowel disease (IGF ...

[0069] 30. The modified antibody or modified antibody fragment for use according to item 26 or 28, or the method according to item 27 or 28, wherein the disease comprises cells selected from lymphoma cells, myeloma cells, renal cancer cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, small cell lung cancer cells, testicular cancer cells, pancreatic cancer cells, liver cancer cells, melanoma, head and neck cancer cells, and any cells that are deregulated and grow and divide at a rapid pace, causing cancer; preferably cells selected from breast cancer cells, small cell lung cancer cells, lymphoma cells, colorectal cancer cells, and head and neck cancer cells. [Brief explanation of the drawings]

[0070] [Figure 1] Schematic diagram of an antibody conjugation technique using the compounds of the present invention. A vector capable of interacting with the Fc region of an antibody binds to the Fc region, thereby bringing the reactive moiety into close proximity with the side chain of a lysine residue exposed on the surface of the antibody. Reaction between the side chain of the lysine residue and the reactive moiety results in the covalent attachment of the payload to the antibody (via a linker) and concomitant release of the vector. [Figure 2] 1 shows the synthesis of compound 29, a compound containing a labeling moiety (DOTA) as the payload and a PEG10 spacer. a) 1. HATU, DMF, DIEA (pre-activation for 3-5 min), 2. compound 1, 3. 20% piperidine in DMF (yield: 42% over 2 steps), b) 1. HATU, DMF, DIEA (pre-activation for 3-5 min), 2. compound 7, 3. TFA (+ HPLC purification). [Figure 3]Fluorescence polarization (FP) binding assays show binding isotherms for the fluorescein derivative of the Fc-III ligand (Fc-III-FAM) at a concentration of 5 nM to the therapeutic monoclonal antibodies trastuzumab, alemtuzumab, bevacizumab, and rituximab. The lines are fits to the data using the Hill equation, yielding the half-maximal effective concentration (EC50). The Fc-binding ligand Fc-III-FAM was confirmed to bind with high affinity to each antibody (trastuzumab: 14 nM, alemtuzumab: 13 nM, bevacizumab: 7 nM, rituximab: 11 nM). [Figure 4] 1 is a graph showing a competitive FP binding assay. The tendency of the Fc binding vectors of Example 1 (compounds 1 (Fc-III), 2, 9-11, 15, and 16) to bind to the Fc region of trastuzumab relative to Fc-III-FAM was evaluated. The lines are fits to the data using the Hill equation, yielding half-maximal inhibitory concentrations (IC50). The results are also shown in Table 3. [Figure 5] 1 shows the synthesis of compounds 17 and 19, i.e., fluorescein-carbonate and DOTA-carbonate derivatives: a) EtN in CHCN at 40°C, b) DMAP in CHCl at 25°C, c) TFA / CHCl (1 / 3, v / v), d) DIPEA in CHCN at 25°C, e) DIPEA in CHCN / DMF (1 / 1, v / v) at 25°C. [Figure 6] Figure 1 shows high-resolution mass spectrometry (HRMS) of trastuzumab and trastuzumab modified with compound 31, i.e., trastuzumab-DOTA conjugate. Peaks D0 to D3 correspond to trastuzumab fragments with various degrees of conjugation. HRMS measurements were performed after deglycosylation of the samples. [Figure 7] Figure 1 shows HRMS of trastuzumab-DOTA conjugates digested into Fab and Fc regions using GingisKhan® enzyme. Peaks D0-D2 correspond to trastuzumab with different levels of conjugation. [Figure 8]This graph shows the affinity of trastuzumab-DOTA conjugates and trastuzumab for SK-BR-3 (HER2+) and MD-MB-231 (HER2-) cells. For SK-BR-3 cells, antibody or antibody conjugate concentrations ranged from 0.003 to 30 μg / mL (1 / 10 dilutions were made). For MD-MB-231 cells, only 3 and 30 μg / mL were used. Trastuzumab and trastuzumab-DOTA conjugates were stained with a secondary rat anti-human IgG Fc antibody conjugated to Alexa 488. Dead cells were excluded using DRAQ7. Error bars: SD (n=2). [Figure 9] FIG. 1 shows the synthesis of compound 38, a reactive conjugate containing a PEG20 spacer and a labeling moiety (fluorescein (FL)) as the payload. a) 1. HATU, DMF, DIEA (pre-activation for 3-5 min), 2. compound 1, 3. 20% piperidine in DMF (yield: 50% over two steps), b) 1. HATU, DMF, DIEA (pre-activation for 3-5 min), 2. compound 10, 3. TFA (+HPLC purification; yield: 19%). [Figure 10] Figure 1 shows non-reducing SDS-PAGE analysis of trastuzumab-FL conjugates prepared by reacting compounds 35-41 with trastuzumab (IgGT). The conjugates after reduction (A) or IdeS protease digestion (B) were analyzed using Coomassie blue staining and fluorescence. [Figure 11] Figure 1 shows a graph depicting BT-474 cells incubated with 10 μg / ml FITC-trastuzumab (conjugate 12, random conjugation, dashed line) and FL-trastuzumab (conjugate 11, solid line) and increasing concentrations of unlabeled trastuzumab. The plotted data represent the average MFI scores from two independent experiments. The maximum MFI for each antibody was normalized to 1. [Figure 12]Figure 1 shows non-reducing SDS-PAGE analysis of trastuzumab-FL conjugates prepared by reacting compounds 38 and 40 with trastuzumab (IgGT), commercial trastuzumab (Herceptin®) (IgGH), alemtuzumab (IgGA), bevacizumab (IgGB), and rituximab (IgGR). The conjugates after IdeS protease digestion were analyzed using fluorescence and Coomassie blue staining. [Figure 13] FIG. 1 is a schematic diagram of reactive conjugate immobilization on a solid support. [Figure 14] FIG. 1 is a schematic diagram of an antibody conjugation approach using a peptide conjugate containing a DBCO group (compound 43) and any payload containing an azide group. DETAILED DESCRIPTION OF THE INVENTION

[0071] 1.Definition The term "payload" as used herein characterizes a substance (e.g., a naturally occurring substance or a synthetic substance) that can provide a new function when it is attached (conjugated) to an antibody or antibody fragment. In some embodiments, the term "payload" as used herein should be understood as a labeling moiety (e.g., a chromophore, a fluorophore, a radiolabeled moiety) that allows and / or facilitates detection and / or visualization of the complementary moiety (e.g., an antibody) to which it is attached. For example, the labeling moiety can be detected and / or visualized by functional (physiological) imaging techniques known in the art, such as computed tomography (CT), positron emission tomography (PET), etc. In some embodiments, the term "payload" as used herein should be understood as a pharmacologically active substance that can inhibit or prevent the function of a cell and / or kill a cell. In some embodiments, the term "payload" should be understood as synonymous with other terms commonly used in the art, such as "cytotoxic agent," "toxin," or "drug" used in the field of cancer therapy. Alternatively, the payload is a moiety selected from moieties that include a conjugation group. The payload may include groups derived from functional groups such as carboxylic acids, primary amines, secondary amines, hydroxyl groups, thiol groups, etc., that allow for covalent attachment of the payload to the remainder of the compound (e.g., reactive moiety Y in formula (1)).

[0072] The term "peptide" as used herein can be understood as a compound comprising a consecutive sequence of at least three amino acids linked together by peptide bonds. The term "peptide bond" in this context is meant to encompass (backbone) amide bonds as well as modified bonds that can be obtained when non-natural amino acids are introduced into a peptide sequence. In this case, the modified bond replaces the (backbone) amide bond that is formed in the consecutive peptide sequence by reacting the amino and carboxyl groups of two amino acid residues (NH-CR). 1 -COOH + NH2-CR 2 -COOH→NH2-CR 1 -(C=O)-NH-CR 2For example, the modified bond can be an ester (NH2-CR 1 -(C=O)-O-CR 2 -COOH), thioester (NH2-CR 1 -(C=O)-S-CR 2 -COOH or NH2-CR 1 -(C=S)-O-CR 2 -COOH), carbamide (NH2-CR 1 -NH-(C=O)-NH-CR 2 -COOH), thiocarbamide (NH2-CR 1 -NH-(C=S)-NH-CR 2 -COOH) or triazole bond (e.g., NH2-CR 1 -C≡CH + N3-CR 2 -COOH→NH2-CR 1 -X-CR 2 -COOH (wherein X represents a 1,4-disubstituted-1,2,3-triazole moiety). Preferably, the amino acids forming the continuous peptide sequence are linked to each other by backbone amide bonds. Peptides may be linear or branched. In one aspect, peptides may be cyclic, e.g., made from a linear chain of amino acids modified to form a ring, e.g., undergo "head-to-tail" cyclization, or made from a linear chain of amino acids having side chains covalently linked to each other, e.g., by disulfide bond formation or any other modification. Here, amino acids include both naturally occurring amino acids as well as unnatural (synthetic) amino acids, as described below.

[0073] As used herein, the phrase "labeling moiety" (or synonymous with "label" or "labeling group") refers to a moiety that contains a group that allows and / or facilitates detection and / or visualization by visual or instrumental means of a complementary moiety (e.g., an antibody) to which it is attached. Examples of labeling moieties include radiolabels (e.g., radionuclides), contrast agents for magnetic resonance imaging (MRI), and light-absorbing or light-emitting chemicals, such as chromophores and fluorophores.

[0074] As used herein, the phrase "drug-derived moiety" refers to a moiety that corresponds to a natural drug, except that it has a structural modification that allows it to be attached to a reactive group or linker contained in the compounds of the present invention. Depending on the functional groups available in the natural drug, attachment can be achieved either by using one of the functional groups already present in the natural drug, or by modifying the natural drug by incorporating a new functional group. Depending on the result, the drug can be used for attachment in its unmodified form, or the drug can be chemically modified to contain a functional group that allows for covalent attachment to a reactive moiety or linker contained in the compounds of the present invention. As used herein, the phrase "drug-derived moiety" is meant to encompass both meanings.

[0075] In a similar manner, the term "derivative" is used in reference to other moieties that characterize the presence of a covalent bond required for attachment to an adjacent moiety or that have been chemically modified to include a functional group that allows for covalent attachment to an adjacent moiety. In other words, the term "derivative" may characterize a moiety that is attached to an adjacent moiety, which differs from the molecule derived only by the structural element responsible for attachment to the adjacent moiety. This may include, for example, a covalent bond formed by an existing functional group after removal of one hydrogen atom to provide the necessary free valence for attachment, or a covalent bond and an adjacent functional group newly introduced for this purpose.

[0076] The expression "natural drug" characterizes a compound whose therapeutic efficacy has been established by in vitro and / or in vivo testing. In a preferred embodiment, the natural drug is a compound whose therapeutic efficacy has been established by clinical trials. More preferably, the natural drug is a drug that is already on the market. The type of therapeutic efficacy established and the appropriate tests to be applied depend on the type of medical indication being treated.

[0077] When referring to specific classes of drug molecules, such as antineoplastic agents, topoisomerase inhibitors, RNA polymerase II inhibitors, DNA cleaving agents, antimitotic or microtubule disrupting agents, antimetabolites, kinase inhibitors, immunomodulators, or anti-infective agents, these terms are intended to have their generally accepted meaning in the pharmaceutical arts, as reflected, for example, in Mosby's Medical Dictionary, Mosby, Elsevier, 10th Edition (2016) or Oxford Textbook of Oncology, David J. Kerr, OUP Oxford, 3rd Edition (2016).

[0078] As used herein, the term "chelating agent" refers to a molecule containing two or more electron donor atoms capable of forming coordinate bonds with a single central metal ion, e.g., a radionuclide. Typically, chelating agents coordinate metal ions through oxygen or nitrogen donor atoms, or both. After the initial coordinate bond is formed, each successive donor atom that binds creates a ring containing the metal ion. A chelating agent may be bidentate, tridentate, tetradentate, etc., depending on whether it contains two, three, four, or more donor atoms capable of binding to a metal ion. However, the mechanism of chelation is not fully understood and depends on the chelating agent and / or the radionuclide. For example, DOTA is believed to coordinate radionuclides through its carboxylate and amino groups (donor groups), thus forming complexes with high stability (Dai et al., Nature Com. 2018, 9, 857). The term "chelating agent" should be understood to include chelating agents as well as their salts. Chelators having carboxylic acid groups, such as DOTA, TRITA, HETA, HEXA, EDTA, DTPA, etc., can be derivatized to convert one or more carboxylic acid groups to amide groups for attachment to, for example, a compound, i.e., a reactive moiety or linker, or, for example, the compound can be derivatized to allow attachment to a compound through one of the CH2 groups in the chelate ring.

[0079] As used herein, the term "radionuclide" refers to an atom having an unstable nucleus, a nucleus characterized by the addition of excess energy to newly created radiative particles or atomic electrons within the nucleus. Radionuclides can be naturally occurring or artificially produced. In some embodiments, the radionuclides used in the present invention are medically useful radionuclides, including positively charged ions of radioactive metals such as, for example, Y, In, Cu, Lu, Tc, Re, Co, and Fe. Preferably, the radionuclide is 89 Zr, 111 In, 64 Cu, 177 Lu, 68 Ga, 99m Tc, 203 Pb, 72 As, 55 Co, 97 Ru, 201 Tl, 152 Tb, 133 Xe, 86 Y, and Al 18 F, more preferably 89 Zr, 111 In, 64 Cu, 177 Lu, 68 Ga, and 99m Tc, especially 111 In is selected.

[0080] As used herein, the term "chromophore" refers to an organic or metallo-organic compound capable of absorbing electromagnetic radiation in the range of 350 nm to 1100 nm, or any subrange thereof, e.g., 350 to 500 nm or 500 to 850 nm, or 350 to 850 nm.

[0081] As used herein, the term "fluorophore" refers to a compound that, when excited by exposure to light of a particular wavelength, emits light at a different (higher) wavelength. Fluorophores are typically described in terms of their emission profile or "color." For example, green fluorophores such as Cy3 or FITC generally emit in the wavelength range of 515-540 nm, while red fluorophores such as Cy5 or tetramethylrhodamine generally emit in the wavelength range of 590-690 nm. As used herein, the term "fluorophore" should be understood to encompass, among other things, organic fluorescent dyes such as fluorescein, rhodamine, or AMCA, as well as biological fluorophores.

[0082] As used herein, the phrase "pharmaceutically acceptable salts" refers to derivatives (including reactive conjugates) of the disclosed compounds, in which the parent compound is modified by making acid or base salts thereof. Pharmaceutically acceptable salts include, for example, non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids or bases. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, PA, 1985, p. 1418; SM Berge, LM Bighley, and DC Monkhouse, "Pharmaceutical Salts," J. Pharm. Sci. 66 (1), pp. 1-19 (1977); PH Stahl and CG Wermuth (eds.), Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zurich, Wiley-VCH, 2008; and AK Bansal et al., Pharmaceutical Technology, 3(32), 2008. Pharmaceutical salts can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. For reactive conjugates, this can be done before or after incorporating the drug moiety into the compounds of the invention. Unless the context states otherwise, all references to the compounds of the invention (conjugates, modified antibodies, etc.) should also be understood to refer to pharmaceutically acceptable salts of the respective compounds.

[0083] As used herein, the phrase "reactive moiety" refers to a moiety that can readily react with another molecule, e.g., a binding partner on a nucleophile. This is in contrast to a moiety that requires the addition of a catalyst or very impractical reaction conditions (i.e., a "non-reactive" or "inert" moiety). In particular, the phrase "reactive moiety" refers to a portion of a reactive conjugate that reacts with the side chain of Lys of an antibody, preferably trastuzumab (Herceptin® available from Roche), at a molar ratio of 2:1 conjugate to trastuzumab when stirred at 1000 rpm in 50 mM NaHCO3, pH 9.0, for 2 hours at room temperature, resulting in reaction (e.g., attachment of a payload to trastuzumab) of at least 25% of the conjugate, preferably at least 50% of the conjugate, and more preferably at least 70% of the conjugate. Attachment of a payload to trastuzumab can be determined by high-resolution mass spectrometry according to the methods described below in Section 9.3.5.

[0084] As used herein, the phrase "side chain of an amino acid" may refer to the moiety attached to the α-carbon of an amino acid. For example, the side chain of Ala is methyl, the side chain of Phe is phenylmethyl, the side chain of Cys is thiomethyl, the side chain of Tyr is 4-hydroxyphenylmethyl, etc. Both naturally occurring and non-naturally occurring side chains are encompassed by this definition. In the case of unnatural amino acids, the side chain may also be located at a different position, for example, attached to the backbone nitrogen in peptoid structures or to the β-carbon in some forms of β-amino acids.

[0085] As used herein, the term "amino acid" refers to a compound containing or derived from at least one amino group and at least one acidic group, preferably a carboxyl group. The distance between the amino and acidic groups is not particularly limited. α-, β-, and γ-amino acids are suitable, with α-amino acids, particularly α-aminocarboxylic acids, being particularly preferred. The term encompasses both naturally occurring amino acids and synthetic amino acids not found in nature. Hereinafter, references to amino acids can be made by the three-letter amino acid code (Arg, Phe, Ala, Cys, Gly, Gln, etc.) or by the one-letter amino acid code (R, F, A, C, G, Q, etc.). Hereinafter, amino acid sequences will be written from the N-terminus to the C-terminus (left to right).

[0086] As used herein, the term "trifunctional" refers to a compound or moiety having three functional groups that can form, or have formed, three covalent bonds with adjacent moieties. Thus, the term "trifunctional amino acid" refers to a compound that contains or is derived from at least one amino group, an acid group (e.g., a carboxyl group), and a compound that contains another functional group, such as an amino group or a carboxyl group.

[0087] The term "C-terminus" as used herein refers to the C-terminus of an amino acid (peptide) chain. Attachment to the "C-terminus" means that a covalent bond is formed between an acid group in the main chain (backbone) of the amino acid residue and a binding partner. For example, attachment of a group "X" to the C-terminus of an amino acid residue Axx results in an ester or amide type structural element -C(O)-X, where the carbonyl group is derived from the acid group of Axx.

[0088] The term "N-terminus" as used herein refers to the N-terminus of an amino acid (peptide) chain. Binding to the "N-terminus" means that a covalent bond is formed between the amino group in the main chain (backbone) of the amino acid residue and the binding partner (replacing one hydrogen atom). For example, binding of the group "X" to the N-terminus of the amino acid residue Axx results in the structural element X-NH- (wherein the amino group is derived from Axx).

[0089] As used herein, the phrase "capable of interacting with the fragment crystallizable (Fc) region of an antibody or fragment thereof" refers to a vector capable of binding to the Fc region of an antibody or antibody fragment as defined previously herein. The interaction / binding can result in a targeting effect, i.e., a partial increase in the concentration of reactive moieties near the side chains of amino acids (e.g., lysine residues) of the antibody or antibody fragment. The interaction (binding) of a vector with the Fc region of an antibody or antibody fragment can be assessed by using fluorescence polarization techniques known in the art and described further below. In some embodiments, the phrase "a compound capable of interacting with the Fc region of an antibody or fragment thereof" refers to a compound that retains at least 20%, preferably at least 50%, and more preferably at least 80% of the binding affinity of the ligand "Fc-III" to the Fc region of IgG, as described by DeLano et al. (Science 2000, 287, pp. 1279-1283) and measured by fluorescence polarization. A compound capable of interacting with the Fc region of an antibody or fragment thereof may have superior binding affinity to the Fc region compared to Fc-III.

[0090] As used herein, the term "antibody" (also referred to interchangeably as "immunoglobulin" (Ig)) encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), veneered antibodies, and small immune proteins, provided that they contain at least one fragment crystallizable (Fc) region. Antibodies are proteins produced by the immune system that can recognize and bind to a specific antigen. A target antigen generally has several binding sites, also called epitopes, recognized by complementarity-determining regions on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to a desired target antigen or a portion thereof. The antibody may be an IgG, e.g., IgG1, IgG2, IgG3, or IgG4. Preferably, the antibody is an IgG protein, more preferably an IgG1, IgG2, or IgG4 protein. Most preferably, the antibody is an IgG1 protein. The antibody may be human or derived from other species. Preferably, the antibody is a human antibody.

[0091] The expression "monoclonal antibody" as used herein characterizes antibodies that are identical because they are produced by one type of immune cell and are all clones of a single parent cell.

[0092] As used herein, the phrase "antibody fragment" refers to a molecule comprising at least one polypeptide chain derived from an antibody that is not full-length but has at least a fragment crystallizable region that allows it to interact with a ligand.

[0093] As used herein, the phrase "commercially formulated antibody" refers to a marketed formulation comprising a therapeutic antibody and one or more excipients. Preferably, the commercially formulated antibody is a formulation marketed in the European Union. Examples of commercially formulated antibodies include Humira®, Lemtrada®, Campath®, Tecentriq®, Bavencio®, Simulect®, LymphoScan®, Xilonix®, Scintimun®, Avastin®, Zinplava®, Blincyto®, Libtayo®, Erbitux®, hPAM4-Cide®, Zenapax®, Darzalex®, Prolia®, Unituxin®, Imfinzi®, Panorex®, Empliciti®, Gamifant®, Renca®, and others. Rex®, Remicade®, Besponsa®, Yervoy®, CEA-Cide®, Poteligeo®, Tysabri®, Portrazza®, Theracim®, Opdivo®, Arzerra®, Lartruvo®, Omnitarg®, Vaxira®, Cyramza®, MabThera®, Rituxan®, Sylvant®, Bexxar®, Herceptin®, Kadcyla®, Stelara®, HuMax-EGFr®, HuMax-CD4®, and biosimilars thereof. Information regarding commercially formulated antibodies can be found, for example, in Allgemeine and Spezielle Pharmakologie und Toxicologie, Thomas Karow and Ruth Lang-Roth, Karow, 27th Edition (2018).

[0094] Preferably, the commercially formulated antibody is Herceptin® (a trastuzumab-containing formulation) (available from Roche), which has been approved for marketing in the European Union by the European Medicines Agency (EMA) under the authorization numbers EU / 1 / 00 / 145 / 001 and EU / 1 / 00 / 145 / 002, or MabThera® (a rituximab-containing formulation), which has been approved for marketing in the European Union by the EMA under the authorization numbers EU / 1 / 98 / 067 / 001, EU / 1 / 98 / 067 / 002, EU / 1 / 98 / 067 / 003 and EU / 1 / 98 / 067 / 004.

[0095] As used herein, the expression "Fc fusion protein" refers to a protein comprising at least an Fc-containing antibody fragment, i.e., an immunoglobulin-derived portion comprising at least one Fc region, and a portion derived from a second, non-immunoglobulin protein. The Fc-containing antibody fragment forms part of, and is thus incorporated into, the Fc fusion protein. The Fc-containing antibody fragment may be derived from the above-mentioned antibodies, in particular IgG, e.g., IgG1, IgG2, IgG3, IgG4. Preferably, the Fc-containing portion is derived from an IgG1 protein, more preferably a human IgG1 protein. The non-Ig protein may be derived from a therapeutic protein, e.g., erythropoietin (EPO), thrombopoietin (THPO), such as THPO-binding peptides, growth hormones, interferons (IFN), such as IFNα, IFNβ, or IFNγ, platelet-derived growth factor (PDGF), interleukins (IL), such as IL1α or IL1β, transforming growth factors (TGF), such as TGFα or TGFβ, or derived from a receptor, in particular a ligand-binding fragment of the extracellular domain of a receptor, e.g., The therapeutic protein may be derived from cluster of differentiation 2 (CD2), CD4, CD8, CD11, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD58 (LFA3), CD80, CD86, CD147, CD164, IL2 receptor, IL4 receptor, IL6 receptor, IL12 receptor, epidermal growth factor (EGF) receptor, vascular endothelial growth factor (VEGF) receptor, epithelial cell adhesion molecule (EpCAM), or cytotoxic T-lymphocyte-associated protein 4 (CTLA4). Examples of Fc fusion proteins include belatacept (Nulojix®), aflibercept (Eyla®), rilonacept (Arcalyst®), romiplostim (NPlate®), abatacept (Orencia®), alefacept (Amevine®), and etanercept (Enbrel®).

[0096] The term "cancer," as used herein, refers to a physiological condition in mammals characterized by deregulated cell proliferation. A tumor contains one or more cancer cells. Examples of cancer include carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Further examples of cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer, including gastrointestinal cancer, gastrointestinal stromal tumor, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, thyroid cancer, and liver cancer.

[0097] As used herein, the phrase "solid phase matrix" (or synonymous with "solid phase support," "solid phase," or "solid phase material") characterizes a material that is insoluble, or may be made insoluble by a subsequent reaction. Representative examples of solid phase materials include polymeric or glass beads, microparticles, tubes, sheets, plates, slides, wells, and tapes.

[0098] The term "alkyl group" as used herein refers to a linear or branched hydrocarbon group having 1 to 20 carbon atoms, preferably a methyl or ethyl group, or a cycloalkyl group having 3 to 20 carbon atoms, preferably 5 to 8 carbon atoms. The cycloalkyl group may consist of a single ring or may be formed by two or more condensed rings.

[0099] As used herein, the term "aryl" refers to the radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared among the ring arrangement) having 6 to 14 ring carbon atoms and 0 heteroatoms provided in the aromatic ring system. In some embodiments, an aryl group has 6 ring carbon atoms (e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (e.g., anthracyl). As used herein, the term "aryl" is meant to encompass ring systems in which an aryl ring is fused to one or more carbocyclyl or heterocyclyl groups, the radical or point of attachment being on the aryl ring (in such cases, the number of carbon atoms designates the number of carbon atoms in the aryl ring system). Unless otherwise specified, an aryl group can be unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more (e.g., 1 to 5) substituents. Non-limiting examples of aryl groups include radicals derived from benzene, naphthalene, anthracene, biphenyl, and the like.

[0100] As used herein, the term "heteroaryl" refers to a radical of a 5- to 14-membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in the ring arrangement) having ring carbon atoms and 1 to 4 heteroatoms provided in the aromatic ring system, each heteroatom independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon atom or a nitrogen atom, if valence permits. Heteroaryl polycyclic ring systems may contain one or more heteroatoms in one or both rings. As used herein, the term "heteroaryl" is meant to encompass ring systems in which a heteroaryl ring is fused to one or more carbocyclyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring (in such cases, the number of ring members designates the number of ring members in the heteroaryl ring system). The term "heteroaryl" is also meant to include ring systems in which a heteroaryl ring is fused to one or more aryl groups, and the point of attachment is on either the aryl or heteroaryl ring (in such cases, the number of ring members designates the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system).

[0101] As used herein, the phrase "substituted aryl group" refers to an aryl group in which one or more hydrogen atoms are each independently replaced with a substituent. Non-limiting examples of substituents include -Z, -R, -OR, -SR, -NR, -NR, -CZ, -CN, -OCN, -SCN, -NO, -C(O)R, -C(O)NR, -SO, -S(O)R, -C(S)R, -C(O)OR, and -C(O)SR, where each Z is independently a halogen (i.e., -F, -Cl, -Br, or -I) and each R is independently -H, -C 1~20 Alkyl or alkoxyl, -C 6~20 Aryl or -C 5~14 Heteroaryl. The above heteroaryl groups may be similarly substituted.

[0102] The expression "divalent arylene group" refers to a divalent moiety derived from an optionally substituted aryl or heteroaryl group, as defined above, in which two hydrogen atoms have been replaced by a covalent bond that allows for bonding to an adjacent moiety. Divalent arylene-type disulfide bridges (e.g., groups of the formula -SX) are also useful. 3 -S- / -SX 4 -S-(wherein, X 3 / X 4 represents a divalent arylene group) can be obtained by side chain-to-side chain cyclization according to techniques known in the art (see Stefanucci et al., ACS Med. Chem. Lett. 2017, 8, 449-454 and Beard et al., Bioorg. & Med. Chem. 2018, 26, 3039-3045).

[0103] As used herein, the phrase "divalent xylene group" refers to a divalent moiety derived from one of the three isomers of dimethylbenzene (i.e., ortho-xylene, meta-xylene, para-xylene) in which one hydrogen atom of each methyl group has been replaced by a covalent bond that allows for bonding to adjacent moieties. Preferably, the divalent xylene group is a divalent meta-xylene group. Divalent xylene-type disulfide bridges (e.g., those of the formula -SX 3 -S- / -SX 4 -S-(wherein, X 3 / X 4 represents a divalent xylene group) can be obtained, for example, by side-chain cyclization in the presence of dibromo-xylene, as described by Stefanucci et al., ACS Med. Chem. Lett. 2017, 8, 449-454.

[0104] As used herein, the phrase "divalent maleimide group" refers to a divalent moiety derived from maleimide in which the hydrogen atoms at positions 2 and 3 are each replaced by a covalent bond that allows for binding to an adjacent moiety. 3 -S- / -SX 4 -S-(wherein, X 3 / X4 represents a divalent maleimide group) can be obtained, for example, by side chain-to-side chain cyclization in the presence of 2,3-dibromomaleimide or another suitable reagent, as described by Kuan et al., Chem. Eur. J. 2016, 22, 17112-17129.

[0105] As used herein, the expression "divalent acetone group" refers to a divalent moiety derived from acetone (ACE) in which one hydrogen atom of each methyl group has been replaced by a covalent bond that allows for bonding to adjacent moieties. 3 -S- / -SX 4 -S-(wherein, X 3 / X 4 represents a divalent ACE group) can be obtained, for example, by side-chain to side-chain cyclization in the presence of dibromoacetone or dichloroacetone (see, for example, Assem et al., Angew. Chem. Int. Ed. Engl. 2015, 54(30), 8665-8668).

[0106] As used herein, the phrase "a group capable of modulating the electron density and stability of X" refers to a group capable of modulating (increasing or decreasing) the properties (electron density / stability) of a neighboring group (X), such as the moiety (F2) in formula (3b). The modulating group (M) can, for example, withdraw electrons or donate electrons to a neighboring group through inductive and / or mesomeric effects (see International Union of Pure and Applied Chemistry, Compendium of Chemical Technology, Gold Book 2012, pp. 477-480). Preferably, the inductive and mesomeric effects result in a shift in electron density distribution toward the modulating group, thereby modulating the electron density and stability of a neighboring group (e.g., F2). The modulation of electron density can be determined, for example, by measuring the shift of the carbon atom of the carbonate group and comparing it with the shift of a reference compound, such as compound 31. 13 The reactivity of the carbonate signal can be determined by C NMR spectroscopy. A change in the NMR shift of the carbonate signal (compared to that of a reference compound) to a higher ppm value indicates a decrease in electron density and thus a decrease in stability. A change in the NMR shift of the carbonate signal (compared to that of a reference compound) to a lower ppm value indicates an increase in electron density and an increase in stability. The modulation of electron density can be used to optimize the reactivity and stability of the conjugates of the present invention.

[0107] According to embodiments of the present invention, the group capable of modulating the electron density and stability of X is selected such that, in the absence of further reagents, the conjugate is stable to degradation (e.g., hydrolysis), meaning that the conjugate, when mixed with water / DMSO (95 / 5, v / v) at a concentration of 1 mg / mL, pH 9, and stirred at 500 rpm at 25° C. for 1 hour, exhibits less than 50% degradation, preferably less than 25% degradation, more preferably less than 10% degradation, in particular less than 5% degradation, as determined by HPLC.

[0108] As used herein, the phrase "electron-withdrawing group" refers to a group or substituent that can withdraw electrons from the moiety to which it is attached, i.e., reduce the electron density of the moiety compared to the same moiety bearing a hydrogen atom instead of the electron-withdrawing group. Typical electron-withdrawing groups include, but are not limited to, cyano, nitro, haloalkyl, carboxyl, aryl, sulfonyl, and the like. Electron-withdrawing groups can exert their electron-withdrawing effect through inductive and / or mesomeric effects (as described above). As used herein, the phrase "electron-withdrawing" is meant to encompass both meanings. Electron-withdrawing groups / substituents are known in the art and are described, for example, by Carey & Sundberg, Advanced Organic Chemistry, Part A: Structure and Mechanisms, 4th Edition.

[0109] As used herein, the expression "leaving group" refers to an atom or group (which may be charged or uncharged) that separates from an atom or molecule in what is considered to be the remainder or major part of the molecule that participates in a particular reaction, e.g., a nucleophilic substitution reaction (Pure Appl. Chem. 1994, 66, 1134). Examples of leaving groups include thiophenolate, phenolate, carboxylate, and sulfonate.

[0110] Where the present description refers to "preferred" embodiments / features, combinations of these "preferred" embodiments / features should also be considered as disclosed, provided that such combinations of "preferred" embodiments / features make technical sense.

[0111] Hereinafter, in this description and claims of the invention, use of the terms "comprising" and "including" should be understood to mean that additional, unrecited elements may be present in addition to the recited elements. However, these terms should also be understood to similarly disclose the term "consisting of" as a more limited embodiment in which additional, unrecited elements may be present, provided this is technically meaningful.

[0112] Unless otherwise specified, or unless the context states otherwise, references to "substituted" or "optionally substituted" groups include F, Cl, Br, I, CN, NO, NH, NH-C 1~6 Alkyl, N(C 1~6 alkyl)2, -XC 1~6 Alkyl, -XC 2~6 Alkenyl, -XC 2~6 Alkynyl, -XC 6~14 Aryl, -X-(5-14 membered heteroalkyl having 1-3 heteroatoms selected from N, O, S), where X is to be understood as a reference to the presence (or, in some cases, the optional presence) of at least one substituent selected from a single bond, -(CH)-, -O-, -S-, -S(O)-, -S(O)-, -NH-, -CO-, or any combination thereof including, for example, -C(O)-NH-, -NH-C(O)-. The number of substituents is not particularly limited and can range from 1 to the maximum valence that can be saturated with the substituents. It is typically 1, 2, or 3, usually 1 or 2, and most typically 1.

[0113] Unless otherwise specified, all valencies of individual atoms of compounds or moieties described herein are saturated. In particular, they are saturated by the indicated binding partners. If no binding partners or too few binding partners are indicated, the remaining valencies of each atom are saturated by the corresponding number of hydrogen atoms.

[0114] Unless otherwise specified, chiral compounds and moieties may exist in the form of pure stereoisomers or in the form of mixtures of stereoisomers, including 50:50 racemates. In the context of the present invention, reference to a particular stereoisomer should be understood as a reference to the compound or moiety, where the specified stereoisomer is present in at least 90% enantiomeric excess (ee), more preferably at least 95% ee, and most preferably 100% ee, where % ee is the ratio of (|RS|) / (R+S) *100% (where R and S represent the molar amounts of the respective enantiomers).

[0115] Unless otherwise specified or dictated by context, all connections between adjacent amino acid groups are formed by peptide (amide) bonds.

[0116] Unless the context states otherwise and / or an alternative meaning is expressly provided herein, all terms are intended to have their generally accepted meaning in the art as reflected by the IUPAC Gold Book (as of November 1, 2019) or the Dictionary of Chemistry, Oxford, 6th Edition.

[0117] 2. Overview The present invention is based on the surprising discovery that regioselective attachment of a payload to an antibody or antibody fragment can be achieved using the compounds of the invention, more particularly that said regioselective attachment can be achieved in a single step, e.g. without the need for an additional chemical reaction to cleave the covalent bond between the vector and the antibody or antibody fragment.

[0118] 3. Compound of formula (1) The present invention relates to a compound represented by general formula (1): PYSV (1) The present invention relates to a compound represented by the formula:

[0119] The compound of formula (1) comprises a vector V capable of interacting with (having binding affinity for) the Fc region of an antibody or fragment thereof, where the antibody fragment is optionally incorporated into an Fc fusion protein, a spacer S having a length Z, a reactive moiety Y, and a payload P.

[0120] 3.1 Payload (P) The payload used is not particularly limited, and any payload, such as a labeled molecule and / or a pharmaceutically active molecule, can be used as long as it can be bound to a reactive moiety.

[0121] According to one embodiment, the payload comprises: (i) Below: a labeling moiety that may comprise a radionuclide, preferably a chelating agent such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexyldiethylenetriaminepentaacetic acid (CH-X-DTPA), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) or desferrioxamine (DFO), where the chelating agent optionally chelates the radionuclide; · Chromophores; fluorophores, such as fluorescein or rhodamine; and · 125 I, 123 I, 131 I, 18 F, 11 C. 15 O. 18 Labeling moieties containing radionuclides such as F, e.g. 125 I, 123 I or 131 Moieties derived from 4-hydroxyphenylpropionic acid (also known as Bolton-Hunter reagent) containing radionuclides such as I A portion selected from: (ii) a moiety selected from moieties comprising a conjugation group that allows for subsequent attachment of a payload as specified under items (i) and (ii), which may be a moiety selected from the group consisting of an optionally substituted conjugated diene; an optionally substituted tetrazine; an optionally substituted alkyne or azide; an optionally substituted dibenzocyclooctyne (DBCO); an optionally substituted trans-cyclooctene (TCO), an optionally substituted bicyclo[6.1.0]nonyne (BCN); an optionally substituted aldehyde; an optionally substituted ketone; and an optionally substituted hydrazine; (iii) Below: · DNA alkylating agents, e.g., antineoplastic agents such as duocarmycins; Topoisomerase inhibitors, for example, doxorubicin; RNA polymerase II inhibitors, for example, alpha-amanitin; DNA cleaving agents, e.g., calicheamicin; antimitotic or microtubule-disrupting agents, such as taxanes, auristatins or maytansinoids; ·Antimetabolites; Kinase inhibitors such as ipatasertib; Immunomodulators; A portion selected from anti-infective agents A moiety derived from a drug selected from and radioisotopes and / or pharmaceutically acceptable salts thereof. Includes.

[0122] According to one embodiment, the payload (P) is a chelating agent that optionally chelates a radionuclide, preferably diethylenetriaminepentaacetic acid (DTPA), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), cyclohexyldiethylenetriaminepentaacetic acid (CHX-DTPA), desferrioxamine (DFO), 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-tetraacetic acid (NODAGA), ), 1,4,7,10-tetraazacyclododecane-1-glutamic acid-4,7,10-triacetic acid (DOTAGA), 2,2'-(1,4,7-triazacyclononane-1,4-diyl)diacetic acid (NO2A), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminediacetic acid, triethylenetetraminehexaacetic acid (TTHA), 1,4,8,11-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA ...1,4,8,11-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,8,11-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,8,11-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,8,11-tetraazacyclododecane-1,4,7,10-tetraacetic acid (TTHA), 1,4,8,11- tetradecane (CYCLAM), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetamide (DO3AM), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), 1,5,9-triazacyclododecane (TACD), (3a1s, 5a1s)-dodecahydro-3a,5a,8a,10a-tetraazapyrene (cis-glyoxal-cyclam), 1,4,7-triazacyclononane (TACN), 1,4,7,10-tetraazacyclododecane (cyclene), tri(hydroxypyridinone) (THP), 3-(((4,7-bis((hydroxy(hydroxymethyl)phosphoryl)methyl)-1,4,7-triazonan-1-yl)methyl)(hydroxy)phosphoryl)propanoic acid (NOPO), 3,6,9,15-tetraazabicyclo[9.3.1]Pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), 2,2',2'',2''''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetic acid (TRITA), 2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetamide (TRITAM), 2,2',2''-(1,4,7,10-tetraazacyclotridecane-1,4,7-triyl)triacetamide (TRITRAM), trans-N-dimethyl-cyclam, 2,2',2''-(1,4,7-tria ... trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cyclam, trans-N-dimethyl-cycl Chelating agents that are moieties derived from (nonane-1,4,7-triyl)triacetamide (NOTAM), oxocyclam, dioxocyclam, 1,7-dioxa-4,10-diazacyclododecane, bridged cyclam (CB-cyclam), triazacyclononanephosphinate (TRAP), dipyridoxyl diphosphate (DPDP), meso-tetra-(4-sulfanotophenyl)porphine (TPPS4), ethylenebishydroxyphenylglycine (EHPG), hexamethylenediaminetetraacetic acid, dimethylphosphinomethane (DMPE), methylenediamine diphosphate, dimercaptosuccinic acid (DMPA), or derivatives thereof.

[0123] According to one preferred embodiment, the payload is a chelating agent that optionally chelates a radionuclide, which is a moiety derived from DTPA, DOTA, DFO, NOTA, PCTA, CH-X-DTPA, NODAGA, or DOTAGA, preferably a moiety derived from DTPA, DOTA, DFO, NOTA, PCTA, CH-X-DTPA, or NODAGA, more preferably a moiety derived from DTPA, DOTA, DFO, or PCTA. Most preferably, the chelating agent is DTPA.

[0124] According to one embodiment, the chelating agent is 124 I, 131 I, 86 Y, 90 Y, 177 Lu, 111 In, 188 Re,55 Co, 64 Cu, 67 Cu, 68 Ga, 89 Zr, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 72 As, 211 At, 225 Ac, 223 Ra, 97 Ru, 149 Tb, 152 Tb, 161 Tb, 99m Tc, 226 Th, 227 Th, 201 Tl, 89 Sr, 44 / 43 Sc, 47 Sc, 153 Sm, 133 Xe and Al 18 From F, preferably 89 Zr, 111 In, 64 Cu, 177 Lu, 68 Ga, 99m Tc, 203 Pb, 72 As, 55 Co, 97 Ru, 201 Tl, 152 Tb, 133 Xe, 86 Y, and Al 18 From F, more preferably 89 Zr, 111 In, 64 Cu, 177 Lu, 68 Ga, and 99m Tc, in particular 111 In, which chelates the radionuclide.

[0125] In one preferred embodiment, the payload is 111 It is DTPA that chelates In.

[0126] In another preferred embodiment, the payload comprises: ·111 DOTA, PCTA, DTPA or CH-X-DTPA, most preferably those that chelating In 111 moiety (i) derived from CH-X-DTPA that chelates In; · 64 NOTA, NODAGA or PCTA, most preferably, which chelates Cu; 64 a NOTA-derived moiety that chelates Cu (i); · 89 DOTA, DFO, DFO' or DFO-cyclo', which chelates Zr, most preferably 89 Zr-chelating moiety (i) derived from DFO is selected from.

[0127] According to one embodiment, the payload is a moiety selected from those containing conjugation groups that allow subsequent attachment of the payloads identified under items (i) and (ii). This may be a moiety containing a conjugation group suitable for "click chemistry" to rapidly and reliably form a covalent bond by reacting with another moiety containing a "click chemistry" partner group (i.e., a payload containing a conjugation partner group) via, for example, strain-promoted cycloaddition, [2+3] dipolar cycloaddition, or Diels-Alder cycloaddition.

[0128] In one embodiment, the moiety is a moiety comprising a conjugation group selected from the group consisting of an optionally substituted conjugated diene, an optionally substituted tetrazine, an optionally substituted alkyne or azide, an optionally substituted dibenzocyclooctyne (DBCO), an optionally substituted trans-cyclooctene (TCO), an optionally substituted bicyclo[6.1.0]nonyne (BCN), an optionally substituted aldehyde, an optionally substituted ketone, and an optionally substituted hydrazine.

[0129] In one embodiment, the moiety is a moiety containing a conjugation group capable of reacting to form a covalent bond in the absence of a metal catalyst ("metal-free"), as described, for example, by Becer et al., "Click Chemistry beyond Metal-Catalyzed Cycloaddition," Angewandte Chemie Int. Ed. 2009, 48(27), pp. 4900-4908. Examples of conjugation groups capable of reacting in the absence of a metal catalyst include electron-deficient alkynes, strained alkynes such as cyclooctynes, tetrazines, and azides. Preferably, the moiety is a moiety containing a conjugation group selected from azide (N3), TZ, TCO, BCN, and DBCO, more preferably BCN or DBCO, and most preferably DBCO.

[0130] According to one embodiment, the payload is a drug-derived moiety. The following are examples of drugs that can be used as payloads in the compounds of the present invention: (A) DNA alkylating agents, such as duocarmycins (including synthetic analogs: adozelesin, carzelesin, bizelesin, KW-2189, and CBI-TMI), nitrogen mustard analogs (e.g., cyclophosphamide, chlorambucil, melphalan, chlormethine, ifosfamide, trofosfamide, prednimustine, bendamustine, chlornaphazine, estramustine, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitolactol, novembitine, fenesterine, uracil) antineoplastic agents such as mustard), alkyl sulfonates (e.g., busulfan, treosulfan, mannosulfan, improsulfan, and piposulfan), ethyleneimines (e.g., thiotepa, triaziquone, carboquone); nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin, chlorozotocin, fotemustine, nimustine, ranimustine), epoxides (e.g., etoglucide), other alkylating agents (e.g., mitobronitol, pipobromantemozolomide, dacarbazine); (B) Topoisomerase inhibitors, e.g., doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin, etoposide, etoposide phosphate, irinotecan and its metabolites such as SN-38, teniposide, topotecan, resveratrol, epipodophyllin (e.g., 9-aminocamptothecin, camptothecin, crisnatol, daunomycin, mitoxantrone, novanthrone, retinoic acid (retinol), 9-nitrocamptothecin (RFS2000)); (C) RNA polymerase II inhibitors, e.g., alpha-amanitin, other amatoxins; (D) DNA cleaving agents, e.g., calicheamicin; (E) Antimitotic or microtubule-disrupting agents, such as vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine, navelbine, vinflunide, vintafolide); taxanes (e.g., paclitaxel, docetaxel, paclitaxel poliglumex, cabazitaxel) and their analogs, maytansinoids (e.g., DM1, DM2, DM3, DM4, maytansine, and ansamitocin) and their analogs, cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); epothilones, eleutherobin, discodermolide, bryostatin, dolostatin, auristatins (e.g., monomethylauristatin E, monomethylauristatin F), tubulysins, cephalostatins; pancratistatin, sarcodictine, spongistatin, demecolcine, mitomycin; (F) Antimetabolites, such as DHFR inhibitors (e.g., methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid) or other folic acid analogs, e.g., raltitrexed, pemetrexed, pralatrexate); IMP dehydrogenase inhibitors (e.g., mycophenolic acid, tiazofurin, ribavirin, EICAR); ribonucleotide reductase inhibitors (e.g., hydroxyurea, deferoxamine); pyrimidine analogs (e.g., cytarabine, fluorouracil, 5-fluorouracil, and and its metabolites, tegafur, carmofur, gemcitabine, capecitabine, azacitidine, decitabine, fluorouracil combination drugs, tegafur combination drugs, trifluridine combination drugs, cytosine arabinoside, ancitabine, floxuridine, doxifluridine), uracil analogs (e.g., 6-azauridine, deoxyuridine); cytosine analogs (e.g., enocitabine); purine analogs (e.g., azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine, cladribine, clofarabine, nelarabine); folic acid supplements such as folinic acid; (G) Kinase inhibitors, such as ipatasertib, BIBW2992 (anti-EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, afatinib, vemurafenib, crizotinib, regorafenib, masitinib, dabrafenib, trametinib, ibrutinib, ceritinib, lenvatinib, nintedanib, and sedim Ranib, palvocidib, osimertinib, alectinib, rociletinib, cobimetinib, midostaurin, olmutinib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, tivozanib, ispinesib, temsirolimus, everolimus, ridaforolimus; (H) immunomodulatory agents, e.g., immunostimulants, immunosuppressants, cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolondanazole, dexamethasone, prednisone, triamcinolone acetonide, beclomethasone propionate), DHEA, hydroxychloroquine, meloxicam, methotrexate, mofetil, mycophenylate, sirolimus, tacrolimus, everolimus, fingolimod, ibrutinib; (I) Anti-infective agents, such as antibacterial agents, antimycobacterial agents, and antiviral agents. Non-limiting examples of antibiotics used in antibiotic-antibody drug conjugates are rifalogs, rafamycin derivatives.

[0131] According to one embodiment, the payload is a moiety derived from exatecan, PNU-159682, (alpha)amanitin, duocarmycin, auristatin, maytansine, tublysin, calicheamicin, SN-38, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, a pyrrolobenzodiazepine, a pyrrole-based kinesin spindle protein (KSP) inhibitor, an indolino-benzodiazepine dimer, or a radioisotope and / or a pharmaceutically acceptable salt thereof.

[0132] In some aspects of the invention, it may be preferable to use payloads with a certain level of hydrophobicity, for example in the case of chelating agents that chelate radionuclides, to avoid and / or prevent possible aggregation phenomena. For example, the addition / increase in the number of PEG units in the linker between the antibody and the payload can overcome high aggregation phenomena.

[0133] This attachment of the payload to the reactive group can be achieved by a linking group (or "linker"). In the context of the present disclosure, this linking group may be considered part of the payload. Thus, in some embodiments, the payload has the following formula (2): P 1 -L-- *' (2) (In the formula, P 1 will return the above payload, e.g. 177 represents a chelating agent or drug-derived moiety that optionally chelates a radionuclide, such as Lu-DOTA; L represents a linker, *' indicates a covalent bond to a reactive moiety) is expressed by

[0134] The linker is preferably a divalent group containing one or more atoms selected from carbon, nitrogen, oxygen, and sulfur.

[0135] In certain embodiments, the linker is (a1) an alkylene group having 1 to 12 carbon atoms, preferably an alkylene group having 2 to 6 carbon atoms, such as an ethylene group or a propylene group; (b1) a polyalkylene oxide group having 2 or 3 carbon atoms and having 1 to 36 repeating units; preferably a group of the formula -NH-(CH2CH2O) n1 -CHCH- (wherein n1 is an integer of 0 to 35, for example, 1 to 20); and (c1) a peptide group having 2 to 12 amino acids You can choose from:

[0136] In a more particular embodiment, the linker is (a1) an alkylene group having 2 to 6 carbon atoms (-(CH2) 2~6 -); (b1) Formula -NH-(CH2CH2O) n1 a polyalkylene group of -CH2CH2- (wherein n1 is an integer of 0 to 35); and (c1) optionally cleavable peptide linkers comprising 2 to 12 amino acids, preferably cleavable peptide linkers comprising Val-Cit, Val-Ala, Val-Cit-PABC or Val-Cit-PABC-DMEA units; You can choose from:

[0137] The linker may be a cleavable or non-cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0138] The cleavable linker may be a linker that can specifically release a payload upon internalization in a target cell. It may utilize inherent properties of the target cell, e.g., tumor cell, to selectively release the payload from the modified antibody or modified antibody fragment, namely, (1) protease sensitivity (enzyme-triggerable release linker system), (2) pH sensitivity, (3) glutathione sensitivity, or (4) glucoronidase sensitivity. In certain embodiments, the linker is a cleavable linker containing a valine-citrulline (Val-Cit) or valine-alanine (Val-Ala) dipeptide, which can serve as a substrate for intracellular cleavage by cathepsin B (CatB).

[0139] In another specific embodiment, the linker is a cleavable linker containing a self-immolative moiety that can release the payload by an elimination or cyclization-based mechanism. An example of a cleavable linker containing a self-immolative moiety is the para-aminobenzyloxycarbonyl (PABC) linker, such as that used in the bremituximab-vedotin conjugate Adcetris® (Younes et al., N. Engl. J. Med. 2010, 363, 1812-1821; Jain et al., Pharm. Res. 2015, 32(11), 3526-3540). The PABC-containing linker contains a protease-sensitive Val-Cit-PABC dipeptide linker unit that can be recognized and cleaved by CatB. This linker unit can be attached to the reactive moiety (and, after antibody modification, to the antibody) via a maleimidocaproyl moiety. Such linkers can help avoid steric clashes during substrate recognition by CatB. After enzymatic cleavage of the citrulline-PABC amide bond, the resulting PABC-substituted payload spontaneously undergoes 1,6-elimination, releasing the free payload into the target cell as the product. Thus, the group according to formula (2) may represent a group consisting of a payload moiety derived from vedotin, i.e., monomethyl auristatin E, linked to a reactive moiety via a linker containing a Val-Cit-PABC unit.

[0140] In another particular embodiment, the linker is a cleavable linker comprising a C-terminal dipeptide unit that can act as a highly specific substrate for the exopeptidase activity of CatB (exo-CatB). Examples of exo-CatB cleavable linker systems are described in WO2019 / 096867A1. In particular, the linker L may comprise a C-terminal dipeptide unit ("Axx-Ayy" or "Ayy-Axx") as defined in claim 1, 2, or 3 of WO2019 / 096867A1.

[0141] 3.2 Reactive Moiety (Y) The compounds of the invention comprise a reactive moiety (Y) that can react with the side chain of an amino acid exposed on the surface of an antibody or antibody fragment (e.g., by nucleophilic substitution). Preferably, the reactive moiety is capable of reacting with a side chain lysine. This reaction results in the covalent attachment of a payload (P) to the antibody or antibody fragment, with the concomitant release of a spacer (S) and a vector (V). The reactive moiety (Y) reacts with the side chain of an amino acid exposed on the surface of the antibody or antibody fragment to form a covalent bond, and the covalent bond within Y or between Y and S is spontaneously cleaved, releasing the peptide (without the need for further chemical reactions such as hydrolysis or reduction).

[0142] The reactive moiety comprises a reactive center (RC) that can react with the side chain of an amino acid, preferably the side chain of a lysine residue, for example, by a nucleophilic substitution reaction. Preferably, the reactive center is electrophilic. Non-limiting examples of electrophilic reactive centers that can react with the side chain of an amino acid include C=O and C=S. Preferred reactive centers are carbonyl (C=O) or thiocarbonyl (C=S), with carbonyl (C=O) being particularly preferred.

[0143] Covalently attached to one side of the reactive center is a moiety (F1) that connects the reactive center (RC) to a payload (P), and covalently attached to the other side of the reactive center is a moiety (F2) that connects the reactive center (RC) to a vector (V) via a spacer (S). Thus, the reactive moiety (Y) has the following formula (3a): ** --F1-RC-F2-- * (3a) (In the formula, R C is a reactive center, preferably an electrophilic reactive center, more preferably a group selected from C=O and C=S, most preferably C=O; F1 represents a single covalent bond, an atom, or a group of atoms; preferably an atom selected from O and S, or a group of atoms comprising one or more atoms selected from C, N, O, and S; more preferably an atom selected from O and S; F2 represents an atom or a group of atoms; preferably an atom selected from O and S, or a group of atoms containing one or more atoms selected from C, N, O and S; more preferably an atom selected from O and S; ** indicates binding to the payload (P), * indicates a bond to a spacer (S) It can be expressed as:

[0144] F1 and F2 may be the same atom or group of atoms. However, preferably, the atom or group of atoms comprising F2 makes it a better / preferred leaving group than F1 in a nucleophilic substitution reaction. This ensures that when the reactive center reacts with the side chain of an amino acid residue on an antibody or antibody fragment, for example, the side chain of a lysine residue, by nucleophilic substitution, F2 is the preferred leaving group; thereby, the payload is attached to the antibody or antibody fragment and not to the vector / spacer construct.

[0145] According to one embodiment, the reactive moiety of formula (3a) is represented by the following formulae (4a) to (4m):

[0146] [ka]

[0147] (In the formula, ** indicates binding to the payload (P), * indicates a bond to a spacer (S) is represented by one of the following:

[0148] To ensure that F2 is a better or preferred leaving group than F1 in a nucleophilic substitution reaction, particularly when F1 and F2 are the same atom or group of atoms, F2 can be linked to a modifying group (M) (where M is a group that can modulate the electronegativity and / or stability of the neighboring moiety F2, for example, by withdrawing or donating electrons).

[0149] Thus, in certain embodiments, the reactive moiety (Y) has the following formula (3b): ** --(F1-RC-F2)-(M)-- * (3b) (In the formula, RC, F1, F2, ** , and * is as defined in formula (3a) above, and M represents a group capable of modifying the electron density and stability of F2, preferably a group capable of withdrawing electrons. is expressed by

[0150] In certain embodiments, M is represented by the following formula (3c): ***' --M'-BC-- * (3c) (In the formula, M' is an aryl group having 6, 10, or 14 ring members and 1, 2, or 3 fused rings, respectively, which may be substituted with one or more substituents, or a heteroaryl group having 5 to 20 ring members, 1, 2, or 3 fused rings, and 1 to 4 heteroatoms independently selected from N, O, and S; preferably a phenyl group, a naphthyl group, a pyridyl group, a quinolinyl group, an isoquinolinyl group, or a benzotriazolyl group, which may be substituted with one or more substituents, and each substituent is preferably -F, -Br, -Cl, -I, -NO2, -CN, -C 1~6 Alkyl, -C 1~6 Alkoxy, -C 1~6 Amides, for example, selected from -C(=O)NH2 and combinations thereof, such as -CCl3, -CF3 or -CH2NO2; B is a single covalent bond, O, S, NR' (wherein R' is a hydrogen atom, OH, an alkyl or cycloalkyl group, C 2~6 Alkenylene, C 2~6 Alkynylene, general formula: -(CH2) n1 -(H 1 ) x1 -(CH2) n2 -(H 2 ) x2 -(CH2) n3-(H 3 ) x3 -(CH2) n4 - (3c') (In the formula, n1, n2, n3, and n4 each represent an integer independently selected from 0 to 10 such that n1+n2+n3+n4 is 10 or less; x1, x2, and x3 are each independently selected from 0 and 1; H 1 , H 2 and H 3 are each an atom independently selected from N, O and S, However, if x1+x2=2, then n2≧1; if x2+x3=2, then n3≧0; if x1+x3=2, then n2≧1 or n3≧1; if x1+x2+x3 is 3, then n2≧1 and n3≧1. represents a group having the formula: or any combination thereof; preferably a single covalent bond, NH or C 1~10 an alkylene group; more preferably a single covalent bond; C represents C═O, C═S, or C(═NR″) (wherein R″ represents a hydrogen atom, OH, an alkyl or cycloalkyl group, S═O, or S(═O)2; preferably C═O). and; * indicates a covalent bond to the spacer (S); ***' indicates a covalent bond to F2) is expressed by

[0151] According to one embodiment, in formula (3b), the moiety (F1-RC-F2) is represented by one of the formulae (4a') to (4m') and / or M is represented by the following formulae (5a) to (5j'):

[0152] [ka]

[0153] [ka]

[0154] [ka]

[0155] (In the formula, * indicates a covalent bond to the spacer (S), ** indicates a covalent bond to the payload (P), *** indicates a covalent bond to the modifying group (M), ***' indicates a covalent bond to F2) is independently represented by one of

[0156] In a preferred embodiment, the reactive moiety has the following formulae (6a) to (6l'):

[0157] [ka]

[0158] [ka]

[0159] [ka]

[0160] (In the formula, * indicates a covalent bond to the spacer (S), ** indicates covalent attachment to the payload (P) is represented by one of

[0161] Most preferably, the reactive moiety is represented by one of formulas (6a), (6b) and (6m), in particular formula (6a).

[0162] 3.3 Spacer (S) The compounds of the present invention comprise a spacer (S) having a length Z, such that when the vector interacts with the Fc region of an antibody or its fragment, the reactive moiety can react with the side chains of amino acid residues exposed on the surface of the antibody or antibody fragment, resulting in regioselective attachment of the payload (and, optionally, a linker) to the antibody or antibody fragment. The spacer is attached to the vector (V) through a functional group (e.g., amino group, carboxyl group) of the vector's chemical structure. When the vector is a peptide, the spacer is attached to the N-terminus or C-terminus of the peptide (described further below). For example, the spacer can be attached to an amino or carboxyl function at the N-terminus or C-terminus of the polypeptide backbone, or to an N- or C-terminal amino acid side chain. In particular, in the case of non-peptide vector molecules, the binding affinity (K d It is preferred to identify an attachment point for the spacer (S) such that there is no significant (less than 20%) decrease in the .mu.m (expressed as .mu.m).

[0163] The length Z refers to the length of the spacer in its native conformation (not its maximally extended length), which the spacer can adopt when linked to a vector and reactive moiety as part of the construction of a reactive conjugate of the invention.

[0164] A suitable length for length Z can be determined by using computer modeling (Molecular Operating Environment (MOE) available from Chemical Computing Group, Inc.) or X-ray crystallography to calculate the appropriate distance in angstroms (Å) between the binding site of the vector and a target amino acid, e.g., a lysine or cysteine ​​residue, most preferably lysine, on the Fc domain of an antibody or fragment thereof. In the case of a polymer, length Z can be determined by applying the worm-like chain (WLC) model, which is further described below. The three-dimensional structure of the Fc-III / Fc region complex at 2.7 Å resolution is available under the PDB identifier 1DN2 (DeLano et al., Science 2000, vol. 287, no. 5456, pp. 1279-1283).

[0165] In one embodiment, the length Z is 13 to 30 Å, preferably 14 to 25 Å, and more preferably 16 to 18 Å.

[0166] The inventors believe that a length Z in the range of 13 to 30 Å detailed above can result in targeting of one or more lysine residues found in the Fc region of an antibody or antibody fragment (a region highly conserved among antibodies, particularly IgG antibodies), e.g., at positions 317, 326, 338, 340, and 439, particularly positions 317 and / or 326, resulting in reaction of the reactive moiety and attachment of the payload with a high degree of regioselectivity. In certain embodiments, a high degree of regioselectivity is achieved when the payload loading ratio Fc / F(ab)2 is greater than 1.0, greater than 1.5, greater than 2.0, particularly greater than 2.5. The degree of regioselectivity can be determined by measuring the payload loading ratio between the Fc region and the F(ab)2 region (selectivity Fc / F(ab)2), as further described below.

[0167] The spacer may be any group having the above length Z that can link the vector and the reactive moiety. Preferably, it is chemically inert.

[0168] In one embodiment, the spacer is preferably (a2) a polyalkylene oxide group having 6 to 36 repeating units, for example, 8 to 24 repeating units; preferably, a polyalkylene oxide group represented by the following formula (7): -X 1 -(CH2CH2O) n2 -CH2CH2-X 2 - (7) (In the formula, X 1 is NH, O or S; preferably NH; X 2 If is covalently linked to the vector, X 2 is NH or C=O, preferably C=O; n2 is an integer of 4 to 28, preferably 6 to 20, more preferably 8 to 12, and particularly preferably 10. A group represented by: (b2) A peptide group having 6 to 25 amino acids in the main chain, for example, 9 amino acids in the main chain, each of which is preferably selected from Pro, Gly, Ala, Asn, Asp, Thr, Glu, Gln, and Ser; more preferably, Pro, Gly, or Ser.

[0169] In formula (7), in some embodiments, X 2 is covalently linked to the vector, X 1 is covalently linked to the reactive moiety; in some other embodiments, X 1 is covalently linked to the vector, X 2 In certain embodiments, the attachment point of the spacer to the vector as well as X 1 and X 2 are each independently selected so that the bond to the vector forms an amide bond. For example, if the vector is linked to the spacer via the N-terminus (i.e., via the amino group of the N-terminal amino acid), then X 2 may be selected to be C=O, and when the vector is linked to the spacer via the C-terminus (i.e., via the carboxyl group of the C-terminal amino acid), X 1(or X 2 ) may be selected to be NH.

[0170] According to one embodiment, the spacer comprises a polyethylene oxide group having 4 to 36 repeating units, preferably 6 to 28 repeating units, more preferably 7 to 24 repeating units, for example 10 or 20 repeating units. Most preferably, the spacer comprises a polyethylene oxide group having 10 repeating units.

[0171] 3.4 Vector (V) The compounds of the present invention comprise a vector (V) (or "ligand") capable of interacting with (binding to) the fragment crystallizable (Fc) region of an antibody or fragment thereof, where the antibody fragment is optionally incorporated into an Fc fusion protein. The interaction of the vector with the Fc region results in an increased concentration of reactive moieties near the side chains of surface-exposed amino acids of the antibody or antibody fragment, resulting in covalent attachment of a payload to the side chains. In some embodiments, the interaction of the vector with the Fc region results in a targeting effect insofar as the reactive moieties react with the side chains of specific surface-exposed amino acids of the antibody or antibody fragment (e.g., the lysine residue at position 317), resulting in regioselective attachment of the payload to the antibody or antibody fragment.

[0172] Vectors capable of interacting with the Fc region of an antibody or a fragment thereof are known in the art and are described, for example, in Choe et al., Materials 2016, 9, 994. Suitable vectors are also disclosed in WO2018 / 199337A1. Non-limiting examples of vectors capable of interacting with the Fc region of an antibody or a fragment thereof include protein Z and Fc-III. In particular, the cyclic peptide Fc-III has a dissociation constant K of about 16 nm. d has been described as a peptide vector / ligand with high affinity for the Fc region of IgG protein (DeLano et al., Science 2000, 287, 1279-1283).

[0173] In one embodiment, the vector used in the compounds of the present invention is a peptide comprising a sequence of 11 to 17 amino acids, preferably 13 to 17 amino acids. In certain embodiments, the spacer is attached to the vector (i.e., the above-described peptide sequence) via its N- or C-terminus. In some further specific embodiments, the vector is not separately further modified for attachment of the spacer to the N- or C-terminus.

[0174] According to one preferred embodiment, the vector has the following formulas (8a) and (8b):

[0175] [ka]

[0176] (In the formula, Bxx, Cxx, Dxx, Exx, and Fxx each independently represent an amino acid; Axx is an amino acid, a dicarboxylic acid, or a group represented by the following formula (9a): ---Axx1-Axx2-Axx3--- (9a) (In the formula, Axx1 represents a single covalent bond or an amino acid such as Arg; Axx2 represents an amino acid such as Gly or Cys; Axx3 represents an amino acid such as Asp or Asn) represents a peptide moiety represented by: Gxx is an amino acid or a group represented by the following formula (9b): ---Gxx1-Gxx2-Gxx3--- (9b) (In the formula, Gxx1 represents an amino acid such as Thr; Gxx2 represents an amino acid such as Tyr or Cys; Gxx3 represents a single covalent bond or an amino acid such as His) represents a peptide moiety represented by: The side chain of Axx2 in formula (9a) may be covalently bonded to the side of Gxx2 in formula (9b) to form a ring; when Axx2 is Cys and Gxx2 is Cys, preferably the side chains of Axx2 and Gxx2 are linked together to form a ring of formula -(SX 4 -S)- (wherein X represents a single covalent bond or a divalent group containing one or more atoms selected from carbon, nitrogen, and oxygen, such as a divalent maleimide group, a divalent acetone group, or a divalent arylene group (e.g., a divalent xylene group), and preferably X 4 represents a single covalent bond); Hxx represents a single covalent bond or a trifunctional amino acid such as a diamino-carboxylic acid; The Z1 is a group covalently attached to the C-terminus of Gxx when Hxx is a single covalent bond selected from -N(H)(R), where R represents a hydrogen atom, an alkyl group or a cycloalkyl group, and a moiety derived from a compound containing a conjugation group selected from biotin, DBCO, TCO, BCN, alkyne, azide, bromoacetamide, maleimide, and thiol; a group covalently attached to the C-terminus of Hxx when Hxx is a trifunctional amino acid and Y' is attached to the side chain of Hxx, preferably N(H)(R), where R represents a hydrogen atom, an alkyl group or a cycloalkyl group when Z1 is covalently attached to the C-terminus of Hxx; or When Hxx is a trifunctional amino acid and Y' is bonded to the C-terminus of Hxx, it represents a hydrogen atom bonded to the side chain of Hxx; The Z2 is a group covalently attached to the N-terminus of Axx when Hxx is a single covalent bond selected from a hydrogen atom, a carbonyl-containing group such as an acetyl group, and a group containing a conjugation moiety such as biotin; a group covalently attached to the N-terminus of Hxx when Hxx is a trifunctional amino acid and Y' is attached to the side chain of Hxx selected from a hydrogen atom and a carbonyl-containing group such as an acetyl group; or When Hxx is a trifunctional amino acid and Y' is bonded to the N-terminus of Hxx, it represents a hydrogen atom bonded to the side chain of Hxx; Y' is present only if Hxx is a trifunctional amino acid, which is Z 1 When Z2 is bonded to the C-terminus of Hxx in formula (8a), or when Z2 is bonded to the N-terminus of Hxx in formula (8b), the side chain of Hxx, Z 1 When is bonded to the side chain of Hxx in formula (8a), it is Z 2 When attached to the side chain of Hxx in formula (8b), it represents a moiety covalently attached to the N-terminus of Hxx; Y' is preferably derived from a compound containing a conjugation group selected from biotin, DBCO, TCO, BCN, alkyne, azide, bromoacetamide, maleimide, and thiol; X 3 represents a single covalent bond or a divalent group containing one or more atoms selected from carbon, nitrogen, and oxygen, such as a divalent maleimide group, a divalent acetone group, or a divalent arylene group (e.g., a divalent xylene group), and preferably X 3 represents a single covalent bond; **** indicates a covalent bond to the spacer (S) is a peptide represented by one of

[0177] In certain embodiments, the moiety Y′ has the following formula (9c): Y 1 -L 1 -- ****' (9c) (In the formula, Y 1 is a moiety derived from a conjugation group selected from biotin, DBCO, TCO, BCN, alkyne, azide, bromoacetamide, maleimide, and thiol; L 1 is preferably a divalent group containing one or more atoms selected from C, N, O and S, more preferably a polyethylene oxide group having 1 to 12 repeating units, for example, 4 repeating units; and ****'indicates a covalent bond to Hxx) is expressed by

[0178] The linker is preferably a divalent group containing one or more atoms selected from carbon, nitrogen, oxygen, and sulfur.

[0179] In one embodiment, the linker L 1 teeth, (a1) an alkylene group having 1 to 12 carbon atoms, preferably an alkylene group having 2 to 6 carbon atoms, such as an ethylene group or a propylene group; (b1) a polyalkylene oxide group having 2 or 3 carbon atoms and having 1 to 36 repeating units; preferably a group of the formula -NH-(CH2CH2O) n1 -CHCH- (wherein n1 is an integer of 0 to 35, for example, 1 to 20); and (c1) a peptide group having 2 to 12 amino acids You can choose from:

[0180] According to one preferred embodiment, at least one of Axx, Bxx, Cxx, Dxx, Exx, Fxx, Gxx and Hxx in formulas (8a) and (8b) is defined as follows: Axx represents an amino acid selected from Ala, 2,3-diamino-propionic acid (Dap), Asp, Glu, 2-aminosuberic acid, α-aminobutyric acid, Asn, and Gln, a dicarboxylic acid selected from succinic acid, glutaric acid, and adipic acid; preferably Ala, Asp, or Asn; more preferably Asp; or a peptide moiety of formula (9a) (wherein Axx1 is a single covalent bond, Axx2 is Cys, and Axx3 is Asp); Bxx represents an amino acid selected from Trp, Phe, Tyr, phenylglycine (Phg), 3-benzothiopen-2-yl-L-alanine, 3-naphthalen-2-yl-L-alanine, 3-biphenyl-4-yl-L-alanine, and 3-naphthalen-1-yl-L-alanine; preferably Trp; Cxx represents an amino acid selected from His, Ala, 3-pyridin-2-yl-L-alanine, meta-tyrosine (mTyr) and Phe; preferably His, Ala or mTyr; more preferably His; Dxx represents an amino acid selected from Ala, Abu, Gly, Leu, Ile, Val, Met, cyclohexylalanine (Cha), Phe, Thr, Cys, Tyr, and norleucine (Nle); preferably, Ala, Nle, or Leu; more preferably, Leu; Exx represents an amino acid selected from Ala, Gly, Asn, Ser, Abu, and Asp; preferably, Ala or Gly; more preferably, Gly; Fxx represents an amino acid selected from Ala, Glu, Asp, Gln, His, Arg, Ser, and Asn; preferably, Asp or Glu; more preferably, Glu; Gxx represents an amino acid selected from Thr, Ser, Ala, Asn, Val, 2-aminobutyric acid (Abu), Ile, Met, Leu, Pro, Gln, and Cys; preferably Thr or Ser; more preferably Thr; or a peptide moiety of formula (9b) wherein Gxx1 is Thr, Gxx2 is Cys, and Gxx3 is a single covalent bond; Hxx represents an amino acid selected from Dap, Dab, Lys, Orn and homo-lysine (homo-Lys), preferably an amino acid selected from Dap, Dab, Lys, Orn and homo-Lys.

[0181] According to one embodiment, the ligand V capable of interacting with the Fc region of an antibody or antibody fragment has the following formulae (8a') to (8d'):

[0182] [ka]

[0183] is a peptide represented by one of

[0184] In the above formulas (8a'), (8b'), (8c') and (8d'), Z1, Z2, X 3 , X 4 and **** is as described above with respect to equations (8a) and (8b).

[0185] In certain embodiments, the disulfide bridge between the cysteine ​​residues in the above formula (i.e., formula -(SX 3 -S)- or -(SX 4 The -S)- disulfide bridges) can each independently be replaced by a divalent group suitable for inter-side chain cyclization (sometimes referred to as "cysteine ​​re-bridging"; see, e.g., Stefanucci et al., Scientific Reports 2019, 9:5771). Examples of suitable divalent groups include divalent xylene groups, divalent maleimide groups, divalent triazole-containing groups, divalent carbonyl-containing groups (e.g., divalent acetone groups), divalent succinimide groups (which can be obtained by reacting a cysteine ​​side chain with, for example, an aryloxymaleimide reagent; see Marculescu et al., Chem. Commun. 2014, 50, 7139), divalent thioether groups (which can be obtained by reacting a cysteine ​​side chain with, for example, a bis-sulfone or arylsulfone reagent; see Brocchini et al., Nat. Protoc. 2006, 1, 2241-2252), and divalent pyridazinedione groups (which can be obtained by reacting a cysteine ​​side chain with, for example, a dibromopyridazinedione reagent; see Chudamasa et al., Chem. Commun. 2011, 47, 8781-8783). In particular, the disulfide bridges can each independently be replaced by a divalent triazole-containing group that can be obtained by "click" chemistry. In this case, the cysteine ​​residues (forming the bridge in the above formula) can be replaced by amino acids bearing side chains containing functional groups suitable for click chemistry, i.e., alkyne or azide groups, that can react to form divalent triazole moieties (e.g., 1,4-disubstituted-1,2,3-triazole moieties).

[0186] Preferably, the vector is a peptide represented by formula (8a') or (8b').

[0187] According to one embodiment, the compounds of the present invention are 1 -(O-(C=O)-O)-P, VS 1 -(O-(C=O))-P, VS 1 -(S-(C=O))-P, VS 1 -(S-(C=O)-O)-P, VS 1 -(O-(C=S)-O)-P, VS 1 -(O-(C=O)-S)-P, VS 1 -(S-(C=O)-S)-P, VS 1 -(S-(C=S)-O)-P, VS 1 -(O-(C=S)-S)-P, VS 1 -(S-(C=S))-P, VS 1 -(O-(C=O)-NH)-P, VS 1 -(S-(C=S)-S)-P, VS 1 -(MO-(C=O)-O)-P, VS 1 -(MO-(C=O))-P, VS 1 -(MS-(C=O))-P, VS 1 -(MS-(C=O)-O)-P, VS 1 -(MO-(C=S)-O)-P, VS 1 -(MO-(C=O)-S)-P, VS 1 -(MS-(C=O)-S)-P, VS 1 -(MS-(C=S)-O)-P, VS 1 -(MO-(C=S)-S)-P, VS 1 -(MS-(C=S))-P, VS 1 -(MO-(C=O)-NH)-P, VS 1 -(MS-(C=S)-S)-P, VS 1 -(O-(C=O)-O)-LP 1 , VS 1 -(O-(C=O))-LP 1 , VS 1-(S-(C=O))-LP 1 、VS 1 -(S-(C=O)-O)-LP 1 、VS 1 -(O-(C=S)-O)-LP 1 、VS 1 -(O-(C=O)-S)-LP 1 、VS 1 -(S-(C=O)-S)-LP 1 、VS 1 -(S-(C=S)-O)-LP 1 、VS 1 -(O-(C=S)-S)-LP 1 、VS 1 -(S-(C=S))-LP 1 、VS 1 -(O-(C=O)-NH)-LP 1 、VS 1 -(S-(C=S)-S)-LP 1 、VS 1 -(MO-(C=O)-O)-LP 1 、VS 1 -(MO-(C=O))-LP 1 、VS 1 -(MS-(C=O))-LP 1 、VS 1 -(MS-(C=O)-O)-LP 1 、VS 1 -(MO-(C=S)-O)-LP 1 、VS 1 -(MO-(C=O)-S)-LP 1 、VS 1 -(MS-(C=O)-S)-LP 1 、VS 1 -(MS-(C=S)-O)-LP 1 、VS 1 -(MO-(C=S)-S)-LP 1 、VS 1 -(MS-(C=S))-LP 1 、VS 1 -(MO-(C=O)-NH)-LP 1 、&&VS 1 -(MS-(C=S)-S)-LP1 (In the formula, V, P, P 1 and L are as defined above, and S 1 is a spacer S as defined above, preferably V, S 1 , P / P 1 At least one, e.g., two, three, four, or more than four, of L, L, and M are defined as follows: (α) V is a peptide of formula (8a) or (8b), preferably a peptide of formula (8a′) or (8b′); (β)S 1 teeth, (a2) a polyalkylene oxide group having 6 to 36 repeating units; preferably, a polyalkylene oxide group represented by the following formula (7): -X 1 -(CH2CH2O) n2 -CH2CH2-X2- (7) (In the formula, X 1 is NH, O or S; preferably NH; X 2 If is covalently linked to the vector, X 2 is NH or C=O, preferably C=O; n2 is an integer of 4 to 28, preferably 6 to 20, and more preferably 10. a group represented by (b2) A peptide group having 6 to 25 amino acids in the main chain, each amino acid being preferably selected from Pro, Gly, Ala, Asn, Asp, Thr, Glu, Gln, and Ser; more preferably, Pro, Gly, or Ser. is a group selected from (γ)P or P 1 teeth, (γ1) respectively, 89 Zr, 111 In, 64 Cu, 177 Lu, 68 Ga, and 99m From Tc, preferably 89 Zr, 111 In, 64Cu, which may be chelated to a radionuclide selected from NOTA, DOTA, NODAGA, DTPA, (γ2)N3, TZ, TCO, DBCO, BCN, (γ3) auristatin (e.g., MMAE) or PNU-159582 is the part that comes from; (δ)L is (a1) an alkylene group having 2 to 6 carbon atoms (-(CH2) 2~6 -), (b1) Formula -NH-(CH2CH2O) n1 a polyalkylene group of -CH2CH2- (wherein n1 is an integer of 0 to 35), and (c1) a linker that is optionally cleavable, comprising 2 to 12 amino acids, preferably selected from cleavable peptide linkers comprising a Val-Cit unit, a Val-Ala unit, a Val-Cit-PABC unit, or a Val-Cit-PABC-DMEA unit; and (ε) M is a group of formula (5a) or (5e), ​​preferably a group of formula (5a). is a compound represented by a formula selected from

[0188] According to a preferred embodiment, in the above formula, V, S1 and M are defined as follows: (α) V is a peptide of formula (8a′) or (8b′); (β)S 1 is expressed by the following equation (7): -X 1 -(CH2CH2O) n2 -CH2CH2-X 2 - (7) (In the formula, X 1 is NH, O or S; preferably NH; X 2 If is covalently linked to the vector, X 2 is NH or C=O, preferably C=O; n2 is an integer of 6 to 20, preferably 10. is a group represented by (ε)M is a group of formula (5a).

[0189] (γ)P 1 When (γ)P1 is a moiety derived from an auristatin, e.g., MMAE, (δ)L preferably represents a cleavable linker comprising a Val-Cit, Val-Ala, or Val-Cit-PABC unit, more preferably a Val-Cit-PABC unit. When (γ)P1 is a moiety derived from PNU-159582, (δ)L preferably represents a cleavable linker comprising a Val-Cit-PABC-DMEA unit.

[0190] According to one embodiment, the compound of the present invention is V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MO-(C=O)-O)-P, V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MO-(C=O))-P, V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MS-(C=O))-P, V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MS-(C=O)-O)-P, V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MO-(C=S)-O)-P, V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MO-(C=O)-S)-P, V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MS-(C=O)-S)-P, V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MS-(C=S)-O)-P, V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MO-(C=S)-S)-P, V 1 -NH-(CH2CH2O) n2-CH2CH2-NH-(M-S-(C=S))-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=O)-NH)-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=S)-S)-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(O-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=S))-L-P 1 、V 1 -NH-(CH2CH2O) n2-CH2CH2-NH-(O-(C=O)-NH)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=S)-O)-L-P 1 、V-NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=S))-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=O)-NH)-L-P1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=S)-S)-L-P 1 、P-(O-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-((C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-((C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(O-(C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-((C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(NH-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V2 、P 1 -L-((C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=S)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=S)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(NH-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、V-AA 6-25 -(M-O-(C=O)-O)-P、V-AA 6-25-(MO-(C=O))-P、V-AA 6-25 -(MS-(C=O))-P、V-AA 6-25 -(MS-(C=O)-O)-P、V-AA 6-25 -(MO-(C=S)-O)-P、V-AA 6-25 -(MO-(C=O)-S)-P、V-AA 6-25 -(MS-(C=O)-S)-P、V-AA 6-25 -(MS-(C=S)-O)-P、V-AA 6-25 -(MO-(C=S)-S)-P、V-AA 6-25 -(MS-(C=S))-P、V-AA 6-25 -(MO-(C=O)-NH)-P、V-AA 6-25 -(MS-(C=S)-S)-P、V-AA 6-25 -(O-(C=O)-O)-LP 1 、V-AA 6-25 -(O-(C=O))-LP 1 、V-AA 6-25 -(S-(C=O))-LP 1 、V-AA 6-25 -(S-(C=O)-O)-LP 1 、V-AA 6-25 -(O-(C=S)-O)-LP 1 、V-AA 6-25 -(O-(C=O)-S)-LP 1 、V-AA 6-25 -(S-(C=O)-S)-LP 1 、V-AA 6-25 -(S-(C=S)-O)-LP 1 、V-AA 6-25 -(O-(C=S)-S)-LP 1 、V-AA 6-25 -(S-(C=S))-LP 1 、V-AA 6-25 -(O-(C=O)-NH)-LP 1 、V-AA 6-25 -(S-(C=S)-S)-LP 1 、V-AA 6-25 -(MO-(C=O)-O)-LP 1 、V-AA 6-25 -(MO-(C=O))-LP1 , V-AA 6-25 -(MS-(C=O))-LP 1 , V-AA 6-25 -(MS-(C=O)-O)-LP 1 , V-AA 6-25 -(MO-(C=S)-O)-LP 1 , V-AA 6-25 -(MO-(C=O)-S)-LP 1 , V-AA 6-25 -(MS-(C=O)-S)-LP 1 , V-AA 6-25 -(MS-(C=S)-O)-LP 1 , V-AA 6-25 -(MO-(C=S)-S)-LP 1 , V-AA 6-25 -(MS-(C=S))-LP 1 , V-AA 6-25 -(MO-(C=O)-NH)-LP 1 , and V-AA 6~25 -(MS-(C=S)-S)-LP 1 (In the formula, V, P, P 1 and L is as defined above, and V 1 is a peptide of formula (8b), and V 2 is a peptide of formula (8a), preferably V / V 1 / V 2 , n2 / AA, P / P 1 At least one, e.g., two, three, four, or more than four, of L, L, and M are defined as follows: (α) V is a peptide of formula (8a) or (8b), preferably of formula (8a′) or (8b′); V 1 is a peptide of formula (8b'), and V 2 is a peptide of formula (8a'); (β)n2 is an integer of 6 to 20, preferably 10; or each amino acid (AA) is independently selected from Pro, Gly, Ala, Asn, Asp, Thr, Glu, Gln and Ser, preferably from Pro, Gly and Ser; (γ)P or P 1teeth, (γ1) respectively, 89 Zr, 111 In, 64 Cu, 177 Lu, 68 Ga, and 99m From Tc, preferably 89 Zr, 111 In, 64 Cu, which may be chelated to a radionuclide selected from NOTA, DOTA, NODAGA, DTPA, (γ2)N3, TZ, TCO, DBCO, BCN, (γ3) auristatin (e.g., MMAE) or PNU-159582 is the part that comes from; (δ)L is (a1) an alkylene group having 2 to 6 carbon atoms (-(CH2) 2~6 -), (b1) Formula -NH-(CH2CH2O) n1 a polyalkylene group of -CH2CH2- (wherein n1 is an integer of 0 to 35), and (c1) optionally cleavable peptide linkers comprising 2 to 12 amino acids, preferably cleavable peptide linkers comprising Val-Cit, Val-Ala, Val-Cit-PABC or Val-Cit-PABC-DMEA units; is a linker selected from: (ε) M is a group of formula (5a) or (5e), ​​preferably a group of formula (5a). is a compound represented by a formula selected from

[0191] According to a preferred embodiment, in the above formula, V / V 1 / V 2 , n2 / AA and M are defined as follows: (α) V is a peptide of formula (8a′) or (8b′); V 1 is a peptide of formula (8b'), and V 2 is a peptide of formula (8a'); (β)n2 is an integer of 6 to 20, preferably 10; or each amino acid (AA) is independently selected from Pro, Gly, Ala, Asn, Asp, Thr, Glu, Gln and Ser, preferably from Pro, Gly and Ser; and (ε)M is a group of formula (5a).

[0192] (γ)P 1 When (γ)P1 is a moiety derived from an auristatin, e.g., MMAE, (δ)L preferably represents a cleavable linker comprising a Val-Cit, Val-Ala, or Val-Cit-PABC unit, more preferably a Val-Cit-PABC unit. When (γ)P1 is a moiety derived from PNU-159582, (δ)L preferably represents a cleavable linker comprising a Val-Cit-PABC-DMEA unit.

[0193] According to one embodiment, the compound of the present invention is V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MO-(C=O)-O)-P, V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MO-(C=O))-P, V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MS-(C=O))-P, V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MS-(C=O)-O)-P, V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MO-(C=S)-O)-P, V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MO-(C=O)-S)-P, V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MS-(C=O)-S)-P, V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MS-(C=S)-O)-P, V 1 -NH-(CH2CH2O) 6-20-CH2CH2-NH-(M-O-(C=S)-S)-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=S))-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=O)-NH)-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=S)-S)-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(S-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20-CH2CH2-NH-(S-(C=S))-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=O)-NH)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(S-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=S)-O)-L-P 1 、V-NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=S))-L-P 1 、V1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=O)-NH)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=S)-S)-L-P 1 、P-(O-(C=O)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-((C=O)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-((C=O)-S-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(O-(C=O)-S-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-S-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-S-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-((C=S)-S-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(NH-(C=O)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-S-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P1 -L-(O-(C=O)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=O)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=O)-S-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=O)-S-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=S)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=O)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=O)-S-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=S)-S-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P[[ID=**48**]] 1 -L-(S-(C=S)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=S)-S-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P 1 -L-(NH-(C=O)-O-M)-NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、及びP 1 -L-(S-(C=S)-S-M)-NH-(CH2CH2O) 6~20 -CH2CH2-(C=O)-V It should be noted that there may be some inaccuracies in the original text as "及びP" seems a bit out of place in the context. If possible, it would be beneficial to double-check the source for a more accurate translation.2 (In the formula, V, P, P 1 and L is as defined above, and V 1 is a peptide of formula (8b), and V 2 is a peptide of formula (8a), preferably V 1 / V 2 , P / P1, L, and M, at least one of which, for example, two, three, four, or more than four, are defined as follows: (α)V 1 is a peptide of formula (8b'), and V 2 is a peptide of formula (8a'); (γ)P or P 1 teeth, (γ1) respectively, 89 Zr, 111 In, 64 Cu, 177 Lu, 68 Ga, and 99m From Tc, preferably 89 Zr, 111 In, 64 Cu, which may be chelated to a radionuclide selected from NOTA, DOTA, NODAGA, DTPA, (γ2)N3, TZ, TCO, DBCO, BCN, (γ3) auristatin (e.g., MMAE) or PNU-159582 is the part that comes from; (δ)L is (a1) an alkylene group having 2 to 6 carbon atoms (-(CH2) 2~6 -), (b1) Formula -NH-(CH2CH2O) n1 a polyalkylene group of -CH2CH2- (wherein n1 is an integer of 0 to 35), and (c1) optionally cleavable peptide linkers comprising 2 to 12 amino acids, preferably cleavable peptide linkers comprising Val-Cit, Val-Ala, Val-Cit-PABC or Val-Cit-PABC-DMEA units; is a linker selected from: (ε) M is a group of formula (5a) or (5e), ​​preferably a group of formula (5a). is a compound represented by a formula selected from

[0194] According to a preferred embodiment, in the above formula, V 1 / V 2 and M is defined as follows: (α)V 1 is a peptide of formula (8b'), and V 2 is a peptide of formula (8a'); and (ε)M is a group of formula (5a).

[0195] (γ)P 1 When (γ)P1 is a moiety derived from an auristatin, e.g., MMAE, (δ)L preferably represents a cleavable linker comprising a Val-Cit, Val-Ala, or Val-Cit-PABC unit, more preferably a Val-Cit-PABC unit. When (γ)P1 is a moiety derived from PNU-159582, (δ)L preferably represents a cleavable linker comprising a Val-Cit-PABC-DMEA unit.

[0196] According to one embodiment, the compound of the present invention is V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MO-(C=O)-O)-P, V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MO-(C=O))-P, V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MS-(C=O))-P, V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MS-(C=O)-O)-P, V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MO-(C=S)-O)-P, V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MO-(C=O)-S)-P, V 1-NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=O)-S)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=S)-O)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=S)-S)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=S))-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=O)-NH)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=S)-S)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(O-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10-CH2CH2-NH-(S-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(S-(C=S))-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(O-(C=O)-NH)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(S-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=S)-O)-L-P 1 、V-NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=S)-O)-L-P 1 、V1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=S))-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=O)-NH)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=S)-S)-L-P 1 、P-(O-(C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-((C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-((C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(O-(C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-((C=S)-S-M)-NH-(CH2CH2O) 10-CH2CH2-(C=O)-V 2 、P-(NH-(C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=S)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=S)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=S)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=S)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V2 , P 1 -L-(NH-(C=O)-OM)-NH-(CH2CHO) 10 -CH2CH2-(C=O)-V 2 , and P 1 -L-(S-(C=S)-SM)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 (In the formula, V, P, P 1 and L is as defined above, and V 1 is a peptide of formula (8b), and V 2 is a peptide of formula (8a), preferably V 1 / V 2 , P / P 1 At least one, e.g., two, three, four, or more than four, of L, L, and M are defined as follows: (α)V 1 is a peptide of formula (8b'), and V 2 is a peptide of formula (8a'); (γ)P or P 1 teeth, (γ1) respectively, 89 Zr, 111 In, 64 Cu, 177 Lu, 68 Ga, and 99m From Tc, preferably 89 Zr, 111 In, 64 Cu, which may be chelated to a radionuclide selected from NOTA, DOTA, NODAGA, DTPA, (γ2)N3, TZ, TCO, DBCO, BCN, (γ3) auristatin (e.g., MMAE) or PNU-159582 is the part that comes from; (δ)L is (a1) an alkylene group having 2 to 6 carbon atoms (-(CH2) 2~6 -), (b1) Formula -NH-(CH2CH2O) n1 a polyalkylene group of -CH2CH2- (wherein n1 is an integer of 0 to 35), and (c1) optionally cleavable peptide linkers comprising 2 to 12 amino acids, preferably cleavable peptide linkers comprising Val-Cit, Val-Ala, Val-Cit-PABC or Val-Cit-PABC-DMEA units; is a linker selected from: (ε) M is a group of formula (5a) or (5e), ​​preferably a group of formula (5a). is a compound represented by a formula selected from

[0197] According to a preferred embodiment, in the above formula, V 1 / V 2 and M is defined as follows: (α)V 1 is a peptide of formula (8b'), and V 2 is a peptide of formula (8a'); and (ε)M is a group of formula (5a).

[0198] (γ)P 1 When (γ)P1 is a moiety derived from an auristatin, e.g., MMAE, (δ)L preferably represents a cleavable linker comprising a Val-Cit, Val-Ala, or Val-Cit-PABC unit, more preferably a Val-Cit-PABC unit. When (γ)P1 is a moiety derived from PNU-159582, (δ)L preferably represents a cleavable linker comprising a Val-Cit-PABC-DMEA unit.

[0199] In one embodiment, the compound of formula (1) is

[0200] [ka]

[0201] wherein P represents a moiety derived from a payload as defined above, preferably a chelator that optionally chelates a radionuclide, more preferably DTPA, DOTA, DFO, NOTA, PCTA, CH-X-DTPA, NODAGA or DOTAGA; and Y′ represents a moiety derived from a compound containing a conjugation group, preferably selected from biotin, DBCO, TCO, BCN, alkyne, azide, bromoacetamide, maleimide and thiol, more preferably selected from biotin, DBCO, BCN and azide. The repeat number of the spacer polyethylene oxide moiety in the above compound (i.e., 9) can be replaced by any of 5 to 35, preferably 7 to 19, with a spacer having 9 polyethylene oxide repeat units being the most preferred option.

[0202] In one embodiment, the compound of formula (1) is

[0203] [ka]

[0204] [ka]

[0205] [ka]

[0206] [ka]

[0207] [ka]

[0208] [ka]

[0209]

change

[0210]

change

[0211]

change

[0212]

change

[0213]

change

[0214]

change

[0215]

change

[0216]

change

[0217]

change

[0218]

change

[0219] [ka]

[0220] [ka]

[0221] [ka]

[0222] [ka]

[0223] [ka]

[0224] [ka]

[0225] [ka]

[0226] [ka]

[0227] [ka]

[0228] [ka]

[0229] is selected from.

[0230] In one embodiment, the repeat number of the spacer polyethylene oxide moiety in the above compound (i.e., 9) can be replaced by any of 5 to 35, preferably 7 to 19, with a spacer having 9 polyethylene oxide repeat units being the most preferred option.

[0231] In a preferred embodiment, the compound of formula (1) is

[0232] [ka]

[0233] [ka]

[0234] [ka]

[0235] [ka]

[0236] [ka]

[0237] is selected from.

[0238] The repeat number of the spacer polyethylene oxide moiety in the above compound (i.e., 9) can be replaced by any of 5 to 35, preferably 7 to 19, with a spacer having 9 polyethylene oxide repeat units being the most preferred option.

[0239] In a more preferred embodiment, the compound of formula (1) is

[0240] [ka]

[0241] [ka]

[0242] is selected from.

[0243] In the above compounds, DFO represents a desferrioxamine group that is attached to the rest of the molecule via its amino group to form a thiourea group together with the thiocarbonyl-containing group to which it is attached. The repeat number of the spacer polyethylene oxide moiety in the above compounds (i.e., 9) can be replaced by any of 5 to 35, preferably 7 to 19, with a spacer having 9 polyethylene oxide repeat units being the most preferred option.

[0244] 4. Kits for site-specific modification of antibodies or antibody fragments In some aspects, the invention relates to kits comprising the above-described compounds and a buffer, which can be used for the regioselective modification (e.g., labeling) of antibodies or fragments thereof, where the antibody fragment is optionally incorporated into an Fc-fusion protein, in particular for the regioselective modification of therapeutic antibodies.

[0245] The compounds of the invention and buffering agent (together forming a kit) can be provided individually, e.g., in separate primary containers (which can be shipped to the customer in a single box), which can be stored for extended periods of time without degradation. The compounds and buffering agent can be formulated and apportioned for a given amount of antibody or fragment thereof to be modified. In some aspects, the compounds of the invention are provided as solids (e.g., lyophilized powders, or noncovalently adsorbed or covalently attached to a solid phase matrix as further described below) or as solutions in suitable solvents, such as water-miscible, polar, aprotic solvents (e.g., DMF, DMSO), which can be mixed with the buffering agent immediately prior to antibody or antibody fragment modification.

[0246] The buffer used in the kit of the present invention is not particularly limited. Preferably, the buffer has a pH of 5.5 to 11, more preferably 7.5 to 9.5. The buffer can be selected from, for example, 2-bis(2-hydroxyethyl)aminoacetic acid (bicine), carbonate-bicarbonate, tris(hydroxymethyl)methylaminopropanesulfonic acid (TAPS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Preferably, the buffer is a carbonate-bicarbonate buffer or bicine buffer having a pH of 7.5 to 9.5, for example, about 9.0.

[0247] According to one embodiment, the compounds of the present invention are immobilized on a solid matrix (solid support), e.g., on beads. The compounds can be immobilized using methods known in the art, such as high-affinity (e.g., biotin-streptavidin, biotin-neutravidin) binding, "click" chemistry (as defined by Kolb et al., "Click Chemistry: Diverse Chemical Function from a Few Good Reactions," Angewandte Chemie Int. Ed. 2001, 40(11), pp. 2004-2021), or hydrazone ligation. Preferably, the solid matrix is ​​an inert matrix, such as a polymer gel, that contains a three-dimensional structure, lattice, or network of material. More preferably, the solid matrix is ​​a material used for affinity chromatography, such as a xerogel. Such gels shrink upon drying to a compressed solid containing only the gel matrix. When the dried xerogel is resuspended in liquid, the gel matrix absorbs the liquid, expands, and returns to a gel state. Examples of xerogels that can be suitably used in the present invention include polymer gels such as cellulose, cross-linked dextran gels (e.g., Sephadex (registered trademark)), agarose, cross-linked agarose, polyacrylamide gels, and polyacrylamide-agarose gels.

[0248] In one embodiment, the compound is immobilized on a solid matrix by a conjugation group Y' in formula (8a), for example, by biotin-streptavidin or biotin-neutravidin binding (where Y' in formula (8a) represents, for example, a biotin-containing group), by click chemistry (where Y' represents, for example, a DBCO-, azide-, or alkyne-containing group), by tetrazine ligation (where Y' in formula (8a) represents, for example, a TCO- or TZ-containing group), by reaction of a thiol with maleimide, or by reaction of a thiol with acetamide (where Y' in formula (8a) represents, for example, a maleimide- or (chloro)acetamide-containing group).

[0249] 5. Use of Reactive Conjugates in Methods for Site-Selective Modification of Antibodies or Antibody Fragments The compounds of the invention can be used in methods for site-selective modification of antibodies or fragments thereof, where the antibody fragment is optionally incorporated into an Fc-fusion protein. The methods result in modified antibodies or modified antibody fragments (e.g., ADCs) that can be used in methods for diagnosing, monitoring, e.g., over time, e.g., monitoring the effectiveness of treatment, imaging, or treating diseases as further described below.

[0250] In one embodiment, the method comprises reacting (contacting) an antibody or fragment thereof, which may be included in the kit described above, with a compound. The reaction mixture can be purified by techniques known in the art, such as gel permeation chromatography using a suitable solvent.

[0251] When a compound of the invention is immobilized on a solid phase matrix, the immobilized compound is contacted with a sample containing the antibody or antibody fragment to be modified, and the solid phase matrix is ​​then washed with a suitable solvent that removes substantially all material in the sample other than the antibody bound to the solid phase matrix. Finally, the solid phase matrix is ​​washed with another suitable solvent, such as glycine buffer at pH 2.5, which releases the modified antibody / antibody fragment (e.g., ADC) from the solid phase matrix.

[0252] The methods of the invention can be applied to any antibody (e.g., IgG protein), antibody fragment, or Fc-fusion protein, provided that it comprises an Fc region for interaction with ligand V. In one embodiment, the antibody to be modified is a monoclonal antibody (mAb), preferably adalimumab, aducanumab, alemtuzumab, altumomab pentetate, atezolizumab, anetumab, avelumab, bapineuzumab, basiliximab, bectumomab, bermekimab, besilesomab, bevacizumab, bezlotoxumab, brentuximab, brentuximab vedotin, brodalumab, blinatumomab, catumaxomab, cemiplimab, cetuximab, simpanemab, clivatuzumab, clivatuzumab tetraxeta , crenezumab tetraxetan, daclizumab, daratumumab, denosumab, dinutuximab, durvalumab, edrecolomab, elotuzumab, emapalumab, enfortumab, enfortumab vedotin, epratuzumab, epratuzumab-SN-38, etaracizumab, gemtuzumab, gemtuzumab ozogamicin, girentuximab, goslanemab, ibritumomab, inebilizumab, infliximab, inotuzumab, inotuzumab ozogamicin, ipilimumab, isatuximab, ixekizumab, J591 PSMA antibody, labetuzumab, lecanemab, mogamulizumab, necitumumab, nimotuzumab, natalizumab, nivolumab, ocrelizumab, ofatumumab, olaratumab, oregovomab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, polatuzumab vedotin, prasinezumab, racotumomab, ramucirumab, rituximab, siltuximab, sacituzumab, sacituzumab govitecan, semolinemab, siltuximab, solanexin tuzumab, tacatuzumab, teprotumumab, tirabonemab, tocilizumab, tositumomab, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, TS23, ustekinumab, vedolizumab, votumumab, zagotenemab, zalutumumab, zanolimumab, fragments and derivatives thereof; more preferably, an antibody selected from the group consisting of atezolizumab, durvalumab, pembrolizumab, rituximab or trastuzumab.

[0253] In one embodiment, the antibody or fragment thereof to be modified is a commercially formulated antibody, preferably a commercially formulated antibody with manufacturing approval delivered by the EMA or the U.S. Food and Drug Administration (FDA). According to one embodiment, the commercially formulated antibody is selected from the group consisting of Humira®, Lemtrada®, Campath®, Tecentriq®, Bavencio®, Simulect®, LymphoScan®, Xilonix®, Scintimun®, Avastin®, Zinplava®, Blincyto®, Libtayo®, Erbitux®, hPAM4-Cide®, Zenapax®, Darzalex®, Prolia®, Unituxin®, Imfinzi®, Panorex®, Empliciti®, Gamifant®, Rencarex®, Remicad®, and others. e®, Besponsa®, Yervoy®, CEA-Cide®, Poteligeo®, Tysabri®, Portrazza®, Theracim®, Opdivo®, Arzerra®, Lartruvo®, Omnitarg®, Vaxira®, Cyramza®, MabThera®, Rituxan®, Sylvant®, Bexxar®, Herceptin®, Kadcyla®, Stelara®, HuMax-EGFr®, HuMax-CD4®, and biosimilars thereof; preferably selected from MabThera® and Herceptin®.

[0254] Commercially available antibodies are often formulated with histidine for stability. When a commercially available antibody is mixed with a reactive conjugate, histidine is expected to react in a competitive manner on the reactive center, thus degrading the reactive conjugate and resulting in a decrease in yield for the ADC. However, the inventors surprisingly found that the yield for the compounds of the present invention is not affected or significantly affected. Without wishing to be bound by theory, the inventors believe that this is due to an increased reaction rate between the compounds of the present invention and amino acids, such as lysine or cysteine, on the side chains of antibodies or antibody fragments. This favorable rate is likely associated with a dramatic increase in the local concentration of reactive centers near the target amino acid upon binding to the Fc fragment of the vector.

[0255] In one embodiment, the antibody fragment to be modified is incorporated into an Fc-fusion protein, preferably selected from belatacept, aflibercept, ziv-aflibercept, dulaglutide, rilonacept, romiplostim, abatacept, and alefacept.

[0256] 6. Modified Antibodies or Modified Antibody Fragments

[0257] The modified antibodies and modified antibody fragments obtained (or obtained) by reacting the compounds of the present invention with antibodies or antibody fragments (wherein the antibody fragment is optionally incorporated into an Fc fusion protein) comprise one or more payloads attached to the antibody or fragment thereof via a divalent group, which is a group derived from the reactive moiety Y in formula (1) (i.e., which corresponds to the reactive moiety Y in formula (1) reacted with a side chain of an amino acid exposed on the surface of the antibody or fragment thereof).

[0258] According to one embodiment, the modified antibody or modified antibody fragment has the following formula (10): (PW) p -A (10) (In the formula, P is the payload as defined above, preferably a moiety identified under items (i)-(iii) above; W is F1-RC', where F1 is bound to P, RC' is a moiety derived from the reactive center (RC) bound to A, and RC is as defined in formulas (3a) and (3b); A is a moiety derived from an antibody or antibody fragment, optionally incorporated into an Fc fusion protein, said antibody or antibody fragment being as defined above; and p is an integer of 1 to 4, preferably 1 to 2. is expressed by

[0259] In instances where the reactive moiety reacts with the side chain of Lys, attachment of the payload to the antibody or antibody fragment occurs via a nitrogen-containing group, such as an amide group, a urethane group, a thiourethane group, or a dithiourethane group. For example, when a compound of the invention contains a reactive moiety of formula (4a) or (4d) (or formula (4a') or (4d')), the divalent group W in formula (10) is a urethane group whose nitrogen atom forms part of the Lys side chain. When a compound contains a reactive moiety of formula (4e), (4f), or (4j) (or formula (4e'), (4f'), or (4j')), for example, the divalent group W is a thiourethane group.

[0260] p represents the degree of conjugation (DoC; sometimes referred to as the "drug-antibody ratio" (DAR)) of the modified antibody or modified antibody fragment.

[0261] According to one embodiment, the modified antibody or modified antibody fragment has the following formula (11): (P 1 -LW) p -A (11) (In the formula, P 1 , L, W, A and p are as defined above) is expressed by

[0262] 7. Use of Modified Antibodies or Modified Antibody Fragments for Diagnostic and / or Therapeutic Purposes The modified antibodies and modified antibody fragments obtained (or obtained) by reacting the compounds of the present invention with antibodies or antibody fragments (wherein the antibody fragment is optionally incorporated into an Fc-fusion protein) can be used to diagnose and / or treat diseases, particularly cancer. Treatment may be therapeutic and / or prophylactic treatment, the purpose of which is to prevent, reduce, or halt an undesired physiological change or disorder. In some instances, treatment may prolong the survival of a subject compared to expected survival if not receiving treatment.

[0263] The disease treated by the engineered antibody or engineered antibody fragment (e.g., ADC) may be any disease that would benefit from treatment, including chronic and acute disorders or diseases, as well as conditions that predispose to the disorder. In some examples, the disease is a neoplastic disease, such as cancer, that can be treated by targeted destruction of tumor cells. Non-limiting examples of cancers that can be treated include benign and malignant tumors, whether solid or liquid; leukemia and lymphoid malignancies, as well as breast cancer, ovarian cancer, gastric cancer, endometrial cancer, salivary gland cancer, lung cancer, kidney cancer, colon cancer, thyroid cancer, pancreatic cancer, prostate cancer, or bladder cancer. The disease may be a disease of neurons, glia, astrocytes, hypothalamus or other glands, macrophages, epithelium, stroma, and blastocyst cavities; or an inflammatory, vascular, or immune disease. An exemplary disease is a solid malignant tumor.

[0264] According to one embodiment, the disease or treatment thereof is Alzheimer's disease, amyotrophic lateral sclerosis, cerebral arteriosclerosis, encephalopathy, Huntington's disease, multiple sclerosis, Parkinson's disease, progressive multifocal leukoencephalopathy, systemic lupus erythematosus, systemic sclerosis, angina pectoris (including unstable angina), aortic aneurysm, atherosclerosis, heart transplant, cardiac toxicity diagnosis, coronary artery bypass graft, heart failure (including systolic dysfunction resulting in atrial fibrillation), high cholesterol In some embodiments, the inflammatory bowel disease is selected from the group consisting of hemoglobinemia, ischemia, myocardial infarction, thromboembolism, thrombosis, ankylosing spondylitis, autoimmune cytopenia, autoimmune myocarditis, Crohn's disease, graft-versus-host disease, granulomatosis with polyangiitis, idiopathic thrombocytopenic purpura, juvenile arthritis, juvenile-onset diabetes mellitus (type 1 diabetes), lupus, microscopic polyangiitis, multiple sclerosis, plaque psoriasis, psoriasis, psoriatic arthritis, rheumatoid arthritis, ulcerative colitis (UC), uveitis, and vasculitis.

[0265] According to one embodiment, the disease to be treated is selected from lymphoma cells, myeloma cells, renal cancer cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, small cell lung cancer cells, testicular cancer cells, pancreatic cancer cells, liver cancer cells, melanoma, head and neck cancer cells, and any cells that are deregulated and grow and divide at a rapid pace, causing cancer; preferably, cells selected from breast cancer cells, small cell lung cancer cells, lymphoma cells, colorectal cancer cells, and head and neck cancer cells.

[0266] According to one embodiment, the modified antibody or modified antibody fragment is used in a method of diagnosing, monitoring, e.g., monitoring the effectiveness of treatment over time, imaging, and / or treating a disease by administering the modified antibody or modified antibody fragment to a subject (e.g., a patient).

[0267] The molecule can be administered to a subject at once or over a series of treatments. Depending on the type and severity of the disease and / or the payload and / or the antibody or antibody fragment, about 0.1 μg / kg to 1 mg / kg of drug can be used as an initial candidate dosage for initial administration in first-in-human trials, e.g., by one or more individual administrations or continuous infusion. Typical daily dosages can range from about 0.1 mg to 50 mg or more, or from about 0.5 to about 30 mg, e.g., 0.5 to about 25 mg per kg of patient body weight. However, typical dosages will depend on a variety of factors, including the particular payload (active agent), the subject's age, weight, general health, sex, and diet; whether administration is for imaging, monitoring, or treatment; and other factors well known in the medical community.

[0268] When treating cancer, the observed therapeutic effect may be a reduction in the number of cancer cells; a reduction in tumor size; an inhibition or delay of cancer cell invasion into peripheral organs; an inhibition of tumor growth; and / or an alleviation of one or more symptoms associated with the cancer.

[0269] According to a preferred embodiment, the modified antibody or modified antibody fragment is administered by injection, such as parenterally, intravenously, subcutaneously, intramuscularly, etc.

[0270] According to a further embodiment, the modified antibody or modified antibody fragment is used in a method of diagnosing, monitoring, e.g., over time, e.g., monitoring the effectiveness of treatment, imaging, and / or treating cancer, and is administered simultaneously with one or more other therapeutic agents, such as a chemotherapeutic agent, radiation therapy, immunotherapy agent, autoimmune disorder agent, anti-infective agent, or one or more other modified antibodies or modified antibody fragments. The other therapeutic agents can also be administered before or after the modified antibody or modified antibody fragment.

[0271] 8. Preparation of Compounds of the Invention Below, methods are provided for preparing ligands, spacers, payload-linkers, and compounds (reactive conjugates) and for their use in the site-selective modification of therapeutic antibodies or therapeutic proteins (e.g., Fc fusion proteins). The compounds of the present invention can be synthesized using standard chemical methods as well as Fmoc-based solid-phase peptide synthesis (SPPS), including on-resin peptide coupling and convergent strategies. The introduction of various payloads and the immobilization of compounds on solid-phase matrices are also exemplified below. General strategies and methods that can be used to prepare the compounds of the present invention are known to those skilled in the art and are illustrated in Figures 2, 5, and 9. [Example]

[0272] 9. Working Example 9.1 List of abbreviations used in the examples: ACN: acetonitrile DCM: dichloromethane DIC: diisopropylcarbodiimide DIEA: Diisopropylethylamine DMF: dimethylformamide DMSO: dimethyl sulfoxide FL or FITC: Fluorescein HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HPLC: High-performance liquid chromatography HRMS: High resolution mass spectrometry PBS: phosphate buffered saline SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis SPPS: Solid Phase Peptide Synthesis TFA: Trifluoroacetic acid TIS: Triisopropylsilane UPLC: Ultra-high performance liquid chromatography WLC: Worm Chain

[0273] 9.2 Starting Materials and Chemicals: The main starting materials and chemicals used in the following examples are listed below: > Resins for solid-phase peptide synthesis (Fmoc-Rink Amide AM resin, 4-Fmoc-hydrazinobenzoyl AM Novagel™) and protected amino acids, N,N-diisopropylcarbodiimide (DIC), piperazine, from Novabiochem (Switzerland), unless otherwise indicated; > Synthesis solvents, deprotection reagents, cleavage reagents from Merck or Fischer Scientific AG (Switzerland); > Maleimidopropionic acid, 4-nitrophenyl chloroformate, TFA, TIS, and DIEA from Sigma-Aldrich (Switzerland); > Amino acids from Bachem AG (Switzerland), Novabiochem, and Aapptec (USA); > Solvents and chemicals for high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) from Macherey-Nagel (Switzerland); > Fluorescently labeled peptide Fc-III-FAM from Genscript (USA); > GingisKhan®, Fabalactica®, and Fabricator® proteases from Genovis (Sweden); > IdeS® protease from Promega (Switzerland); > EndoS® protease from BioConcept (Switzerland); > Biotin-PEG4-amine and PEG linker from BroadPharm (USA); > Herceptin® (commercial trastuzumab) manufactured by Roche (Switzerland); > p-SCN-Bn-CHX-A''-DTPA.3HCl, p-SCN-Bn-PCTA.3HCl manufactured by Macrocyclics (USA); > p-NCS-Bz-DFO manufactured by Chematech (France).

[0274] Biosimilar monoclonal IgG1 antibodies (trastuzumab, alemtuzumab, bevacizumab, rituximab) were produced by culturing recombinant CHO cell lines in the laboratory of Dr. G. Hagens at the University of Applied Sciences (HES-SO Valais / wallis, Switzerland).

[0275] GingisKhan and Fabalactica are cysteine ​​proteases that site-specifically cleave IgG1 above the hinge, thereby generating two Fab fragments and one Fc fragment. Fabricator is a cysteine ​​protease that site-specifically digests antibodies below the hinge, generating F(ab')2 and Fc / 2 fragments.

[0276] 9.3 Method: The following methods were used to evaluate the compounds and conjugates of the present invention:

[0277] 9.3.1 Determining the spacer length The length of the spacer (part S in formula (1)) introduced at the N-terminus of the Fc-binding vector was calculated by using the worm-like chain (WLC) model (Rubinstein and Colby (2003), Polymer Physics, Oxford University Press), which considers the spacer as a continuously flexible rod and has been shown to be a suitable model for biopolymers: <R 2 >=2×L p ×L (In the formula, L pwhere is the persistence length (correlation length in the chain direction), and L is the path length (length of the fully extended chain). For the polyethylene glycol spacer, a persistence length value of 3.8 Å was used (Kienberger et al., Single Molecules 2000, 1(2), pp. 123-128). For the SGGPPPPPP spacer, a length of 20.8 Å was estimated based on procedures described in the literature (Mahoney et al., Nature Chemical Biology 1997, 4(12), pp. 953-960; Garbuio et al., Chemistry: A European Journal 2015, 21(30), pp. 10747-10753).

[0278] 9.3.2 Saturation FP binding assay Saturation fluorescence polarization (FP) measurements were performed on a SpectraMax Paradigm multimode detection platform (available from Molecular Devices) on flat-bottom 384-well Corning microplates (Merck KGaA) using excitation and emission wavelengths of 485 nm and 535 nm, respectively. The acquisition time was 700 ms and the read height was 1 mm. All reagents used in the assay were diluted in PBS containing 0.05% Tween 20.

[0279] The fluorescently labeled peptide Fc-III-FAM (structure shown below) was mixed with a series of dilutions of IgG1 in PBS containing 0.05% Tween to a final peptide concentration of 5 nM. Samples were incubated at 27°C for 15 min, and fluorescence anisotropy was measured in triplicate.

[0280] [ka]

[0281] Fc-III is a 13-mer cyclic peptide known to bind with high affinity to the Fc region of IgG antibodies (DeLano et al., Science 2000, 287, 1279-1283; Nilsson et al., Protein Eng. 1987, 1, 107-113). The fluorescently labeled peptide Fc-III-FAM was prepared by GenScript® using standard SPPS techniques and a convergent strategy.

[0282] 9.3.3 Competitive FP Binding Assay Competitive FP measurements were performed on a SpectraMax Paradigm multimode detection platform (Molecular Devices) in flat-bottom 384-well Corning microplates (Merck KGaA) using excitation and emission wavelengths of 485 nm and 535 nm, respectively. The acquisition time was 700 ms and the read height was 1 mm. All reagents used in the assay were diluted in PBS containing 0.05% Tween 20.

[0283] Increasing concentrations of peptide to be measured were mixed with Fc-III-FAM peptide and added to IgG1 in a total volume of 80 μL. The final concentration of Fc-III-FAM remained constant at 5 nM, while the final concentration of IgG1 ranged from 10 to 30 nM. The mixture was incubated at 27 °C for 15 min, and the fluorescent signal was red in a Spectramax Paradigm. All sample preparations were performed in PBS, pH 7.4 or 7.0, containing 0.05% Tween. Each experiment was performed in triplicate.

[0284] 9.3.4 Peptide and Conjugate Concentration Determination Peptide samples were prepared by dissolving purified peptides or reactive conjugates in DMSO. Concentrations were adjusted to 1000 ppm for Trp (ε = 5500 M) in 1x PBS, pH 7.4. -1 cm -1 ) residue, p-SCN-Bn-CHX-A''-DTPA(ε=13000M -1 cm -1), p-SCN-Bn-PCTA (ε = 13000 M -1 cm -1 ), p-NCS-Bz-DFO (ε = 21000M -1 cm -1 ) absorbance at 280 nm, or FITC (ε = 73000 M -1 cm -1 ) was determined using the absorbance at 496 nm.

[0285] 9.3.5 High resolution mass spectrometry Prior to HRMS analysis, antibody-payload conjugates were desalted against 50 mM ammonium acetate buffered at pH 7.0 using four cycles of concentration / dilution in a microconcentrator (Vivaspin, 30 kD cutoff, Sartorius, Germany). Deglycosylation of the conjugate was achieved by incubation (37°C, 1 hour or overnight) of 1 unit of Endo S per μg of conjugate in the formulation buffer.

[0286] Direct infusion HRMS for peptide / conjugate analysis was performed on a QExactive HF Orbitrap-FT-MS (Thermo Fisher Scientific, Germany) connected to a chip-based automated nanoelectrospray device (Triversa Nanomate, Advion, USA). Electrospray ionization was performed at a capillary voltage of 1.4 kV and a nitrogen nanoflow of 0.15 psi. MS experiments were performed in positive ion mode with a nominal resolution of 45,000. Data deconvolution was performed with Protein Deconvolution (Thermo Fisher Scientific, USA) using the Xtract algorithm with a fit coefficient of 90%.

[0287] For both intact mass measurements (LC-MS) and middle-down analysis (LC-HCDMS / MS), samples were separated on an Acquity UPLC protein column BEH C4 (300 Å, 1.7 μm, 1 × 150 mm, Waters, USA) using a Dionex Ultimate 3000 analytical RSLC system (Dionex, Germany) connected to a HESI source (Thermo Fisher Scientific, Germany). Separation was performed using a 90 μL / min flow rate by applying a gradient of solvent B from 15 to 45% in 2 min, followed by 45 to 60% in 10 min, followed by column wash and re-equilibration steps. Solvent A consisted of water containing 0.1% formic acid, and solvent B consisted of acetonitrile containing 0.1% TFA.

[0288] Eluting proteoforms were analyzed on a high-resolution QExactive HF-HT-Orbitrap-FTMS benchtop instrument (Thermo Fisher Scientific, Germany). For intact mass MS1 scans, scans were performed in protein mode with a resolution of 15,000 and 10 μ-scan averaging. Middle-down analysis for binding site localization was performed in PRM mode, isolating the species at 1356 m / z for Fc / 2-mod, with an isolation width of 300 Th, a resolution of 240,000, and 10 μ-scan averaging. HCD (high-energy collision-induced dissociation) was used as the fragmentation method at normalized collision energies of 12, 15, and 18%.

[0289] Intact mass data were analyzed using Protein Deconvolution (Thermo Fischer Scientific, USA) using the Respect algorithm with a 99% noise removal confidence and an average mass identification accuracy of 20 ppm. Middle-down data were deconvoluted using MASH Suite software (Ge research group, University of Wisconsin). Data acquired using three different NCE values ​​were integrated to generate fragmentation maps with assigned b and y fragments using ProSight Lite software (Kelleher research group, Northwestern University) with a mass tolerance of 15 ppm.

[0290] 9.3.6 Determination of the degree of binding based on HRMS analysis The average degree of coupling (DoC) was calculated using the HRMS data and the following equation 1 (equivalent 1): These results were derived from the relative peak intensities of the deconvoluted mass spectra.

[0291]

number

[0292] (In the formula, I(DoC k ) is the relative peak intensity of the conjugate with k binding molecules per antibody.

[0293] 9.3.7 SDS-PAGE Reducing or non-reducing Bis-Tris SDS-PAGE was performed on Bolt 4-12% Bis-Tris Plus gels (ThermoFisher, Germany). Loading buffer was added to the antibody conjugate (non-reducing Bolt sample buffer, ThermoFisher), and the samples were heated to 70°C for 10 min. For reducing SDS-PAGE, reducing buffer was added to the samples before loading buffer. Gels were run at a constant voltage (200 V) for 25-30 min using Bolt MES running buffer. Fluorescence was visualized on a FluoroM bioimaging system (Syngene, United Kingdom) followed by staining with Coomassie blue.

[0294] Example 1 Preparation and characterization of Fc-binding vectors The Fc-binding vector and vector spacer constructs described herein (portions V or SV of Formula (1)) were prepared using standard Fmoc / tBu-based SPPS, including on-resin coupling, and convergent strategies. The ligands prepared in Example 1 are shown in Table 1 below (underlined bold indicates the presence of a disulfide bond between the side chains of each Cys residue). The spacer length was calculated using the WLC model as described above.

[0295] [Table 1]

[0296] Peptides were prepared by standard Fmoc / tBu-based SPPS using Rink Amide AM resin (loading: 0.57 mmol / g) and a Liberty Blue™ automated microwave peptide synthesizer (available from CEM Corp., Germany).

[0297] The coupling reaction for amide bond formation was carried out at room temperature for 4 min using 0.2 M Fmoc-amino acids preactivated with 0.5 M DIC and 1 M OxymaPure® in DMF. Fmoc deprotection was carried out using 10% piperazine (v / v) in DMF.

[0298] After completion of the synthesis, the peptide was manually cleaved from the resin by treatment with TFA / TIS / water (90 / 5 / 5, v / v / v) with gentle agitation for 1.5 h at room temperature. After filtration and evaporation of the cleavage mixture with a nitrogen stream, the crude peptide was precipitated with cold diethyl ether, centrifuged, and washed with cold diethyl ether. The peptide was dried, dissolved in ultrapure water / ACN, frozen, and lyophilized.

[0299] For disulfide bond formation, the crude lyophilized peptide was resuspended in a mixture of DMSO / ACN / water (2 / 3 / 3, v / v / v). Water was then added until the peptide was dissolved (approximately 35-50 mL). The resulting solution was adjusted to pH 8.5 with NH4HCO3 or NaHCO3 (concentration: 0.1-0.5 mM). The progress of the oxidation was monitored via analytical UPLC-MS. After completion of the reaction, salts were removed using a Sep-Pak C18 Plus Long Cartridge (820 mg of sorbent per cartridge, particle size: 55-105 μm, available from Waters, Switzerland), and the peptide was lyophilized.

[0300] Peptides were purified by preparative reverse-phase HPLC on a Kinetex® XB-C18 column (100 Å, 5 μm, 100 × 21.2 mm, Phenomenex Helvetia) using solvent systems A (0.1% TFA in water) and B (0.1% TFA in ACN) at a flow rate of 35 mL / min and a gradient ranging from 15 to 55% B over 25 min. Peptide elution was monitored at a wavelength of 214 nm. Appropriate fractions were analyzed by UPLC-MS before being concentrated and lyophilized.

[0301] For the synthesis of compounds 3-12 and 15-16, Fmoc-NH-(CH2-CH2-O) n A solution of -CH2-CH2-COOH (where n = 2, 4, 6, 8, 10, 12, 15, 20, 24, or 36, 1.3 eq, 4.7 μmol) and HATU (1.2 eq, 4.33 μmol) in DMF was stirred for 1 min, and DIEA (2 eq, 7.16 μmol) was added. After a 3-min preactivation period, the Fc-binding peptide (compound 1, 13, or 14) in DMF (1 eq, 3.58 μmol) was added to the reaction mixture and stirred at room temperature for 1–2 h. The completion of the reaction was monitored by UPLC-MS. The peptide was then precipitated with cold diethyl ether. Fmoc deprotection was performed using 20% ​​piperidine (v / v) in DMF at room temperature for 30 min, followed by precipitation of the peptide with cold diethyl ether (Figure 2a). The peptide was isolated after HPLC purification (described in the immediately preceding paragraph).

[0302] Peptide purity was determined on a Waters Acquity UPLC system connected to a Micromass Quattro micro API mass spectrometer equipped with a Kinetex® XB-C18 column (100 Å, 1.7 μm, 50 × 2.1 mm, Phenomenex Helvetia) using solvent systems A (0.1% TFA in water) and B (0.1% TFA in ACN) at a flow rate of 0.6 mL / min with a gradient of 2 to 98% B over 4 min. Peptide elution was monitored at a wavelength of 214 nm. The results are shown in the table below.

[0303] [Table 2]

[0304] Example 2 Saturation FP binding assay The tendency of the Fc-binding ligand Fc-III-FAM (structure shown above) to bind to the Fc regions of the IgG1 antibodies trastuzumab, alemtuzumab, bevacizumab, and rituximab was evaluated in the saturation FP binding assay described above. The results are shown in Figure 3. The Fc-binding ligand Fc-III-FAM was confirmed to bind with high affinity to each antibody (trastuzumab: 14 nM, alemtuzumab: 13 nM, bevacizumab: 7 nM, rituximab: 11 nM).

[0305] Example 3 Competitive FP binding assay The tendency of the Fc-binding ligands prepared in Example 1 (compounds 1, 2, 9-11, 13, 15, and 16) to bind to the Fc region of trastuzumab relative to Fc-III-FAM was evaluated in the competitive FP binding assay described above. The results are shown in Table 3 below and Figure 4.

[0306] [Table 3]

[0307] These results confirm that the Fc-binding ligands of Example 1 (compounds 1, 2, 9, 10, and 11) and Fc-III-FAM compete for the same binding site on the Fc region of trastuzumab. Furthermore, the results demonstrate that N-terminal modification of the Fc-III peptide (compound 1) with a spacer moiety (e.g., a peptide spacer such as Ser-(Gly)2-(Pro)6 or a polyethylene glycol spacer) does not affect the binding of the modified peptide to the Fc region of the antibody. Notably, compounds 2, 9, 10, and 11 exhibited high affinity for trastuzumab in a competitive FP binding assay against Fc-III-FAM (Figure 4).

[0308] On the other hand, modification of the Fc-III C-terminal sequence in compounds 15 and 16 (i.e., replacement of Val-Trp-Cys-Thr (VWCT) with Trp-Ala-Cys-Thr (WACT) or Val-Trp-Ala-Thr (VWAT)) reduced peptide binding to the antibody. In the following, compounds or conjugates bearing the modified C-terminal sequence (WACT or VWAT) are used as negative controls.

[0309] Example 4 Preparation of DOTA-, FL-, and DBCO-carbonate derivatives and FL-thioester derivatives, i.e., compounds 17, 18, 19, 20, 21, 22, and 23 Compounds 17, 18, 19, 20, 21, 22, and 23 (moiety PY of formula (1)) were prepared according to the procedures described below and are shown in Figure 5. The structures of each of compounds 17 to 23 are shown in the table below.

[0310] [Table 4A]

[0311] [Table 4B]

[0312] Preparation of Compound 17:

[0313] [ka]

[0314] To a solution of 2.0 g of 2-(2-Boc-aminoethoxy)ethanol 1 (9.6 mmol) in 70 mL of acetonitrile, 5.2 g of N,N'-disuccinimidyl carbonate (19 mmol, 2.0 eq) was added, followed by 2.7 mL of triethylamine (19 mmol, 2.0 eq), and the suspension was stirred at 40 °C for 1 h 30 min. The solvent was removed under vacuum. The residue was dissolved in DCM and filtered through a silica cartridge, eluting with dichloromethane / ethyl acetate 80 / 20 to give crude 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl (2,5-dioxopyrrolidin-1-yl)carbonate (purity >80%, yield: 99%). LCMS: m / z = 247 [M-BOC+H]+, 369 [M+Na]+. 1H NMR (CDCl3): δ 4.52 - 4.40 (m, 2H), 3.77 - 3.68 (m, 2H), 3.55 (t, 2H), 3.32 (dd, 2H), 2.84 (s, 4H), 1.44 (s, 9H).

[0315] A solution of 1.5 g of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl (2,5-dioxopyrrolidin-1-yl)carbonate (3.4 mmol, 2.0 eq) in 12 mL of DCM was treated with 0.35 g of tert-butyl 4-hydroxybenzoate (1.7 mmol) followed by 0.43 g of 4-(dimethylamino)pyridine (3.4 mmol, 2 eq). The reaction mixture was stirred at room temperature for 30 minutes. 50 mL of water was added and extracted with 3 × 10 mL of dichloromethane. The organic layer was concentrated in vacuo. The residue was purified by flash chromatography (cyclohexane / ethyl acetate, 90 / 10 to 60 / 40) to give 0.64 g of tert-butyl-4-[2-[2-(tertbutoxycarbonylamino)ethoxy]ethoxycarbonyloxy]benzoate as a colorless oil (purity >98%, yield: 88%). LCMS: m / z = 326 [M-BOC+H]+, 448 [M+Na]+. 1H NMR (DMSO) δ 7.96 (d, 2H), 7.38 (d, 2H), 6.83 (s, 1H), 4.41 - 4.28 (m, 2H), 3.75 - 3.60 (m, 2H), 3.43 (t, J = 6.0 Hz, 2H), 3.09 (q, 2H), 1.54 (s, 9H), 1.37 (s, 9H)

[0316] To a solution of 0.67 g of the compound obtained above (1.5 mmol) in 6.3 mL of DCM, 2.1 ml of TFA (27 mmol, 17 eq) was added at 0° C., and the reaction mixture was stirred at room temperature for 3 hours. The mixture was concentrated in vacuo to give 0.73 g of the compound 4-[2-(2-aminoethoxy)ethoxycarbonyloxy]benzoic acid; 2,2,2-trifluoroacetic acid as a white solid (purity >80%, yield: 97%). LCMS: m / z = 270 [M+H]+. 1H NMR (DMSO) δ 8.01 (d, 1H), 7.87 (s, 2H), 7.37 (d, 2H), 4.38 (dd, 2H), 3.75 (dd, 2H), 3.65 (t, 2H), 3.06 - 2.97 (m, 2H).

[0317] To a solution of 0.70 g of DOTA-tris(tBu) ester NHS ester (0.83 mmol) in 3.5 mL of ACN, 0.88 mL of DIEA (5.0 mmol, 6.0 eq) was added, followed by 0.44 g of 4-[2-(2-aminoethoxy)ethoxycarbonyloxy]benzoic acid (0.92 mmol, 1.1 eq). The reaction mixture was stirred at room temperature for 10 min. (Note: Solubilized solids appeared immediately after sonication.) The solution was diluted with 3.5 mL of water and purified by flash chromatography on a C18 cartridge (water / ACN, 90 / 10 to 0 / 100). The fractions were collected, concentrated under vacuum, and lyophilized to give 0.66 g of compound 17 (4-[2-[2-[[2-[4,7,10-tris(2-tert-butoxy-2-oxo-ethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]ethoxycarbonyloxy]benzoic acid) as a white solid (purity >95%, yield: 93%). LCMS: m / z = 824 [M+H]+, 413 [M / 2+H]+. 1H NMR (DMSO) δ 8.56 (s, 1H), 7.95 (d, 2H), 7.27 (d, 2H), 4.31 (s, 2H), 3.66 (s, 2H), 3.48 - 3.42 (m, 2H), 3.35 - 3.25 (m, 8H), 3.00 (s, 2H), 2.75 (s, 8H), 2.63 (s, 4H), 1.37 (s, 27H).

[0318] Preparation of Compound 18:

[0319] [ka]

[0320] To a solution of 1.0 g of 5-hydroxy-2-nitrobenzoic acid (5.4 mmol) in 14 mL of toluene, 7.6 mL of 2-methylpropan-2-ol (80 mmol, 15 eq) was added and the reaction mixture was heated at 85 °C. 4.5 mL of N,N-dimethylformamide dineopentyl acetal (16 mmol, 3.0 eq) was added slowly, and the reaction mixture was stirred at 85 °C for 3 h. The reaction was cooled, and then 10 mL of a saturated aqueous solution of NaHCO was added, and the aqueous layer was extracted with 3 × 5 mL of ethyl acetate. The combined organic layers were washed with 10 mL of water and concentrated under vacuum to give 1.1 g of crude tert-butyl 5-hydroxy-2-nitrobenzoate as a yellow oil (purity: 89%, yield: 73%). LCMS: m / z = 238 [MH]-. 1H NMR (DMSO): δ 8.00 (d, 1H), 7.00 (dd, 1H), 6.92 (d, 1H), 1.50 (s, 9H).

[0321] A solution of 0.35 g of crude tert-butyl 5-hydroxy-2-nitrobenzoate (1.3 mmol) in 5.0 mL of DCM was added to 0.90 g of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl (2,5-dioxopyrrolidin-1-yl)carbonate (2.6 mmol, 2.0 eq, prepared as described above), followed by 0.46 mL of DIEA (2.6 mmol, 2.0 eq). The reaction mixture was stirred at room temperature for 30 min. The mixture was purified by flash chromatography (cyclohexane / ethyl acetate 90 / 10 to 40 / 60) to afford 0.18 g of 5-((11,11-dimethyl-9-oxo-2,5,10-trioxa-8-azadodecanoyl)oxy)-2-nitrobenzoic acid as a yellow oil (purity: 99%, yield: 29%). LCMS: m / z = 315 [M-Boc-(t-Bu)+H]+, 371 [M-Boc+H]+, 493 [M+Na]+. 1H NMR (DMSO): δ 8.15 (d, 1H), 7.77 (d, 1H), 7.69 (dd, 1H), 6.84 (s, 1H), 4.39 - 4.32 (m, 2H), 3.71 - 3.65 (m, 2H), 3.47 - 3.39 (m, 2H), 3.14 - 3.05 (m, 2H), 1.50 (s, 9H), 1.37 (s, 9H).

[0322] To a solution of 0.60 mL of TFA (7.8 mmol, 21 equivalents) in 1.8 mL of DCM, 0.17 g of 5-((11,11-dimethyl-9-oxo-2,5,10-trioxa-8-azadodecanoyl)oxy)-2-nitrobenzoic acid (0.37 mmol) was added, and the reaction mixture was stirred at room temperature for 2 hours. 0.30 mL of TFA was added, and the mixture was stirred at room temperature for 30 minutes. The solvent was evaporated in vacuo to give 0.23 g of crude 5-[2-(2-aminoethoxy)ethoxycarbonyloxy]-2-nitrobenzoic acid; 2,2,2-trifluoroacetic acid as a yellow oil (purity: 67%, yield: quantitative). LCMS: m / z = 315 [M+H]+. 1H NMR (DMSO): δ 8.13 (d, 1H), 7.78 (d, 1H), 7.67 (dd, 1H), 4.46 - 4.35 (m, 2H), 3.01 (q, 2H).

[0323] To a solution of 0.22 g of 5-((11,11-dimethyl-9-oxo-2,5,10-trioxa-8-azadodecanoyl)oxy)-2-nitrobenzoic acid (0.34 mmol, 1.15 equivalents) in 1.3 mL of acetonitrile was added 0.25 g of tri-tert-butyl 2,2′,2″-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (0.30 mmol), followed by 0.31 mL of N,N-diisopropylethylamine (1.8 mmol, 6.0 equivalents), and the reaction mixture was stirred at room temperature for 10 minutes. 1.5 mL of water was added and the solution was purified by C18 flash chromatography (water / acetonitrile 95 / 5 to 0 / 1) to give 85 mg of compound 18 (2-nitro-5-[2-[2-[[2-[4,7,10-tris(2-tert-butoxy-2-oxo-ethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]ethoxycarbonyloxy]benzoic acid) as a clear yellow solid (purity >80%, yield: 26%). LCMS: m / z = 701 [M-3(t-Bu)+H]+, 757 [M-2(t-Bu)+H]+, 813 [M-(t-Bu)+H]+, 869 [M+H]+. 1H NMR (DMSO): δ 8.62 (s, 1H), 7.69 (d, 1H), 7.39 (d, 1H), 7.23 (dd, 1H), 4.38 - 4.31 (m, 2H), 3.70 - 3.67 (m, 2H), 1.42 (s, 6H), 1.41 (s, 27H).

[0324] Preparation of Compound 19:

[0325] [ka]

[0326] To a solution of 0.71 g of 4-[2-(2-aminoethoxy)ethoxycarbonyloxy]benzoic acid; 2,2,2-trifluoroacetic acid (1.2 mmol, 1.2 equiv.) in 4.0 mL of ACN, 0.40 g of fluorescein isothiocyanate isomers (1.0 mmol) and 4.0 mL of dimethylformamide were added, followed by 1.1 mL of N,N-diisopropylethylamine (6.0 mmol, 6.0 equiv.). The mixture was stirred at room temperature for 10 minutes. The solvent was evaporated under vacuum. The residue was purified by C 18 Purification by flash chromatography (water / acetonitrile 95 / 5 to 0 / 1) gave 0.38 g of compound 19 (4-[2-[2-[(3′,6′-dihydroxy-3-oxo-spiro[isobenzofuran-1,9′-xanthen]-5-yl)carbamothioylamino]ethoxy]ethoxycarbonyloxy]benzoic acid) as an orange solid (purity: 98%, yield: 56%). LCMS: m / z = 657 [MH]-, 659 [M+H]+. 1H NMR (DMSO): δ 13.07 (s, 1H), 10.24 - 9.95 (m, 3H), 8.26 (s, 1H), 8.16 (s, 1H), 7.98 (d, 2H), 7.74 (d, 1H), 7.35 (d, 2H), 7.18 (d, 1H), 6.67 (d, 2H), 6.61 - 6.53 (m, 4H), 4.42 - 4.37 (m, 2H), 3.80 - 3.66 (m, 6H).

[0327] Preparation of Compound 20:

[0328] [ka]

[0329] To a solution of 194 mg of tri(ethylene glycol)bis(chloroformate) (0.690 mmol, 2.0 eq) and 0.070 mL of DIEA (0.420 mmol, 1.2 eq) in anhydrous DCM (1.20 mL) was added 3-amino-1-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-1-propanone (100 mg, 0.350 mmol, 1.0 eq) dropwise over 10 minutes at 0°C. The reaction was stirred at room temperature. After 10 minutes, a solution of 0.3 mL of DIEA (1.73 mmol, 5.0 eq) in anhydrous DCM (1.20 mL) and 336 mg of tert-butyl 4-hydroxybenzoate (1.73 mmol, 5.0 eq) were added to the reaction mixture. The reaction was stirred at room temperature for 30 minutes. A saturated aqueous solution of ammonium chloride was then added to the reaction mixture, and the mixture was extracted with DCM (2 × 5 mL). The combined organic extracts were dried over MgSO. After filtration, the solvent was removed under vacuum, and the residue was purified by flash chromatography (cyclohexane / ethyl acetate, 40 / 60 to 10 / 90) to give 67.6 mg of tert-butyl 4-[2-[2-[2-[[3-(2-azatricyclo[10.4.0.04,9]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl)-3-oxo-propyl]carbamoyloxy]ethoxy]ethoxy]ethoxycarbonyloxy]benzoate. (Purity: 80%, Yield: 23%). LCMS: m / z = 673.3 [M+H].

[0330] A solution of 67.6 mg of the tert-butyl ester compound in 1:1 DCM / TFA was stirred at room temperature for 5 hours. The solution was concentrated under reduced pressure and the residue was purified by C 18Purification by flash chromatography (water / ACN modified with 0.1% TFA, 80 / 20 to 20 / 80) gave 40.3 mg of compound 20 (purity: 80%, yield: 59%). LCMS: m / z = 615 [M-H], 617 [M+H]. 1H NMR (CDCl3): δ 8.18 - 8.07 (m, 2H), 8.04 - 7.95 (m, 2H), 7.70 - 7.25 (m, 8H), 5.16 (s, 2H), 4.47 - 4.37 (m, 2H), 4.31 - 4.21 (m, 2H), 3.94 - 3.62 (m, 8H), 3.44 - 3.32 (m, 2H).

[0331] Preparation of Compound 21:

[0332] [ka]

[0333] To a solution of 6-hydroxy-2-naphthoic acid (941 mg, 5.00 mmol) in 2-methyltetrahydrofuran (20.0 mL) was added a solution of 2-tert-butyl-1,3-diisopropylisourea (4.00 mL, 15.0 mmol) in 2-methyltetrahydrofuran (5.00 mL). The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was filtered through a plug of silica and rinsed with ethyl acetate. The filtrate was washed with saturated aqueous NaHCO3 solution and brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by normal phase chromatography (Biotage Isolera, 40 g, Silicycle SiliaSep cartridge) using 0-40% ethyl acetate in heptane to give the desired compound (715 mg, 59% yield, 99% purity) as an orange oil. ESI: m / z = 243 (MH) - . 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.59 (s, 9H), 7.14-7.21 (m, 2H), 7.75 (d, 1H), 7.82 (dd, 1H), 7.95 (d, 1H), 8.40 (s, 1H), 10.15 (br s, 1H).

[0334] To a solution of tert-butyl 6-hydroxy-2-naphthoate (200 mg, 0.82 mmol) and tert-butyl (2-(2-((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)ethoxy)ethyl)carbamate (567 mg, 1.64 mmol) in dichloromethane (15.0 mL) was added 4-dimethylaminopyridine (200 mg, 1.64 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was washed with water, and the aqueous layer was washed with dichloromethane. The combined organic layers were concentrated under reduced pressure. The residue was purified by normal phase chromatography (Biotage Isolera, 60 g, Silicycle SiliaSep cartridge) using 10-90% ethyl acetate in heptane to afford the title compound (190 mg, 49% yield, 98% purity) as a colorless solid. ESI: m / z = 498 (M+Na) + . 1 H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.38 (s, 9H), 1.61 (s, 9H), 3.11 (q, 2H), 3.46 (t, 2H), 3.68-3.72 (m, 2H), 4.34-4.39 (m, 2H), 6.81-6.86 (m, 1H), 7.53 (dd, 1H), 7.90 (d, 1H), 7.96-8.05 (m, 2H), 8.22 (d, 1H), 8.60 (br s, 1H).

[0335] To a solution of tert-butyl 6-((11,11-dimethyl-9-oxo-2,5,10-trioxa-8-azadodecanoyl)oxy)-2-naphthoate (190 mg, 0.400 mmol) in dichloromethane (10.0 mL) was added trifluoroacetic acid (1.00 mL), and the reaction mixture was stirred at room temperature for 22 hours. The mixture was concentrated under reduced pressure. The residue was dissolved in N,N-dimethylformamide (2.00 mL) and acetonitrile (2.00 mL), and then fluorescein isothiocyanate isomer 1 (204 mg, 0.520 mmol) was added, followed by DIPEA (343 μL, 1.97 mmol). The reaction mixture was stirred at room temperature for 90 minutes. This material was purified by reverse-phase chromatography (Biotage Isolera, 60 g, C18 SNAP Ultra Biotage cartridge) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (90:10 to 0:100). The product-containing fractions were freeze-dried to give the desired compound (180 mg, 64% yield, 71% purity). ESI: m / z = 707 (M−H). - . 1 H NMR (400 MHz, DMSO-d6) δ [ppm] = 3.68-3.83 (m, 6H), 4.41-4.46 (m, 2H), 6.52-6.70 (m, 6H), 7.19 (d, 1H), 7.50 (dd, 1H), 7.75 (d, 1H), 7.88 (d, 1H), 8.01 (s, 2H), 8.14-8.24 (m, 2H), 8.28 (d, 1H), 8.65 (s, 1H), 10.06 (br s, 1H), 10.11 (br s, 2H), 13.13 (br s, 1H).

[0336] To a solution of 6-(((2-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)thioureido)ethoxy)ethoxy)carbonyl)oxy)-2-naphthoic acid (70.0 mg, 0.099 mmol) in N,N-dimethylformamide (1.00 mL) was added N-hydroxysuccinimide (34.0 mg, 0.300 mmol), followed by EDCI.HCl (57.0 mg, 0.300 mmol). The mixture was stirred at room temperature for 4 hours and then purified on a 60 g C18 column using an eluent of 5-95% acetonitrile (0.1% formic acid) in water (0.1% formic acid). The desired fractions were combined and freeze-dried to give the title compound (55.0 mg, 69% yield, 95% purity). ESI: m / z = 806 (M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ [ppm] = 2.94 (s, 4H), 3.68-3.84 (m, 6H), 4.42-4.48 (m, 2H), 6.51-6.63 (m, 4H), 6.66 (d, 2H), 7.19 (d, 1H), 7.61 (dd, 1H), 7.75 (d, 1H), 7.99 (d, 1H), 8.08 (dd, 1H), 8.16 (d, 2H), 8.28 (d, 1H), 8.34 (d, 1H), 8.91 (s, 1H), 10.00-10.15 (m, 3H).

[0337] Preparation of Compound 22:

[0338] [ka]

[0339] To a solution of 6-hydroxyquinoline-2-carboxylic acid (750 mg, 3.96 mmol) in tert-butanol (40.0 mL) was added a solution of 2-tert-butyl-1,3-diisopropylisourea (3.20 mL, 11.9 mmol) in tert-butanol (5.00 mL). The reaction mixture was stirred at room temperature for 3 days. The reaction mixture was concentrated under reduced pressure, and the residue was suspended in ethyl acetate and filtered through a plug of silica, rinsing with ethyl acetate. The filtrate was concentrated under reduced pressure and then purified by normal phase chromatography (Biotage Isolera, 40 g, Silicycle SiliaSep cartridge) using 5-50% ethyl acetate in heptane to give the desired compound (378 mg, 39% yield, 95% purity) as an orange oil. ESI: m / z = 244 (MH) - . 1 H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.60 (s, 9H), 7.21 (d, 1H), 7.40 (dd, 1H), 7.94 (d, 1H), 7.99 (d, 1H), 8.27 (d, 1H), 10.42 (br s, 1H).

[0340] To a solution of tert-butyl 6-hydroxyquinoline-2-carboxylate (200 mg, 0.820 mmol) and tert-butyl (2-(2-((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)ethoxy)ethyl)carbamate (706 mg, 2.04 mmol) in dichloromethane (15.0 mL) was added 4-dimethylaminopyridine (199 mg, 1.64 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was washed with water, and the aqueous layer was washed with dichloromethane. The combined organic layers were concentrated under reduced pressure. The residue was purified by normal phase chromatography (Biotage Isolera, 40 g, Silicycle SiliaSep cartridge) using 10–90% ethyl acetate in heptane to give the desired compound (278 mg, 71% yield, 94% purity) as a colorless oil. ESI: m / z = 499 (M+Na) + . 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.38 (s, 9H), 1.62 (s, 9H), 3.07-3.16 (m, 2H), 3.46 (t, 2H), 3.68-3.74 (m, 2H), 4.35-4.41 (m, 2H), 6.80-6.87 (m, 1H), 7.79 (dd, 1H), 8.00 (d, 1H), 8.10 (d, 1H), 8.22 (d, 1H), 8.56 (d, 1H).

[0341] To a solution of tert-butyl 6-((11,11-dimethyl-9-oxo-2,5,10-trioxa-8-azadodecanoyl)oxy)quinoline-2-carboxylate (290 mg, 0.61 mmol) in dichloromethane (5.00 mL) was added trifluoroacetic acid (1.50 mL), and the reaction mixture was stirred at room temperature for 26 hours. The mixture was concentrated under reduced pressure. The residue was dissolved in N,N-dimethylformamide (2.00 mL) and acetonitrile (2.00 mL), and then fluorescein isothiocyanate isomer 1 (237 mg, 0.610 mmol) was added, followed by DIPEA (530 μL, 3.04 mmol). The reaction mixture was stirred at room temperature for 2 hours. This material was purified by reverse-phase chromatography (Biotage Isolera, 60 g, C18 SNAP Ultra Biotage cartridge) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (90:10 to 100:0). The product-containing fractions were freeze-dried to give the title compound (150 mg, 35% yield, 91% purity). ESI: m / z = 710 (M+H). + . 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 3.68-3.83 (m, 6H), 4.41-4.46 (m, 2H), 6.52-6.69 (m, 6H), 7.18 (d, 1H), 7.72-7.80 (m, 2H), 7.97 (d, 1H), 8.12-8.22 (m, 3H), 8.27 (d, 1H), 8.53 (d, 1H), 10.00-10.20 (m, 3H), 13.49 (br s, 1H).

[0342] To a solution of 6-(((2-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)thioureido)ethoxy)ethoxy)carbonyl)oxy)quinoline-2-carboxylic acid (70.0 mg, 0.099 mmol) in N,N-dimethylformamide (1.00 mL) was added N-hydroxysuccinimide (34.0 mg, 0.300 mmol), followed by EDCI.HCl (57.0 mg, 0.300 mmol). The mixture was stirred at room temperature for 2 hours and then purified on a 60 g C18 column using an eluent of 5-95% acetonitrile (0.1% formic acid) in water (0.1% formic acid). The desired fractions were combined and freeze-dried to give the title compound (55.0 mg, 69% yield, 92% purity). ESI: m / z = 807 (M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ [ppm] = 2.94 (s, 4H), 3.68-3.85 (m, 6H), 4.43-4.48 (m, 2H), 6.52-6.63 (m, 4H), 6.66 (d, 2H), 7.19 (d, 1H), 7.75 (d, 1H), 7.87 (dd, 1H), 8.09 (d, 1H), 8.13-8.21 (m, 1H), 8.25-8.33 (m, 3H), 8.72 (d, 1H), 10.00-10.13 (m, 3H).

[0343] Preparation of Compound 23:

[0344] [ka]

[0345] To a solution of 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propanoic acid (300 mg, 1.29 mmol) in dichloromethane (3.00 mL) was added EDCI.HCl (296 mg, 1.54 mmol), followed by 1-hydroxypyrrolidine-2,5-dione (177 mg, 1.54 mmol). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with dichloromethane and washed with water. The organic layer was passed through a 15.0 mL Telos phase separation cartridge and concentrated to give the desired product (313 mg, 79% purity) as a colorless oil. It was used in the next step without purification. ESI: m / z = 353 (M+Na) + , 231 (M-Boc+H) + . 1 H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.38 (s, 9H), 2.82 (s, 4H), 2.92 (t, 2H), 3.04-3.09 (m, 2H), 3.40 (t, 2H), 3.69 (t, 2H), 6.71-6.75 (m, 1H). The NMR spectrum contains unknown impurities: 2.50(t), 3.50(t).

[0346] To a suspension of 2,5-dioxopyrrolidin-1-yl 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propanoate (200 mg, 0.606 mmol) in dichloromethane (4.00 mL) was added 4-mercaptohydrocinnamic acid (88.4 mg, 0.485 mmol), followed by 4-dimethylaminopyridine (148 mg, 1.21 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was washed with 10% aqueous citric acid and then with water. The organic layer was passed through a 15.0 mL Telos phase separation cartridge, and the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase chromatography (Biotage Isolera, 30 g, C18 SNAP Ultra Biotage cartridge) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (80:20 to 20:80). The appropriate fractions were freeze-dried to give the desired product (94.0 mg, 29% yield over two steps, 97% purity) as a white solid. ESI: m / z = 298 (M-Boc+H). + . 1 H NMR (400 MHz, CDCl3) δ [ppm] = 1.44 (s, 9H), 2.70 (t, 2H), 2.85-2.91 (m, 2H), 2.99 (t, 2H), 3.27-3.32 (m, 2H), 3.49 (t, 2H), 3.76 (t, 2H), 4.94 (br s, 1H), 7.27 (d, 2H) (overlapped with CHCl3 peak), 7.36 (d, 2H).

[0347] To a solution of 3-(4-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)propanoyl)thio)phenyl)propanoic acid (180 mg, 0.453 mmol) in dichloromethane (2.25 mL) was added trifluoroacetic acid (0.59 mL, 7.70 mmol) and the reaction mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to give the desired product (205 mg, 80% purity) as a pale yellow oil. It was used in the next step without purification. ESI: m / z = 298 (M+H) + . 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 2.57 (t, 2H), 2.87 (t, 2H), 2.96-3.01 (m, 4H), 3.58 (t, 2H), 3.74 (t, 2H), 7.31-7.36 (m, 4H), 7.76 (br s, 3H).

[0348] To a solution of 3-(4-((3-(2-aminoethoxy)propanoyl)thio)phenyl)propanoic acid trifluoroacetate (max. 0.453 mmol) in N,N-dimethylformamide (3.70 mL) and acetonitrile (3.70 mL) was added fluorescein isothiocyanate isomer 1 (176 mg, 0.453 mmol), followed by DIPEA (0.12 mL, 0.680 mmol). The reaction mixture was stirred at room temperature for 1 h and then concentrated under reduced pressure. This material was purified by reverse-phase chromatography (Biotage Isolera, 60 g, C18 SNAP Ultra Biotage cartridge) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (95:5 to 20:80). The product-containing fractions were freeze-dried to give the desired compound (145 mg, 51% yield over two steps, 68% purity) as an orange solid. ESI: m / z = 687 (M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ [ppm] = 2.55 (t, 2H) (overlapping with DMSO peak), 2.85 (t, 2H), 2.99 (t, 2H), 3.61 (t, 2H), 3.67-3.72 (m, 2H), 3.76 (t, 2H), 6.55-6.69 (m, 6H), 7.18 (d, 1H), 7.29-7.34 (m, 4H), 7.74 (d, 1H), 8.10 (br s, 1H), 8.26 (s, 1H), 10.05 (br s, 1H), 10.13 (br s, 2H), 12.16 (br s, 1H).

[0349] To a solution of 3-(4-((3-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)thioureido)ethoxy)propanoyl)thio)phenyl)propanoic acid (140 mg, 0.204 mmol) in N,N-dimethylformamide (4.70 mL) was added N-hydroxysuccinimide (117 mg, 1.02 mmol), followed by EDCI.HCl (196 mg, 1.02 mmol). Stirring was continued at room temperature for 1 h. The reaction mixture was directly purified by reverse-phase chromatography (Biotage Isolera, 60 g, C18 SNAP Ultra Biotage cartridge) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (80:20 to 30:70). The product-containing fractions were freeze-dried to give the desired compound (25.3 mg, 16% yield, 81% purity) as an orange solid. ESI: m / z = 784 (M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ [ppm] = 2.81 (s, 4H), 2.96-3.06 (m, 6H), 3.62 (t, 2H), 3.68-3.71 (m, 2H), 3.76 (t, 2H), 6.56 (dd, 2H), 6.61 (d, 2H), 6.68 (d, 2H), 7.18 (d, 1H), 7.33 (d, 2H), 7.39 (d, 2H), 7.74 (d, 1H), 8.08 (br s, 1H), 8.26 (d, 1H), 10.03 (br s, 1H), 10.13 (br s, 2H).

[0350] Example 5 Preparation of DOTA-containing reactive conjugates The Fc-binding vectors prepared in Example 1 were converted into reactive conjugates of formula (1) by coupling compound 17 (or compound 19) to the N-terminus of each Fc-binding vector (FIG. 2b). The structures of the DOTA-containing reactive conjugates prepared in Example 5 are shown in the following table.

[0351] [Table 5A]

[0352] [Table 5B]

[0353] To prepare the reactive conjugate, a solution of the carbonate derivative (1.2 eq, compound 17) in DMF was added to HATU (1.1 eq) and stirred for 1 min, followed by the addition of DIEA (2 eq). After 3 min, the preactivated carbonate derivative for the Fc-binding vector and the reaction mixture were stirred at room temperature for 2–4 h. The reaction completion was monitored by UPLC-MS. If the reaction was not complete, an additional amount of the preactivated carbonate derivative (approximately 1–3 eq) was added, and the mixture was further stirred for 1–2 h. The reactive conjugate was precipitated with cold diethyl ether and purified by HPLC (as described above).

[0354] The tert-butyl protecting group of the DOTA moiety was then removed by treatment with TFA / TIS / water (95 / 2.5 / 2.5, v / v / v) for 2.5 h at room temperature, followed by precipitation with cold diethyl ether and purification by HPLC (as described above).

[0355] The purity of the reactive conjugate was determined on a Waters Acquity UPLC system connected to a Micromass Quattro micro API mass spectrometer equipped with a Kinetex® XB-C18 column (100 Å, 1.7 μm, 50 × 2.1 mm, Phenomenex Helvetia) using solvent systems A (0.1% TFA in water) and B (0.1% TFA in ACN) at a flow rate of 0.6 mL / min with a gradient of 2 to 98% B over 4 min. Elution of the conjugate was monitored at a wavelength of 214 nm. The results are shown in the table below.

[0356] [Table 6]

[0357] Indium chelation at the DOTA moiety was carried out by dissolving InCl in ultrapure water (1.5 eq, 14.2 nmol, 2 μL) and mixing with the reactive conjugate described above (9.45 nmol, 5 μL) in 50 mM sodium acetate buffer, pH 5 (3 μL) and incubating for 5–30 min at 37°C. In chelation was monitored and analyzed by UPLC-MS.

[0358] Example 6 Preparation of trastuzumab-DOTA conjugates The tendency of the reactive conjugates of Example 5 to react with antibodies was evaluated using trastuzumab as a model system. To prepare the trastuzumab-DOTA conjugate, 2 eq of the reactive conjugate prepared in Example 5 (compounds 24-33, 1.62 nmol, 0.86 μL) in DMF was added to a solution of trastuzumab (1 eq, 0.81 nmol, commercial trastuzumab Herceptin® available from Roche, buffer-exchanged into phosphate-buffered saline (PBS) prior to conjugation) diluted in 50 mM NaHCO, pH 9.0, and the reaction mixture (24 μL) was stirred at room temperature for 2 hours.

[0359] After DOTA conjugation, the reaction buffer was diluted with 0.1 M glycine, pH 2.5, or exchanged into 0.1 M glycine, pH 2.5 using a 30 kDa MWCO Vivaspin® 500 centrifugal concentrator. The antibody conjugate was then purified by gel filtration chromatography using a pre-equilibrated Bio-Spin P-30 column (bed height: 3.7 cm, total length: 5 cm, available from Bio-Rad, USA) and then eluted with 0.1 M glycine, pH 2.5. The purified antibody conjugate fraction was neutralized with 1 M PBS, pH 8.5.

[0360] The conjugation of the DOTA moiety with trastuzumab was assessed by HRMS analysis (described above). An exemplary HRMS spectrum of a trastuzumab-DOTA conjugate prepared by reacting compound 31 with trastuzumab is shown in Figure 6. The sample exhibited +517 Da adducts (D1-D3), characteristic of DOTA incorporation.

[0361] The payload loading ratio (selectivity) between Fc and F(ab)2 was assessed by digesting the conjugates with GingisKhan protease (1 unit per μg of antibody conjugate in the presence of 2 mM cysteine, 0.1 M Tris, pH 8.0, for 1 hour at 37°C) followed by HRMS analysis (described above). An exemplary HRMS spectrum of the digested conjugate is shown in Figure 7. Peaks D0-D2 correspond to the number of conjugated DOTA moieties, while G0F / G0F, G0F / G1F, and G1F / G1F correspond to different glycans on the Fc domain.

[0362] The degree of conjugation (DoC) of the trastuzumab-DOTA conjugate was assessed based on the results of HRMS analysis (described above), the results of which are shown in Table 6 below.

[0363] [Table 7]

[0364] These results indicate that compounds 27 to 33 (reactive conjugates) were able to produce trastuzumab-DOTA conjugates with excellent selectivity to the Fc region of the antibody. In particular, compounds 27, 30, and 31 produced trastuzumab-DOTA conjugates with excellent selectivity and yield.

[0365] Trastuzumab-DOTA conjugates were analyzed by peptide mapping using HRMS to determine the conjugation sites of the DOTA moieties on the antibody (data not shown). It was found that in most conjugates, Lys317 in the Fc region was nearly quantitatively labeled, but labeling of Lys326 was additionally observed in higher DoC conjugates carrying three DOTA moieties per Fc region.

[0366] Example 7 Affinity of trastuzumab-DOTA conjugate and trastuzumab to SK-BR-3 (HER2+) and MD-MB-231 (HER2-) cells The tendency of trastuzumab-DOTA conjugates to bind to adenocarcinoma cells was assessed by measuring the affinity of the conjugates for SKBR-3 (HER2+) and MDA-MB-231 (HER2-) breast adenocarcinoma cell lines. In particular, the affinity of a trastuzumab-DOTA conjugate (similar to conjugate 8, DoC = 0.89) prepared as described in Example 6 was measured using flow cytometry by incubating the trastuzumab-DOTA conjugate and (unlabeled) trastuzumab with SKBR-3 or MDA-MB-231 cells. A fluorescent secondary antibody specific for trastuzumab was then added, and binding was measured by fluorescence. The results are shown in Figure 8.

[0367] As can be seen in Figure 8, the median fluorescence intensity (MFI) increased in a dose-response manner when using unlabeled trastuzumab and the trastuzumab-DOTA conjugate, confirming that DOTA conjugation did not affect antibody binding to SKBR-3 cells. The decrease in mean fluorescence intensity of trastuzumab and the conjugate at a concentration of 30 μg / mL (SKBR-3 cells) can be explained by the high concentration of primary antibody. No binding to MDA-MB-231 was observed in either sample (negative control).

[0368] Example 8 Preparation of FL-containing reactive conjugates The Fc-binding vectors prepared in Example 1 were converted into reactive conjugates of formula (1) (compounds 35-42) by coupling compound 19 to the N-terminus of each Fc-binding ligand according to the same procedure as described in Example 5 above (FIG. 9b). The FL-containing reactive conjugates prepared in Example 8 are shown in the table below.

[0369] [Table 8]

[0370] The purity of the reactive conjugate was determined by UPLC-MS (described above) and the results are shown in the table below.

[0371] [Table 9]

[0372] Example 9 Preparation of trastuzumab-FL conjugates The tendency of the reactive conjugates of Example 8 to react with antibodies was evaluated using trastuzumab as a model system. Trastuzumab-FL conjugates were prepared according to the same procedure as described in Example 6 above using compounds 35-41. The obtained trastuzumab-FL conjugates were analyzed by SDS-PAGE (FIG. 10).

[0373] It was found that compounds 36-39 resulted in efficient trastuzumab labeling with good selectivity for the Fc region (lanes 2-5 in Figure 10). No trastuzumab labeling was observed when compounds 40 and 41 were used (negative control, lanes 6 and 7).

[0374] To prepare trastuzumab-FITC (random) conjugates, 10 eq of FITC in DMSO (0.47 μmol, 25.5 μL) was added to a solution of trastuzumab (1 eq, 47 nmol, commercial trastuzumab Herceptin® available from Roche, buffer-exchanged into phosphate-buffered saline (PBS) prior to conjugation) diluted in 50 mM NaHCO, pH 9.0, and the reaction mixture (1.4 mL) was stirred at room temperature for 16 h.

[0375] After FITC conjugation, the reaction buffer was diluted with 0.1 M glycine, pH 2.5. The antibody conjugate was then purified by gel filtration chromatography using a pre-equilibrated column (bed height: 5.0 cm) manually packed with Bio-Spin P-30 fine beads, and then eluted with 0.1 M glycine, pH 2.5. The purified antibody conjugate fraction was neutralized with 1 M phosphate buffer, pH 8.5.

[0376] Conjugation of the moiety to trastuzumab was assessed by HRMS analysis (described above). The results of HRMS analysis of trastuzumab-FITC and trastuzumab-Fl are shown in Table 9 below.

[0377] [Table 10]

[0378] Example 10 Affinity of trastuzumab-FL and -FITC conjugates (11, 12) for BT-474 (HER2+) and MDA-MB33 (HER2-) cells The tendency of trastuzumab-FL, -FITC conjugates to bind to adenocarcinoma cells was assessed by measuring the affinity of the conjugates for BT-474 (HER2+) and MDA-MB33 (HER2-) breast adenocarcinoma cell lines. In particular, the affinity of trastuzumab-FITC conjugates prepared as described in Example 9 (MS data shown in Table 9) was measured using flow cytometry by incubating trastuzumab-FL, -FITC conjugates and (unlabeled) trastuzumab with SKBR-3 or MDA-MB-231 cells.

[0379] As seen in Figure 11, the mean fluorescence index (MFI) of the FITC- and Fl-conjugated antibodies decreased in a dose-response manner after the addition of unlabeled trastuzumab (competing antibody). At equimolar concentrations of labeled and unlabeled antibodies (10 μg / ml), a decrease in MFI approaching 70% was observed for conjugate 12, whereas a decrease in MFI approaching 50% was observed for conjugate 11. These results suggest that fluorescein conjugation does not affect antibody binding to HER2 cells, but the random labeling of conjugate 12 does affect antibody affinity. No binding to MDA-MB33 was observed in either sample (negative control, data not shown).

[0380] Example 11 Preparation of antibody-FL conjugates using trastuzumab, commercial trastuzumab, alemtuzumab, bevacizumab, and rituximab The tendency of the reactive conjugates of the present invention to react with various antibodies was evaluated using trastuzumab, commercially available trastuzumab (Herceptin®), alemtuzumab, bevacizumab, and rituximab. Antibody-FL conjugates were prepared using compound 38 and the aforementioned antibodies according to the same procedure as described in Example 6 above. The conjugates were analyzed by SDS-PAGE (FIG. 12).

[0381] It was found that compound 38 resulted in efficient antibody labeling (lanes 1, 3, 5, 7, and 9 in Figure 12). No trastuzumab labeling was observed when compound 40 was used (lanes 2, 4, 6, 8, and 10).

[0382] Example 12 Preparation of DBCO-containing reactive conjugates and trastuzumab-DBCO conjugates The Fc-binding ligands (compound 10) prepared in Example 1 were converted to the corresponding reactive conjugates of formula (1) by coupling compound 20 to the N-terminus of each Fc-binding ligand. The structures of the DBCO-containing reactive conjugates prepared in Example 12 are shown in the table below.

[0383] [Table 11]

[0384] The purity of the reactive conjugate was determined by UPLC-MS (described above) and the results are shown in the table below.

[0385] [Table 12]

[0386] A trastuzumab-DBCO conjugate was prepared using compound 43 and commercial trastuzumab (Herceptin®) according to the same procedure as described in Example 6 above. The conjugate was digested using GingisKhan and analyzed by HRMS as described above. The results are shown in the table below.

[0387] [Table 13]

[0388] Example 13 Preparation of immobilized FL-containing reactive conjugates and solid-phase modification of trastuzumab Reactive conjugates immobilized on solid supports were prepared and their propensity to react with trastuzumab was assessed.

[0389] Biotinylated Fc-binding vectors were prepared by standard Fmoc / tBu-based SPPS using 4-Fmoc-hydrazinobenzoyl AM NovaGel™ (loading: 0.61 mmol / g) and a Liberty Blue™ automated microwave peptide synthesizer (available from CEM Corp., Germany). The coupling reaction for amide bond formation was carried out at room temperature for 4 minutes using 0.2 M Fmoc-amino acids preactivated with 0.5 M DIC and 1 M OxymaPure® in DMF. Fmoc deprotection was carried out using 10% piperazine (v / v) in DMF.

[0390] After completion of the synthesis, the peptide was purified by resuspending the resin in DMF and adding 1.4 eq of CuII(AcO)2 * The peptide was manually cleaved from the resin by mixing with HO, 3.5 eq. biotin-PEG-NH, and 3 eq. pyridine. The reaction was stirred at room temperature for 4 h. The cleavage mixture was filtered, and the peptide was precipitated with water and filtered. The pellet was dissolved in a cleavage cocktail (TFA / TIS / water, 90:5:5), and the peptide side chain was deprotected by stirring at room temperature for 2 h. The mixture was concentrated, and the crude peptide (compound 41) was precipitated with cold diethyl ether, centrifuged, washed with cold diethyl ether, dried, dissolved in ultrapure water / acetonitrile, lyophilized, and purified by HPLC.

[0391] Fmoc-NH-(PEG) 20A solution of -COOH (1.3 eq, 4.7 μmol) and HATU (1.2 eq, 4.33 μmol) in DMF was stirred for 1 min, and DIEA (10 eq, 35.8 μmol) was added. After 3 min of preactivation, a biotinylated Fc-binding peptide (compound 44) ​​in DMF (1 eq, 3.58 μmol) was added to the reaction mixture and stirred at room temperature for 1–2 h to prepare compound 45. Completion of the reaction was monitored by UPLC-MS. The peptide was then precipitated with cold diethyl ether. Fmoc deprotection was carried out using 20% ​​piperidine (v / v) in DMF for 30 min at room temperature, followed by precipitation of the peptide with cold diethyl ether and purification by HPLC.

[0392] The biotinylated Fc-binding ligand was converted to a reactive conjugate (compound 46) by coupling compound 19 to the N-terminus of compound 45, following the same procedure as described above in Example 5. The structures of the compounds prepared in Example 13 are shown in the table below.

[0393] [Table 14]

[0394] To immobilize the biotinylated reactive conjugate on the solid support, NeutrAvidin agarose resin (Thermo Fisher Scientific) was packed into a column (Fisher Scientific) and washed with binding buffer (0.1 M phosphate buffer, 0.15 M sodium chloride, pH 7.2). Compound 46 (2.1 nmol) was incubated with washed NeutrAvidin agarose beads (40 μl beads: 7.5 μg peptide) at room temperature for 30 minutes (Figure 13).

[0395] The beads were washed four times with binding buffer, then 50 mM bicine, pH 9.0, was added to raise the pH. Trastuzumab in PBS, pH 7.0 (2.1 nmol) was added to the beads, and the mixture was stirred at room temperature for 2 hours, followed by three to four washes with binding buffer. The labeled trastuzumab was eluted (100 μl, 0.1 M glycine, pH 2.5) into a collection tube containing neutralization buffer (1 M phosphate buffer, pH 8.5) at a volume ratio of 1:10. The elution step was repeated, and the fractions were combined. The eluted labeled trastuzumab buffer was then exchanged with PBS, pH 7.0, using a Vivaspin® 500 centrifugal concentrator with a 30 kDa MWCO.

[0396] The antibody was then analyzed by SDS-PAGE. The gel showed one fluorescent band, indicating successful conjugation of the FL moiety with trastuzumab.

[0397] Example 14 Preparation of other payload-carbonate-containing reactive conjugates The Fc-binding vector prepared in Example 1 was used to bind various payloads (DTPA, PCTA, DFO) to NH2-carbonate-PEG. 10 -Fc-III to give reactive conjugates of formula (1) (compounds 47-49). The structures of these payload-containing reactive conjugates are shown in the table below.

[0398] [Table 15]

[0399] The purity of the reactive conjugate was determined by UPLC-MS (described above) and the results are shown in the table below.

[0400] [Table 16]

[0401] NH2-carbonate-PEG 10 - Preparation of Fc-III:

[0402] [ka]

[0403] Step 1. DIEA was added to a solution of 4-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxycarbonyloxy]benzoic acid (2.35 mg, 6.4 μmol, 1.3 equiv) in DMF (0.65 mL) at room temperature. After stirring at room temperature for 1 minute, HATU.HPF6 (2.81 mg, 5.4 μmol, 1.1 equiv) was added to the reaction mixture. After stirring at room temperature for 3 minutes, a solution of compound 7 (10.0 mg, 4.9 μmol, 1.0 equiv) in DMF (0.65 mL) was added to the reaction mixture. After stirring at room temperature for 18 hours, two drops of 0.1% TFA in water were added. Purification on C18 (12 g, 30-70% ACN + 0.1% TFA in water + 0.1% TFA over 12 CV) afforded BocHN-carbonate-PEG. 10 -Fc-III (2.4 mg, 1.0 μmol, UV purity 95%, 20% yield) was obtained as a white powder after freeze-drying. UPLC-MS: Rt = 2.78 min, m / z = 1147 [M-Boc + 2H] 2+ , 1195[M-2H] 2- .

[0404] Step 2. TFA was added to a solution of BocHN-carb-PEG10-FcIII (23.9 mg, 8.3 μmol, 1.0 equiv.) in DCM (0.5 mL). The reaction mixture was stirred at room temperature for 1.5 hours and then concentrated in vacuo. A mixture of ACN / water (1:1, 5 mL) was added, and the mixture was freeze-dried to give HN-carbonate-PEG as a white powder. 10 -Fc-III (23.7 mg, 8.3 μmol, UV purity 99%, quantitative yield). UPLC-MS: Rt=2.20 min, m / z=1147[M+2H] 2+ , 1145[M-2H] 2- .

[0405] DTPA-carbonate-PEG 10 - Preparation of Fc-III: p-SCN-Bn-CHX-A''-DTPA.3HCl (4.47 mg, 6.0 μmol, 1.0 equiv.) was added to NH2-carbonate-PEG 10 To a solution of p-SCN-Bn-CHX-A''-DTPA.3HCl (0.90 mg, 1.2 μmol, 0.2 equiv.) in DMF (0.3 mL) was added p-SCN-Bn-CHX-A''-DTPA.3HCl (0.90 mg, 1.2 μmol, 0.2 equiv.) and triethylamine (0.5 μL, 3.6 μmol, 0.6 equiv.) were added, and the reaction mixture was stirred at room temperature for 18 hours. Purification by preparative HPLC (30-60% ACN + 0.1% FA in water + 0.1% FA) afforded DTPA-carbonate-PEG. 10 -Fc-III (1.4 mg, 0.52 μmol, 8.7% yield) was obtained as a white powder after freeze-drying.

[0406] PCTA-carbonate-PEG 10 - Preparation of Fc-III: p-SCN-Bn-PCTA.3HCl (4.15 mg, 6.5 μmol, 1.05 equiv.) was dissolved in NH2-carbonate-PEG 10 To a solution of 1p-Fc-III (14.9 mg, 6.2 μmol, 1.0 equiv) in DMF (0.1 mL) was added at room temperature. The reaction mixture was stirred at room temperature for 5 minutes, and then triethylamine (4.2 μL, 30.0 μmol, 5.0 equiv) was added. After stirring at room temperature for 3 hours, 1p-SCN-Bn-PCTA.3HCl (4.15 mg, 6.5 μmol, 1.05 equiv) was added to the reaction mixture at room temperature. After stirring at room temperature for 16 hours, PCTA-carbonate-PEG was purified by preparative HPLC (28-37% ACN + 0.1% TFA in water + 0.1% TFA). 10 -Fc-III (2.53 mg, 8.97 μmol, 14% yield) was obtained as a white powder after freeze-drying.

[0407] DFO-carbonate-PEG 10 - Preparation of Fc-III: DIEA (10 μL, 80.0 μmol, 16.0 equiv.) was added to NH2-carbonate-PEG 10 To a solution of DFO-Fc-III (12.42 mg, 4.9 μmol, 1.0 equiv.) and DFO-NHS (8.2 mg, 5.9 μmol, 1.2 equiv.) in DMF (0.4 mL) was added at room temperature. After stirring at room temperature for 3.5 h, ACN / water / TFA (1:1:0.5%, 0.2 mL) was added and the reaction mixture was stirred at room temperature for 5 min. Purification by preparative HPLC (25-60% ACN + 0.1% FA in water + 0.1% FA) afforded DFO-carbonate-PEG. 10 -Fc-III (1.6 mg, 0.45 μmol, UV purity 86%, 9% yield) was obtained as a white powder after freeze-drying.

[0408] Example 15 Preparation of trastuzumab-DTPA / PCTA / DFO conjugate The tendency of the reactive conjugates of Example 14 to react with antibodies was evaluated using trastuzumab. Trastuzumab-DTPA / PCTA / DFO conjugates were prepared according to the same procedure as described in Example 6 above using compounds 47-49. The obtained trastuzumab-DTPA / PCTA / DFO conjugates were analyzed by HRMS (Table 16).

[0409] [Table 17]

[0410] Example 16 Preparation of Fl-containing reactive conjugates with various chemical or reactive modifiers The Fc-binding vector prepared in Example 1 was converted into a reactive conjugate by coupling FL-carbonate-naphthalene / -carbonate-isoquinoline / -CH2CH2-thioester (compounds 21-23) to the N-terminus of Fc-binding ligand 7 according to the procedure described below. The structures of these payload-containing reactive conjugates prepared in Example 16 are shown in the table below.

[0411] [Table 18]

[0412] Preparation of Compound 50 (Naphthalene): To a solution of 2,5-dioxopyrrolidin-1-yl 6-(((2-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)thioureido)ethoxy)ethoxy)carbonyl)oxy)-2-naphthoate (19.0 mg, 0.023 mmol) and compound 7 (40.0 mg, 0.019 mmol) in N,N-dimethylformamide (1.00 mL) was added DIPEA (10.0 μL, 0.057 mmol) at room temperature. The mixture was stirred for 3 h and then purified on a 60 g C18 column using an eluent of 5–95% acetonitrile (0.1% formic acid) in water (0.1% formic acid). The desired fractions were combined and freeze-dried. The resulting material was purified by elution with 2,5-dioxopyrrolidin-1-yl 6-(((2-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)thioureido)ethoxy)ethoxy)carbonyl)oxy)-2-naphthoate (11.0 mg, 0.014 mmol) and TFA.PEG. 10This was combined with a similar batch obtained from the reaction of -FcIII (30.0 mg, 0.014 mmol) and DIPEA (7.00 μL, 0.042 mmol) in N,N-dimethylformamide (1.00 mL) and further purified on a 60 g C18 column using an eluent of 20-60% acetonitrile (0.1% formic acid) in water (0.1% formic acid). The desired fractions were combined and freeze-dried to give the title compound (11.9 mg, 13% overall yield, 95% purity). UPLC-MS:R t =1.95 minutes, 94.5%. ESI:m / z=911.8[M+3H] / 3 + .

[0413] Preparation of Compound 51 (Isoquinoline): 2,5-Dioxopyrrolidin-1-yl 6-(((2-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)thioureido)ethoxy)ethoxy)carbonyl)oxy)quinoline-2-carboxylate (11.0 mg, 0.014 mmol) with TFA.PEG 10 To a solution of α-FcIII (30.0 mg, 0.014 mmol) in N,N-dimethylformamide (1.00 mL) was added DIPEA (7.00 μL, 0.042 mmol) at room temperature. The mixture was stirred for 2 h and then purified on a 60 g C18 column using an eluent of 20-60% acetonitrile (0.1% formic acid) in water (0.1% formic acid). The desired fractions were combined and freeze-dried to give the title compound (8.70 mg, 23% yield, 93% purity) as a yellow powder. UPLC-MS:R t =1.93 minutes. ESI:m / z=912.0[M+3H] / 3 + .

[0414] Preparation of Compound 52 (thioester): To a solution of 2,5-dioxopyrrolidin-1-yl 3-(4-((3-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)thioureido)ethoxy)propanoyl)thio)phenyl)propanoate (12.8 mg, 0.0163 mmol) and compound 7 (21.1 mg, 0.00980 mmol) in N,N-dimethylformamide (1.00 mL) was added DIPEA (8.52 μL, 0.0489 mmol). Stirring was continued at room temperature for 2 hours. The reaction was directly purified by reverse-phase chromatography (Biotage Isolera, 60 g, C18 SNAP Ultra Biotage cartridge) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (80:20 to 30:70). The product-containing fractions were freeze-dried to give the desired compound (4.88 mg, 4% yield, 87% purity) as a yellow solid. UPLC4-MS:R t =1.67 minutes. ESI:m / z=904[M+3H] / 3 + .

[0415] Example 17 Preparation of trastuzumab-FL conjugates The tendency of the reactive conjugates of Example 16 to react with antibodies was evaluated using trastuzumab. Trastuzumab-FL conjugates were prepared according to the same procedure as described in Example 6 above using compounds 50-52. The obtained trastuzumab-FL conjugates were analyzed by HRMS (Table 18).

[0416] [Table 19]

Claims

1. The following formula (1): PYSV (1) [In the formula, P is a payload, said payload being a substance that provides a new function when attached to an antibody or antibody fragment; Y is a reactive moiety capable of reacting with the side chain of an amino acid; V is a vector capable of interacting with the fragment crystallizable (Fc) region of an antibody or fragment thereof; S is a spacer having a length Z, wherein Z is of a length such that when vector V interacts with the Fc region of an antibody or antibody fragment thereof, reactive moiety Y can react with a side chain of an amino acid residue on the antibody or antibody fragment; The reactive moiety Y is represented by the following formulas (6a) to (6l′): 【Chemistry 1A】 【Chemistry 1B】 【Chemistry 1C】 【1D】 is represented by one of During the ceremony, * indicates a covalent bond to the spacer (S), ** indicates a covalent bond to the payload (P); The spacer is (a2) The following equation (7): -X 1 -(CH 2 CH 2 O) n2 -CH 2 CH 2 -X 2 - (7) (In the formula, X 1 is NH, O or S; X 2 is NH or C=O; n2 is an integer between 8 and 12) a group represented by (b2) A peptide group having 6 to 25 amino acids in the backbone, each amino acid being selected from Pro, Gly, Ala, Asn, Asp, Thr, Glu, Gln, and Ser. is a group selected from A compound represented by:

2. The payload is (i) Below: ・Labeling part; - chromophores; a fluorophore; and - A labeling moiety containing a radionuclide A portion selected from: (ii) a moiety selected from a moiety containing a conjugation group including a conjugated diene; a tetrazine (TZ); an alkyne or an azide; a dibenzocyclooctyne (DBCO); a trans-cyclooctene (TCO), a bicyclo[6.1.0]nonyne (BCN); an aldehyde; a ketone; and a hydrazine; (iii) Below: -Antineoplastic agents; Topoisomerase inhibitors; - RNA polymerase II inhibitors; DNA cleaving agents; -Antimitotic or microtubule disrupting agents; -Antimetabolites; - kinase inhibitors; - immunomodulators; -Anti-infective agents; A moiety derived from a drug selected from and radioisotopes and / or pharmaceutically acceptable salts thereof 10. The compound of claim 1, comprising a moiety selected from:

3. 3. The compound of claim 1 or 2, wherein the payload is a chelating agent.

4. The compound of claim 3, wherein the chelating agent chelates a radionuclide.

5. The chelating agents are DTPA, CH-X-DTPA, DFO, 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-tetraacetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid (DOTAGA), 2,2'-(1,4,7-triazacyclononane-1,4-diyl)diacetic acid (NO2A), DOTA, 1,4,7- Triazacyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminediacetic acid, triethylenetetraminehexaacetic acid (TTHA), 1,4,8,11-tetraazacyclotetradecane (CYCLAM), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,8,11-tetraazabicyclo[6.6.2]Hexadecane-4,11-diacetic acid (CB-TE2A), 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetamide (DO3AM), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), 1,5,9-triazacyclododecane (TACD), (3a1s,5a1s)-dodecahydro-3a,5a,8a,10a-tetraazapyrene (cis-glyoxal-cyclam), 1,4,7-triazacyclononane (T ACN), 1,4,7,10-tetraazacyclododecane (cyclene), tri(hydroxypyridinone) (THP), 3-(((4,7-bis((hydroxy(hydroxymethyl)phosphoryl)methyl)-1,4,7-triazonan-1-yl)methyl)(hydroxy)phosphoryl)propanoic acid (NOPO), PCTA, 2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetic acid (TRITA), 2,2',2'',2''''- (1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetamide (TRITAM), 2,2',2''-(1,4,7,10-tetraazacyclotridecane-1,4,7-triyl)triacetamide (TRITRAM), trans-N-dimethylcyclam, 2,2',2''-(1,4,7-triazacyclononane-1,4,7-triyl)triacetamide (NOTAM), oxocyclam, dioxocyclam, 1,7-dioxa-4,1 4. The compound of claim 3, wherein the moiety is derived from 0-diazacyclododecane, bridged cyclam (CB-cyclam), triazacyclononanephosphinate (TRAP), dipyridoxyl diphosphate (DPDP), meso-tetra-(4-sulfanotophenyl)porphine (TPPS4), ethylenebishydroxyphenylglycine (EHPG), hexamethylenediaminetetraacetic acid, dimethylphosphinomethane (DMPE), methylenediphosphate, dimercaptosuccinic acid (DMPA), or a derivative thereof.

6. The radioactive nuclide 124 I, 131 I, 86 Y, 90 Y, 177 Lu, 111 In, 188 Re, 55 Co, 64 Cu, 67 Cu, 68 Ga, 89 Zr, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 72 As, 211 At, 225 Ac, 223 Ra, 97 Ru, 149 Tb, 152 Tb, 161 Tb, 99m Tc, 226 Th, 227 Th, 201 Tl, 89 Sr, 44 / 43 Sc, 47 Sc, 153 Sm, 133 Xe and Al 18 6. The compound of any one of claims 2 to 5, selected from F.

7. 3. The compound of claim 1 or 2, wherein the payload is a moiety derived from exatecan, PNU-159682, amanitin, duocarmycin, auristatin, maytansine, tublysin, calicheamicin, SN-38, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, a pyrrolobenzodiazepine, a pyrrole-based kinesin spindle protein (KSP) inhibitor, an indolino-benzodiazepine dimer, or a radioisotope and / or a pharmaceutically acceptable salt thereof.

8. P is the following formula (2): P 1 -L-- *' (2) (In the formula, P 1 is a payload as defined in any one of claims 2 to 7; L is a linker; *' indicates a covalent bond to a reactive moiety (Y) 8. The compound of any one of claims 1 to 7, represented by:

9. The linker (a1) an alkylene group having 1 to 12 carbon atoms; (b1) a polyalkylene oxide group having 2 or 3 carbon atoms and having 1 to 36 repeating units; (c1) a peptide group having 2 to 12 amino acids 9. The compound of claim 8, selected from:

10. The compound according to any one of claims 1 to 9, wherein the vector is a peptide comprising a sequence of 11 to 17 amino acids.

11. The vector has the following formulas (8a) and (8b): 【Chemistry 2】 (In the formula, Bxx, Cxx, Dxx, Exx, and Fxx each independently represent an amino acid; Axx is an amino acid, a dicarboxylic acid, or a group represented by the following formula (9a): ---Axx1-Axx2-Axx3--- (9a) (In formula (9a), Axx1 represents a single covalent bond or amino acid; Axx2 represents an amino acid; Axx3 represents an amino acid) represents a peptide moiety represented by: Gxx is an amino acid or a group represented by the following formula (9b): ---Gxx1-Gxx2-Gxx3--- (9b) (In formula (9b), Gxx1 represents the amino acid; Gxx2 represents the amino acid; Gxx3 represents a single covalent bond or an amino acid) represents a peptide moiety represented by: Hxx represents a single covalent bond or a trifunctional amino acid; Z 1 teeth, a group covalently attached to the C-terminus of Gxx when Hxx is a single covalent bond selected from -N(H)(R), where R represents a hydrogen atom, an alkyl group or a cycloalkyl group, and a moiety derived from a compound containing a conjugation group selected from biotin, DBCO, TCO, BCN, alkyne, azide, bromoacetamide, maleimide, and thiol; a group covalently attached to the C-terminus of Hxx when Hxx is a trifunctional amino acid and Y' is attached to the side chain of Hxx; or When Hxx is a trifunctional amino acid and Y' is bonded to the C-terminus of Hxx, a hydrogen atom bonded to the side chain of Hxx represents Z 2 teeth, a group covalently attached to the N-terminus of Axx when Hxx is a single covalent bond selected from a hydrogen atom, a carbonyl-containing group, and a group containing a conjugation moiety; a group covalently attached to the N-terminus of Hxx when Hxx is a trifunctional amino acid and Y' is attached to the side chain of Hxx selected from a hydrogen atom and a carbonyl-containing group; or When Hxx is a trifunctional amino acid and Y' is bonded to the N-terminus of Hxx, a hydrogen atom bonded to the side chain of Hxx represents Y' is present only if Hxx is a trifunctional amino acid, which is ・Z 1 When bound to the C-terminus of Hxx, or Z 2 When bound to the N-terminus of Hxx, the side chain of Hxx, ・Z 1 When bound to the side chain of Hxx, the C-terminus of Hxx, or ・Z 2 when attached to the side chain of Hxx, represents the moiety covalently attached to the N-terminus of Hxx; Y' is derived from the compound containing the conjugation group; X 3 represents a single covalent bond or a divalent group containing one or more atoms selected from carbon, nitrogen, and oxygen; **** indicates a covalent bond to the spacer (S) 11. The compound of claim 10, which is a peptide represented by one of:

12. Axx2 is Cys and Gxx2 is Cys, and the side chains of Axx2 and Gxx2 are linked together to form a group of the formula -(SX 4 -S)- group (wherein X 4 represents a single covalent bond or a divalent group containing one or more atoms selected from carbon, nitrogen, and oxygen.

13. At least one of Axx, Bxx, Cxx, Dxx, Exx, Fxx, Gxx, and Hxx is defined as follows: Axx represents an amino acid selected from Ala, 2,3-diamino-propionic acid (Dap), Asp, Glu, 2-aminosuberic acid, α-aminobutyric acid, Asn and Gln, a dicarboxylic acid selected from succinic acid, glutaric acid and adipic acid; Bxx represents an amino acid selected from Trp, Phe, Tyr, phenylglycine (Phg), 3-benzothiopen-2-yl-L-alanine, 3-naphthalen-2-yl-L-alanine, 3-biphenyl-4-yl-L-alanine and 3-naphthalen-1-yl-L-alanine; Cxx represents an amino acid selected from His, Ala, 3-pyridin-2-yl-L-alanine, meta-tyrosine (mTyr) and Phe; Dxx represents an amino acid selected from Ala, Abu, Gly, Leu, Ile, Val, Met, cyclohexylalanine (Cha), Phe, Thr, Cys, Tyr, and norleucine (Nle); Exx represents an amino acid selected from Ala, Gly, Asn, Ser, Abu, and Asp; Fxx represents an amino acid selected from Ala, Glu, Asp, Gln, His, Arg, Ser, and Asn; Gxx represents an amino acid selected from Thr, Ser, Ala, Asn, Val, 2-amino-butyric acid (Abu), Ile, Met, Leu, Pro, Gln, and Cys; or a peptide moiety of formula (9b) (wherein Gxx1 is Thr, Gxx2 is Cys, and Gxx3 is a single covalent bond); and Hxx represents an amino acid selected from Dap, Dab, Lys, Orn and homo-lysine (homo-Lys), 13. The compound of claim 11 or 12.

14. The vectors are represented by the following formulas (8a') to (8d'): 【Chemistry 3】 (In the formula, Z 1 , Z 2 , X 3 , X 4 and **** is as defined in claim 11) A peptide represented by one of 14. The compound of any one of claims 1 to 13, wherein

15. below: 【Chemistry 4A】 【Chemistry 4B】 (In the formula, P is as defined in any one of claims 1 to 7, Y' is as defined in claim 11) 15. The compound according to any one of claims 1 to 14, selected from:

16. below: 【Chemistry 5A】 【Chemistry 5B】 【Chemistry 5C】 【5D】 【Chemistry 5E】 【Chemistry 5F】 【5G】 16. The compound according to any one of claims 1 to 15, selected from:

17. 17. A kit for site-specific modification of an antibody or a fragment thereof, comprising a compound according to any one of claims 1 to 16 and a buffer.

18. 18. A kit for site-specific modification of an antibody or a fragment thereof according to claim 17, wherein the compound is immobilized on a solid matrix.

19. A method for site-selective modification of an antibody or a fragment thereof, comprising reacting the antibody or a fragment thereof with a compound according to any one of claims 1 to 16.

20. - the antibody is a monoclonal antibody; or - the antibody fragment is incorporated into an Fc fusion protein; 20. The method of claim 19.

21. Antibodies include adalimumab, aducanumab, alemtuzumab, altumomab pentetate, atezolizumab, anetumab, avelumab, bapineuzumab, basiliximab, bectumomab, bermekimab, besilesomab, bevacizumab, bezlotoxumab, brentuximab, brentuximab vedotin, brodalumab, blinatumomab, catumaxomab, cemiplimab, cetuximab, simpanemab, clivatuzumab, clivatuzumab tetraxetan, crenezumab tetraxetan, david Crizumab, daratumumab, denosumab, dinutuximab, durvalumab, edrecolomab, elotuzumab, emapalumab, enfortumab, enfortumab vedotin, epratuzumab, epratuzumab-SN38, etaracizumab, gemtuzumab, gemtuzumab ozogamicin, girentuximab, goslanemab, ibritumomab, inebilizumab, infliximab, inotuzumab, inotuzumab ozogamicin, ipilimumab, isatuximab, ixekizumab, J591 PSMA antibody, labetuzumab, lecanemab, mogamulizumab, necitumumab, nimotuzumab, natalizumab, nivolumab, ocrelizumab, ofatumumab, olaratumab, oregovomab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, polatuzumab vedotin, prasinezumab, racotumomab, ramucirumab, rituximab, siltuximab, sacituzumab, sacituzumab selected from the group consisting of zumab govitecan, semolinemab, siltuximab, solanezumab, tacatuzumab, tetrotumumab, tirabonemab, tocilizumab, tositumomab, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, TS23, ustekinumab, vedolizumab, votumumab, zagotenemab, zalutumumab, zanolimumab, fragments and derivatives thereof; 21. The method of claim 20.

22. the Fc fusion protein is selected from belatacept, aflibercept, ziv-aflibercept, dulaglutide, rilonacept, romiplostim, abatacept, and alefacept; 21. The method of claim 20.

23. 17. A method for preparing a modified antibody or modified antibody fragment, comprising reacting an antibody or antibody fragment with a compound according to any one of claims 1 to 16.

24. 24. The method of claim 23, wherein the antibody or antibody fragment has the same definition as in claim 20.

25. 17. A method for preparing a composition comprising a modified antibody or modified antibody fragment for use in a method of diagnosing, monitoring, imaging, or treating a disease, said method comprising reacting an antibody or antibody fragment with a compound of any one of claims 1 to 16.

26. 26. The method of claim 25, wherein the disease is a neurological disease, a cardiovascular disease, an autoimmune disease, or cancer.

27. The disease or treatment thereof may be Alzheimer's disease, amyotrophic lateral sclerosis, cerebral arteriosclerosis, encephalopathy, Huntington's disease, multiple sclerosis, Parkinson's disease, progressive multifocal leukoencephalopathy, systemic lupus erythematosus, systemic sclerosis, angina pectoris including unstable angina, aortic aneurysm, atherosclerosis, heart transplant, cardiac toxicity diagnosis, coronary artery bypass graft, heart failure including systolic dysfunction resulting in atrial fibrillation, hypercholesterolemia, ischemia, myocardial infarction, thromboembolism 27. The method of claim 25 or 26, wherein the inflammatory bowel disease is selected from the group consisting of psoriasis, thrombosis, ankylosing spondylitis, autoimmune cytopenia, autoimmune myocarditis, Crohn's disease, graft-versus-host disease, granulomatosis with polyangiitis, idiopathic thrombocytopenic purpura, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), lupus, microscopic polyangiitis, multiple sclerosis, plaque psoriasis, psoriasis, psoriatic arthritis, rheumatoid arthritis, ulcerative colitis (UC), uveitis, and vasculitis.

28. 27. The method of claim 25 or 26, wherein the disease involves cells selected from lymphoma cells, myeloma cells, renal cancer cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, lung cancer cells, testicular cancer cells, pancreatic cancer cells, liver cancer cells, melanoma, head and neck cancer cells, and any cells that are deregulated and grow and divide at a rapid pace, causing cancer.

29. 17. A method for producing a modified antibody or modified antibody fragment, comprising reacting an antibody or antibody fragment with a compound according to any one of claims 1 to 16.

Images