Linkers and antibody drug conjugates for drug conjugates

Abstract

This disclosure provides novel and advantageous synthetic linkers and conjugates capable of covalently binding to one or more isolated polypeptides or one or more antibodies or antibody fragments. These include isolated polypeptides, antibodies, or antibody fragments having heavy-chain variable regions and / or light-chain variable regions that specifically bind to the nectin-4 protein. Also provided are linker-conjugated polypeptides, antibodies, or antibody fragments, as well as pharmaceutical compositions and kits containing them, and methods for producing synthetic linkers and conjugates, and methods for using them therapeutically.

Description

[Technical Field]

[0001] Reference to XML Array Listings The sequence listing submitted with this specification as an XML file named "BIAT-1036WOSequenceListingXML," created on April 24, 2024, is 81 kilobytes in size and is incorporated herein by reference in its entirety. This disclosure relates to a synthetic linker capable of covalently binding to an isolated polypeptide, antibody, or antibody fragment. The synthetic linker can be bound to an isolated polypeptide, antibody, or antibody fragment, and compositions and / or kits containing the same, as well as methods for producing and using the covalently bound polypeptide, antibody, or antibody fragment for therapeutic purposes, are provided. The improved stability of the linker-bound polypeptide, antibody, or antibody fragment of this disclosure offers a significant advantage in reducing the adverse events associated with payload release that are often faced with the use of other types of linkers, which improves the therapeutic effect of the linker-bound polypeptide, antibody, or antibody fragment in therapies such as immunoconjugate therapy. The isolated polypeptide, antibody, or antibody fragment may be conditionally active or unconditionally active. In particular, conditionally or unconditionally active isolated polypeptide, antibody, or antibody fragment may be an anti-nectin-4 polypeptide, an anti-nectin-4 antibody, an anti-nectin-4 antibody fragment, or an anti-nectin-4 multispecific antibody. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are complex molecules consisting of an antibody (such as a monoclonal antibody) conjugated to a biologically active, highly cytotoxic drug moiety via a cleavable (such as an acid-unstable linker, protease linker, or disulfide linker) or non-cleavable linker. The conjugation of a cytotoxic drug moiety to an antibody enables targeted delivery of the drug payload to the surface of affected tissue (such as a tumor surface) while minimizing toxicity to healthy tissue, thus improving the therapeutic scope of ADCs in therapeutic therapies such as cancer treatment.

[0003] A "linker" is a bifunctional or polyfunctional moiety that can be used to bind one or more drug (D) moieties to a polypeptide such as an antibody (Ab) to form an immunoconjugate or antibody-drug conjugate (ADC). In this respect, linkers play a crucial role in the stability and drug payload release profile of ADCs, which are important for their efficacy. One of the challenges in developing safe and effective ADCs is the generation of suitable chemical linkers between cytotoxic drugs and antibodies (such as monoclonal antibodies). Biopharma PEG, What are ADC linkers: Cleavable vs. Non-Cleavable Linkers; biochempeg.com / article / 87.html; 12 / 19 / 2019 (publish date).

[0004] Ideally, linkers should be stable in the circulatory system and specifically release cytotoxic payloads to targets such as tumors. (Su Z. et al., Acta Pharmaceutica Sinica B., 11(12):3889-3907 (2021)). However, existing linkers have a drawback that limits ADC development: they release payloads nonspecifically, resulting in off-target toxicity. (Su Z., et al. (2021)).

[0005] Nectin-4 is a surface molecule belonging to the nectin family of proteins, which includes four members. Nectins are cell adhesion molecules that play important roles in various biological processes during development and adulthood, such as the polarity, proliferation, differentiation, and migration of epithelial, endothelial, immune, and neuronal cells. Nectins are involved in several pathological processes in humans. Nectins are the primary receptors for polio, herpes simplex, and measles viruses. Mutations in the genes encoding nectin-1 (PVRL1) and nectin-4 (PVRL4) cause ectodermal dysplasia syndromes associated with several abnormalities. Nectin-4 is expressed during fetal development. In adult tissues, its expression is more restricted than that of other members of its family.

[0006] Nectin-4 is a tumor-associated antigen in 30%, 49%, and 86% of breast, ovarian, and lung cancers, respectively, and is often associated with invasive tumors. In breast tumors, nectin-4 is primarily expressed in triple-negative cancers. In patients with these cancers, detection of the soluble form of nectin-4 in the serum is associated with poor prognosis. Serum levels of nectin-4 increase during metastatic progression and decrease after treatment. These results suggest that nectin-4 may be a reliable target for cancer treatment.

[0007] Therefore, several anti-nectin-4 antibodies have been described in the prior art. In particular, enfortumab vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) that targets nectin-4 and is currently in clinical study for the treatment of patients with solid tumors. [Overview of the project] [Means for solving the problem]

[0008] This disclosure aims to provide an antibody-drug conjugate comprising an anti-nectin-4 antibody or antibody fragment and a novel linker, which has reduced or minimal side effects and is suitable for therapeutic and diagnostic applications, particularly for the diagnosis and treatment of cancer. Some anti-nectin-4 antibodies or antibody fragments may have higher binding activity or affinity to nectin-4 in the tumor microenvironment compared to binding activity or affinity to nectin-4 in the non-tumor microenvironment. The anti-nectin-4 antibodies or antibody fragments of this disclosure typically have at least equivalent efficacy to known anti-nectin-4 antibodies. Furthermore, because the anti-nectin-4 antibodies or antibody fragments of the present invention have relatively low binding affinity to nectin-4 in normal tissues such as the non-tumor environment and the microenvironment, they may exhibit reduced side effects compared to monoclonal anti-nectin-4 antibodies known in the art. These advantages may provide more selective targeting of nectin-4 expressed in tumors, and as a result of their increased binding activity to nectin-4 present in the tumor microenvironment, may enable the use of higher doses of the anti-nectin-4 antibody or antibody fragment of the present invention, thereby enabling more effective therapeutic treatment without a corresponding increase in undesirable side effects.

[0009] In one embodiment, the present disclosure relates to the conjugate of formula I, [ka] Equation I Provided, in the formula L, [ka] Represented by, where R1 is a nucleophilic functional group capable of binding to an antibody, and the functional group is C1~C 10 Alkyl halides, C1-C 10 Alkyl alcohols, phenols, C1-C 10 Alkylthiols, and C1-C 10 Selected from the group consisting of alkyl azides, where R2 is a substituted or unsubstituted C1-C 20 Alkylene, or substituted or unsubstituted (PEG) represented by the following:n is a part of: [Chem.] where n is from 1 to 20; X is a substituted nitrophenyl represented by: [Chem.] where R3 is C1 - C 20 alkylene; R4 is a triazole ring having a C = C double bond in the ring; Y is a glycoside or a glycoside derivative; D is a drug; The wavy line indicates the site of the covalent bond. Y is bonded to X at the site of the wavy line bonded to the nitrophenyl ring, D is bonded to X at the site of the wavy line bonded to the carbonyl, and R2 is bonded to R4.

[0010] In the above - mentioned embodiments, R1 can be a C1 - C6 alkyl halide having a halide selected from the group consisting of Br, I, Cl, and F, a C1 - C2 alkyl alcohol, 4 - phenol, or a C1 - C2 alkyl thiol.

[0011] In each of the embodiments of paragraphs

[0009] -

[0010] , R1 can be - CH2 - Br.

[0012] In each of the embodiments of paragraphs

[0009] -

[0011] , R2 can be an unsubstituted C1 - C 20 alkylene.

[0013] In one embodiment of paragraph

[0012] , R2 can be an unsubstituted C2 - C5 alkylene.

[0014] In each of the above - mentioned embodiments of paragraphs

[0009] -

[0011] , R2 can be an unsubstituted (PEG) n where n can be 8.

[0015] In each of the embodiments described in paragraphs

[0009] to

[0014] , R3 may be -CH2-.

[0016] In each of the embodiments described in paragraphs

[0009] to

[0015] , the glycoside or glycoside derivative Y may be selected from the following structures. [ka]

[0017] In each of the embodiments described in paragraphs

[0009] to

[0016] above, the glycoside or glycoside derivative Y may be a glucuronide.

[0018] In each of the embodiments described in paragraphs

[0009] to

[0017] above, the triazole ring is [ka] Linker intermediate (L) having the formula represented by int ) may be formed together with, where R1 may be a nucleophilic functional group capable of binding to an antibody, and the functional group may be C1~C 10 Alkyl halides, C1-C 10 Alkyl alcohols, phenols, C1-C 10 Alkylthiols, and C1-C 10 Selected from the group consisting of alkyl azides, R2 is either substituted or non-substituted C1-C 20 Alkylene, or substituted or unsubstituted (PEG) represented by the following: n It could be a part, [ka] In the formula, n can be 1 to 20, and R7 can be an azide group or an acetylene group, where the nitrogen atom of the azide group or the carbon atom of the acetylene group can be a ring member in the triazole ring. Considering that the acetylene group can react with the azide group in a click reaction, as will be described later in this specification, it is conceivable that the triazole ring of R4 can be formed by reacting the azide group bonded to R3 with the acetylene group at R7, or vice versa, i.e., by reacting the acetylene group bonded to R3 with the azide group at R7.

[0019] In each of the embodiments described in paragraphs

[0009] to

[0018] above, the triazole ring is [ka] It can be formed with an azide group or acetylene group bonded to L, which can be represented by (wherein p can be 2 to 5), or L, [ka] It can be expressed as follows (where n can be 8):

[0020] In each of the embodiments described in paragraphs

[0009] to

[0019] above, the linker is [ka] The linker may have a structure represented by, [ka] It may have a structure represented by [this].

[0021] In each of the embodiments described in paragraphs

[0009] to

[0020] above, the conjugate has the following structure: [ka] It may be an SPC07 having, or The conjugate has the following structure: [ka] It may be an SPC19 having the following characteristics.

[0022] In one embodiment of paragraph

[0018] , if R2 is a substituted or unsubstituted (PEG)n, Y may not be a galactoside.

[0023] In each of the embodiments described in paragraphs

[0009] to

[0018] , the conjugate has the following structure: [ka] It may be an SPC17 having the following characteristics.

[0024] In any one of the embodiments described in paragraphs

[0009] to

[0023] above, drug D may be selected from the group consisting of auristatin, drastatin, meitansinoid, calicheamicin, pyrrolobenzodiazepine, anthracycline, ribonuclease, and DNA endonuclease.

[0025] In the particular embodiment described in paragraph

[0024] above, drug D may be an auristatin selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

[0026] In another embodiment, this disclosure is, [ka] Linker intermediate (L) having the formula represented by int ) provides, and in the formula R1 is C1~C 10 Alkyl halides, C1-C 10 Alkyl alcohols, phenols, C1-C 10 Alkylthiols, and C1-C 10 A nucleophilic functional group capable of binding to an antibody selected from the group consisting of alkyl azides, R2 is either substituted or non-substituted C1-C 20 Alkylene, or a substituted or unsubstituted (PEG)n portion represented by the following: [ka] In the formula, n is between 1 and 20. R7 is either an azide group or an acetylene group.

[0027] In one embodiment of paragraph

[0026] , R1 may be a C1-C6 alkyl halide, a C1-C2 alkyl alcohol, a 4-phenol, or a C1-C2 alkylthiol having a halide selected from the group consisting of Br, I, Cl, and F.

[0028] In each of the embodiments described in paragraphs

[0026] to

[0027] , R1 may be -CH2-Br.

[0029] In each of the embodiments described in paragraphs

[0026] to

[0028] , R2 is non-substituted C1 to C 20 It may be an alkylene, or R2 may be an unsubstituted (PEG)n, where n may be 8.

[0030] In one embodiment of paragraph

[0029] , R2 may be an unsubstituted C2-C5 alkylene.

[0031] In each of the embodiments described in paragraphs

[0026] to

[0030] , R7 may be an azide group or an acetylene group.

[0032] In one embodiment described in paragraph

[0031] above, the linker is [ka] It may have a structure represented by the formula, where p can be 2 to 5.

[0033] In one embodiment of the above paragraph

[0032] , the linker is [ka] The linker may have a structure represented by, [ka] It may have a structure represented by [this].

[0034] In each of the embodiments described in paragraphs

[0026] to

[0028] above, the linker is [ka] It may have a structure represented by the formula, where n can be 8.

[0035] In another embodiment, the present disclosure relates to an immunoconjugate of formula II, [ka] Formula II Provided, in the formula: L, [ka] It is expressed by the formula, where R1 is C1~C 10 Alkyl halides, C1-C 10 Alkyl alcohols, phenols, C1-C 10 Alkylthiols, and C1-C 10 A nucleophilic functional group capable of binding to an antibody selected from the group consisting of alkyl azides, R2 is either substituted or non-substituted C1-C 20 Alkylene, or a substituted or unsubstituted (PEG)n portion represented by the following: [ka] In the formula, n is between 1 and 20; X [ka] It is a substituted nitrophenyl represented by, where R3 is C1~C20 It is an alkylene; R4 is a triazole ring having a C=C double bond in the ring; Y is a glycoside or a glycoside derivative; D is a drug; The wavy lines indicate the covalent bond sites; m is between 1 and 10. In Formula II, the term "antibody" refers to an antibody or an antibody fragment.

[0036] In the embodiments described in paragraph

[0035] above, R1 may be a C1-C6 alkyl halide, a C1-C2 alkyl alcohol, a 4-phenol, or a C1-C2 alkylthiol having a halide selected from the group consisting of Br, I, Cl, and F.

[0037] In each of the embodiments described in paragraphs

[0035] to

[0036] , R1 may be -CH2-Br.

[0038] In each of the embodiments described in paragraphs

[0035] to

[0037] , R2 is non-substituted C1 to C 20 It could be alkylene.

[0039] In a particular embodiment of paragraph

[0038] above, R2 may be an unsubstituted C2-C5 alkylene.

[0040] In each of the embodiments described in paragraphs

[0035] to

[0037] , R2 may be an unsubstituted (PEG)n, where n is 8.

[0041] In each of the embodiments described in paragraphs

[0035] to

[0040] , R3 may be -CH2-.

[0042] In each of the embodiments described in paragraphs

[0035] to

[0041] above, the glycoside or glycoside derivative Y may be selected from the following structures. [ka]

[0043] In each of the embodiments described in paragraphs

[0035] to

[0042] , the glycoside or glycoside derivative Y may be a glucuronide.

[0044] In each of the embodiments described in paragraphs

[0035] to

[0043] above, L is [ka] It can be expressed by the formula, where p can be 2 to 5, or L can be [ka] It can be expressed as follows, where n can be 8.

[0045] In one embodiment of the above paragraph

[0044] , the linker is [ka] The linker may have a structure represented by, [ka] It may have a structure represented by [this].

[0046] In any one of the embodiments described in paragraphs

[0035] to

[0045] above, drug D may be selected from the group consisting of auristatin, drastatin, meitansinoid, calicheamicin, pyrrolobenzodiazepine, anthracycline, ribonuclease, and DNA endonuclease.

[0047] In a particular embodiment of paragraph

[0046] above, drug D may be an auristatin selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

[0048] In one embodiment described in paragraphs

[0035] to

[0044] above, [ka] teeth, [ka] It has a structure selected from among.

[0049] In another embodiment of paragraph

[0044] above, if R2 is a substituted or unsubstituted (PEG)n, Y may not be a galactoside.

[0050] In each of the embodiments described above in

[0035] to

[0049] , the antibody has a heavy chain variable region (where: ) comprising three complementarity-determining regions (CDRs) having sequences H1, H2, and H3. The H1 sequence can be GFTFSSYNX1N (sequence number 1); The H2 sequence can be YISSSSSTIYYADSVKG (sequence number 2); The H3 sequence can be AYYYGX2DX3 (sequence number 3); Here, X1 can be M or D; X2 can be M or D; X3 can be V or K, provided that the heavy and light chain variable regions are not, in combination, sequence numbers 18 and 31; Furthermore, a light chain variable region containing three CDRs having sequences L1, L2, and L3 (where: The L1 sequence can be X4ASQGISGWX5A (sequence number 4); The L2 sequence can be AASTLQS(sequence ID 5); The L3 sequence could be QQANSX6PX7T (sequence number 6), Here, X4 can be R or H; X5 can be L or E; X6 can be F or E; X7 can be P or D, provided that X1, X2, X3, X4, X5, X6, and X7 cannot be M, M, V, R, L, F, and P at the same time. This may be an anti-nectin-4 antibody or antibody fragment containing an isolated polypeptide having the following properties.

[0051] In the embodiments described above in paragraph

[0050] , the antibody or antibody fragment may include a heavy chain variable region and a light chain variable region that specifically bind to nectin-4, wherein the heavy chain variable region includes three complementarity-determining regions having sequences H1, H2, and H3, where: The H1 sequence can be GFTFSSYNX1N (sequence number 1); The H2 sequence can be YISSSSSTIYYADSVKG (sequence number 2); The H3 sequence can be AYYYGX2DX3 (sequence number 3); Here, X1 can be M or D; X2 can be M or D; X3 can be V or K; The light chain variable region includes three complementarity-determining regions having sequences L1, L2, and L3, where: The L1 sequence can be X4ASQGISGWX5A (sequence number 4); The L2 sequence can be AASTLQS(sequence ID 5); The L3 sequence could be QQANSX6PX7T (sequence number 6), Here, X4 may be R or H; X5 may be L or E; X6 may be F or E; X7 may be P or D; however, X1, X2, X3, X4, X5, X6, and X7 may not be M, M, V, R, L, F, and P simultaneously, respectively, and the heavy chain and light chain variable regions may not be sequence numbers 18 and 31 in combination.

[0052] In each of the embodiments described in paragraphs

[0050] to

[0051] above, the H1 sequence may be selected from GFTFSSYNMN (sequence number 7) and GFTFSSYNDN (sequence number 8). The H3 sequence may be selected from AYYYGMDV (sequence number 9), AYYYGDDV (sequence number 10), and AYYYGMDK (sequence number 11).

[0053] In each of the embodiments described in paragraphs

[0050] to

[0052] above, the L1 sequence may be selected from RASQGISGWLA (sequence number 12), RASQGISGWEA (sequence number 13), and HASQGISGWLA (sequence number 14). The L3 sequence may be selected from QQANSFPPT (sequence number 15), QQANSEPPT (sequence number 16), and QQANSFPDT (sequence number 17).

[0054] In each of the embodiments described in paragraphs

[0050] to

[0053] above, the antibody or antibody fragment may include a heavy chain variable region having a sequence selected from SEQ ID NOs: 18 to 30.

[0055] In each of the embodiments described in paragraphs

[0050] to

[0054] above, the antibody or antibody fragment may include a light chain variable region having a sequence selected from SEQ ID NOs: 31 to 43.

[0056] In each of the embodiments described in paragraphs

[0050] to

[0055] above, the antibody or antibody fragment may include a heavy chain variable region and a light chain variable region having any pair of sequences selected from SEQ ID NOs: 19 and 32, SEQ ID NOs: 20 and 33, SEQ ID NOs: 21 and 34, SEQ ID NOs: 22 and 35, SEQ ID NOs: 23 and 36, SEQ ID NOs: 24 and 37, SEQ ID NOs: 25 and 38, SEQ ID NOs: 26 and 39, SEQ ID NOs: 27 and 40, SEQ ID NOs: 28 and 41 and SEQ ID NOs: 29 and 42.

[0057] In each of the embodiments described in paragraphs

[0050] to

[0056] above, the antibody or antibody fragment may include a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a combination of amino acid sequences selected from one of sequence numbers 18 to 30 in combination with one of sequence numbers 31 to 43; provided that the heavy chain and light chain variable regions are not sequence numbers 18 and 31 in combination; and the antibody or antibody fragment may specifically bind to human nectin-4 protein.

[0058] In each of the embodiments described in paragraphs

[0050] to

[0057] above, the antibody or antibody fragment may include a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with each of the pairs of amino acid sequences selected from SEQ ID NOs: 19 and 32, SEQ ID NOs: 20 and 33, SEQ ID NOs: 21 and 34, SEQ ID NOs: 22 and 35, SEQ ID NOs: 23 and 36, SEQ ID NOs: 24 and 37, SEQ ID NOs: 25 and 38, SEQ ID NOs: 26 and 39, SEQ ID NOs: 27 and 40, SEQ ID NOs: 28 and 41, and SEQ ID NOs: 29 and 42; however, the heavy chain and light chain variable regions may not be SEQ ID NOs: 18 and 31 in combination; and the antibody or antibody fragment may specifically bind to human nectin-4 protein.

[0059] In each of the embodiments described in paragraphs

[0050] to

[0058] above, the antibody or antibody fragment may specifically bind to nectin-4, or in particular to human nectin-4 protein.

[0060] In yet another embodiment, the antibody or antibody fragment may be multispecific and may bind specifically to nectin-4, or in particular to human nectin-4 protein and CD3, the antibody may include a heavy chain variable region and a light chain variable region, the heavy chain variable region including three complementarity-determining regions having sequences H1, H2, and H3, where: The H1 sequence may be selected from sequence numbers 7 and 8. The H2 sequence can be sequence number 2. The H3 sequence may be selected from sequence number 9, sequence number 10, and sequence number 11; The light chain variable region consists of sequences L1, L2, and L3 (where: The L1 sequence can be X4ASQGISGWX5A (sequence number 4); The L2 sequence can be AASTLQS(sequence ID 5); The L3 sequence could be QQANSX6PX7T (sequence number 6), Here, X4 may be R or H; X5 may be L or E; X6 may be F or E; X7 may be P or D; however, X1, X2, X3, X4, X5, X6 and X7 cannot be M, M, V, R, L, F and P at the same time, respectively), and Six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9 (where: The L4 sequence can be GFTFNTYAMN (sequence number 44), The L5 sequence could be RIRSKYNNYATYYADSVKD (sequence number 45), The L6 array is HX 11 NTE 12 NSX 13 VSWFX 14 It could be Y (sequence number 46), The L7 sequence is RSSTGAVTTSNYX 15 It could be N (sequence number 47), The L8 sequence could be GTNKRAP (sequence number 48), The L9 sequence can be ALWYSNLWV (sequence number 49), Here, X 11 However, it can be G or S, and X 12 However, it could be G or P, and X 13 However, it could be Y or K, and X 14 However, it could be A or Q, and X 15 (However, it could be A or D) It may include three complementarity determination regions having [specific characteristics].

[0061] In another embodiment of the antibody or antibody fragment of paragraph

[0060] , the L6 sequence may be selected from any one of sequence numbers 50 to 53, and the L7 sequence may be selected from sequence numbers 54 and 55.

[0062] In a preferred embodiment, the antibody or antibody fragment of paragraphs

[0060] to

[0061] may include a heavy chain variable region comprising three complementarity-determining regions, H1, H2, and H3, where: The H1 sequence may be selected from sequence numbers 7 and 8. The H2 sequence can be sequence number 2. The H3 sequence may be selected from sequence number 9, sequence number 10, and sequence number 11; The light chain variable region has three complementarity-determining regions that may have sequences L1, L2, and L3 (where: The L1 sequence may be selected from sequence numbers 12, 13, and 14. The L2 sequence can be sequence number 5. The L3 sequence may be selected from SEQ ID NOs. 15, 16, and 17, as well as six anti-CD3 complementarity determination regions L4, L5, L6, L7, L8, and L9 (where: The L4 sequence can be GFTFNTYAMN (sequence number 44), The L5 sequence could be RIRSKYNNYATYYADSVKD (sequence number 45), The L6 sequence may be selected from HGNFGNSYVSWFAY (sequence number 50), HSNFGNSKVSWFAY (sequence number 51), HGNFPNSKVSWFQY (sequence number 52), and HSNFGNSKVSWFAY (sequence number 53). The L7 sequence can be selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and RSSTGAVTTSNYDN (SEQ ID NO: 55). The L8 sequence could be GTNKRAP (sequence number 48), The L9 sequence may be ALWYSNLWV (sequence number 49). Includes.

[0063] In another preferred embodiment, the antibody or antibody fragment of paragraphs

[0060] to

[0062] may include a heavy chain variable region comprising three complementarity-determining regions, H1, H2, and H3, where: The H1 sequence can be sequence number 7. The H2 sequence can be sequence number 2. The H3 sequence can be selected from sequence numbers 9, 10, and 11. The light chain variable region consists of three complementarity-determining regions having sequences L1, L2, and L3 (where: The L1 sequence may be selected from sequence numbers 12 and 13. The L2 sequence can be sequence number 5. The L3 sequence may be sequence number 15), as well as six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9 (where: The L4 sequence can be GFTFNTYAMN (sequence number 44), The L5 sequence could be RIRSKYNNYATYYADSVKD (sequence number 45), The L6 sequence may be selected from HGNFGNSYVSWFAY (sequence number 50), HSNFGNSKVSWFAY (sequence number 51), HGNFPNSKVSWFQY (sequence number 52), and HSNFGNSKVSWFAY (sequence number 53). The L7 sequence can be selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and RSSTGAVTTSNYDN (SEQ ID NO: 55). The L8 sequence could be GTNKRAP (sequence number 48), The L9 sequence may be ALWYSNLWV (sequence number 49). It may include.

[0064] In each of the embodiments described in paragraphs

[0060] to

[0063] above, the antibody or antibody fragment may include a heavy chain variable region having a sequence selected from SEQ ID NOs: 18, 25, 27, and 29.

[0065] In each of the embodiments described in paragraphs

[0060] to

[0064] above, the antibody or antibody fragment may include a light chain variable region having a sequence selected from SEQ ID NOs: 56 to 60, provided that the heavy chain and light chain variable region are not a combination of SEQ ID NOs: 18 and 56.

[0066] In each of the embodiments described in paragraphs

[0060] to

[0065] above, the antibody or antibody fragment may include a heavy chain variable region having a sequence selected from SEQ ID NOs: 18, 25, 27, and 29, and a light chain variable region having a sequence selected from SEQ ID NOs: 56 to 60, provided that the heavy chain and light chain variable region are not a combination of SEQ ID NOs: 18 and 56.

[0067] In each of the embodiments described in paragraphs

[0060] to

[0066] above, the antibody or antibody fragment may include a heavy chain variable region and a light chain variable region having one pair of sequences selected from SEQ ID NOs. 25 and SEQ ID NOs. 57, SEQ ID NOs. 27 and SEQ ID NOs. 58, SEQ ID NOs. 29 and SEQ ID NOs. 59, and SEQ ID NOs. 29 and SEQ ID NOs. 60.

[0068] In each of the embodiments described in paragraphs

[0050] to

[0063] above, the antibody or antibody fragment of the present disclosure may include a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a combination of amino acid sequences selected from one of SEQ ID NOs. 18, 25, 27, and 29 in combination with one of SEQ ID NOs. 56 to 60, provided that the heavy chain and light chain variable regions are not a combination of SEQ ID NOs. 18 and 56; the isolated polypeptide may specifically bind to human nectin-4 protein.

[0069] In each of the embodiments described in paragraphs

[0050] to

[0063] above, the antibody or antibody fragment may include a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a pair of amino acid sequences selected from SEQ ID NOs. 25 and 57, SEQ ID NOs. 27 and 58, SEQ ID NOs. 29 and 59, and SEQ ID NOs. 29 and 60; the antibody or antibody fragment may specifically bind to human nectin-4 protein.

[0070] In each of the embodiments described in paragraphs

[0060] to

[0069] above, the antibody or antibody fragment may be a multispecific antibody or antibody fragment that specifically binds to nectin-4, particularly human nectin-4 protein.

[0071] In preferred embodiments of paragraphs

[0060] to

[0070] , a bispecific antibody or antibody fragment may specifically bind to nectin-4 and CD3, particularly to human nectin-4 protein and CD3.

[0072] In each of the embodiments described in paragraphs

[0050] to

[0071] above, the antibody or antibody fragment may have a higher binding affinity to nectin-4 protein, particularly human nectin-4 protein, at values ​​of conditions in the tumor microenvironment compared to different values ​​of the same conditions occurring in the non-tumor microenvironment. In one embodiment, the conditions may be pH.

[0073] In each of the embodiments described in paragraphs

[0050] to

[0072] above, the antibody or antibody fragment may have at least 70% antigen-binding activity at pH 6.0 compared to the same antigen-binding activity of the parent antibody or antibody fragment at pH 6.0, and the antibody or antibody fragment may have less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% antigen-binding activity at pH 7.4 compared to the same antigen-binding activity of the parent antibody or antibody fragment at pH 7.4. The antigen-binding activity may be able to bind to the nectin-4 protein.

[0074] In each of the embodiments described in paragraphs

[0050] to

[0073] above, the antibody or antibody fragment may have a higher binding affinity to nectin-4 protein, particularly human nectin-4 protein, at the pH in the tumor microenvironment compared to the pH occurring in the non-tumor microenvironment. The pH in the tumor microenvironment may be in the range of 5.0 to 6.8, and the pH in the non-tumor microenvironment may be in the range of 7.0 to 7.6.

[0075] In each of the embodiments described above, the antigen-binding activity of the antibody or antibody fragment can be measured by an ELISA assay.

[0076] In yet another embodiment, the Disclosure provides an immunoconjugate comprising either the antibody or antibody fragment of the Disclosure described above. In the immunoconjugate, the antibody or antibody fragment may be conjugated with a drug selected from chemotherapeutic agents, radioatoms, cell proliferation inhibitors, and cytotoxic agents.

[0077] In yet another embodiment, the Disclosure provides a pharmaceutical composition comprising either the antibody or antibody fragment or immunoconjugate described above in the Disclosure, together with a pharmaceutically acceptable carrier.

[0078] A single dose of the pharmaceutical composition may contain approximately 135 mg, 235 mg, 335 mg, 435 mg, 535 mg, 635 mg, 735 mg, 835 mg, 935 mg, 1035 mg, 1135 mg, 1235 mg, or 1387 mg of antibody, antibody fragment, or immunoconjugate.

[0079] A single dose of the pharmaceutical composition may contain an amount of antibody or antibody fragment, or immunoconjugate, in the range of 135-235 mg, 235-335 mg, 335-435 mg, 435-535 mg, 535-635 mg, 635-735 mg, 735-835 mg, 835-935 mg, 935-1035 mg, 1035-1135 mg, 1135-1235 mg, or 1235-1387 mg.

[0080] Each of the above pharmaceutical compositions may further contain an immune checkpoint inhibitor molecule. The immune checkpoint inhibitor molecule may be an antibody or antibody fragment against an immune checkpoint. The immune checkpoint may be selected from LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, CTLA4, PD-1, PD-L1, GITR, B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS, or the immune checkpoint may be CTLA4, PD-1, or PD-L1.

[0081] Each of the above pharmaceutical compositions may further contain an antibody or antibody fragment against an antigen selected from PD1, PD-L1, CTLA4, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4, and WNT proteins. The WNT protein may be selected from WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16.

[0082] In yet another embodiment, the Disclosure provides a diagnostic or therapeutic kit comprising any of the antibodies or antibody fragments, immunoconjugates, or pharmaceutical compositions described above.

[0083] In yet another embodiment, the Disclosure provides a pharmaceutical composition comprising any of the linkers, conjugates, and / or immunoconjugates of the Disclosure described above, together with a pharmaceutically acceptable carrier.

[0084] This disclosure also provides the use of any one of the above embodiments of linkers, conjugates, and / or immunoconjugates for the treatment of cancer.

[0085] This disclosure further provides a diagnostic or therapeutic kit comprising a linker, conjugate, and / or immunoconjugate of any one of the embodiments described above, and instructions for using the linker, conjugate, and / or immunoconjugate for the diagnosis or treatment of cancer. [Brief explanation of the drawing]

[0086] [Figure 1] SPC07 shows the structure of the conjugate including the linker of this disclosure.

[0087] [Figure 2A-2B] The amino acid sequences of the light chain variable region (Figure 2A) and heavy chain variable region (Figure 2B) of the conditionally active anti-nectin-4 antibodies (BA-143-01-VK and BA-143-01-VH) and wild-type anti-nectin-4 antibodies (BAP143-00-VK and BAP143-00-VH) of this disclosure are shown.

[0088] [Figure 3A-3B]The PK analysis of BA-143-00-MC, BA-143-00-SPC04, BA-143-00-SPC07, BA-143-00-SPC08, BA-143-01-MC, BA-143-01-SPC04, BA-143-01-SPC07, and BA-143-01-SPC08 by pH affinity ELISA at 0.17 hours to 168 hours is shown. The results are summarized in Tables 3 and 4.

[0089] [Figure 4A-4B] The PK analysis by MMAE affinity ELISA for BA-143-00-MC, BA-143-00-SPC04, BA-143-00-SPC07, BA-143-00-SPC08, BA-143-01-MC, BA-143-01-SPC04, BA-143-01-SPC07, and BA-143-01-SPC08 at 0.17 to 168 hours is shown. The results are summarized in Tables 5 and 6.

[0090] [Figure 5A] Table 10-4 shows the average tumor volume for groups 1 (vehicle), 2 (BA-143-00-MC), 4 (BA-143-00-SPC07), 6 (BA-143-01-MC), 8 (BA-143-01-SPC07), 10 (BA-B12-MC), and 12 (BA-B12-SPC07).

[0091] [Figure 5B] Table 7-7 shows the mean tumor volumes for Group 1 (Vehicle), Group 2 (BA-185.01-00502-02-MC), Group 3 (BA-185.01-00502-02-SPC07), Group 4 (BA-185.01-00502-02-SPC17), and Group 5 (BA-185.01-00502-02-SPC19).

[0092] [Figure 5C]Table 7-8 shows the average tumor volume for Group 1 (Vehicle), Group 2 (BA-104.01-00523-01-MC), Group 3 (BA-104.01-00523-01-SPC07), Group 4 (BA-104.01-00523-01-SPC17), and Group 5 (BA-104.01-00523-01-SPC19).

[0093] [Figure 5D] Tables 7-9 and 7-10 show the average tumor volumes for Group 1 (Vehicle), Group 2 (BA-143-00-MC), Group 3 (BA-143-00-SPC07), Group 4 (BA-143-00-SPC17), and Group 5 (BA-143-00-SPC19).

[0094] [Figure 5E] Table 7-11 shows the tumor volumes for Group 1 (Vehicle), Group 2 (BA-091-00-2E2-MC), Group 3 (BA-091-00-2E2-SPC07), Group 4 (BA-091-00-2E2-SPC17), and Group 5 (BA-091-00-2E2-SPC19).

[0095] [Figure 6A] This indicates that there was no significant weight loss (more than 10%) in any of the groups.

[0096] [Figure 6B] This shows the relative weight changes in each of the different groups.

[0097] [Figure 7] This shows the tumor size of each group at different time points after treatment.

[0098] [Figure 8A] This shows that there was no difference in DAR levels in human serum for BA-143-00-MC, BA-143-00-SPC04, BA-143-00-SPC07, and BA-143-00-SPC08.

[0099] [Figure 8B]This shows the difference in %DAR change between BA-143-00-MC and groups BA-143-00-SPC04, BA-143-00-SPC07, and BA-143-00-SPC08.

[0100] [Figure 9A] The binding activity of BA-143-00-MC, BA-143-00-SPC07, BA-143-01-MC, and BA-143-01-SPC07 to human nectin-4 is measured by pH affinity ELISA assay.

[0101] [Figure 9B] The binding activity of BA-143-00-MC, BA-143-00-SPC07, BA0143-01-MC, and BA-143-01-SPC07 to cyno-nectin-4 is measured by pH affinity ELISA assay.

[0102] [Figure 10A] BA-143-00-MC, BA-143-00-SPC04, BA-143-00-SPC07, BA-143-00-SPC08, BA-143-01-MC, BA-143-01-SPC04, BA-143-01-SPC07, and BA-143-01-SPC08 are shown to bind specifically to nectin 4 and to a similar degree at pH 6.0.

[0103] [Figure 10B]BA-143-00-MC, BA-143-00-SPC04, BA-143-00-SPC07, BA-143-00-SPC08, BA-143-01-MC, BA-143-01-SPC04, BA-143-01-SPC07, and BA-143-01-SPC08 specifically bind to nectin-4, and it is shown that BA-143-01-MC, BA-143-01-SPC04, BA-143-01-SPC07, and BA-143-01-SPC08 exhibit reduced binding activity to nectin-4 at pH 7.4 compared to BA-143-00-MC, BA-143-00-SPC04, BA-143-00-SPC07, and BA-143-00-SPC08.

[0104] [Figure 11A] It is shown that BA-143-00-MC, BA-143-00-SPC04, BA-143-00-SPC07, BA-143-00-SPC08, BA-143-01-MC, BA-143-01-SPC04, BA-143-01-SPC07, and BA-143-01-SPC08 specifically bind to human nectin-4 and bind to the same extent at pH 6.0.

[0105] [Figure 11B] BA-143-00-MC, BA-143-00-SPC04, BA-143-00-SPC07, BA-143-00-SPC08, BA-143-01-MC, BA-143-01-SPC04, BA-143-01-SPC07, and BA-143-01-SPC08 specifically bind to human nectin-4, and it is shown that BA-143-01-MC, BA-143-01-SPC04, BA-143-01-SPC07, and BA-143-01-SPC08 exhibit reduced binding activity to human nectin-4 at pH 7.4 compared to BA-143-00-MC, BA-143-00-SPC04, BA-143-00-SPC07, and BA-143-00-SPC08.

[0106] [Figures 12A-12B]BA-143-00-MC, BA-143-00-SPC04, BA-143-00-SPC07, BA-143-00-SPC08, BA-143-01-MC, BA-143-01-SPC04, BA-143-01-SPC07, and BA-143-01-SPC08 specifically bind to human nectin 4, and 0.1 μl / ml of BA-143- The results show that 01-MC, BA-143-01-SPC04, BA-143-01-SPC07, and BA-143-01-SPC08 each exhibited reduced binding activity to human nectin 4 at pH 7.4 compared to BA-143-00-MC, BA-143-00-SPC04, BA-143-00-SPC07, and BA-143-00-SPC08.

[0107] [Figure 13] The inhibition of different doses of BA-143-00-SPC17 and BA-143-00-SPC19 is plotted as concentration-response luminescence signals.

[0108] [Figure 14A] This shows that there was no difference in DAR levels in human serum at days 0, 7, and 14 among BA-143-00-MC, BA-143-00-SPC17, and BA-143-00-SPC19, respectively.

[0109] [Figure 14B] This shows the difference in %DAR change between BA-143-00-MC and groups BA-143-00-SP17 and BA-143-00-SPC19.

[0110] [Figure 15A] The mean tumor volumes for the vehicle group (G1), BA-143-00-SPC17 (G6), and BA-143-00-SPC19 (G8) are shown.

[0111] [Figure 15B] The relative body weight changes for the vehicle group (G1), BA-143-00-SPC17 (G6), and BA-143-00-SPC19 (G8) are shown.

[0112] [Figure 16A] Shows the average weight of different treatment groups and does not include groups showing significant weight loss (more than 10%).

[0113] [Figure 16B] Shows the weight change (%) of different groups.

[0114] [Figure 16C] Shows the average tumor volume of different groups.

[0115] [Figure 17] Shows the effect on the tumor volume of xxT47D xenograft mice by treatment with representative CAB ADCs and WT ADCs of the present disclosure.

[0116] [Figure 18] Shows the protein sequences of the heavy and light chain variable regions of a representative conditionally active anti - nectin - 4 antibody of the present disclosure and the heavy and light chain variable regions of a benchmark wild - type antibody.

[0117] [Figure 19A] When measured by ELISA, shows a higher binding activity of CAB nectin - 4×CAB CD3 at the tumor microenvironment pH compared to physiological pH.

[0118] [Figure 19B] Shows the different binding affinities of CAB nectin - 4×CAB CD3 and WT nectin - 4×WT CD3 at a pH range of 6.0 - 7.4.

[0119] [Figure 19C] Shows the in - vivo efficacy of CAB nectin 4×CAB CD3 compared to isotype×WT CD3 and WT nectin - 6×WT CD3.

[0120] [Figures 20A-20B]The protein sequences of the heavy chain and light chain variable regions of a representative conditionally active nectin-4×CD3 bispecific antibody of this disclosure, as well as the heavy chain and light chain variable regions of a wild-type antibody, are shown. The heavy chains (Figure 20A) are BA-150-19-01-01-BF1-VH (SEQ ID NO: 18), BA-150-30-33-16-BF11-VH (SEQ ID NO: 25), BA-150-30-33-16-BF19-VH (SEQ ID NO: 27), BA-150-30-03-12-BF11-VH (SEQ ID NO: 29), and BA-150-30-03-12-BF19-VH (SEQ ID NO: 29). The light chains (Figure 20B) are BA-150-19-01-01-BF1-LC (SEQ ID NO: 56), BA-150-30-33-16-BF11-LC (SEQ ID NO: 57), BA-150-30-33-16-BF19-LC (SEQ ID NO: 58), BA-150-30-03-12-BF11-LC (SEQ ID NO: 59), and BA-150-30-03-12-BF19-LC (SEQ ID NO: 60).

[0121] [Figure 21A] SPC17 shows the structure of the conjugate including the linker of this disclosure.

[0122] [Figure 21B] SPC19 shows the structure of the conjugate including the linker of this disclosure.

[0123] [Figure 22A]SPC02, 2-(1-(4-((2-bromoacetamido)methyl)benzyl)-1H-1,2,3-triazol-4-yl)-1-(3-nitro-4-(((3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-(( The structure of 1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate is shown.

[0124] [Figure 22B] SPC04, 2-(1-(5-(2-bromoacetamide)pentyl)-1H-1,2,3-triazol-4-yl)-1-(3-nitro-4-(((3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R, The structure of 2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate is shown.

[0125] [Figure 22C]SPC05, 2-(1-(1-bromo-2-oxo-6,9,12-trioxa-3-azatetradecane-14-yl)-1H-1,2,3-triazol-4-yl)-1-(3-nitro-4-(((3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethyl((S)-1-(((S)-1-(((3R,4S,5S)-1-( The structure of (S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate is shown.

[0126] [Figure 22D] SPC06, 2-(1-(10-(2-bromoacetamide)decyl)-1H-1,2,3-triazol-4-yl)-1-(3-nitro-4-(((3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R, The structure of 2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate is shown.

[0127] [Figure 22E]SPC08, 2-(1-(10-(2-bromoacetamide)decyl)-1H-1,2,3-triazol-4-yl)-1-(3-nitro-4-(((3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R, The structure of 2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate is shown. [Modes for carrying out the invention]

[0128] definition To facilitate understanding of the examples provided herein, certain frequently occurring terms are defined herein.

[0129] In relation to the measured quantity, the term “approximately” as used herein refers to the normal variation of the measured quantity that would be expected by a person skilled in the art by performing the measurement and exercising a level of care commensurate with the purpose of the measurement and the precision of the measuring instrument used. Unless otherwise specified, “approximately” refers to a variation of + / - 10% of the given value.

[0130] As used herein, the term “alkyl” refers to branched or unbranched saturated hydrocarbon groups having 1 to 20 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, and hexadecyl. Alkyl groups may be cyclic or acyclic. Alkyl groups may be branched or unbranched. Alkyl groups may also be substituted or unsubstituted. For example, an alkyl group may be substituted with one or more groups, including but not limited to alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing 1 to 6 (e.g., 1 to 4) carbon atoms. The term alkyl is also used for C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C 10 Alkyl, etc. (up to C1~C 20 It may contain alkyl groups.

[0131] As used herein, the term "alkylene" refers to divalent saturated aliphatic groups (such as ethylene, which are derived from alkenes by opening a double bond or from alkanes by removing two hydrogen atoms from different carbon atoms).

[0132] As used herein, the term “affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, “binding affinity” as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those used herein. Specific descriptive and exemplary embodiments for measuring binding affinity are described below.

[0133] As used herein, the term “affinity-mature” antibody refers to an antibody having one or more modifications in one or more heavy chain or light chain variable regions compared to an unmodified parent antibody, such modifications resulting in improved antibody affinity for an antigen.

[0134] As used herein, the term “amino acid” preferably refers to any organic compound containing an amino group (--NH2) and a carboxyl group (--COOH), either as a free group or after condensation as part of a peptide bond. "The 20 naturally encoded alpha-amino acids that form polypeptides" is understood in the art to mean alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine ​​(cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0135] As used herein, the term “antibody” refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules capable of binding to the epitopes of antigens, e.g., Fab, Fab', (Fab')2, Fv, and SCA fragments. These antibody fragments, which retain some ability to selectively bind to antigens (e.g., polypeptide antigens) from which they are derived, can be prepared using methods well known in the art (see, e.g., Harlow and Lane, op. cit.), and are further described below. Antibodies can be used to isolate fractional amounts of antigen by immunoaffinity chromatography. Various other uses of such antibodies are for diagnosing and / or staging diseases (e.g., neotissue formation), and for therapeutic applications to treat diseases such as neotissue formation, autoimmune diseases, AIDS, cardiovascular diseases, and infections. Chimeric antibodies, human-like antibodies, humanized or fully human antibodies are particularly useful for administration to human patients.

[0136] Fab fragments consist of monovalent antigen-binding fragments of antibody molecules and are produced by digesting the entire antibody molecule with the enzyme papain to obtain fragments consisting of intact light chains and parts of heavy chains.

[0137] The Fab' fragment of an antibody molecule can be obtained by treating the entire antibody molecule with pepsin and then reducing it to produce a molecule consisting of intact light chains and parts of the heavy chain. Two Fab' fragments are obtained for each antibody molecule treated in this way.

[0138] The (Fab')2 fragment of an antibody can be obtained by treating the entire antibody molecule with the enzyme pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0139] An Fv fragment is defined as a recombinant fragment containing a variable region of the light chain and a variable region of the heavy chain expressed as two separate chains.

[0140] As used herein, the term “antibody fragment” refers to a molecule other than an intact antibody, which contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0141] As used herein, the terms “anti-nectin-4 antibody,” “nectin-4 antibody,” and “antibody that binds to nectin-4” refer to an antibody capable of binding to nectin-4 with sufficient affinity so that it is useful as a diagnostic and / or therapeutic agent when the antibody targets nectin-4. In one embodiment, the degree of binding of the anti-nectin-4 antibody to unrelated non-nectin-4 proteins is less than about 10% of the antibody binding to nectin-4, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to nectin-4 has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant of M). In certain embodiments, the anti-nectin-4 antibody binds to an epitope of nectin-4 that is conserved among nectin-4 from different species, for example, the extracellular domain of nectin-4.

[0142] The term "nectin-4" has its general meaning in the art and includes human nectin-4, particularly the natural sequence polypeptide, isoforms, chimeric polypeptide, all homologs, fragments, and precursors of human nectin-4. The amino acid sequence of natural nectin-4 includes the NCBI reference sequence: NP_112178.2.

[0143] As used herein, the term “binding” refers to the interaction between the variable region or Fv of an antibody and an antigen, and the interaction depends on the presence of a specific structure within the antigen (e.g., an antigenic determinant or epitope). As used herein, the terms “specifically binding” or “binding specifically” mean that an antibody variable region (Fv) binds to or associates with a particular antigen (i.e., “that antigen”) more frequently, more rapidly, for a longer duration, and / or with higher affinity than it binds to or associates with other proteins. For example, an antibody variable region (Fv) specifically binds to that particular antigen with higher affinity, binding activity (binding activity), more readily, and / or for a longer duration than it binds to other antigens. In another example, an antibody variable region (Fv) binds to a particular cell surface protein (antigen) with substantially higher affinity than it binds to a related protein or other cell surface protein or antigen commonly recognized by a polyreactive native antibody (i.e., by a native antibody known to bind to a variety of naturally occurring antigens in humans). However, “specific binding” does not require “selective binding,” i.e., exclusive binding to a particular antigen, and there is no binding to or detection of binding to any other antigen. For example, "specific binding" of an antibody variable region or Fv (or other binding region) to an antigen means that the antibody variable region or Fv binds to the antigen with a dissociation constant (Kd) of, for example, 100 nM or less, or 50 nM or less, or 20 nM or less, or 15 nM or less, or 10 nM or less, or 5 nM or less, or 2 nM or less, or 1 nM or less.

[0144] As used herein, the terms “cancer” and “malignant” refer to or describe physiological conditions in mammals that are typically characterized by unregulated cell proliferation / growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin and non-Hodgkin lymphomas), blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia, and other lymphoproliferative disorders, as well as various types of head and neck cancers.

[0145] As used herein, the terms “proliferative disorder” and “proliferative disorder” refer to disorders related to a certain degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer.

[0146] As used herein, the term “chemotherapeutic agent” refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, metsuredopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomellamine; acetogenins (especially bratacin and bratacinone); Δ-9-tetrahydrocannabinol (dronabinol, MARINOL®); β-rapacone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; calistatin; CC-106 5 (including its synthetic analogues adzeresin, karzeresin, and bizeresin); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin; duocalmycin (including synthetic analogues, KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodicin; spongstatin; chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifospha Nitrogen mustards such as mid, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobuenvicin, fenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; engine antibiotics (e.g., calichemycin, especially calichemycin γ1I and calichemycin ωI1 (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); CDP323, oral α-4 integrin inhibitors; dinemycin including dinemycin A; esperamicin;(and neocartinostatin chromophores and related pigment proteins, enediin antibiotic chromophores), acrasinomycin, actinomycin, autoramycin, azaserin, bleomycin, kactinomycin, carabicin, caminomycin, cartinophylline, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (adriamycin®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (Doxil®), liposomal doxorubicin TLC) D-99 (MYOCET®), including pegylated liposomal doxorubicin (CAELYX®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epotilon, and 5-fluorouracil (5-FU); denopterin, methotrexate, pteropterin, Folic acid analogs such as trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisantren; edatrexate; defofamine; demecolsin; diazicone; elformitin; eriptinium acetate; epotilone;Etoglucid; gallium nitrate; hydroxyurea; lentinan; ronidynin; mytansinoids such as mytansin and anthamitosin; mitogwazone; mitoxantrone; mopidammol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural) Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadiquan; 2,2',2'-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, Beraclin A, Loridine A and Anguidin); Urethane; Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Thiotepa; Taxoids, e.g., Paclitaxel (TAXOL®), Albumin-Modified Nanoparticle Formulations of Paclitaxel (ABRAXANE®), and Docetaxel (TAXOTERE (Registered Trademark)); Chlorambucil; 6-Thiogunine; Mercaptopurine; Methotrexate; Platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; Vinca, which prevents microtubule formation by tubulin polymerization, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); Etoposide (VP-16); Ifosfamide; Mitoxantrone; Leucovorin; Novantrone; Edatrexate; Daunomycin; Aminopterin; Ibandronate; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMF®); Retinoids such as retinoic acid, including bexarotene (TARGRETIN®);Bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tildronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, in particular those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTI N(registered trademark) vaccines and VAXID(registered trademark) vaccines; topoisomerase 1 inhibitors (e.g., LURTOTECAN(registered trademark)); rmRH (e.g., Abarelix(registered trademark)); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT(registered trademark), Pfizer); perifosin, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); Lutezomib (VELCADE®); CCI-779; tipifarnib (R11577); olafenib, ABT510; oblimersen sodium (GENASENSE®); pixantrone; Bcl-2 inhibitors such as EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such as ronafarnib (SCH 6636, SARASAR®); and any pharmaceutically acceptable salts, acids, or derivatives of the above; as well as combinations of two or more of the above, such as CHOP (an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone);Examples include FOLFOX (an abbreviation for a treatment regimen using oxaliplatin (ELOXATIN®) in combination with 5-FU and leucovorin).

[0147] Chemotherapy agents as defined herein include “anti-hormone agents” or “endocrine therapeutic agents” that act to modulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth. These may be hormones themselves, but are not limited to: anti-estrogen agents having a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), doxifen, doroxifen, raloxifene (EVISTA®), trioxyfen, keoxyfen, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogen agents without agonist properties, such as fulvestrant (FASLODEX®) and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); steroidal aromatase inhibitors such as formestan and exemestane (AROMASIN®), and anastrazole (ARIMIDEX®), letrozole (FEMARA®), and others. Aromatase inhibitors including nonsteroidal aromatase inhibitors such as aminoglutethimide, as well as other aromatase inhibitors including borozol (RIVISOR®), megestrol acetate (MEGASE®), fadrozol, and 4(5)-imidazole; luteinizing hormone-releasing hormone agonists including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and triptorelin; megestrol acetate and medroxypro acetate This includes progestins such as gesterone, estrogens such as diethylstilbestrol and premarin, and sex steroids including androgens / retinoids such as fluoxymesterone, all trans-retionic acids and fenretinide; onapristone; anti-progesterone agents; estrogen receptor down-regulators (ERDs); anti-androgens such as flutamide, nilutamide and bicalutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above.

[0148] As used herein, the term “chimeric” antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a particular source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0149] As used herein, the term “conditionally active antibody” refers to an anti-nectin-4 antibody that is more active under conditions within the tumor microenvironment compared to conditions within the non-tumor microenvironment. Conditions within the tumor microenvironment include lower pH, higher concentrations of lactate and pyruvate, hypoxia, lower concentrations of glucose, and slightly higher temperature compared to the non-tumor microenvironment. For example, a conditionally active antibody may be substantially inactive at normal body temperature but active at higher temperatures within the tumor microenvironment. In yet another embodiment, a conditionally active antibody may be less active in normal oxygenated blood but more active in the hypoxic environment present in tumors. In yet another embodiment, a conditionally active antibody may be less active at the normal physiological pH of 7.0–7.6 but more active at the acidic pH of 5.0–6.8, or 6.0–6.8, present in the tumor microenvironment. Other conditions within the tumor microenvironment are known to those skilled in the art and may also be used as conditions in this disclosure, under which anti-nectin-4 antibodies have different binding affinities to nectin-4.

[0150] As used herein, the term “cell growth inhibitor” refers to a compound or composition that stops cell growth either in vitro or in vivo. Therefore, cell growth inhibitors may significantly reduce the percentage of cells in the S phase. Further examples of cell growth inhibitors include agents that block cell cycle progression by inducing G0 / G1 arrest or M phase arrest. The humanized anti-Her2 antibody trastuzumab (HERCEPTIN®) is an example of a cell growth inhibitor that induces G0 / G1 arrest. Classical M phase blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors (such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin). Certain drugs that stop G1, such as tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and DNA alkylating agents such as ara-C, also cause S-phase arrest. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WBSaunders, Philadelphia, 1995), e.g., p. 13. Taxanes (paclitaxel and docetaxel) are both anticancer drugs derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semi-synthetic analog of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel stabilize microtubules by promoting microtubule assembly from tubulin dimers and inhibiting depolymerization, leading to inhibition of intracellular mitosis.

[0151] As used herein, the term “cytotoxic agent” refers to a substance that inhibits or blocks cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212 Radioactive isotopes of Lu); chemotherapeutic agents or chemotherapy drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes such as nucleases and their fragments; antibiotics; toxins such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin (including their fragments and / or variants); and various antitumor or anticancer agents disclosed below.

[0152] As used herein, the term “diabody” refers to a small antibody fragment having two antigen-binding sites, which are the same polypeptide chain (V H -V L ) Light chain variable domain (V L ) bound to the heavy chain variable domain (V H ) includes. By using a linker that is too short to allow pairing between two domains on the same chain, the domains are made to pair with complementary domains on another chain, generating two antigen-binding sites.

[0153] As used herein, the term “detectably labeled” refers to any substance whose detection or measurement, by physical or chemical means, directly or indirectly, indicates the presence of an antigen in a sample. Typical examples of useful detectable labels include, but are not limited to,: molecules or ions that are directly or indirectly detectable based on absorbance, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties; molecules or ions that are detectable by radioactivity; and molecules or ions that are detectable by nuclear magnetic resonance or paramagnetism. Among the molecules that are indirectly detectable based on absorbance or fluorescence are, for example, various enzymes that convert suitable substrates, for example, from non-light-absorbing molecules to light-absorbing molecules, or from non-fluorescent molecules to fluorescent molecules.

[0154] As used herein, the term “diagnosis” refers to determining the susceptibility of an object to a disease or disorder, determining whether an object is currently suffering from a disease or disorder, making a prognostic diagnosis of an object suffering from a disease or disorder (e.g., identifying a pre-metastatic or metastatic cancerous state, the stage of cancer, or the response of cancer to a treatment), and guiding a treatment plan (e.g., monitoring the object’s condition to provide information regarding the effectiveness or efficacy of a treatment). In some embodiments, the diagnostic methods of this disclosure are particularly useful in detecting early-stage cancer.

[0155] As used herein, the term “diagnostic agent” refers to a molecule that can be detected directly or indirectly and used for diagnostic purposes. Diagnostic agents may be administered to a subject or sample. Diagnostic agents may be provided on their own or conjugated to a vehicle such as a conditionally active antibody.

[0156] As used herein, the term “effector function” refers to the biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0157] As used herein, the term “effective dose” of a drug refers, for example, to a pharmaceutical formulation, to the amount effective in the dosage and duration required to achieve the desired therapeutic or prophylactic outcome.

[0158] As used herein, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes both the natural sequence (or wild-type) Fc region and the variant (or mutant) Fc region. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0159] As used herein, the term "framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR or H1-3 in the heavy chain and L1-3 in the light chain). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally follow this order: V H (or V L ): Appears in FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0160] The terms "full-length antibody," "intact antibody," or "whole antibody" refer to the antigen-binding variable region (V). H or V LThis refers to antibodies containing a light chain constant domain (CL) and a heavy chain constant domain, CH1, CH2, and CH3. The constant domain may be a natural sequence constant domain (e.g., human natural sequence constant domain) or an amino acid sequence variant thereof. Depending on the amino acid sequence of the constant domain of their heavy chains, different "classes" may be assigned to full-length antibodies. There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0161] As used herein, the term “function-conserving variant” refers to a given amino acid residue in a protein or enzyme that has been altered without altering the overall conformation and function of the polypeptide, and includes, but is not limited to, amino acid substitutions by amino acids having similar properties (e.g., polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity, aromaticity, etc.). Amino acids other than those indicated as conserved may result in proteins that differ in such a way that the percentage of protein or amino acid sequence similarity between any two proteins with similar functions may vary, for example, from 70% to 99% when the similarity is determined according to an alignment scheme such as the clustering method based on the MEGALIGN algorithm. A “function-conserving variant” also includes polypeptides having at least 60% amino acid identity, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95%, when determined by the BLAST or FASTA algorithm, and which have the same or substantially similar properties or functions as the native or parent protein being compared.

[0162] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used synonymously and refer to cells into which exogenous nucleic acids have been introduced (including the offspring of such cells). Host cells include “transformed cells” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the number of passages. Offspring may not have exactly the same nucleic acid content as the parent cells and may contain mutations. Mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included herein.

[0163] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence that corresponds to an antibody produced by a human or human cell, or an amino acid sequence derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0164] As used herein, the term “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from a non-human CDR and amino acid residues derived from a human FR. In certain embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR corresponds to that of a non-human antibody, and all or substantially all of the FR corresponds to that of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0165] As used herein, the term “immunoconjugate” refers to an antibody conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic agents.

[0166] As used herein, the terms “individual” or “subject” refer to mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0167] As used herein, the term “inhibition of cell growth or proliferation” means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0168] As used herein, the term “isolated” antibody refers to an antibody isolated from its natural environment. In some embodiments, antibodies are purified to a purity of 95% or greater than 99%, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase high-performance liquid chromatography (HPLC)). For an overview of antibody purity assessment methods, see, for example, Flatman et al., J. Chromatogr. B, vol. 848, pp. 79-87, 2007.

[0169] As used herein, the term “isolated nucleic acid encoding an anti-nectin-4 antibody” refers to such nucleic acid molecules in a single vector or separate vectors, and one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules present in one or more locations within a host cell.

[0170] As used herein, the term “metastasis” refers to all nectin-4-involved processes that support cancer cells dispersing from a primary tumor, infiltrating lymphatic vessels and / or blood vessels, circulating through the bloodstream, and proliferating in distal lesions (metastases) in normal tissues elsewhere in the body. In particular, it refers to cellular events of tumor cells that underlie metastasis and are stimulated or mediated by nectin-4, such as proliferation, migration, anchorage independence, evasion of apoptosis, or secretion of angiogenic factors.

[0171] As used herein, the term “microenvironment” means any part or region of a tissue, organ, or body that has immutable or transient, physical or chemical differences from other regions of that tissue, organ, or body. In the case of a tumor, as used herein, the term “tumor microenvironment” means the environment in which the tumor resides, which includes non-cellular regions within the tumor and regions directly outside the tumor tissue but not related to the intracellular compartments of the cancer cells themselves. The tumor and the tumor microenvironment are closely related and constantly interacting. The tumor can alter its microenvironment, and the microenvironment can influence the growth and spread of the tumor. Typically, the tumor microenvironment has a low pH in the range of 5.0–6.8, or 5.8–6.8, or 6.2–6.8. In contrast, the normal physiological pH is in the range of 7.0–7.6. The tumor microenvironment is also known to have lower concentrations of glucose and other nutrients but higher concentrations of lactate compared to plasma. Furthermore, the tumor microenvironment may have a temperature 0.3–1°C higher than the normal physiological temperature. The tumor microenvironment is described in Gillies et al., “MRI of the Tumor Microenvironment,” Journal of Magnetic Resonance Imaging, vol. 16, pp. 430-450, 2002, which is incorporated herein by reference in its entirety. The term “non-tumor microenvironment” refers to the microenvironment in sites other than tumors.

[0172] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies within that population are identical and / or bind to the same epitope, except for variant antibodies that, for example, contain naturally occurring mutations or may arise during the production of a monoclonal antibody preparation, such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which contain different antibodies that are typically specific to different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is specific to a single determinant of the antigen. Therefore, the modifier “monoclonal” should not be interpreted as indicating an antibody characteristic such as that obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies used in accordance with this disclosure may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0173] As used herein, the term "naked antibody" refers to an antibody that is not conjugated with a heterogeneous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in pharmaceutical formulations.

[0174] As used herein, the term “packaging instructions” refers to the instructions that are customarily included in the market packaging of such therapeutic products, including information relating to indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings.

[0175] With respect to the reference polypeptide sequence used herein, the term “percent (%) amino acid sequence identity” is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved in various ways within the scope of skill in the art using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. However, for the purposes herein, the % amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office (Washington DC, 20559) and is registered under U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program must be compiled for use with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0176] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity between a given amino acid sequence B and a given amino acid sequence A (or, as can be expressed, a given amino acid sequence A having a specific % amino acid sequence identity with or containing a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y (In the formula, X is the number of amino acid residues that were scored as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B). It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding section.

[0177] As used herein, the term “pharmaceutical preparation” refers to a preparation that is in a form that enables the biological activity of the active ingredient contained herein, and that does not contain any additional toxic ingredients that would be unacceptable to the subject to which the preparation is administered.

[0178] As used herein, the term “pharmaceutically acceptable carrier” refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0179] As used herein, the terms “purified” and “isolated” refer to antibodies or nucleotide sequences as disclosed herein, and mean that the indicated molecules exist in the substantial absence of other biomolecules of the same type. As used herein, the term “purified” preferably means the presence of at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of biomolecules of the same type. An “isolated” nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that substantially does not contain other nucleic acid molecules that do not encode the polypeptide; however, the molecule may contain several additional bases or portions that do not adversely affect the fundamental characteristics of the composition.

[0180] As used herein, the term “recombinant antibody” refers to an antibody expressed by a recombinant host cell containing nucleic acid encoding the antibody (e.g., a chimeric, humanized, or human antibody or its antigen-binding fragment). Examples of "host cells" for producing recombinant antibodies include: (1) mammalian cells, e.g., Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 and NS0 cells), baby hamster kidney (BHK), Hela and Vero cells; (2) insect cells, e.g., sf9, sf21 and Tn5; (3) plant cells, e.g., plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, e.g., those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or those belonging to the genus Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, e.g., Escherichia coli cells or Bacillus subtilis Examples include subtilis cells.

[0181] As used herein, the term “single-stranded Fv” (“scFv”) refers to covalently bonded V H ::V LIt is a heterodimer, which typically encodes a gene linked by a peptide coding linker, V H and V L It is expressed from a gene fusion containing [the specified gene]. "dsFv" is V stabilized by a disulfide bond. H :V L It is a heterodimer. Divalent and polyvalent antibody fragments, such as divalent sc(Fv)2, can be spontaneously formed by the non-covalent association of monovalent scFv or can be produced by covalently bonding monovalent scFv with a peptide linker.

[0182] The term “therapeutic dose” of the antibodies in this disclosure means an amount of antibody sufficient to treat the cancer in a reasonable benefit-risk ratio applicable to any medical treatment. However, it will be understood that the total daily dose of the antibodies and compositions in this disclosure will be determined by the attending physician within reasonable medical judgment. The level of a particular therapeutic dose for any particular patient depends on a variety of factors, including the disorder being treated and its severity; the activity of the particular antibody used; the particular composition used, the patient’s age, weight, overall health, sex, and diet; the timing, route of administration, and excretion rate of the particular antibody used; the duration of treatment; drugs used in combination with or concurrently with the particular antibody used; and similar factors well known in the medical field. For example, it is well known in the art to start administration of a compound at a level lower than necessary to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0183] As used herein, the terms “treatment,” “to treat,” or “to treat” refer to a clinical intervention in an attempt to alter the natural course of the individual being treated, and may be carried out either for prevention or in the course of clinicopathology. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of this disclosure are used to delay the onset of disease or to delay the progression of disease.

[0184] As used herein, the term “tumor” refers to all tumor cell proliferation and growth, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as used herein.

[0185] As used herein, the terms “variable region” or “variable domain” refer to domains of the antibody heavy chain or light chain involved in the binding of the antibody to an antigen. (V) H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single V H or V L The domain may be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds to a specific antigen may be derived from an antibody that binds to the antigen. H or V L The domains are isolated, and each is a complementary V L or V HYou can screen domain libraries. For example, see Portolano et al., J.Immunol., vol.150, pp.880-887, 1993; Clarkson et al., Nature, vol.352, pp.624-628, 1991.

[0186] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of a host cell into which they are introduced. Certain vectors can induce the expression of nucleic acids to which they are operably ligated. Such vectors are referred to herein as “expression vectors.”

[0187] Detailed description of the invention For illustrative purposes, the principles of this disclosure are explained by reference to various exemplary embodiments. While specific embodiments of this disclosure are described herein, those skilled in the art will readily understand that the same principles are equally applicable to and can be used in other systems and methods. Before describing in detail the embodiments disclosed herein, it should be understood that this disclosure is not limited in its application to the details of any specific embodiments shown. Furthermore, the terminology used herein is for illustrative purposes only, not limiting purposes. Moreover, while specific methods are described by reference to the steps shown herein in a particular order, in many cases these steps can be performed in any order as can be understood by those skilled in the art; therefore, novel methods are not limited to the specific arrangement of the steps disclosed herein.

[0188] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise specified in the context. Furthermore, the terms “a” (or “an”), “one or more,” and “at least one” may be used synonymously herein. The terms “comprising,” “including,” “having,” and “constructed from” may also be used synonymously.

[0189] Unless otherwise indicated, all numbers used in this specification and the claims, such as quantities, molecular weights, percentages, ratios, and reaction conditions of components, should be understood to be modified by the term "approximately," whether or not the term "approximately" is present. Therefore, unless otherwise stated, the numerical parameters described herein and in the claims are approximations that may vary depending on the desired properties to be obtained by this disclosure. Each numerical parameter should be interpreted, at least in consideration of the reported number of significant figures and by applying the usual rounding method, not as an attempt to limit the application of the doctrine of equivalents to the claims. Although the numerical ranges and parameters representing the broad scope of this disclosure are approximations, the numerical values ​​shown in specific examples are reported as accurately as possible. However, each numerical value inherently contains some degree of error, which is necessarily due to the standard deviation found in the respective test measurements.

[0190] It should be understood that each component, compound, substituent, or parameter disclosed herein is disclosed for use alone or in combination with any one or more other components, compounds, substituents, or parameters disclosed herein.

[0191] Each quantity / value or range of quantity / value disclosed herein for each component, compound, substituent, or parameter should be interpreted as being disclosed in combination with any other quantity / value or range of quantity / value disclosed herein for any other component, compound, substituent, or parameter disclosed herein, and therefore, any combination of two or more quantities / values ​​or ranges of quantity / value disclosed herein for any other component, compound, substituent, or parameter should also be understood as being disclosed in combination with each other for the purposes of this specification.

[0192] It is further understood that each lower limit of each range disclosed herein should be interpreted as being disclosed in combination with each upper limit of each range disclosed herein for the same component, compound, substituent, or parameter. Accordingly, a disclosure of two ranges should be interpreted as a disclosure of four ranges derived by combining each lower limit of each range with each upper limit of each range. A disclosure of three ranges should be interpreted as a disclosure of nine ranges derived by combining each lower limit of each range with each upper limit of each range, and so on. Furthermore, any specific amount / value of a component, compound, substituent, or parameter disclosed herein or in the examples should be interpreted as a disclosure of either a lower or upper limit of a range, and thus may be combined with any other lower or upper limit or specific amount / value of the same component, compound, substituent, or parameter disclosed elsewhere in this application to form a range for that component, compound, substituent, or parameter.

[0193] A. Linker This disclosure is, [ka] We provide a linker (L) having the formula represented by, where R1 is C1~C 10 Alkyl halides, C1-C 10 Alkyl alcohols, phenols, C1-C 10 Alkylthiols, C1-C 10 Alkylcarboxylic acids, and C1-C 10A nucleophilic functional group capable of binding to an antibody selected from the group consisting of alkyl azides, R2 is either substituted or non-substituted C1-C 20 Alkylene, substituted or unsubstituted C8-C 10 Alkylene-phenyl-alkylene, or a substituted or unsubstituted (PEG)n portion represented by the following: [ka] In the formula, n is between 1 and 20. R7 is either an azide group or an acetylene group.

[0194] In one embodiment of the linker described above, R1 may be a C1-C6 alkyl halide, a C1-C2 alkyl alcohol, a 4-phenol, or a C1-C2 alkylthiol having a halide selected from the group consisting of Br, I, Cl, and F.

[0195] In the above embodiment of the linker, R1 may be -CH2-Br.

[0196] In any one of the above linker embodiments, R2 is unsubstituted C1~C 20 It could be alkylene.

[0197] In the above embodiment of the linker, R2 may be an unsubstituted C2-C5 alkylene.

[0198] In any one of the above linker embodiments, R2 is non-substituted (PEG) n This is possible, where n is 8.

[0199] In any one of the above embodiments of the linker, R7 may be an azide group.

[0200] In any one of the above embodiments, the linker is [ka] The structure may be represented by the formula, where p can be 1 to 20, preferably 2 to 5.

[0201] In the above embodiment, the linker is [ka] It may have a structure represented by [this].

[0202] In another embodiment described above, the linker is: [ka] It may have a structure represented by [this].

[0203] In another embodiment described above, the linker is: [ka] It may have a structure represented by the formula, where n is 8.

[0204] The linker is C1-C, either substituted or unsubstituted. 20 Alkyl, or any substituted or unsubstituted (PEG)n portion represented by the following: [ka] The formula may include n, where n is between 1 and 20.

[0205] The “linker” in this disclosure may be a bifunctional or polyfunctional component or portion that can be used to link one or more drug (D) portions (payloads) to a polypeptide such as an antibody (Ab) to form a drug conjugate, immunoconjugate, or antibody-drug conjugate (ADC). The linkers in this disclosure play a crucial role in the stability of drug conjugates and ADCs and provide a targeted payload release (release of one or more drug portions) profile that is important for the efficacy of the ADC drug. The linkers in this disclosure are stable in the circulatory system and reduce systemic off-target toxicity by specifically releasing cytotoxic payloads to targets such as tumors.

[0206] It is desirable to generate a linker of the present disclosure that can covalently bind one or more drug portions to an antibody or an antibody fragment thereof.

[0207] Specifically, the linkers of this disclosure can covalently bind an antibody (Ab) or a fragment thereof to form an ADC without the drawback of nonspecific release of the payload in order to optimize the therapeutic range of the ADC. The linkers of this disclosure may be directly or indirectly covalently bound to the drug portion described herein.

[0208] As described above, the linker of the present disclosure includes an antibody-binding group or a nucleophilic functional group capable of binding to an antibody at one end and a reactive group at the other end, which is complementary to the acetylene or azide group of the tether group ( "X") described herein. Preferably, the reactive group reacts with an acetylene or azide group bonded to R3 of the tether group by acetylene-azide cycloaddition to yield a conjugate useful for conjugating to an antibody or antibody fragment of the present disclosure, which is an azide or acetylene at R7. This cycloaddition reaction between the acetylene moiety and the azide moiety is an example of "click reaction" or "click chemistry", which is described by Kolb H.C. and K.B. Sharpless, Drug Discov. Today, 8:1128-37 (2003), the disclosure of which is incorporated herein by reference in its entirety. The click reaction or click chemistry is chemoselective and can complement other conjugation chemistries such as the thiol-maleimide reaction.

[0209] The antibody-binding group or nucleophilic functional group capable of binding to an antibody may include, but is not limited to, C1-C 10 alkyl halide, C1-C 10 alkyl alcohol, phenol, C1-C 10 alkyl thiol, C1-C 10 alkyl carboxylic acid, and C1-C 10 alkyl azide. Preferably, the antibody-binding group or nucleophilic functional group capable of binding to an antibody is a C1-C6 alkyl halide, and the halide is selected from the group consisting of Br, I, Cl, and F, C1-C2 alkyl alcohol, 4-phenol, or C1-C2 alkyl thiol.

[0210] B. Drug Conjugate The tether group ( "X") described herein may include an acetylene or azide group useful for conjugating with the linker of the present disclosure. In some cases, as described herein and as applicable throughout the present disclosure, the substituents of substituted alkyl, substituted aryl, substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl are substituted or unsubstituted C1-C20 Alkyl, substituted or unsubstituted C6 - C 25 Aryl, substituted or unsubstituted C2 - C 25 Heteroaryl, substituted or unsubstituted C3 - C 25 Cycloalkyl, and substituted C2 - C 25 Heterocyclyl, and / or PEG n (where n is 1 - 25).

[0211] As described herein and as applicable throughout the present disclosure, substituted or unsubstituted C1 - C 20 Alkyl can be straight - chained or branched - chained. The alkyl group contains 1 - 20 carbon atoms, for example, 1 - 10 carbon atoms, 1 - 8 carbon atoms, 1 - 6 carbon atoms, 1 - 5 carbon atoms, 1 - 4 carbon atoms, 1 - 3 carbon atoms, or 1 - 2 carbon atoms. For example, substituted or unsubstituted C1 - C 20 Alkyl is substituted or unsubstituted alkylene, for example, substituted or unsubstituted C1 - C 10 Alkylene, or substituted or unsubstituted C1 - C8 alkylene, substituted or unsubstituted C1 - C6 alkylene, substituted or unsubstituted C1 - C5 alkylene, substituted or unsubstituted C1 - C4 alkylene, substituted or unsubstituted C1 - C3 alkylene, or substituted or unsubstituted C1 - C2 alkylene. In some cases, the substituted alkyl can contain substituents such as cleavable moieties, for example, enzymatically cleavable moieties such as glycosides or glycoside derivatives.

[0212] As described herein and as applicable throughout the present disclosure, substituted or unsubstituted aryl can contain phenyl. In some cases, the substituted aryl is substituted phenyl, where the substituent contains a cleavable moiety such as an enzymatically cleavable moiety such as a glycoside or glycoside derivative.

[0213] Similarly, in some cases, substituents on a substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl may include cleavable moieties, such as enzymatically cleavable moieties like glycosides or glycoside derivatives.

[0214] Preferably, the tether group ("X") is [ka] It is expressed by the formula, where R3 is C1~C 20 It may be an alkylene; R4 may be a triazole ring having a C=C double bond in the ring. The triazole ring of R4 may be formed by a click reaction between the azide group and the acetylene group.

[0215] This disclosure relates to the conjugate of formula I, [ka] Equation I Provided, in the formula: L, [ka] It is represented by, and R1 is C1~C 10 Alkyl halides, C1-C 10 Alkyl alcohols, phenols, C1-C 10 Alkylthiols, and C1-C 10 It may be a nucleophilic functional group that can bind to an antibody selected from the group consisting of alkyl azides, R2 is either substituted or non-substituted C1-C 20 Alkylene, substituted or unsubstituted C8-C 10 Alkylene-phenyl-alkylene, or substituted or unsubstituted (PEG) represented as follows: n It could be a part, [ka] In the formula, n can be between 1 and 20; X is a tether group. Preferably, X is [ka] It is expressed by the formula, where R3 is C1~C 20 It could be an alkylene; R4 could be a triazole ring having a C=C double bond in the ring; Y may be a glycoside or a glycoside derivative; D could be a drug; The wavy lines indicate the locations of covalent bonds.

[0216] In one embodiment of the above conjugate, R1 may be a C1-C6 alkyl halide, a C1-C2 alkyl alcohol, a 4-phenol, or a C1-C2 alkylthiol having a halide selected from the group consisting of Br, I, Cl, and F.

[0217] In any one of the above embodiments of the conjugate, R1 may be -CH2-Br.

[0218] In any one of the above conjugate embodiments, R2 is unsubstituted C1~C 20 It could be alkylene.

[0219] In a particular embodiment of the above conjugate, R2 may be an unsubstituted C2-C5 alkylene.

[0220] Alternatively, in any one of the above embodiments of the conjugate, R2 is non-substituted (PEG) n This is possible, where n is 8.

[0221] In any one of the above embodiments of the conjugate, R3 may be -CH2-.

[0222] In any one of the above embodiments of the conjugate, glycoside Y may be selected from the following structures. [ka]

[0223] In any one of the above embodiments of the conjugate, the glycoside or glycoside derivative Y may be a glucuronide.

[0224] In any one of the above embodiments of the conjugate, the triazole ring of R4 is [ka] It can be formed with an azide group bonded to L, which can be represented by the formula (where p is 2 to 5).

[0225] Alternatively, in any one of the above embodiments of the conjugate, the triazole ring is [ka] It can be formed with an azide group bonded to L, which can be represented by the formula (where n is 8).

[0226] In any one of the above conjugate embodiments, R2 is either substituted or unsubstituted (PEG) n If this is the case, then Y is not a galactoside.

[0227] In any one of the above-described embodiments of the conjugate, drug D may be selected from the group consisting of auristatin, drastatin, meitansinoid, calicheamicin, pyrrolobenzodiazepine, anthracycline, ribonuclease, and DNA endonuclease.

[0228] In any one of the above-described embodiments of the conjugate, drug D may be an auristatin selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

[0229] Examples of conjugates in this disclosure include, but are not limited to, the following structures:

[0230] In any one of the above embodiments of the conjugate, the conjugate is SPC07 [ka] It may have a structure represented by, Conjugate is SPC19 [ka] It may have a structure represented by, Conjugate is SPC17 [ka] It may have a structure represented by, Conjugate is SPC02 [ka] It may have a structure represented by, Conjugate is SPC04 [ka] It may have a structure represented by, Conjugate is SPC05 [ka] It may have a structure represented by, Conjugate is SPC06 [ka] It may have a structure represented by, Conjugate is SPC08 [ka] It may have a structure represented by [this].

[0231] D. Method for preparing linkers and drug conjugates The Disclosure also provides methods for preparing the linkers and drug conjugates of the Disclosure, as illustrated in Examples 1 to 6 described below.

[0232] In the preparation of linkers or drug conjugates according to this disclosure, compounds represented by formula III are also provided: [ka] In the formula, R 5a and R 5b However, independently or simultaneously, these are hydrogen, a substituted or unsubstituted C1-C6 acyl, or a substituted or unsubstituted C1-C6 alkyl; R6 [ka]

[0233] In the above embodiment, R6 is [ka] If R 5a The acetyl group is R 5b is hydrogen; R6 [ka] If R 5a The acetyl group is R 5b Is it a methyl group? or R6 [ka] If R 5a and R 5b Each of them is hydrogen.

[0234] E. Immunoconjugates This disclosure relates to the immunoconjugate of formula II, [ka] Formula II Provided, in the formula: L, [ka] It is represented by, and R1 is C1~C 10 Alkyl halides, C1-C 10 Alkyl alcohols, phenols, C1-C 10 Alkylthiols, and C1-C 10 It may be a nucleophilic functional group that can bind to an antibody selected from the group consisting of alkyl azides, R2 is either substituted or non-substituted C1-C 20 Alkylene, substituted or unsubstituted C8-C 10 Alkylene-phenyl-alkylene, or substituted or unsubstituted (PEG) represented as follows: n It could be a part, [ka] In the formula, n can be between 1 and 20; X [ka] It can be a substituted nitrophenyl represented by the formula, where R3 is C1~C 20 It could be an alkylene; R4 could be a triazole ring having a C=C double bond in the ring; Y may be a glycoside or a glycoside derivative; D could be a drug; The wavy lines indicate the covalent bond sites; m can be between 1 and 10.

[0235] In any one of the embodiments of the immunoconjugate described herein, the antibody may include the antibodies described in sections G and H below.

[0236] In one embodiment of the above immunoconjugate, R1 may be a C1-C6 alkyl halide, a C1-C2 alkyl alcohol, a 4-phenol, or a C1-C2 alkylthiol having a halide selected from the group consisting of Br, I, Cl, and F.

[0237] In any one of the above embodiments of the immunoconjugate, R1 may be -CH2-Br.

[0238] In any one of the above embodiments of the immunoconjugate, R2 is unsubstituted C1-C 20 It could be alkylene.

[0239] In certain embodiments of the immunoconjugate described above, R2 may be an unsubstituted C2-C5 alkylene.

[0240] Alternatively, in any one of the above embodiments of the immunoconjugate, R2 may be unsubstituted (PEG)n, where n is 8.

[0241] In any one of the above embodiments of the immunoconjugate, R3 may be -CH2-.

[0242] In any one of the above embodiments of the immunoconjugate, the glycoside or glycoside derivative Y may be selected from the following structures. [ka]

[0243] In any one of the above embodiments of the immunoconjugate, the glycoside or glycoside derivative Y may be a glucuronide.

[0244] In any one of the above embodiments of the immunoconjugate, the triazole ring at R4 is [ka] It can be formed with an azide group or acetylene group bonded to L represented by the formula (where p is 2 to 5).

[0245] Alternatively, in any one of the above embodiments of the immunoconjugate, the triazole ring at R4 is [ka] It can be formed with an azide group or acetylene group bonded to L represented by (where n is 8 in the formula).

[0246] In any one of the above embodiments of the immunoconjugate, R2 is a substituted or unsubstituted (PEG) n If this is the case, then Y is not a galactoside.

[0247] In any one of the above embodiments of the immunoconjugate, drug D may be selected from the group consisting of auristatin, drastatin, meitansinoid, calicheamicin, pyrrolobenzodiazepine, anthracycline, ribonuclease, and DNA endonuclease.

[0248] In any one of the above embodiments of the immunoconjugate, drug D may be an auristatin selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

[0249] In any one of the above embodiments of the immunoconjugate, the conjugate is [ka] It may have the structure of SPC07 represented by, or Conjugate is [ka] It may have the structure of SPC19 represented by, or Conjugate is [ka] It may have the structure of SPC17 represented by, or Conjugate is [ka] It may have the structure of SPC02 represented by, or Conjugate is [ka] It may have the structure of SPC04 represented by, or Conjugate is [ka] It may have the structure of SPC05 represented by, or Conjugate is [ka] It may have the structure of SPC06 represented by, or Conjugate is [ka] It may have the structure of SPC08 represented by [this].

[0250] F. Isolated anti-nectin-4 polypeptide In one embodiment, the present disclosure provides an isolated polypeptide (which may be an antibody or antibody fragment in some embodiments described below) comprising a heavy chain variable region that specifically binds to nectin-4, or in particular to human nectin-4 protein. The heavy chain variable region comprises three complementarity-determining regions (CDRs) having sequences H1, H2, and H3, wherein: The H1 sequence is GFTFSSYNX1N (sequence number 1); The H2 sequence is YISSSSSTIYYADSVKG (sequence number 2); The H3 sequence is AYYYGX2DX3 (sequence number 3); Here, X1 is M or D; X2 is M or D; X3 is V or K, provided that the heavy chain and light chain variable regions are not, in combination, sequence numbers 18 and 31.

[0251] The H1 sequence can be selected from GFTFSSYNMN (sequence number 7) and GFTFSSYNDN (sequence number 8). The H3 sequence can be selected from AYYYGMDV (sequence number 9), AYYYGDDV (sequence number 10), and AYYYGMDK (sequence number 11).

[0252] In another embodiment, the present disclosure provides an isolated polypeptide comprising a light chain variable region that specifically binds to human nectin-4. The light chain variable region comprises three complementarity-determining regions having sequences L1, L2, and L3, wherein: The L1 sequence is X4ASQGISGWX5A (sequence number 4); The L2 sequence is AASTLQS(sequence ID 5); The L3 sequence is QQANSX6PX7T (sequence number 6), Here, X4 is R or H; X5 is L or E; X6 is F or E; X7 is P or D, provided that the heavy chain and light chain variable regions are not, in combination, sequence numbers 18 and 31.

[0253] The L1 sequence can be selected from RASQGISGWLA (sequence number 12), RASQGISGWEA (sequence number 13), and HASQGISGWLA (sequence number 14). The L3 sequence can be selected from QQANSFPPT (sequence number 15), QQANSEPPT (sequence number 16), and QQANSFPDT (sequence number 17).

[0254] In another embodiment, the present disclosure provides an isolated polypeptide that specifically binds to nectin-4, or in particular to human nectin-4 protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity-determining regions having sequences H1, H2, and H3, where: The H1 sequence is GFTFSSYNX1N (sequence number 1); The H2 sequence is YISSSSSTIYYADSVKG (sequence number 2); The H3 sequence is AYYYGX2DX3 (sequence number 3); Here, X1 is M or D; X2 is M or D; X3 is V or K; The light chain variable region includes three complementarity-determining regions having sequences L1, L2, and L3, where: The L1 sequence is X4ASQGISGWX5A (sequence number 4); The L2 sequence is AASTLQS(sequence ID 5); The L3 sequence is QQANSX6PX7T (sequence number 6), Here, X4 is R or H; X5 is L or E; X6 is F or E; X7 is P or D; however, X1, X2, X3, X4, X5, X6, and X7 cannot be M, M, V, R, L, F, and P simultaneously, and the heavy chain and light chain variable regions cannot be sequences 18 and 31 in combination.

[0255] [Table 1] JPEG2026520270000077.jpg255170JPEG2026520270000078.jpg27170

[0256] The isolated polypeptide may contain a heavy chain variable region having a sequence selected from SEQ ID NOs: 18-30, provided that the heavy chain and light chain variable regions are not combined to be SEQ ID NOs: 18 and 31.

[0257] The isolated polypeptide may contain a light chain variable region having a sequence selected from SEQ ID NOs: 31-43, provided that the heavy chain and light chain variable region are not, in combination, SEQ ID NOs: 18 and 31.

[0258] [Table 2] JPEG2026520270000080.jpg255162JPEG2026520270000081.jpg255169JPEG2026520270 000082.jpg255163JPEG2026520270000083.jpg255164JPEG2026520270000084.jpg89170

[0259] In a preferred embodiment, the isolated polypeptide of the present disclosure includes a heavy-chain variable region and a light-chain variable region having any pair of sequences selected from SEQ ID NOs: 19 and 32, SEQ ID NOs: 20 and 33, SEQ ID NOs: 21 and 34, SEQ ID NOs: 22 and 35, SEQ ID NOs: 23 and 36, SEQ ID NOs: 24 and 37, SEQ ID NOs: 25 and 38, SEQ ID NOs: 26 and 39, SEQ ID NOs: 27 and 40, SEQ ID NOs: 28 and 41, and SEQ ID NOs: 29 and 42.

[0260] In another embodiment, the isolated polypeptide of the present disclosure comprises a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a combination of amino acid sequences selected from one of SEQ ID NOs. 18-30 in combination with one of SEQ ID NOs. 31-43; provided that the heavy chain and light chain variable regions are not SEQ ID NOs. 18 and 31 in combination, and the isolated polypeptide specifically binds to human nectin-4 protein.

[0261] In yet another embodiment, the isolated polypeptide of the present disclosure may include a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with each pair of amino acid sequences selected from SEQ ID NOs: 19 and 32, SEQ ID NOs: 20 and 33, SEQ ID NOs: 21 and 34, SEQ ID NOs: 22 and 35, SEQ ID NOs: 23 and 36, SEQ ID NOs: 24 and 37, SEQ ID NOs: 25 and 38, SEQ ID NOs: 26 and 39, SEQ ID NOs: 27 and 40, SEQ ID NOs: 28 and 41, provided that the heavy chain and light chain variable regions are not combined to be SEQ ID NOs: 18 and 31 and SEQ ID NOs: 29 and 42; the isolated polypeptide specifically binds to human nectin-4 protein.

[0262] In yet another embodiment, the isolated polypeptide of the present disclosure specifically binds to nectin-4, or in particular human nectin-4 protein and CD3, and comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three complementarity-determining regions having sequences H1, H2, and H3, where: The H1 sequence is selected from sequence numbers 7 and 8. The H2 sequence is sequence number 2, The H3 sequence is selected from sequence number 9, sequence number 10, and sequence number 11; The light chain variable region includes three complementarity-determining regions having sequences L1, L2, and L3, where: The L1 sequence is X4ASQGISGWX5A (sequence number 4); The L2 sequence is AASTLQS(sequence ID 5); The L3 sequence is QQANSX6PX7T (sequence number 6), Here, X4 is R or H; X5 is L or E; X6 is F or E; X7 is P or D; however, X1, X2, X3, X4, X5, X6, and X7 cannot be M, M, V, R, L, F, and P simultaneously, respectively. Six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9 (where: The L4 sequence is GFTFNTYAMN (sequence number 44), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 45), The L6 array is HX 11 NTE 12 NSX 13 VSWFX 14 Y (sequence number 46), The L7 sequence is RSSTGAVTTSNYX 15 N is (sequence number 47), The L8 sequence is GTNKRAP (sequence number 48), The L9 sequence is ALWYSNLWV (sequence number 49), Here, X 11 However, it is G or S, and X 12 However, it is G or P, and X 13However, it is either Y or K, and X 14 However, it is A or Q, and X 15 However, it is either A or D.

[0263] In another embodiment of the isolated polypeptide having nine CDRs, the L6 sequence is selected from any one of SEQ ID NOs. 50-53, and the L7 sequence is selected from SEQ ID NOs. 54 and 55.

[0264] In a preferred embodiment, the isolated polypeptide specifically binds to nectin-4 and CD3 and comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three complementarity-determining regions, H1, H2, and H3, where: The H1 sequence is selected from sequence numbers 7 and 8. The H2 sequence is selected from sequence number 2. The H3 sequence is selected from sequence numbers 9, 10, and 11. The light chain variable region consists of three complementarity-determining regions having sequences L1, L2, and L3 (where: The L1 sequence is selected from sequence numbers 12, 13, and 14. The L2 sequence is sequence number 5, The L3 sequence is selected from SEQ ID NOs. 15, 16, and 17, as well as six anti-CD3 complementarity determination regions L4, L5, L6, L7, L8, and L9 (where: The L4 sequence is GFTFNTYAMN (sequence number 44), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 45), The L6 sequence is HGNFGNSYVSWFAY (SEQ ID NO: 50), selected from HSNFGNSKVSWFAY (SEQ ID NO: 51), HGNFPNSKVSWFQY (SEQ ID NO: 52), and HSNFGNSKVSWFAY (SEQ ID NO: 53). The L7 sequence was selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and RSSTGAVTTSNYDN (SEQ ID NO: 55). The L8 sequence is GTNKRAP (sequence number 48), The L9 sequence is ALWYSNLWV (sequence number 49). Includes.

[0265] In another preferred embodiment, the isolated polypeptide specifically binds to nectin-4 and CD3 and comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three complementarity-determining regions, H1, H2, and H3, where: The H1 sequence is selected from sequence number 7. The H2 sequence is selected from sequence number 2. The H3 sequence is selected from sequence numbers 9, 10, and 11, and the light chain variable region consists of three complementarity-determining regions L1, L2, and L3 (where, The L1 sequence is selected from sequence numbers 12, 13, and 14. The L2 sequence is sequence number 5, The L3 sequence is sequence number 15), and Six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9 (where: The L4 sequence is GFTFNTYAMN (sequence number 44), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 45), The L6 sequence is selected from HGNFGNSYVSWFAY (sequence number 50), HSNFGNSKVSWFAY (sequence number 51), HGNFPNSKVSWFQY (sequence number 52), and HSNFGNSKVSWFAY (sequence number 53). The L7 sequence was selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and RSSTGAVTTSNYDN (SEQ ID NO: 55). The L8 sequence is GTNKRAP (sequence number 48), The L9 sequence is ALWYSNLWV (sequence number 49). Includes.

[0266] Exemplary anti-nectin-4 isolated polypeptide with 9 CDRs

[0267] Each of the above-listed "exemplary anti-nectin-4 isolated polypeptides" having H1, H2, H3, L1, L2, and L3 sequences may further comprise one of the following combinations of L4, L5, L6, L7, L8, and L9. [Table 3]

[0268] In each of the embodiments described above, the isolated polypeptide having nine CDRs includes a heavy chain variable region having a sequence selected from SEQ ID NOs: 18, 25, 27, and 29.

[0269] In each of the above embodiments, the isolated polypeptide having nine CDRs includes a light chain variable region having a sequence selected from SEQ ID NOs. 56 to 60.

[0270] In a particular embodiment, the isolated polypeptide having nine CDRs comprises one of the heavy chain variable sequences of SEQ ID NOs. 18, 25, 27, and 29, and one of the light chain variable sequences of SEQ ID NOs. 56-60.

[0271] [Table 4]

[0272] In a particular preferred embodiment, the isolated polypeptide having nine CDRs of the present disclosure includes a heavy chain variable region and a light chain variable region having one pair of sequences selected from SEQ ID NOs. 25 and SEQ ID NOs. 57, SEQ ID NOs. 27 and SEQ ID NOs. 58, SEQ ID NOs. 29 and SEQ ID NOs. 59, and SEQ ID NOs. 29 and SEQ ID NOs. 60.

[0273] In another embodiment, an isolated polypeptide having nine CDRs of the present disclosure comprises a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a combination of amino acid sequences selected from one of SEQ ID NOs. 18, 25, 27, and 29 in combination with one of SEQ ID NOs. 56-60; however, the heavy chain and light chain variable regions are not the combination of SEQ ID NOs. 18 and 56; and the isolated polypeptide specifically binds to human nectin-4 protein.

[0274] In another embodiment of the present disclosure, an isolated polypeptide having nine CDRs comprises a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a pair of amino acid sequences selected from SEQ ID NOs. 25 and 57, SEQ ID NOs. 27 and 58, SEQ ID NOs. 29 and 59, and SEQ ID NOs. 29 and 60; the isolated polypeptide specifically binds to human nectin-4 protein.

[0275] In each of the embodiments described above, the isolated polypeptide of the Disclosure that specifically binds to nectin-4, or in particular to human nectin-4 protein and CD3, may also include the sequences described herein for specific binding to nectin-4, and single-chain fragment variable (scFv) of any known CD3 antibody. In this embodiment of the Disclosure, the isolated polypeptide binds to CD3 regardless of conditionally active nectin-4 binding. For example, in one embodiment, the isolated polypeptide of the Disclosure that specifically binds to nectin-4, or in particular to human nectin-4 protein and CD3, comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region comprising three complementarity-determining regions having sequences H1, H2, and H3, wherein: The H1 sequence is selected from sequence numbers 7 and 8. The H2 sequence is sequence number 2, The H3 sequence is selected from sequence number 9, sequence number 10, and sequence number 11; The light chain variable region consists of three complementarity-determining regions having sequences L1, L2, and L3 (where: The L1 sequence is X4ASQGISGWX5A (sequence number 4); The L2 sequence is AASTLQS(sequence ID 5); The L3 sequence is QQANSX6PX7T (sequence number 6), Herein, X4 is R or H; X5 is L or E; X6 is F or E; X7 is P or D; however, X1, X2, X3, X4, X5, X6 and X7 cannot be M, M, V, R, L, F and P simultaneously, respectively), and includes an scFv containing six anti-CD3 complementarity determining regions of any known CD3 antibody.

[0276] The heavy chain variable regions and light chain variable regions of this disclosure were obtained from parent antibodies using the methods disclosed in U.S. Patent No. 8,709,755 and U.S. Patent No. 8,859,467, respectively. These methods for generating the heavy chain variable regions and light chain variable regions, as well as the methods for generating the antibodies and antibody fragments disclosed in U.S. Patent No. 8,709,755 and U.S. Patent No. 8,859,467, are incorporated herein by reference.

[0277] G. Anti-nectin-4 antibody The isolated polypeptide may be an antibody or an antibody fragment. Antibodies and antibody fragments containing these heavy chain variable regions and light chain variable regions may bind specifically to nectin-4, or in particular to human nectin-4. Antibodies or antibody fragments containing a combination of one of these heavy chain variable regions and one of these light chain variable regions have been found to have higher binding to nectin-4 at pH in the tumor microenvironment (e.g., pH 5.0 to 6.8, preferably pH 6.0 to 6.8) than at pH in the non-tumor microenvironment (e.g., pH 7.0 to 7.6). As a result, anti-nectin-4 antibodies or antibody fragments have higher binding to nectin-4 in the tumor microenvironment compared to their binding to nectin-4 in a typical normal tissue microenvironment. In one embodiment, binding is measured by affinity.

[0278] In any of the isolated polypeptides described herein, embodiments of antibodies and antibody fragments, conditionally active isolated polypeptides, antibodies, or antibody fragments may be less active or substantially inactive under normal physiological conditions (such as the non-tumor microenvironment) and more active under abnormal conditions (such as the tumor microenvironment) compared to the activity under normal physiological conditions of the parent or wild-type polypeptide, and the antibody or antibody fragment from which it is derived. As a result, the isolated polypeptides, anti-nectin-4 antibodies, or anti-nectin-4 antibody fragments of this disclosure may have lower binding to nectin-4 under normal physiological conditions (such as the non-tumor microenvironment) compared to the parent or wild-type polypeptide, and the antibody or antibody fragment from which it is derived. For example, a conditionally active isolated polypeptide, anti-nectin-4 antibody, or anti-nectin-4 antibody fragment may be less active or substantially inactive at pH 7.0–7.6 compared to the parent or wild-type polypeptide, antibody, or antibody fragment, but active at a lower pH of 5.0–6.8 compared to the parent or wild-type polypeptide, antibody, or antibody fragment. In some cases, a conditionally active isolated polypeptide, antibody, or antibody fragment is reversibly or irreversibly inactivated under normal physiological conditions (such as a non-tumor microenvironment) compared to the parent or wild-type polypeptide, antibody, or antibody fragment.

[0279] Therefore, the anti-nectin-4 antibodies or antibody fragments of this disclosure are expected to exhibit reduced side effects compared to unconditionally active anti-nectin-4 antibodies due to their reduced binding to nectin-4 within the normal tissue microenvironment. The anti-nectin-4 antibodies or antibody fragments of this disclosure are also expected to have comparable efficacy to monoclonal anti-nectin-4 antibodies known in the art. This combination of features, due to the reduced side effects, may allow for the use of higher doses of these anti-nectin-4 antibodies or antibody fragments, thereby potentially providing a more effective therapeutic option.

[0280] This disclosure provides an antibody or antibody fragment that specifically binds to nectin-4, or in particular to human nectin-4 protein, comprising a heavy chain variable region including three complementarity-determining regions (CDRs) having sequences H1, H2, and H3, wherein: The H1 sequence is GFTFSSYNX1N (sequence number 1); The H2 sequence is YISSSSSTIYYADSVKG (sequence number 2); The H3 sequence is AYYYGX2DX3 (sequence number 3); Here, X1 is M or D; X2 is M or D; X3 is V or K, provided that the heavy chain and light chain variable regions are not, in combination, sequence numbers 18 and 31.

[0281] The H1 sequence can be selected from GFTFSSYNMN (sequence number 7) and GFTFSSYNDN (sequence number 8). The H3 sequence can be selected from AYYYGMDV (sequence number 9), AYYYGDDV (sequence number 10), and AYYYGMDK (sequence number 11).

[0282] In another embodiment, the present disclosure provides an antibody or antibody fragment comprising a light chain variable region that specifically binds to human nectin-4. The light chain variable region comprises three complementarity-determining regions having sequences L1, L2, and L3, wherein: The L1 sequence is X4ASQGISGWX5A (sequence number 4); The L2 sequence is AASTLQS(sequence ID 5); The L3 sequence is QQANSX6PX7T (sequence number 6), Here, X4 is R or H; X5 is L or E; X6 is F or E; X7 is P or D, provided that the heavy chain and light chain variable regions are not, in combination, sequence numbers 18 and 31.

[0283] The L1 sequence can be selected from RASQGISGWLA (sequence number 12), RASQGISGWEA (sequence number 13), and HASQGISGWLA (sequence number 14). The L3 sequence can be selected from QQANSFPPT (sequence number 15), QQANSEPPT (sequence number 16), and QQANSFPDT (sequence number 17).

[0284] In a more specific embodiment, the present disclosure provides an antibody or antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity-determining regions having sequences H1, H2, and H3, where: The H1 sequence is GFTFSSYNX1N (sequence number 1); The H2 sequence is YISSSSSTIYYADSVKG (sequence number 2); The H3 sequence is AYYYGX2DX3 (sequence number 3); Here, X1 is M or D; X2 is M or D; X3 is V or K; the light chain variable region comprises three complementarity-determining regions having sequences L1, L2, and L3, where: The L1 sequence is X4ASQGISGWX5A (sequence number 4); The L2 sequence is AASTLQS(sequence ID 5); The L3 sequence is QQANSX6PX7T (sequence number 6), Here, X4 is R or H; X5 is L or E; X6 is F or E; X7 is P or D; however, X1, X2, X3, X4, X5, X6, and X7 cannot be M, M, V, R, L, F, and P simultaneously, and the heavy chain and light chain variable regions cannot be sequences 18 and 31 in combination.

[0285] The heavy chain variable region may have a sequence selected from sequence numbers 18 to 30, however, the heavy chain and light chain variable regions cannot be in combination with sequence numbers 18 and 31.

[0286] The light chain variable region may have a sequence selected from sequence numbers 31 to 43, provided that the heavy chain and light chain variable region are not, in combination, sequence numbers 18 and 31.

[0287] Exemplary anti-nectin-4 antibody

[0288] In certain embodiments, the anti-nectin-4 antibodies and antibody fragments of the Disclosure include, with respect to an isolated polypeptide, a combination of H1, H2, H3, L1, L2, and L3 CDRs or a combination of heavy chain variable regions (selected from SEQ ID NOs. 18-30) and a light chain variable region (selected from SEQ ID NOs. 31-43). Preferred nectin-4 antibodies and antibody fragments of the Disclosure include preferred combinations of these heavy chain and light chain variable regions with respect to an isolated polypeptide. For example, a preferred antibody or antibody fragment of the Disclosure includes a heavy chain variable region and a light chain variable region having any pair of sequences selected from SEQ ID NOs. 32 and 19, SEQ ID NOs. 33 and 20, SEQ ID NOs. 34 and 21, SEQ ID NOs. 35 and 22, SEQ ID NOs. 36 and 23, SEQ ID NOs. 37 and 24, SEQ ID NOs. 38 and 25, SEQ ID NOs. 39 and 26, SEQ ID NOs. 40 and 27, SEQ ID NOs. 41 and 28, and SEQ ID NOs. 42 and 29.

[0289] The antibodies or antibody fragments of this disclosure may include a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a combination of amino acid sequences selected from one of SEQ ID NOs. 18 to 30 in combination with one of SEQ ID NOs. 31 to 43; however, the heavy chain and light chain variable regions may not be SEQ ID NOs. 18 and 31 in combination; and the antibodies or antibody fragments specifically bind to human nectin-4 protein.

[0290] The antibodies or antibody fragments of this disclosure may include a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with each of the pairs of amino acid sequences selected from SEQ ID NOs. 32 and 19, SEQ ID NOs. 33 and 20, SEQ ID NOs. 34 and 21, SEQ ID NOs. 35 and 22, SEQ ID NOs. 36 and 23, SEQ ID NOs. 37 and 24, SEQ ID NOs. 38 and 25, SEQ ID NOs. 39 and 26, SEQ ID NOs. 40 and 27, SEQ ID NOs. 41 and 28, and SEQ ID NOs. 42 and 29; however, the heavy chain and light chain variable regions may not be SEQ ID NOs. 18 and 31 in combination; and the antibodies or antibody fragments specifically bind to human nectin-4 protein.

[0291] In another embodiment, the antibody or antibody fragment of the present disclosure binds multispecifically to nectin-4, or in particular to human nectin-4 protein and CD3, and comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three complementarity-determining regions having sequences H1, H2, and H3, wherein: The H1 sequence is selected from sequence numbers 7 and 8. The H2 sequence is sequence number 2, The H3 sequence is selected from sequence number 9, sequence number 10, and sequence number 11; The light chain variable region includes three complementarity-determining regions having sequences L1, L2, and L3, where: The L1 sequence is X4ASQGISGWX5A (sequence number 4); The L2 sequence is AASTLQS(sequence ID 5); The L3 sequence is QQANSX6PX7T (sequence number 6), Here, X4 is R or H; X5 is L or E; X6 is F or E; X7 is P or D; however, X1, X2, X3, X4, X5, X6, and X7 cannot be M, M, V, R, L, F, and P simultaneously, respectively. Six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9 (where: The L4 sequence is GFTFNTYAMN (sequence number 44), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 45), The L6 array is HX 11 NTE 12 NSX 13 VSWFX 14 Y (sequence number 46), The L7 sequence is RSSTGAVTTSNYX 15 N is (sequence number 47), The L8 sequence is GTNKRAP (sequence number 48), The L9 sequence is ALWYSNLWV (sequence number 49), Here, X 11 However, it is G or S, and X 12 However, it is G or P, and X13 However, it is either Y or K, and X 14 However, it is A or Q, and X 15 However, it is either A or D.

[0292] In another embodiment of the multispecific antibody or antibody fragment of this disclosure, the L6 sequence is one of SEQ ID NOs. 50 to 53, and the L7 sequence is selected from SEQ ID NOs. 54 and 55.

[0293] Bimorphic bispecific anti-nectin-4×CD3 antibody

[0294] In certain embodiments, the bispecific anti-nectin-4×CD3 antibodies and antibody fragments of the Disclosure include, with respect to an isolated polypeptide, a combination of the H1, H2, H3, L1, L2, L3, L4, L5, L6, L7, L8, and L9 CDRs or a combination of heavy chain variable regions (selected from SEQ ID NOs. 18, 25, 27, and 29) and a light chain variable region (selected from SEQ ID NOs. 56-60), provided that the heavy chain and light chain variable regions are not the combination of SEQ ID NOs. 56. Preferred anti-nectin-4 antibodies and antibody fragments of the Disclosure include, with respect to an isolated polypeptide, preferred combinations of these heavy chain and light chain variable regions.

[0295] In a preferred embodiment, the multispecific antibody or antibody fragment specifically binds to nectin-4, or in particular human nectin-4 protein and CD3, and comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three complementarity-determining regions, H1, H2, and H3, where: The H1 sequence is selected from sequence numbers 7 and 8. The H2 sequence is selected from sequence number 2. The H3 sequence is selected from sequence numbers 9, 10, and 11, and the light chain variable region consists of three complementarity-determining regions L1, L2, and L3 (where: The L1 sequence is selected from sequence numbers 12, 13, and 14. The L2 sequence is sequence number 5, The L3 sequence was selected from SEQ ID NOs. 15, 16, and 17, as well as six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9 (where: The L4 sequence is GFTFNTYAMN (sequence number 44), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 45), The L6 sequence is selected from HGNFGNSYVSWFAY (sequence number 50), HSNFGNSKVSWFAY (sequence number 51), HGNFPNSKVSWFQY (sequence number 52), and HSNFGNSKVSWFAY (sequence number 53). The L7 sequence was selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and RSSTGAVTTSNYDN (SEQ ID NO: 55). The L8 sequence is GTNKRAP (sequence number 48), The L9 sequence is ALWYSNLWV (SEQ ID NO: 49), however, the heavy and light chain variable regions are not a combination of SEQ ID NOs: 18 and 56. Includes.

[0296] In another preferred embodiment, the multispecific antibody or antibody fragment of the present disclosure specifically binds to nectin-4, or in particular human nectin-4 protein and CD3, and comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three complementarity-determining regions, H1, H2, and H3, wherein: The H1 sequence is selected from sequence number 7. The H2 sequence is selected from sequence number 2. The H3 sequence is selected from sequence numbers 9, 10, and 11, and the light chain variable region consists of three complementarity-determining regions L1, L2, and L3 (where: The L1 sequence is selected from sequence numbers 12 and 13. The L2 sequence is sequence number 5, The L3 sequence is sequence number 15), and Six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9 (where: The L4 sequence is GFTFNTYAMN (sequence number 44), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 45), The L6 sequence is selected from HGNFGNSYVSWFAY (sequence number 50), HSNFGNSKVSWFAY (sequence number 51), HGNFPNSKVSWFQY (sequence number 52), and HSNFGNSKVSWFAY (sequence number 53). The L7 sequence was selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and RSSTGAVTTSNYDN (SEQ ID NO: 55). The L8 sequence is GTNKRAP (sequence number 48), The L9 sequence is ALWYSNLWV (SEQ ID NO: 49), however, the heavy and light chain variable regions are not a combination of SEQ ID NOs: 18 and 56. Includes.

[0297] In each of the embodiments described above, the multispecific antibody or antibody fragment of the Disclosure may include a heavy chain variable region having a sequence selected from SEQ ID NOs: 18, 25, 27, and 29.

[0298] In each of the embodiments described above, the multispecific antibody or antibody fragment of the present disclosure may include a light chain variable region having a sequence selected from SEQ ID NOs. 56 to 60.

[0299] In certain embodiments, the multispecific antibody or antibody fragment of the present disclosure comprises a heavy chain variable region having one sequence of SEQ ID NOs: 18, 25, 27, and 29, and a light chain variable region having one sequence of SEQ ID NOs: 56 to 60, provided that the heavy chain and light chain variable region are not a combination of SEQ ID NOs: 18 and 56.

[0300] In certain embodiments, the multispecific antibody or antibody fragment of the present disclosure includes a heavy chain variable region and a light chain variable region having one pair of sequences selected from SEQ ID NOs. 25 and SEQ ID NOs. 57, SEQ ID NOs. 27 and SEQ ID NOs. 58, SEQ ID NOs. 29 and SEQ ID NOs. 59, and SEQ ID NOs. 29 and SEQ ID NOs. 60.

[0301] In another embodiment, the multispecific antibody or antibody fragment of the present disclosure comprises a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a combination of amino acid sequences selected from one of SEQ ID NOs. 18, 25, 27, and 29 in combination with one of SEQ ID NOs. 56-60; however, the heavy chain and light chain variable regions are not the combination of SEQ ID NOs. 18 and 56; and the antibody or antibody fragment specifically binds to human nectin-4 protein.

[0302] In another embodiment, the multispecific antibody or antibody fragment of the present disclosure may include a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a pair of amino acid sequences selected from SEQ ID NOs. 25 and 57, SEQ ID NOs. 27 and 58, SEQ ID NOs. 29 and 59, and SEQ ID NOs. 29 and 60; however, the heavy chain and light chain variable regions may not be a combination of SEQ ID NOs. 18 and 56, and the antibody or antibody fragment specifically binds to human nectin-4 protein.

[0303] In other embodiments, the amino acid sequences of the heavy and light chain variable regions outside the complementarity-determining region may be mutated according to the principles of substitution, insertion, and deletion, as described in this application, to provide these variants. In yet another embodiment, the constant region may be modified to provide these variants. In yet another embodiment, the amino acid sequences of both the heavy and light chain variable regions outside the complementarity-determining region and the constant region may be modified to provide these variants.

[0304] Inducing these variants is guided by the processes described herein. Variants of the heavy and light chain variable regions can be prepared by introducing appropriate modifications to the nucleotide sequences encoding the heavy and light chain variable regions, or by peptide synthesis. Such modifications include, for example, deletions from residues in the amino acid sequences of the heavy and light chain variable regions, and / or insertions into residues and / or substitutions of residues. Any combination of deletions, insertions, and substitutions can be performed to reach the antibody or antibody fragment of the disclosure, as long as the antibody or antibody fragment of the disclosure has the desired properties, for example, antigen binding to human nectin-4 and / or conditional activity.

[0305] Substitution, insertion, and deletion variants In certain embodiments, antibody or antibody fragment variants having one or more amino acid substitutions are provided. The target sites for substitutional mutagenesis include the CDR and framework region (FR). Conservative substitutions are shown in Table 1 under the heading "Conservative Substitutions." More substantial substitutions are provided in Table 1 under the heading "Exemplary Substitutions" and are further described below with respect to classes of amino acid side chains. Amino acid substitutions are introduced into the antibody or antibody fragment of interest, and the product can be screened for desired activity, such as retention / improvement of antigen binding or reduction of immunogenicity. [Table 5]

[0306] Amino acids can be classified according to their general side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0307] Non-conservative substitution involves swapping one member of one of these classes with one of another.

[0308] One type of substitution variant involves substituting one or more complementarity-determining region residues of a parent antibody (e.g., a humanized or human antibody). Generally, variants selected for further testing have alterations (e.g., improvements) to certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated using, for example, a phage display-based affinity maturation technique, e.g., one described herein. Briefly, one or more CDR residues are mutated, the variant antibody is presented on a phage, and screened for specific biological activity (e.g., binding affinity).

[0309] Modifications (e.g., substitutions) may be made in the CDR to improve antibody affinity, for example. Such modifications may be made in the CDR "hotspots," i.e., residues encoded by codons that frequently undergo mutations during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol., vol. 207, pp. 179-196, 2008), and / or in the SDR(a-CDR), resulting in variant V. H or V LHowever, binding affinity is tested. Affinity maturation by constructing a secondary library and then re-selecting from it is described, for example, in Hoogenboom et al. (Methods in Molecular Biology, vol. 178, pp. 1-37, 2001). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-specific mutagenesis). A secondary library is then constructed. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves CDR-specific techniques in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine canning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

[0310] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such modifications do not substantially reduce the ability of the antibody or antibody fragment to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made within a CDR. Such modifications may be outside the CDR "hotspot" or SDR. Variant V provided above H and V L In certain embodiments of the sequence, each CDR is either unmodified or contains one, two, or three or fewer amino acid substitutions.

[0311] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells, Science, vol. 244, pp. 1081-1085, 1989. In this method, a group of residues or target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody or antibody fragment and the antigen is affected. Further substitutions may be introduced at amino acid positions that are functionally sensitive to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex identifies contact points between the antibody or antibody fragment and the antigen. Such contact residues and adjacent residues may be targeted as candidate substitutions or removed. Variants may be screened to determine whether they contain desired properties.

[0312] Amino acid sequence insertions include amino-terminus and / or carboxyl-terminus fusions, which are polypeptides ranging in length from one residue to 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibodies include fusion of the antibody to the N-terminus or C-terminus of an enzyme (e.g., for ADEPT) or polypeptide that increases the serum half-life of the antibody.

[0313] Amino acid sequence modifications of antibodies described herein are anticipated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. V of antibodies derived from non-human animals H and V L Only the CDR inside is used as a human antibody. H and V L When humanized antibodies are produced by simply transplanting them into FR, it is known that their antigen-binding activity is reduced compared to the antigen-binding activity of the original antibodies derived from non-human animals. This is true not only in CDR but also in FR.H and V L Several amino acid residues are thought to be directly or indirectly related to antigen-binding activity. Therefore, these amino acid residues are considered to be related to the V of human antibodies. H and V L Substitution with different amino acid residues derived from FR would reduce binding activity. To address this issue, in antibodies transplanted with human CDR, the V of human antibodies was used. H and V L Within the amino acid sequence of the FR, it is necessary to attempt to identify amino acid residues directly related to antibody binding, amino acid residues that interact with amino acid residues of the CDR, or amino acid residues that maintain the three-dimensional structure of the antibody and are directly related to binding to the antigen. The reduced antigen-binding activity may be increased by substituting the identified amino acids with amino acid residues from the original antibody derived from a non-human animal.

[0314] Modifications and alterations may be made in the structure of the antibody of this disclosure and in the encoding DNA sequence, still yielding a functional molecule encoding an antibody with desired properties.

[0315] When modifying amino acid sequences, the hydroxyl index of amino acids may be considered. The importance of the hydroxyl amino acid index in conferring interactive biological functions to proteins is generally understood in the art. It is accepted that the relative hydroxyl properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interaction between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens. Each amino acid is assigned a hydropathic index based on its hydrophobic and charge properties, which are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0316] A further objective of this disclosure is to also include functionally conserved variants of the antibodies described herein.

[0317] Two amino acid sequences are "substantially homologous" or "substantially similar" if more than 80%, preferably more than 85%, preferably more than 90% of the amino acids are identical or about 90%, preferably more than 95%, of the amino acids compared to the full length of the shorter sequence (i.e., they are functionally identical). Preferably, similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pile-up program or a sequence comparison algorithm such as BLAST or FASTA.

[0318] For example, certain amino acids may be substituted in a protein with other amino acids without significantly impairing their activity. Since the interaction capacity and properties of proteins determine their biological functional activity, certain amino acid substitutions may occur in the protein sequence and, naturally, in its DNA coding sequence, while still resulting in a protein with similar properties. Therefore, it is conceivable that various modifications may be made in the sequences of the antibodies or antibody fragments of this disclosure, or in the corresponding DNA sequences encoding such antibodies or antibody fragments, without significantly impairing their biological activity.

[0319] It is known in the art that a specific amino acid may be substituted with another amino acid having a similar hydroxyl index or score, and this may still result in a protein with similar biological activity, i.e., a protein that is still biologically and functionally equivalent.

[0320] As outlined above, amino acid substitutions are therefore generally based on the relative similarities of amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take various of the above properties into account are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0321] Glycosylated variant In certain embodiments, the anti-nectin-4 antibodies or antibody fragments provided herein are modified to increase or decrease the degree to which the antibody or antibody fragment is glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are generated or removed.

[0322] If an antibody contains an Fc region, the carbohydrate to which it is bound can be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides that are commonly bound by an N-bond to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH, vol.15, pp.26-32, 1997. Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the branched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibodies of this disclosure can be made to produce antibody variants having specific improved properties.

[0323] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the total of all sugar structures (e.g., complex, hybrid, and high-mannose structures) bound to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of the Fc region residue); however, due to slight sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., at positions 294 to 300. Such fucosylated variants may have improved ADCC function. For example, see U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.); U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication No. 2003 / 0157108; International Publication No. 2000 / 61739; International Publication No. 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent Application Publication No. 2002 / 0164328; U.S. Patent Application Publication No. 2004 / 0093621; U.S. Patent Application Publication No. 2004 / 0132140; U.S. Patent Application Publication No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 0110282; U.S. Patent Application Publication No. 2004 / 0109865; International Publication No. 2003 / 085119; International Publication No. 2003 / 084570; International Publication No. 2005 / 035586; International Publication No. 2005 / 035778; International Publication No. 2005 / 053742; International Publication No. 2002 / 031140; Okazaki et al. J.Mol.Biol., vol.336, pp.1239-1249, 2004; Yamane-Ohnuki et al. Biotech.Bioeng., vol.87, pp.614-622, 2004. Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys., vol.249, pp.533-545, 1986; U.S. Patent Application Publication No. 2003 / 0157108 A; and International Publication No. 2004 / 056312 A1, particularly Example 11), and knockout cell lines such as those containing the α-1,6-fucosyltransferase gene, FUT8, and knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng., vol.87, pp.614-622, 2004; Kanda, Y. et al.). See also al., Biotechnol. Bioeng., vol.94, pp.680-688, 2006; and International Publication No. 2003 / 085107).

[0324] Antibody variants containing branched oligosaccharides are also provided, for example, in which the branched oligosaccharide bound to the Fc region of the antibody is bifurcated by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in International Publication 2003 / 011878; U.S. Patent No. 6,602,684; and U.S. Patent Application Publication 2005 / 0123546. Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in International Publication 1997 / 30087; International Publication 1998 / 58964; and International Publication 1999 / 22764.

[0325] Fc region variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an anti-nectin-4 antibody or antibody fragment provided herein, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0326] In certain embodiments, this disclosure envisions antibody variants having some, but not all, effector functions, which would be desirable candidates for applications where the half-life of the antibody in vivo is important, but certain effector functions (such as ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays may be performed to confirm the reduction / removal of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, which are primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., vol. 9, pp. 457-492, 1991. Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see also, e.g., Hellstrom et al., Proc. Nat'l Acad. Sci. USA, vol. 83, pp. 7059-7063, 1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, vol. 82, pp. 1499-1502, 1985; and U.S. Patent No. 5,821,337 (see also, Bruggemann et al., J. Exp. Med., vol. 166, pp. 1351-1361, 1987). Alternatively, non-radioactive assays may be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, Calif.) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA, vol. 95, pp. 652-656, 1998. A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication No. 2006 / 029879 and International Publication No. 2005 / 100402. To evaluate complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al., J.Immunol.Methods, vol.202, pp.163-171, 1996; Cragg, MS et al., Blood, vol.101, pp.1045-1052, 2003; and Cragg, MS, and MJ Glennie, Blood, vol.103, pp.2738-2743, 2004). Determination of FcRn binding and in vivo clearance / half-life may also be performed using methods known in the art (see, e.g., Petkova, S B et al., Int'l.Immunol., vol.18, pp.1759-1769, 2006).

[0327] Examples of antibody or antibody fragment variants with reduced effector function include those having one or more substitutions among Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Examples of such Fc mutants include the so-called "DANA" Fc mutant, which has alanine substitutions at residues 265 and 297, and Fc mutants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581).

[0328] Specific antibody variants exhibiting improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; International Publication No. 2004 / 056312; and Shields et al., J. Biol. Chem., vol. 9, pp. 6591-6604, 2001).

[0329] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 of the Fc region (residue EU numbering).

[0330] In some embodiments, modifications resulting in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC) are made in the Fc region, as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. J. Immunol., vol. 164, pp. 4178-4184, 2000.

[0331] Antibodies that increase the half-life and improve binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J.Immunol., vol.117, pp.587-593, 1976 and Kim et al., J.Immunol., vol.24, p.249, 1994) are described in U.S. Patent Application Publication No. 2005 / 0014934. These antibodies contain an Fc region having one or more substitutions that improve binding to FcRn in the Fc region. Such Fc variants include substitutions in one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substitutions in Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and International Publication No. 94 / 29351.

[0332] Cysteine-modified antibody variant In certain embodiments, it may be desirable to generate a cysteine-modified antibody, such as a “thioMAb” in which one or more residues of an anti-nectin-4 antibody or antibody fragment are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at an accessible site of the antibody. By substituting these residues with cysteine, a reactive thiol group is positioned at an accessible site of the antibody, which can then be used to conjugate the antibody to another part, such as a drug moiety or a linker-drug moiety, as further described herein, to generate an immunoconjugate. In certain embodiments, one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) in the light chain; A118 (EU numbering) in the heavy chain; and 5400 (EU numbering) in the heavy chain Fc region. Cysteine-modified antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541.

[0333] antibody derivative In certain embodiments, the anti-nectin-4 antibodies or antibody fragments provided herein may be further modified to include additional non-proteinoid moieties that are known and readily available in the art. Suitable moieties for derivatization of antibodies or antibody fragments include, but are not limited to, water-soluble polymers. Non-limited examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolipropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymers can have any molecular weight and can be branched or unbranched. The number of polymers bound to the antibody or antibody fragment can vary, and if two or more polymers are bound, they can be the same or different molecules. In general, the number and / or type of polymers used in derivatization is not limited but may be determined based on considerations including the specific properties or functions of the antibody or antibody fragment to be improved, and whether the derivative will be used therapeutically under given conditions.

[0334] In another embodiment, a conjugate of an antibody or antibody fragment and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA, vol. 102, pp. 11600-11605, 2005). The radiation may be of any wavelength, but is not limited, and includes wavelengths that do not damage normal cells but heat the non-proteinaceous moiety to a temperature that kills cells adjacent to the antibody-non-proteinaceous moiety.

[0335] The anti-nectin-4 antibodies or antibody fragments, or variants thereof, of this disclosure have a higher binding affinity to nectin-4 under tumor microenvironment conditions than under non-tumor microenvironment conditions. In one embodiment, both tumor microenvironment and non-tumor microenvironment conditions are pH. Therefore, the anti-nectin-4 antibodies or antibody fragments of this disclosure can selectively bind to nectin-4 at pH approximately 5.0–6.8, but have a lower binding affinity to nectin-4 at pH approximately 7.0–7.6, which is encountered in a normal non-tumor microenvironment. As shown in the following examples, exemplary anti-nectin-4 antibodies or antibody fragments of this disclosure have a higher binding affinity to nectin-4 at pH 6.0 than at pH 7.4.

[0336] In certain embodiments, the anti-nectin-4 antibody or antibody fragment of the present disclosure has a concentration of approximately ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, or 10 -8 M~10 -13 M, or 10 -9 M~10 -13M) has a dissociation constant (Kd) with nectin-4 under the conditions of the tumor microenvironment. In one embodiment, the ratio of the Kd of the antibody or antibody fragment with nectin-4 under non-tumor microenvironment conditions to the Kd under the same conditions in the tumor microenvironment is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0337] In another embodiment, the ratio of the binding activity of an antibody or antibody fragment to nectin-4 under tumor microenvironmental conditions to the binding activity under the same conditions in a non-tumor microenvironment is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0338] In one embodiment, Kd is measured by a radiolabeled antigen-binding assay (RIA) performed on the antibody of interest and its antigen in Fab form using the following assay: The solution binding affinity of Fab to the antigen is measured in the presence of the minimum concentration of the unlabeled antigen in the presence of a series of titrations. 125I) Fab is equilibrated with labeled antigen, and then measured by capturing the bound antigen using a plate coated with anti-Fab antibody (see, e.g., Chen et al., J.Mol.Biol.293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in phosphate-buffered saline (PBS) for 2–5 hours at room temperature (approx. 23°C). Non-adsorbent plates (Nunc #269620) are then coated with 100 pM or 26 pM [ 125 The 1) antigen is mixed with a serial dilution of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, incubation may be continued for a longer time (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., over 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, 150 μl / well of scintillant (MICROSCINT-20®; Packard) is added, and the plate is counted for 10 minutes with a TOPCOUNT® gamma counter (Packard). The concentrations of each Fab that show less than 20% of the maximum binding are selected for use in competitive binding assays.

[0339] According to another embodiment, Kd is measured using a surface plasmon resonance assay with BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an immobilized antigen CM5 chip at approximately 10 response units (RUs). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RUs) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For dynamic measurement, Fab's 2x serial dilution (0.78 nM to 500 nM) is injected at a flow rate of approximately 25 μl / min into PBS containing 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at 25°C. The binding rate (k on ) and dissociation rate (k off The ratio k is calculated using a simple one-to-one Langmuir coupled model (BIACORE® evaluation software version 3.2) by simultaneously fitting coupled and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated using the ratio k off / k on It is calculated as follows. For example, see Chen et al., J.Mol.Biol.293:865-881(1999). The on-rate is 10 by the surface plasmon resonance assay described above. 6 M -1 s -1If it exceeds this, the on-rate can be determined by using a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) at 25°C in the presence of increasing concentrations of antigen, when measured with a spectrophotometer such as an Aviv Instruments spectrophotometer equipped with stop flow or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) equipped with a stirring cuvette.

[0340] The anti-nectin-4 antibodies of this disclosure may be chimeric, humanized, or human antibodies. In one embodiment, anti-nectin-4 antibody fragments, such as Fv, Fab, Fab', Fab'-SH, scFv, diabody, triabody, tetrabody, or F(ab')2 fragments formed from the antibody fragment, and multispecific antibodies are used. In another embodiment, the antibody is a full-length antibody, such as an intact IgG antibody or another antibody class or isotype. For an overview of specific antibody fragments, see Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003. For an overview of the scFv fragment, see, for example, Pluckthuen, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also International Publication No. 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a description of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have increased in vivo half-lives, see U.S. Patent No. 5,869,046.

[0341] The diabodies of this disclosure may be divalent or bispecific. For example, see European Patent No. 404,097; International Publication No. 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993. Examples of triabodies and tetrabodies are also described in Hudson et al., Nat. Med., vol. 9, pp. 129-134, 2003.

[0342] In some embodiments, the Disclosure includes single-domain antibody fragments comprising all or part of the heavy-chain variable domains or all or part of the light-chain variable domains of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, Mass.; U.S. Patent No. 6,248,516 B1).

[0343] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., Escherichia coli or phages), as described herein.

[0344] In some embodiments, the anti-nectin-4 antibody of this disclosure may be a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 6851-6855, 1984). In one example, the chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, the chimeric antibody is a “class-switched” antibody in which the class or subclass of the antibody is modified compared to the class or subclass of the parent antibody. The chimeric antibody includes its antigen-binding fragment.

[0345] In certain embodiments, the chimeric antibody of this disclosure is a humanized antibody. Typically, such a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody includes one or more variable domains in which the CDR (or a portion thereof) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody may optionally also include at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues derived from a non-human antibody (e.g., an antibody from which the CDR residue is derived) to restore or improve the specificity or affinity of the antibody, for example.

[0346] Humanized antibodies and methods for producing them are outlined, for example, in Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008; for example, Riechmann et al., Nature, vol. 332, pp. 323-329, 1988; Queen et al., Proc. Nat'l Acad. Sci. USA, vol. 86, pp. 10029-10033, 1989; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. Further information is available in al., Methods, vol.36, pp.25-34, 2005 (describes SDR(a-CDR) grafts); Padlan, Mol.Immunol., vol.28, pp.489-498, 1991 (describes "resurfacing"); Dall'Acqua et al., Methods, vol.36, pp.43-60, 2005 (describes "FR shuffling"); and Osbourn et al., Methods, vol.36, pp.61-68, 2005 and Klimka et al., Br.J.Cancer, vol.83, pp.252-260, 2000 (describes the "guided selection" method for FR shuffling).

[0347] Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol., vol. 151, p. 2296, 1993); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, vol. 89, p. 4285, 1992; and Presta et al. J. Immunol., vol. 151, p. 2623, 1993); human maturation (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008); and framework regions derived from screening of FR libraries (e.g., Baca et al. See also al., J. Biol. Chem., vol. 272, pp. 10678-10684, 1997 and Rosok et al., J. Biol. Chem., vol. 271, pp. 22611-22618, 1996.

[0348] H. Multispecific antibodies and antibody fragments This disclosure provides multispecific anti-nectin-4 antibodies, such as bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In certain embodiments, one binding specificity is to nectin-4 and the other is to another antigen. In certain embodiments, a bispecific conditionally active antibody may bind to two different epitopes of nectin-4. A multispecific antibody binds to at least nectin-4 and another antigen with higher activity, affinity, and / or binding activity under first physiological conditions than under second physiological conditions. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing nectin-4. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments.

[0349] In some embodiments, the first physiological condition is an abnormal condition, and the second physiological condition is a normal physiological condition. For example, the abnormal condition may be a condition in the tumor microenvironment. The multispecific antibodies of this disclosure may be called conditionally active multispecific antibodies.

[0350] In some embodiments, a conditionally active multispecific antibody is substantially inactive in binding to one or both of its target antigens or epitopes under normal physiological conditions, but is active under abnormal conditions, and optionally has a higher level of activity than the activity of the conditionally active multispecific antibody under normal physiological conditions or the activity of the parent antibody from which it is derived under normal physiological conditions. In another embodiment, a conditionally active multispecific antibody is less active or substantially inactive at pH 7.0–7.6, but active at lower pH 5.0–6.8. In some cases, a conditionally active multispecific antibody is reversibly or irreversibly inactivated under normal physiological conditions. In another example, a conditionally active multispecific antibody may be more active in the lower pH environment found in the tumor microenvironment. Conditionally active multispecific antibodies can be used as drugs, therapeutic agents, or diagnostic agents.

[0351] In some embodiments, a conditionally active multispecific antibody or antibody fragment is less active or substantially inactive under normal physiological conditions (e.g., non-tumor microenvironment) but active under abnormal conditions (e.g., tumor microenvironment) compared to the activity of the parental or wild-type antibody or antibody fragment from which it is derived under normal physiological conditions. As a result, the anti-nectin-4 multispecific antibody or antibody fragment of this disclosure may have lower activity to nectin-4 under normal physiological conditions (e.g., non-tumor microenvironment) compared to the parental or wild-type antibody or antibody fragment from which it is derived. For example, a conditionally active multispecific antibody or antibody fragment is less active or substantially inactive at pH 7.0–7.6 compared to the parental or wild-type antibody or antibody fragment, but active at a lower pH of 5.0–6.8 compared to the parental or wild-type antibody or antibody fragment. In some cases, a conditionally active multispecific antibody or antibody fragment is reversibly or irreversibly inactivated under normal physiological conditions (e.g., non-tumor microenvironment) compared to the parental or wild-type antibody or antibody fragment.

[0352] Techniques for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature, vol. 305, pp. 537-540, 1983), International Publication No. 93 / 08829, and Traunecker et al., EMBO J, vol. 10, pp. 3655-3659, 1991), and the "knob-in-hole" operation (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be produced by manipulating the electrostatic steering effect to create antibody Fc heterodimer molecules (International Publication No. 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, vol. 229, pp. 81-83, 1985); producing bispecific antibodies using a leucine zipper (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp. 1547-1553, 1992); using "diabody" technology to produce bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993); and using single-stranded Fv(scFv) dimers (see, e.g., Gruber et al.) See al., J.Immunol., vol.152, pp.5368-5374, 1994); and they can be prepared by preparing triplicate antibodies, for example, as described in Tutt et al. J.Immunol., vol.147, pp.60-69, 1991.

[0353] Modified antibodies having three or more antigen-binding sites, including an "octopus antibody," are also included herein (see, for example, U.S. Patent Application Publication No. 2006 / 0025576A1).

[0354] The anti-nectin-4 antibodies or antibody fragments of this disclosure can be produced using recombinant methods and compositions, which are described in detail in U.S. Patent Application Publication No. 2016 / 0017040.

[0355] The physical / chemical properties and / or biological activity of the anti-nectin-4 antibodies or antibody fragments of this disclosure can be tested and measured by various assays known in the art. Some of these assays are described in U.S. Patent No. 8,853,369.

[0356] I. Immunoconjugates In another embodiment, the Disclosure also provides an immunoconjugate as described herein, wherein the drug portion is one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzyme-active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), and radioisotopes.

[0357] In one embodiment, the immunoconjugates described herein are, but are not limited to, meitansinoids (U.S. Patent No. 5,208,020, U.S. Patent No. 5,416,064 and European Patent No. 0425235). See Specification B1); Auristatins such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Specifications 5,635,483, 5,780,588, and 7,498,298); Drastatin; Calicheamicin or its derivatives (see U.S. Specifications 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res., vol.53, pp.3336-3342, 1993; and Lode et al., Cancer See Res., vol.58, pp.2925-2928, 1998); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem., vol.13, pp.477-523, 2006; Jeffrey et al., Bioorganic & Med. Chem. Letters, vol.16, pp.358-362, 2006; Torgov et al., Bioconj. Chem., vol.16, pp.717-721, 2005; Nagy et al., Proc. Natl. Acad. Sci. USA, vol.97, pp.829-834, 2000; Dubowchik et al., Bioorg. & Med. Chem. Letters, vol.12, vol.1529-1532, 2002; King et See al., J. Med. Chem., vol. 45, pp. 4336-4343, 2002; and U.S. Patent No. 6,630,579); an antibody-drug conjugate (ADC) conjugated to one or more drug moieties including methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0358] In another embodiment, the immunoconjugates described herein, without limitation, are conjugated to drug moieties that are enzymatically active toxins or fragments thereof, including but not limited to diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, geronin, mitogenin, restrictosin, phenomycin, enomycin, and trichothecenes.

[0359] In another embodiment, as described herein, a radioactive atom is conjugated to the drug moiety to form a radioactive conjugate. Various radioactive isotopes can be used to produce the radioactive conjugate. For example, At 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212 Examples include radioactive isotopes of Lu. When radioactive conjugates are used for detection, they may include radioactive atoms for scintigraphy studies, such as tc99m or I123, or spin-labeled atoms for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, and iron.

[0360] In some embodiments, the immunoconjugates described herein include radioactive agents that may be selected from α-emitters, β-emitters, and γ-emitters. An example of an α-emitter is: 211 At, 210 Bi, 212 Bi, 211 Bi, 223 Ra, 224 Ra, 225 American and 227 It is Th. An example of a β-emitter is, 67 Cu. 90 Y, 131 I, 153 Sm, l66 Ho and 186 It is Re. An example of a γ-emitting material is, 60 Co, 137 Ce, 55 Fe, 54 Mg, 203 Hg, and 133 It is Ba.

[0361] The antibody / antibody fragment conjugates and cytotoxic agents described herein can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethylHCl adipimidoate), active esters (e.g., disaxinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science, vol.238, pp.1098-, 1987. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionucleotides to antibodies. See International Publication No. 94 / 11026. Linkers may be “cleavable linkers” that facilitate the release of cytotoxic drugs into cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res., vol.52, pp.127-131, 1992; U.S. Patent No. 5,208,020) may be used.

[0362] The immunoconjugates described herein expressly refer to conjugates prepared with crosslinking reagents, including but not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0363] An exemplary embodiment of an ADC comprises an antibody or antibody fragment (Ab) that targets tumor cells, a drug moiety (D), and a linker moiety (L) that binds Ab to D. In some embodiments, the antibody is bound to the linker moiety (L) via one or more amino acid residues, such as lysine and / or cysteine.

[0364] An example ADC is Ab-(LD) p The ADC has formula I, where p is between 1 and approximately 20. In some embodiments, the number of drug moieties that can be conjugated to the antibody is limited by the number of free cysteine ​​residues. In some embodiments, the free cysteine ​​residues are introduced into the antibody amino acid sequence by the methods described herein. Exemplary ADCs of formula I include, but are not limited to, antibodies having 1, 2, 3, or 4 modified cysteine ​​amino acids (Lyon et al., Methods in Enzym., vol. 502, pp. 123-138, 2012). In some embodiments, one or more free cysteine ​​residues are already present in the antibody without the use of any manipulation, in which case the existing free cysteine ​​residues can be used to conjugate the antibody to the drug. In some embodiments, the antibody is exposed to reducing conditions before antibody conjugation to generate one or more free cysteine ​​residues.

[0365] A linker is used to conjugate a portion to an antibody to form an immunoconjugate such as an ADC. Other suitable linkers are described in International Publication No. 2017 / 180842.

[0366] Some drug moieties that can be conjugated to antibodies include, but are not limited to, those described in International Publication No. 2017 / 180842.

[0367] The drug portion is not limited to but may include compounds with nucleic acid degradation activity (e.g., ribonucleases or DNA endonucleases).

[0368] In certain embodiments, the immunoconjugate may contain highly radioactive atoms. Various radioisotopes are available for the production of radioconjugate antibodies. For example, At 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212 Examples include radioactive isotopes of Lu. In some embodiments, when an immunoconjugate is used for detection, it may be a radioactive atom for scintigraphy studies, such as Tc 99 or 1 123 or may include spin labeling for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI) such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. Zirconium-89 can be complexed with various metal chelating agents and, for example, conjugated to antibodies for PET imaging (International Publication No. 2011 / 056983).

[0369] Radiolabeling or other labeling can be incorporated into the immunoconjugate by known methods. For example, peptides may be biosynthesized or chemosynthesized using suitable amino acid precursors containing, for example, one or more fluorine-19 atoms instead of one or more hydrogen atoms. In some embodiments, Tc 99 , I 123 Re 186 Re 188 and In 111 Labels such as can be conjugated via cysteine ​​residues in the antibody. In some embodiments, yttrium-90 can be conjugated via lysine residues in the antibody. In some embodiments, iodine-123 can be incorporated using the IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun., vol. 80, pp. 49-57, 1978). "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes certain other methods.

[0370] In certain embodiments, the immunoconjugates described herein may include antibodies conjugated to prodrug-activating enzymes. In some such embodiments, the prodrug-activating enzymes convert prodrugs (e.g., peptidyl chemotherapeutic agents, see International Publication No. 81 / 01145) into active drugs such as anticancer agents. Such immunoconjugates are useful in antibody-dependent enzyme-mediated prodrug therapy ("ADEPT") in some embodiments. Enzymes that can be conjugated to antibodies include, but are not limited to, alkaline phosphatases useful for converting phosphate-containing prodrugs into free drugs; arylsulfatases useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminases useful for converting non-toxic 5-fluorocytosine into the anticancer agent, 5-fluorouracil; proteases such as serratia protease, pyrolysis, subtilisin, carboxypeptidase and cathepsin (cathepsin B and L, etc.) useful for converting peptide-containing prodrugs into free drugs; and D-ami Examples include D-alanyl carboxypeptidases useful for converting prodrugs containing no acid substituents; carbohydrate-cleaving enzymes such as β-galactosidases and neuraminidases useful for converting glycosylated prodrugs into free drugs; β-lactamases useful for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases such as penicillin V amidases and penicillin G amidases useful for converting drugs derivatized with amine nitrogen having a phenoxyacetyl group or a phenylacetyl group, respectively, into free drugs. In some embodiments, the enzymes may be covalently bound to antibodies by recombinant DNA techniques well known in the art. See, for example, Neuberger et al., Nature, vol.312, pp.604-608, 1984.

[0371] The drug load in a conjugate is represented by m, which is the average number of drug moieties per antibody. The drug load may range from 1 to 10 drug moieties per antibody, or from 1 to 5 drug moieties per antibody. The conjugates of this disclosure may have a range of 1 to 10 or 1 to 5 drug moieties. The average number of drug moieties per antibody used to prepare the conjugate from the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC.

[0372] For some antibody-drug conjugates (ADCs), the drug load may be limited by the number of binding sites on the antibody. For example, if the binding is cysteinethiol, as in the specific exemplary embodiments described above, the antibody may have only one or several cysteinethiol groups, or one or several sufficiently reactive thiol groups to which the linker can bind. In certain embodiments, a higher drug load, e.g., n>5, may cause aggregation, insolubility, toxicity, or reduced cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug load of an ADC is in the range of 1 to about 8; about 2 to about 6; or about 3 to about 5. In practice, for certain ADCs, the optimal ratio of drug portion per antibody may be less than 8 and may be about 2 to about 5 (U.S. Patent No. 7,498,298).

[0373] In certain embodiments, fewer drug moieties than the theoretical maximum number are conjugated to the antibody during the conjugation reaction. The antibody may contain lysine residues that do not react with the drug-linker intermediate or linker reagent, for example, as described later. Generally, antibodies do not contain many free and reactive cysteinethiol groups that can be linked to the drug moiety. In fact, most cysteinethiol residues in antibodies exist as disulfide crosslinks. In certain embodiments, the antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partially or entirely reducing conditions to generate reactive cysteinethiol groups. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophiles such as lysine or cysteine.

[0374] The loading (drug / antibody ratio or DAR) of the ADC or immunoconjugate of this disclosure can be controlled in various ways, for example, by (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, or by (ii) limiting the conjugation reaction time or temperature, and / or by (iii) partial or limited reduction conditions of the cysteinethiol modification.

[0375] J. Methods and compositions for diagnosis and detection In certain embodiments, any of the isolated polypeptides or anti-nectin-4 antibodies or antibody fragments provided herein may be used to quantitatively or qualitatively detect the presence of nectin-4 in a biological sample. In certain embodiments, the biological sample includes cells or tissues such as those of the breast, pancreas, esophagus, lungs, and / or brain.

[0376] This disclosure further provides a kit for diagnosis or detection, which includes a linker from any one of the above-described embodiments of the linker, an immunoconjugate from any one of the above-described embodiments of the immunoconjugate, or a pharmaceutical composition from any one of the embodiments of the pharmaceutical composition described later herein, and instructions for using the linker, immunoconjugate and / or pharmaceutical composition for the diagnosis or detection of cancer.

[0377] Further aspects of the present disclosure refer to isolated polypeptides or anti-nectin-4 antibodies or antibody fragments of the present disclosure described herein for diagnosing and / or monitoring cancer or other disease in which nectin-4 expression levels are increased or decreased from normal physiological levels at at least one site in the body.

[0378] In preferred embodiments, the isolated polypeptide or antibody or antibody fragment of the Disclosure may be labeled with a detectable molecule or substance, such as the fluorescent molecule, radioactive molecule or any other label known in the Art. For example, the antibody or antibody fragment of the Disclosure may be labeled with a radioactive molecule. For example, suitable radioactive molecules include, but are not limited to, 123 I, 124 I, 111 In, 186 Re, and 188 Examples of radioactive atoms used in scintigraphy studies include Re. The antibodies or antibody fragments of this disclosure may also be labeled with spin labeling for nuclear magnetic resonance (NMR) imaging, such as iodine-123, iodine-131, indium-1I, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. After administration of the antibody, the distribution of the radiolabeled antibody in the patient is detected. Any suitable known method may be used. Some non-limiting examples include computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence, and ultrasound.

[0379] The isolated polypeptides, antibodies, or antibody fragments described herein may be useful in the diagnosis and staging of cancers and diseases associated with nectin-4 overexpression. Cancers associated with nectin-4 overexpression may include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glioblastoma and neurofibromatosis and other glial cell tumors, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, sarcoma, hematological cancer (leukemia), astrocytoma, and various types of head and neck cancers or other hyperproliferative diseases of nectin-4 expression or overexpression.

[0380] The isolated polypeptides or antibodies or antibody fragments of this disclosure described herein may be useful in diagnosing non-cancer diseases characterized by increased or decreased nectin-4 expression. (Both soluble and cellular nectin-4 forms may be used for such diagnostics. Typically, such diagnostic methods involve the use of biological samples obtained from patients. Biological samples encompass a variety of sample types obtained from subjects that may be used in diagnostic or monitoring assays. Biological samples include, but are not limited to, blood and other fluid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and their offspring. For example, biological samples include individuals suspected of having cancer associated with nectin-4 overexpression, and, in preferred embodiments, cells obtained from tissue samples recovered from glioma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, liver cancer, and endometrial cancer. Biological samples encompass clinical samples, cells in culture, cell supernatant, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0381] In certain embodiments, the Disclosure provides a method for diagnosing cancer associated with nectin-4 overexpression in a subject by detecting nectin-4 on cells from the subject using the antibody of the Disclosure. In particular, the method is 1) A step of bringing a target biological sample into contact with the antibody or antibody fragment according to the present disclosure under conditions suitable for the antibody or antibody fragment to form a complex with cells in the biological sample that express nectin-4; and 2) The step may include detecting and / or quantifying the complex, thereby indicating that the detection of the complex indicates cancer associated with nectin-4 overexpression.

[0382] To monitor cancer progression, the method according to this disclosure may be repeated a number of times to determine whether antibody binding to the sample increases or decreases, thereby determining whether the cancer is progressing, regressing, or stabilizing.

[0383] In certain embodiments, the present invention relates to a method for diagnosing diseases associated with the expression or overexpression of nectin-4. Examples of such diseases include cancer, human immunodeficiency, thrombosis (thrombosis and atherothrombosis), and cardiovascular disease.

[0384] In one embodiment, an anti-nectin-4 antibody or antibody fragment is provided for use in a diagnostic or detection method. In a further embodiment, a method for detecting the presence of nectin-4 in a biological sample is provided. In a further embodiment, a method for quantifying the amount of nectin-4 in a biological sample is provided. In certain embodiments, the method includes contacting a biological sample with the anti-nectin-4 antibody or antibody fragment described herein under conditions that allow binding of the anti-nectin-4 antibody or antibody fragment to nectin-4, and detecting whether a complex is formed between the anti-nectin-4 antibody or antibody fragment and nectin-4. Such methods may be performed in vitro or in vivo. In one embodiment, the anti-nectin-4 antibody or antibody fragment is used to select a subject eligible for a treatment. In some embodiments, the treatment includes administering the anti-nectin-4 antibody or antibody fragment to the subject.

[0385] In certain embodiments, labeled anti-nectin-4 antibodies or antibody fragments are provided. Labels include, but are not limited to, directly detectable labels or moieties (such as fluorescence, color development, high electron density, chemiluminescence, and radioactive labeling), as well as moieties detected indirectly, for example, by enzymatic reactions or molecular interactions, such as enzymes or ligands. Exemplary labels include, but are not limited to, radioactive isotopes. 32 P, 14 C, 125 I, 3 H, and 131 Examples include fluorophores, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases (such as uricase and xanthine oxidase) coupled with enzymes that oxidize pigment precursors using hydrogen peroxide (such as HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin-labeled, bacteriophage-labeled, and stable free radicals.

[0386] K. Pharmaceutical preparations The isolated polypeptides or anti-nectin-4 antibodies or antibody fragments described herein possess cytotoxic activity. This cytotoxic activity extends to several different types of cell lines. Furthermore, when conjugated with cytotoxic agents, these isolated polypeptides or antibodies or antibody fragments of this disclosure may reduce tumor size and exhibit reduced toxicity. Therefore, isolated polypeptides, anti-nectin-4 antibodies, their fragments, or immunoconjugates may be useful in treating proliferative disorders associated with nectin-4 expression. The isolated polypeptides, antibodies, fragments, or immunoconjugates may be used alone or in combination with any suitable agent or other conventional treatment.

[0387] This disclosure provides a pharmaceutical composition comprising any one of the above embodiments of the linker, any one of the above embodiments of the conjugate, or any one of the above embodiments of the immunoconjugate; and a pharmaceutically acceptable carrier.

[0388] In one embodiment of the above-described pharmaceutical composition, the Disclosure provides a single dose of the pharmaceutical composition comprising an amount of immunoconjugate of approximately 135 mg, 235 mg, 335 mg, 435 mg, 535 mg, 635 mg, 735 mg, 835 mg, 935 mg, 1035 mg, 1135 mg, 1235 mg, or 1387 mg.

[0389] In any one of the embodiments of the pharmaceutical composition described above, the Disclosure provides a single dose of a pharmaceutical composition comprising an immunoconjugate in the range of 135-235 mg, 235-335 mg, 335-435 mg, 435-535 mg, 535-635 mg, 635-735 mg, 735-835 mg, 835-935 mg, 935-1035 mg, 1035-1135 mg, 1135-1235 mg, or 1235-1387 mg.

[0390] In any one of the embodiments of the pharmaceutical composition described above, the pharmaceutical composition further comprises an immune checkpoint inhibitor molecule.

[0391] In any one of the embodiments of the pharmaceutical composition described above, the pharmaceutical composition further comprises an immune checkpoint inhibitor molecule, the immune checkpoint inhibitor molecule being an antibody or antibody fragment against an immune checkpoint.

[0392] In any one of the embodiments of the pharmaceutical composition described above, the pharmaceutical composition further comprises an immune checkpoint inhibitor molecule, the immune checkpoint being selected from CTLA4, LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, GITR, B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS.

[0393] In any one of the embodiments of the pharmaceutical composition described above, the pharmaceutical composition further comprises an immune checkpoint inhibitor molecule, wherein the immune checkpoint is CTLA4, PD-1, or PD-L1.

[0394] In any one of the embodiments of the pharmaceutical composition described above, the pharmaceutical composition further comprises an antibody or antibody fragment against an antigen selected from CTLA4, PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4, and WNT proteins.

[0395] This disclosure provides a pharmaceutical composition, one of the embodiments of the above-described pharmaceutical compositions, and a method for treating cancer, comprising the step of administering it to a patient having cancer.

[0396] This disclosure also provides the use of any one of the embodiments of the above-described pharmaceutical compositions for the treatment of cancer.

[0397] This disclosure further provides a kit for treatment, which includes a linker from any of the above embodiments of the linker, a conjugate from any of the above embodiments of the conjugate, or an immunoconjugate from any of the above embodiments of the immunoconjugate, or a pharmaceutical composition from any of the above embodiments of the pharmaceutical composition, and instructions for using the linker, conjugate, immunoconjugate and / or pharmaceutical composition for the treatment of cancer.

[0398] The isolated polypeptides, anti-nectin-4 antibodies or antibody fragments, immunoconjugates, kits, or pharmaceutical compositions described herein may be used to treat diseases associated with nectin-4 expression, overexpression, or activation. Aside from the requirement of nectin-4 expression, there are no particular limitations on the type of cancer or tissue that can be treated. Examples include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, sarcoma, hematological cancer (leukemia), astrocytoma, and various types of head and neck cancers. More preferred cancers are glioma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, liver cancer, and endometrial cancer.

[0399] The isolated polypeptides or anti-nectin-4 antibodies or antibody fragments described herein are potential activators of the innate immune response and can therefore be used in the treatment of human immunodisorders such as sepsis. For example, the anti-nectin-4 antibodies or antibody fragments of this disclosure can also be used as adjuvants for immunization, such as vaccines, and as anti-infective agents against bacteria, viruses, and parasites, for example.

[0400] Isolated polypeptides or anti-nectin-4 antibodies or antibody fragments may be used to protect against, prevent, or treat thrombosis, such as venous and arterial thrombosis and atherothrombosis. For example, anti-nectin-4 antibodies or antibody fragments may also be used to protect against, prevent, or treat cardiovascular disease, as well as to prevent or inhibit the entry of viruses such as Lassa and Ebola viruses, and to treat viral infections.

[0401] In each embodiment of the therapeutic methods described herein, the isolated polypeptide, anti-nectin-4 antibody, antibody fragment, or anti-nectin-4 antibody or antibody fragment immunoconjugate may be delivered in a manner consistent with conventional methods relating to the management of the disease or disorder for which treatment is required. According to the disclosure herein, an effective amount of antibody, antibody fragment, or immunoconjugate is administered to a subject in need of such treatment for a sufficient time and under conditions to prevent or treat the disease or disorder. Accordingly, one aspect of the disclosure relates to a method for treating a disease related to nectin-4 expression, comprising administering a therapeutically effective amount of the antibody, antibody fragment, or immunoconjugate of the disclosure to a subject in need of such treatment.

[0402] For administration, anti-nectin-4 antibodies, antibody fragments, or immunoconjugates may be formulated as pharmaceutical compositions. Pharmaceutical compositions comprising the isolated polypeptide, anti-nectin-4 antibody, antibody fragment, or immunoconjugate of this disclosure may be formulated according to known methods for preparing pharmaceutical compositions. In such methods, the therapeutic molecule is typically combined with a mixture, solution, or composition containing a pharmaceutically acceptable carrier.

[0403] A pharmaceutically acceptable carrier is a material that is acceptable to the recipient patient. Sterile phosphate-buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable pharmaceutically acceptable carriers are well known to those skilled in the art (see, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further contain one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc.

[0404] The form, route of administration, dosage, and regimen of a pharmaceutical composition naturally depend on the condition being treated, the severity of the disease, the patient's age, weight, and sex, etc. These considerations can be taken into account by those skilled in the art to formulate a suitable pharmaceutical composition. The pharmaceutical compositions of this disclosure can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, etc.

[0405] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These may be a specific isotonic sterile saline solution (such as monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, or a mixture of such salts), or a dry, particularly lyophilized, composition that enables the formation of an injectable solution upon addition, for example, sterile water or saline.

[0406] In some embodiments, isotonic agents, sometimes also known as “stabilizers,” are present to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules such as proteins and antibodies, they are often called “stabilizers” because they can interact with the charged groups of amino acid side chains, thereby reducing the potential for intermolecular and intramolecular interactions. Isotonic agents may be present in any amount of 0.1% to 25% by weight, preferably 1% to 5% by weight, of the pharmaceutical composition. Preferred isotonic agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0407] Further excipients may include agents that can function as one or more of the following: (1) volume extenders, (2) dissolution accelerators, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container wall. Such excipients include polyhydric sugar alcohols (listed above); amino acids, e.g., alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine; organic sugars or sugar alcohols, e.g., sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinitose, myonititol, galactose, galactitol, glycerol, cyclitol (e.g., inositol), polyethylene Recall; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; monosaccharides (such as xylose, mannose, fructose, and glucose); disaccharides (such as lactose, maltose, and sucrose); trisaccharides, such as raffinose; and polysaccharides, such as dextrin or dextran.

[0408] Nonionic surfactants or detergents (also known as "wetting agents") may be used to assist in the solubilization of the therapeutic agent and to protect the therapeutic protein from aggregation induced by agitation, which also allows the formulation to be exposed to shear surface loading without causing denaturation of the active therapeutic protein or antibody. Nonionic surfactants may be present in a concentration range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0409] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxomers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0410] The dose used for administration may be adapted depending on various parameters, particularly the method of administration used, the associated lesion, or the desired duration of treatment. To prepare the pharmaceutical composition, an effective amount of antibody or antibody fragment may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0411] Suitable dosage forms for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or propylene glycol aqueous solutions; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi.

[0412] Solutions of the active compound, as a free base or a pharmacokinetically acceptable salt, can be prepared in water, which is suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0413] Anti-nectin-4 antibodies or antibody fragments can be formulated into neutral or salt-formulated compositions. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, or procaine.

[0414] The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Sustained absorption of the injectable composition can be achieved by using absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0415] Sterile injectable solutions are prepared by incorporating the required amount of active compound into a suitable solvent containing, if necessary, one or more components other than those listed above, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredient and any additional desired components from a pre-filtered sterilized solution.

[0416] For direct injection, the preparation of larger or higher concentration solutions is also envisioned, and the use of dimethyl sulfoxide (DMSO) as a solvent is envisioned to provide very rapid penetration and deliver high concentrations of the active agent to small tumor areas.

[0417] During formulation, the solution is administered in a manner compatible with the administered formulation and in a therapeutically effective amount. The formulation can be easily administered in various dosage forms, such as the injectable solution type described above, but drug-release capsules may also be used.

[0418] For example, in the case of parenteral administration in an aqueous solution, the solution should be preferably buffered as needed, and the liquid diluent should first be isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art with regard to this disclosure. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous fluid for infusion, or injected at the proposed injection site (see, e.g., “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in the dose will inevitably occur depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose for each individual subject.

[0419] Antibodies or antibody fragments can be formulated in therapeutic mixtures to deliver approximately 0.0001 to 10.0 milligrams, or approximately 0.001 to 5 milligrams, or approximately 0.001 to 1 milligram, or approximately 0.001 to 0.1 milligrams, or approximately 0.1 to 1.0 or approximately 10 milligrams per dose. Multiple doses may also be administered at selected time intervals.

[0420] In addition to compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; sustained-release capsules; and any other forms currently in use.

[0421] In certain embodiments, the use of liposomes and / or nanoparticles is envisioned for the introduction of antibodies or antibody fragments into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.

[0422] Nanocapsules can generally capture compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) are generally designed using polymers that are biodegradable in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are envisioned for use in this disclosure, and such particles can be readily fabricated.

[0423] Liposomes are dispersed in an aqueous medium and are formed from phospholipids that spontaneously form multilayer concentric bilayer vesicles (also called multilayer vesicles (MLVs)). MLVs generally have a diameter of 25 nm to 4 μm. Sonication of MLVs creates small monolayer vesicles (SUVs) with a diameter ranging from 200 to 500 Å, containing an aqueous solution within their core. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0424] Pharmaceutical formulations containing anti-nectin-4 antibodies or antibody fragments described herein are prepared by mixing such antibodies or antibody fragments having a desired degree of purity with one or more optionally selected pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used, and are not limited to: buffers, e.g., phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); and low molecular weight (less than approximately 10 residues) polypeptides. Examples include: tides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and glucose, mannose, or dextrin; other carbohydrates containing chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., polyethylene glycol (PEG).

[0425] Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Specific exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0426] An example of a lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Examples of aqueous antibody preparations are described in U.S. Patent No. 6,171,586 and International Publication No. 2006 / 044908, the latter of which contains histidine-acetate buffer.

[0427] The formulations described herein may also contain two or more active ingredients as necessary for the specific indication being treated. Preferably, ingredients having complementary activity that does not adversely affect each other may be combined in a single formulation. For example, in addition to the anti-nectin-4 antibody, antibody fragment, or immunoconjugate of this disclosure, it may be desirable to provide an EGFR antagonist (such as erlotinib), an anti-angiogenic agent (such as a VEGF antagonist, which may be an anti-VEGF antibody), or a chemotherapeutic agent (such as a taxoid or platinum agent). Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0428] In one embodiment, the anti-nectin-4 antibody, antibody fragment, or immunoconjugate of the Disclosure is combined in a formulation with another antibody or antibody fragment against an antigen selected from CTLA4, PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4, and WNT proteins (including WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16). The combination may be in the form of two distinct molecules, namely, the anti-nectin-4 antibody, antibody fragment, or immunoconjugate of the Disclosure and another antibody or antibody fragment. Alternatively, the combination may also be in the form of a single molecule, possessing binding affinity to both nectin-4 and other antigens, and thus forming a multispecific (e.g., bispecific) antibody.

[0429] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation technology or by interfacial polymerization. For example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules may be used in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0430] Sustained-release preparations can be prepared. A preferred example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing an antibody or antibody fragment, the matrix of which may be in the form of a molded article, such as a film or microcapsules.

[0431] Preparations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0432] L. Treatment methods and compositions This disclosure provides a method for treating cancer, comprising the step of administering to a patient having cancer one of the embodiments of the immunoconjugate described above or one of the embodiments of the pharmaceutical composition described above.

[0433] This disclosure also provides the use of any one of the above embodiments of the linker, any one of the above embodiments of the immunoconjugate, or any one of the above embodiments of the pharmaceutical composition for the treatment of cancer.

[0434] Any of the isolated polypeptides, anti-nectin-4 antibodies or antibody fragments, pharmaceutical compositions or immunoconjugates described herein may be used in the therapeutic methods described later. In one embodiment, an anti-nectin-4 antibody or antibody fragment is provided for use as a pharmaceutical agent. In a further embodiment, an anti-nectin-4 antibody or antibody fragment is provided for use in treating cancer (e.g., breast cancer, non-small cell lung cancer, pancreatic cancer, brain tumor, pancreatic, brain, kidney, ovarian, stomach, leukemia, endometrial, colon, prostate, thyroid, liver cancer, osteosarcoma, and / or melanoma). In certain embodiments, an anti-nectin-4 antibody or antibody fragment is provided for use in a therapeutic method. In certain embodiments, the Disclosure provides an anti-nectin-4 antibody or antibody fragment for use in a method of treating an individual having cancer, comprising administering an effective amount of the anti-nectin-4 antibody or antibody fragment to the individual. In certain embodiments, the Disclosure provides an anti-nectin-4 antibody or antibody fragment for use in a method of treating an individual having an immunodeficiency (e.g., autoimmune disorder), cardiovascular disease (e.g., atherosclerosis, hypertension, thrombosis), infection (e.g., Ebola virus, Marburg virus), or diabetes, comprising administering an effective amount of the anti-nectin-4 antibody or antibody fragment to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, e.g., as described below, to the individual. In further embodiments, the Disclosure provides an anti-nectin-4 antibody or antibody fragment for use in inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vascular systems (e.g., intratumor vascular systems or tumor-associated vascular systems), and / or inhibiting tumor stromal function.

[0435] In certain embodiments, the Disclosure provides an anti-nectin-4 antibody or antibody fragment for use in a method of inhibiting angiogenesis, cell proliferation, immune function, inflammatory cytokine secretion (e.g., from tumor-associated macrophages), tumor vascularization (e.g., intratumor vascularization or tumor-associated vascularization), and / or tumor stromal function in an individual, the method comprising administering an effective amount of the anti-nectin-4 antibody or antibody fragment to an individual to inhibit angiogenesis, cell proliferation, immune function, inflammatory cytokine secretion (e.g., from tumor-associated macrophages), tumor vascularization (e.g., intratumor vascularization or tumor-associated vascularization), and / or tumor stromal function. The “individual” in any of the above embodiments is preferably a human.

[0436] In further embodiments, the Disclosure provides the use of anti-nectin-4 antibodies or antibody fragments in the manufacture or preparation of pharmaceuticals. In one embodiment, the pharmaceutical is for the treatment of cancer (in some embodiments, breast cancer, non-small cell lung cancer, pancreatic cancer, brain tumors, cancers of the pancreas, brain, kidney, ovary, stomach, leukemia, endometrium, colon, prostate, thyroid, liver, osteosarcoma, and / or melanoma). In further embodiments, the pharmaceutical is for use in a method of treating cancer, comprising administering an effective amount of the pharmaceutical to an individual having cancer. In further embodiments, the pharmaceutical is for use in a method of treating immunodeficiencies (e.g., autoimmune disorders), cardiovascular diseases (e.g., atherosclerosis, hypertension, thrombosis), infections (e.g., Ebola virus, Marburg virus), or diabetes, comprising administering an effective amount of anti-nectin-4 antibodies or antibody fragments to an individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, such as those described below, to the individual. In further embodiments, the agent is for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vascular systems (e.g., intratumor vascular systems or tumor-associated vascular systems), and / or inhibiting tumor stromal function. In further embodiments, the agent is for use in a method of inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vascular development (e.g., intratumor vascular systems or tumor-associated vascular systems), and / or inhibiting tumor stromal function in an individual, which includes administering an effective amount of the agent to the individual to inhibit angiogenesis, inhibit cell proliferation, inhibit immune function, inhibit inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibit tumor vascular systems (e.g., intratumor vascular systems or tumor-associated vascular systems), and / or inhibit tumor stromal function. The “individual” in any of the above embodiments may be a human.

[0437] In further embodiments, the Disclosure provides methods for treating cancer. In one embodiment, the Method comprises administering an effective amount of an anti-nectin-4 antibody or antibody fragment to an individual having such cancer. In one such embodiment, the Method further comprises administering an effective amount of at least one additional therapeutic agent, as described below, to the individual. The “individual” in any of the above embodiments may be a human.

[0438] In further embodiments, the Disclosure provides methods for treating immune disorders (e.g., autoimmune disorders), cardiovascular diseases (e.g., atherosclerosis, hypertension, thrombosis), infections (e.g., Ebola virus, Marburg virus), or diabetes. In one such embodiment, the Method further comprises administering an effective amount of at least one additional therapeutic agent, as described below, to an individual. The “individual” in any of the above embodiments may be a human.

[0439] In a further embodiment, the Disclosure provides methods for inhibiting angiogenesis, cell proliferation, immune function, inflammatory cytokine secretion (e.g., from tumor-associated macrophages), tumor vascularization (e.g., intratumor vascularization or tumor-associated vascularization), and / or tumor stromal function in a solid. In one embodiment, the Method comprises administering an effective amount of an anti-nectin-4 antibody or antibody fragment to a solid to inhibit angiogenesis, cell proliferation, immune function, induce inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibit tumor vascularization (e.g., intratumor vascularization or tumor-associated vascularization), and / or tumor stromal function. In one embodiment, “solid” is a human.

[0440] In further embodiments, the Disclosure provides a pharmaceutical formulation comprising, for example, one of the anti-nectin-4 antibodies or antibody fragments provided herein for use in any of the therapeutic methods described above. In one embodiment, the pharmaceutical formulation comprises one of the anti-nectin-4 antibodies or antibody fragments provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises one of the anti-nectin-4 antibodies or antibody fragments provided herein and at least one additional therapeutic agent, such as those described below.

[0441] In any of the above treatments, the antibodies or antibody fragments of this disclosure may be used alone, as an immune conjugate, or in combination with other agents during treatment. For example, the antibodies of this disclosure may be administered concurrently with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-angiogenic agent. In certain embodiments, the additional therapeutic agent is a VEGF antagonist (in some embodiments, an anti-VEGF antibody, e.g., bevacizumab). In certain embodiments, the additional therapeutic agent is an EGFR antagonist (in some embodiments, erlotinib). In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent and / or a cell proliferation inhibitor. In certain embodiments, the additional therapeutic agent is a taxoid (e.g., paclitaxel) and / or a platinum agent (e.g., carboplatinum). In certain embodiments, the additional therapeutic agent is an agent that enhances the patient's immune system or immune response.

[0442] Such combination therapies as described above include combined administration (two or more therapeutic agents contained in the same or separate formulations) and individual administration, in which case the administration of antibodies or antibody fragments may occur before, simultaneously with, and / or after the administration of additional therapeutic agents and / or adjuvants. Antibodies or antibody fragments may also be used in combination with radiotherapy.

[0443] Anti-nectin-4 antibodies or antibody fragments may be formulated, administered, and given in a manner consistent with good medical practice. Factors relevant to consideration include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to the physician. Antibodies or antibody fragments may, but are not necessarily required, be formulated together with one or more drugs currently used to prevent or treat the disorder in question. The effective dose of such other drugs depends on the amount of antibody or antibody fragment present in the formulation, the type of disorder or treatment, and the other factors mentioned above. These are generally used in the same doses and routes of administration described herein, or in about 1–99% of the doses described herein, or in any dose and route deemed empirically / clinically appropriate.

[0444] For the prevention or treatment of a disease, the appropriate dose of an antibody or antibody fragment (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the type of antibody or antibody fragment, the severity and course of the disease, whether the antibody or antibody fragment is administered for preventive or therapeutic purposes, previous treatments, the patient's medical history and response to the antibody or antibody fragment, and the discretion of the attending physician. The antibody or antibody fragment is preferably administered to the patient in a single dose or over a series of treatments. Depending on the type and severity of the disease, approximately 1 μg of antibody or antibody fragment / kg of patient weight to 40 mg of antibody or antibody fragment / kg of patient weight may be the initial candidate dose for administration to the patient, whether by one or more separate doses or by continuous infusion. A typical daily dose may range from approximately 1 μg of antibody or antibody fragment / kg of patient weight to 100 mg of antibody or antibody fragment / kg of patient weight or more, depending on the factors mentioned above. In the case of repeated administrations over several days or more, treatment will generally continue, depending on the condition, until the desired suppression of disease symptoms occurs. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives approximately 2 to 20 doses of antibody or antibody fragments, or, for example, approximately 6 doses). A higher initial loading dose, followed by one or more lower doses, may be administered. However, other dosing regimens may be useful. The progression of this treatment is readily monitored by conventional techniques and assays.

[0445] Specific doses of the anti-nectin-4 antibody or antibody fragment of this disclosure, which may be administered for the prevention or treatment of the target disease, may be approximately 0.3, 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2, 7.8, 8.4, 9.0, 9.6, or 10.2 mg of antibody or antibody fragment / kg patient weight. In certain embodiments, doses may range from 0.3 to 2.4, 2.4 to 4.2, 4.2 to 6.0, 6.0 to 7.8, 7.8 to 10.2, 10.2 to 12, 12 to 14, 14 to 16, 16 to 18, or 18 to 20 mg of antibody or antibody fragment / kg patient weight. The dose of the antibody or antibody fragment remains the same when administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor or another antibody or antibody fragment, or as an immune conjugate. Furthermore, the polypeptide possessing anti-nectin-4 activity is administered in the same amount as the antibody or antibody fragment.

[0446] A single dose of the pharmaceutical formulations of this disclosure may contain an amount of the anti-nectin-4 antibody or antibody fragment of this disclosure ranging from approximately 45 μg to approximately 13,600 mg, or approximately 45 μg to approximately 5,440 mg. In some embodiments, a single dose of the pharmaceutical formulations of this disclosure may contain an amount of the anti-nectin-4 antibody or antibody fragment of this disclosure ranging from 135 mg to 1,387 mg, or amounts such as 135, 235, 335, 435, 535, 635, 735, 835, 935, 1035, 1135, 1235, 1387 mg. In certain embodiments, the amount of the anti-nectin-4 antibody or antibody fragment of the present disclosure in a single dose of the pharmaceutical formulation is in the range of 135-235, 235-335, 335-435, 435-535, 535-635, 635-735, 735-835, 835-935, 935-1035, 1035-1135, 1135-1235, and 1235-1387 mg. The amount of the antibody or antibody fragment in a single dose of the pharmaceutical formulation remains the same when administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor, or as an immune conjugate, or in combination with another antibody or antibody fragment against another antigen disclosed herein. Furthermore, a polypeptide having anti-nectin-4 activity will be included in the same amount as the antibody or antibody fragment in a single dose of the pharmaceutical formulation.

[0447] For example, an anti-nectin-4 antibody or antibody fragment may be conjugated to an immune checkpoint inhibitor molecule, or may form part of a bispecific antibody with an immune checkpoint inhibitor.

[0448] The combination may be an anti-nectin-4 antibody or antibody fragment disclosed in this application, and an immune checkpoint inhibitor molecule administered as a separate molecule or as a bispecific antibody. Such a bispecific antibody has binding activity to nectin-4 and a second binding activity to immune checkpoints.

[0449] Immune checkpoints can be selected from CTLA4, LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, and GITR (Zahavi and Weiner, International Journal of Molecular Sciences, vol.20, 158, 2019). Further immune checkpoints include B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS (Manni et al., Immune checkpoint blockade and its combination therapy with small-molecule inhibitors for cancer treatment, Bbacan, https: / / doi.org / 10.1016 / j.bbcan.2018.12.002, 2018).

[0450] The immune checkpoint is preferably CTLA4, PD-1, or PD-L1.

[0451] It is understood that any of the above formulations or treatment methods may be carried out using the antibody fragment or immunoconjugate of this disclosure in place of, or in addition to, the anti-nectin-4 antibody.

[0452] Enhancing the host's immune function to eradicate tumors is a subject of increasing interest. Conventional methods include (i) enhancement of APCs, e.g., (a) injection of DNA encoding exogenous MHC alloantigens into the tumor, or (b) transfecting biopsy tumor cells with genes that increase the probability of tumor immune antigen recognition (e.g., immunostimulatory cytokines, GM-CSF, costimulatory molecules B7.1, B7.2), and (iii) adoptive cell immunotherapy, or treatment with activated tumor-specific T cells. Adoptive cell immunotherapy involves isolating tumor-infiltrating host T-lymphocytes and expanding the population in vitro, e.g., through stimulation by IL-2 or the tumor, or both. Furthermore, dysfunctional isolated T cells can also be activated by in vitro application of the anti-PD-L1 antibody of this disclosure. The thus activated T cells can then be re-administered to the host. One or more of these methods can be used in combination with the administration of the antibody, antibody fragment, or immune conjugate of this disclosure.

[0453] Conventional treatments for cancer include: (i) radiotherapy (e.g., radiotherapy, X-ray therapy, irradiation) or the use of ionizing radiation to kill cancer cells and shrink tumors. Radiotherapy may be administered via external beam radiotherapy (EBRT) or via internal close-range radiotherapy; (ii) chemotherapy, or the application of cytotoxic drugs that generally affect rapidly dividing cells; (iii) targeted therapy, or drugs that specifically affect dysregulated proteins in cancer cells (e.g., tyrosine kinase inhibitors imatinib, gefitinib; monoclonal antibodies; photodynamic therapy); (iv) immunotherapy, or enhancement of the host immune response (e.g., vaccines); (v) hormone therapy, or blockade of hormones (e.g., if the tumor is hormone-sensitive); (vi) angiogenesis inhibitors, or blockade of blood vessel formation and growth; and (vii) palliative care, or treatment aimed at improving the quality of care to reduce pain, nausea, vomiting, diarrhea, and bleeding. Analgesics such as morphine and oxycodone, and antiemetics such as ondansetron and aprepitant, can enable more aggressive treatment regimens.

[0454] In the treatment of cancer, any of the conventional therapies described above for cancer immunotherapy may be administered before, after, or concurrently with the administration of anti-nectin-4 antibodies or antibody fragments. Furthermore, anti-nectin-4 antibodies or antibody fragments may be administered before, after, or concurrently with conventional cancer therapies such as the administration of tumor-binding antibodies (e.g., monoclonal antibodies, toxin-conjugated monoclonal antibodies) and / or chemotherapeutic agents.

[0455] M. Products and Kits In another aspect of this disclosure, a product is provided comprising an isolated polypeptide, an anti-nectin-4 antibody or antibody fragment, or an immunoconjugate and other materials useful for the treatment, prevention and / or diagnosis of the above-mentioned disorders. The product includes a container and a label or accompanying information on or associated with the container. Preferred containers include, for example, bottles, vials, syringes, intravenous infusion bags, etc. Containers may be formed from a variety of materials such as glass or plastic. Containers may hold the composition, either by itself or in combination with another composition effective for treating, preventing and / or diagnosing the condition, and may have a sterile access port (for example, the container may be an intravenous infusion bag or a vial with a stopper that can be punctured by a subcutaneous needle). At least one active agent in the composition is an antibody or antibody fragment of this disclosure. The label or accompanying information indicates that the composition is used to treat a selected pathological condition. Furthermore, the product may comprise (a) a first container containing the composition (the composition comprising an antibody or antibody fragment); and (b) a second container containing the composition (the composition comprising a further cytotoxic agent or other therapeutic agent). The product of this embodiment of the present disclosure may further comprise a package insert indicating that the composition may be used to treat a particular medical condition. Alternatively, or in addition, the product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0456] It is understood that any of the above products may include the immunoconjugate of this disclosure in place of, or in addition to, an anti-nectin-4 antibody or antibody fragment.

[0457] Finally, the Disclosure also provides a kit comprising at least one antibody or antibody fragment of the Disclosure. Kits containing the polypeptide, antibody or antibody fragment, or antibody-drug conjugate of the Disclosure are used in the detection of nectin-4 expression (increase or decrease) or in therapeutic or diagnostic assays. The Kits of the Disclosure may comprise antibodies coupled to a solid support, e.g., a tissue culture plate or beads (e.g., Sepharose beads). Kits may be provided containing antibodies for in vitro detection and quantification of nectin-4, e.g., in ELISA or Western blotting. Such antibodies useful for detection may be provided with labeling, such as fluorescent or radiolabeling.

[0458] The kit further includes instructions for its use. In some embodiments, the instructions include instructions required by the U.S. Food and Drug Administration (USFOOD and Drug Administration) for in vitro diagnostic kits. In some embodiments, the kit further includes instructions for diagnosing the presence or absence of cerebrospinal fluid in a sample based on the presence or absence of nectin-4 in the sample. In some embodiments, the kit includes one or more antibodies or antibody fragments. In other embodiments, the kit further includes one or more enzymes, enzyme inhibitors or enzyme activators. In yet another embodiment, the kit further includes one or more chromatographic compounds. In yet another embodiment, the kit further includes one or more compounds used to prepare a sample for a spectroscopic assay. In a further embodiment, the kit further includes comparative reference material for interpreting the presence or absence of nectin-4 according to the intensity, color spectrum, or other physical attributes of an indicator.

[0459] The following examples illustrate, but are not limited to, the anti-nectin-4 antibodies of the present disclosure. Other suitable modifications and adaptations of various conditions and parameters commonly encountered in the art and apparent to those skilled in the art are within the scope of the present disclosure. [Examples]

[0460] [Table 6] JPEG2026520270000089.jpg65170

[0461] Example 1 - Synthesis of SPC07

[0462] Synthesis of (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (SYB-BA2-066) [ka]

[0463] To a solution of (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (100 mg, 0.252 mmol, 1.0 equivalent) in dry MeCN (2.5 mL, 0.1 M), 4-hydroxy-3-nitrobenzaldehyde (63 mg, 0.38 mmol, 1.5 equivalents) and silver oxide (87 mg, 0.38 mmol, 1.5 equivalents) were added at room temperature under a nitrogen atmosphere. The resulting reaction mixture was stirred in the dark at room temperature for 17 hours. TLC showed complete conversion of the donor (CyH /  1:1, KMnO4). The solution was filtered through Celite to remove the solid, and the filtrate was concentrated under reduced pressure. The residue was diluted with  (40 ml) and washed with saturated NaHCO3 (6 × 20 ml), water, and brine. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to obtain (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (109 mg, 0.225 mmol, 89%) as a white solid.

[0464] 1 H NMR (400MHz, CDCl3)δ 9.96(s,1H), 8.29(d,J=2.0Hz, 1H), 8.07(dd,J=8.6, 2.0Hz, 1H), 7.50(d,J=8.6Hz, 1H), 5.45-5.37(m ,2H), 5.35-5.24(m,2H), 4.33(d,J=8.4Hz, 1H), 3.69(s,3H), 2.11(s,3H), 2.06(s,3H), 2.05(s,3H).

[0465] LC-MS(ESI): m / z = 501[M+H2O]

[0466] Synthesis of (2S,3R,4S,5S,6S)-2-(4-(1-hydroxybuta-3-in-1-yl)-2-nitrophenoxy)-6(methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triyltriacetate (SYB-BA2-060) [ka]

[0467] To a suspension of newly activated zinc powder (HCl, H2O, MeOH, Et2O, high vacuum dried, 54 mg, 0.83 mmol, 8.0 equivalents) in dry THF (2 mL, 0.4 M), allyl bromide (123 mg, 0.825 mmol, 8.0 equivalents) was added at 0°C under a nitrogen atmosphere. The reaction mixture was warmed to room temperature and stirred for 1 hour until all zinc was dissolved. The reaction solution was cooled to -15°C, and a solution of (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (50 mg, 0.103 mmol, 1.0 equivalent) in THF (1 mL) was slowly added. The reaction mixture was stirred at -15°C for 30 minutes and quenched by adding saturated NH4Cl aqueous solution (20 mL). The aqueous solution was extracted with toluene (3 x 20 mL). The combined organic layer was washed with H2O (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (12 g, 20 CV, CyH:toluene:95:5~20:80) to obtain (2S,3R,4S,5S,6S)-2-(4-(1-hydroxybuta-3-in-1-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triyltriacetate (14 mg, 1.16 mmol, 53%).

[0468] 1 1H NMR (400 MHz, CDCl3) δ 7.87(ddd,J=5.5, 2.2, 0.6Hz, 1H), 7.58(dddd,J=8.6, 6.3, 2.2, 0.6Hz, 1H ), 7.37(d,J=8.6Hz, 1H), 5.41-5.25(m,3H), 5.24-5.17(m,1H), 4.91(t,J= 6.2Hz, 1H), 4.24-4.18(m,1H), 3.74(s,3H), 2.70-2.57(m,2H), 2.13(s,3H) ), 2.11(t,J=2.6Hz, 1H), 2.07(s,3H), 2.05(s,3H), 2.04(d,J=3.8Hz, 1H).

[0469] LC-MS(ESI): m / z = 541[M+H2O]

[0470] Synthesis of (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(1-(((4-nitrophenoxy)carbonyl)oxy)buta-3-in-1-yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (SYB-BA2-23)

[0471] [ka]

[0472] At 0°C, a solution of (2S,3R,4S,5S,6S)-2-(4-(1-hydroxybuta-3-in-1-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (68 mg, 0.13 mmol, 1 equivalent) and 4-nitrophenyl chloroformate (65 mg, 0.32 mmol, 2.5 equivalents) was added to dichloromethane (2 mL), and pyridine (100 μL, 1.3 mmol, 10 equivalents) was stirred at room temperature for 3 hours. After filtration on a Celite pad, the filtrate was washed with saturated NaHCO3 aqueous solution and extracted three times with Âi. The combined organic layers were dried, filtered, and concentrated under reduced pressure. The residue was purified by FCC (CyH / Â1 95:5~20:80, 20 g) in silica to obtain (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(1-(((4-nitrophenoxy)carbonyl)oxy)buta-3-in-1-yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (60 mg, 87 μmol, 67%).

[0473] R f =0.30 product(KMnO4)

[0474] 1H NMR (400MHz, CDCl3)δ 8.35-8.27(m,2H), 7.96(d,J=2.2Hz, 1H), 7.67(dd,J=8.5, 2.3Hz, 1H), 7.47-7.37(m,3H), 5.83(t,J=6.5Hz, 1H), 5.47-5.22( m,4H), 4.26(d,J=8.8Hz, 1H), 3.76(s,3H), 3.06-2.85(m,2H), 2.15(s,3H), 2.12(t,J=2.6Hz, 1H), 2.10(s,3H), 2.06(s,3H).

[0475] Synthesis of SPC07: A purification optimization procedure in one of three steps. [ka]

[0476] (2S,3R,4S,5S,6S)-2-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (SYB-BA2-026) [ka]

[0477] To a solution of (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(1-(((4-nitrophenoxy)carbonyl)oxy)buta-3-in-1-yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (60 mg, 0.87 mmol, 1 equivalent), monomethyl auristatin E (63 mg, 0.87 mmol, 1 equivalent), DIPEA (23 μL, 0.13 mmol, 1.5 equivalents), and pyridine (0.43 mL, 0.87 mmol, 1 equivalent, 0.2 M in DMF), HOBT (14 mg, 0.43 mmol, 1.05 equivalents) was added.

[0478] The reaction mixture was stirred overnight. LC-MS showed product formation and some remaining SM-MMAE.

[0479] No purification was attempted. The crude reaction product was directly hydrolyzed.

[0480] (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (SYB-BA1-34) [ka]

[0481] The reaction mixture (SYB-BA2-26) was diluted with MeOH (20 mL) and cooled to 0°C. LiOH (8.8 equivalents, 1 M in H2O, 0.76 mL, 0.76 mmol) was added to the resulting solution, and after the reaction, TLC (DCM / MeOH 9 / 1) was performed. The resulting solution was quenched with HCOOH (20 equivalents, 1 M in H2O, 1.1 mL, 1.1 mmol) and concentrated. The reaction product was diluted in water and extracted three times by DCM. LCMS of both the organic and aqueous layers showed that the product remained in the organic layer. The solvent was removed under reduced pressure. The residue in DMF was diluted with water and freeze-dried overnight. The crude mixture was used for the click reaction. If the impurity level is less than 15%, purification at this stage is not necessary, as these impurities will be removed during the reverse-phase purification of the final click reaction.

[0482] General procedure for click response

[0483] To a solution of an alkyne derivative (1.0 equivalent) in DCM (0.05 M), the corresponding azide (1.3 equivalents) was added, followed by the addition of tetrakis(acetonitrile) copper(i) hexafluorophosphate (1.5 equivalents). The solution was stirred at room temperature, and the reaction product was monitored by LC-MS. After 4 hours (LC-MS confirmed that SM had been consumed), the reaction mixture was concentrated under reduced pressure to remove the DCM. Next, the crude product was diluted with DMF (0.05 M), and a solution of ethylenediaminetetraacetate disodium dihydrate (EDTA, 2Na, 2H2O) (4 equivalents) in water (0.15 M) was added at 0°C, and the mixture was stirred at room temperature for 15 minutes. The reaction mixture was injected directly into a reverse-phase column (25 gm), and the crude product was purified using ACN in 0.1% FA in water. The pure fraction (confirmed by LC-MS) was lyophilized to obtain the desired compound as a white solid.

[0484] (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-15-(1-(5-(2-bromoacetamide)pentyl)-1H-1,2,3-triazol-4-yl)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-ol Xopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazapentadecane-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (SYB-BA2-35-SPC07 or SYB-BA1-36-SPC07) [ka]

[0485] After the general procedure for the click reaction, (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl The title product was obtained as a white solid (31 mg, 58%) by adding tyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (44 mg, 39 μmol, 1.0 equivalent) and N-(5-azidopentyl)-2-bromoacetamide (12 mg, 50 μmol, 1.3 equivalents).

[0486] (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11 in anhydrous DCM (6 mL) To a solution of triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (crude product from the reaction) (SYB-BA2-28 assumed 87 μmol, 1.0 equivalent) and N-(5-azidopentyl)-2-bromoacetamide (28.2 mg, 113 μmol, 1.3 equivalents), tetrakis(acetonitrile)copper(I) hexafluorophosphate (48.7 mg, 131 μmol, 1.5 equivalents) was added. The reaction mixture was stirred overnight. DCM was concentrated under reduced pressure, and the residue was dissolved in DMF (1.5 mL). The mixture was treated with a solution of sodium 2,2'-(ethane-1,2-diylbis((carboxymethyl)azandiyl)) diacetate dihydrate (128 mg, 0.350 mmol, 4 equivalents) in H2O (1.5 mL) at 0°C. The reaction mixture was stirred for 15 minutes, and the reaction mixture was directly injected into a reversed-phase column (40 gm, 15 μm). The crude product was purified using ACN (5 / 95-95 / 5, 20 CV) in 0.1% FA containing water. The pure fraction (confirmed by LC-MS) was lyophilized to obtain the desired compound as a white solid (42 mg, 31 μmmol, 35% in 3 steps).

[0487] 1H NMR (400MHz, DMSO)δ 8.24(m,1H), 7.94-7.74(m,2H), 7.66-7.52(m,1H), 7.32-7.25(m,5H), 7.18(m,1H), 5.96-5.75(m ,1H), 5.56-5.15(m,5H), 4.84-4.08(m,6H), 4.08-3.91(m,3H), 3.82(m,2H), 3.59(s,1H), 3.27-3. 11(m,8H), 3.02(m,4H), 2.96-2.65(m,3H), 2.28(s,1H), 2.20-1.92(m,3H), 1.80-1.72(m,5H), 1.6 1-1.12(m,10H), 1.07-0.96(m,8H), 0.92-0.73(m,18H), 0.72-0.57(m,4H), 0.46(d,J=6.5Hz, 1H).

[0488] The LCMS purity was 99%.

[0489] Note: The sequences of MMAE coupling, Ac deprotection, and click reaction can be performed without reverse-phase purification after MMAE coupling or acetyl deprotection. A final product of 40 mg with a purity of >95% (44% in 3 steps) was obtained from 60 mg of activated benzyl alcohol.

[0490] LC-MS(ESI): m / z = 1377[M+H] +

[0491] (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-15-(1-(5-(2-bromoacetamido)pentyl)-1H-1,2,3-triazol-4-yl)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy- 2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazapentadecane-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (SYB-BA2-63-SPC07). (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-15-(1-(5-(2-bromoacetamide)pentyl)-1H-1,2,3-triazol-4-yl)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyro Combine lysine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazapentadecane-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid:(SYB-BA2-06, 31, 35, 37, 44, 53, 55) in ACN / H2O (1 / 1:15 / 15 mL). Then, freeze-dry for 2 days, (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-15-(1-(5-(2-bromoacetamide)pentyl)-1H-1,2,3-triazol-4-yl)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxo Propyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazapentadecane-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (507 mg, 368 μmol) was obtained.

[0492] Example 2 - Linker for SPC07: [ka]

[0493] General procedure for azide substitution A: To a solution of Br compound (1 equivalent) in DMF (0.2 M), sodium azide (1.5 equivalents) was added, and the reaction mixture was stirred at 70°C for 16 hours. It was then washed with saturated NaHCO3 aqueous solution and ethyl acetate. The combined organic layer was dried, filtered, and concentrated. The crude product was used without further purification.

[0494] General procedure B for phthalimide deprotection: A solution of phthalimide compound (1 equivalent) in THF (0.15 M) was mixed with hydrazine monohydrate (40 equivalents). The reaction mixture was stirred at room temperature for 4 hours. THF was removed under reduced pressure, and the residue was washed with saturated aqueous NaHCO3 and ethyl acetate. The combined organic layer was dried, filtered, and concentrated. The crude product was used without further purification.

[0495] Modified procedure B for phthalimide deprotection: To a solution of phthalimide compound (1 equivalent) in THF (0.2 M), hydrazine monohydrate (3 equivalents) was added. The reaction mixture was stirred overnight at room temperature. The phthalic acid hydrazide was removed by filtration and washed with THF. The solvent was removed under reduced pressure to obtain the desired amine. The crude product was used without further purification.

[0496] General procedure for bromoacetyl bromide substitution: To a solution of amine (1 equivalent) in D:DCM (0.20 M), K2CO3 (1.5 equivalents) in water (0.5 mL) was added. Then, 2-bromoacetyl bromide (1.1 equivalents) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 15 hours. Then, it was washed with water and ethyl acetate. The combined organic layer was dried, filtered, concentrated, and purified by column chromatography.

[0497] Following general procedure A for azide substitution of 2-(5-azidopentyl)isoindoline-1,3-dione (SYB-BA2-19), a solution of 2-(5-bromopentyl)isoindoline-1,3-dione (3.00 g, 10.1 mmol, 1 equivalent) and sodium azide (988 mg, 15.2 mmol, 1.5 equivalents) in dimethylformamide (20 mL, 0.5 M) was stirred overnight at 70°C. The reaction mixture was cooled to room temperature, diluted with brine (100 mL), and extracted three times with ethyl acetate (3 × 50 mL). The combined organic layer was washed three times with water, dried over Na₂SO₄, filtered, and concentrated to obtain 2-(5-azidopentyl)isoindoline-1,3-dione (2.58 g, 10.0 mmol, 98% yield).

[0498] 1H NMR (400MHz, CDCl3)δ 7.91-7.78(m,2H), 7.76-7.65(m,2H), 3.78-3.62(m,2H), 3.27(t,J=6.9Hz, 2H), 1.78-1.60(m,4H), 1.50-1.37(m,2H).

[0499] Following the general procedure B for phthalimide deprotection of 5-azidopentane-1-amine (SYB-BA2-38), 2-(5-azidopentyl)isoindoline-1,3-dione (200 mg, 0.770 mmol, 1 equivalent) in THF (3 mL) was mixed with hydrazine hydrate (0.110 mL, 2.32 mmol, 3 equivalents), and the reaction mixture was stirred overnight at room temperature. The precipitate was removed by filtration, washed with THF (1 × 10 mL), and the filtrate was concentrated under reduced pressure to obtain 5-azidopentane-1-amine (85 mg, 0.66 mmol, 86% yield).

[0500] 1H NMR (400MHz, CDCl3) δ 3.27(t,J=6.9Hz, 2H), 2.70(t,J=6.7Hz, 2H), 1.62(p, J=7.0Hz, 2H), 1.53-1.35(m,4H).

[0501] Following the general procedure D for the substitution of N-(5-azidopentyl)-2-bromoacetamide (SYB-BA2-18) with bromoacetyl bromide, potassium carbonate (1.10 g, 7.8 mmol, 2 equivalents) and 2-bromoacetyl bromide (0.38 mL, 4.3 mmol, 1.1 equivalents) were added dropwise at 0°C to a solution of 5-azidopentan-1-amine (500 mg, 3.9 mmol, 1 equivalent) in DCM (20 mL). The reaction mixture was stirred overnight. It was washed with water and ethyl acetate. The combined organic layer was dried over sodium sulfate, filtered, and concentrated.

[0502] Purification of the residue with MPLC(SiO2)(20g, 20CV)(CyH:siRNA:100:0~40:60) yielded N-(5-azidopentyl)-2-bromoacetamide (639mg, 2.57 mmol, 66%).

[0503] Analysis data

[0504] TLC[SiO2](CyH:τ:50:50)

[0505] R f =0.40(UV, KMnO4)

[0506] 1 H NMR (400MHz, CDCl3) δ 6.51(s,1H), 3.88(s,2H), 3.34-3.23(m,4H), 1.68-1.54(m,4H), 1.47-1.37(m,2H).

[0507] Example 3 - Synthesis of SPC17

[0508] (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate SYB-BA2-066 [ka]

[0509] To a solution of (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (100 mg, 0.252 mmol, 1.0 equivalent) in dry MeCN (2.5 mL, 0.1 M), 4-hydroxy-3-nitrobenzaldehyde (63 mg, 0.378 mmol, 1.5 equivalents) and silver oxide (87 mg, 0.38 mmol, 1.5 equivalents) were added at room temperature under a nitrogen atmosphere. The resulting reaction mixture was stirred in the dark at room temperature for 17 hours. TLC showed complete conversion of the donor (cHex / siRNA 1:1, KMnO4). The solution was filtered through Celite to remove the solid, and the filtrate was concentrated under reduced pressure. The residue was diluted with siRNA (40 ml) and washed with saturated NaHCO3 (6 × 20 ml), water, and brine. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to obtain (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (109 mg, 0.225 mmol, 89%) as a white solid.

[0510] 1 H NMR (400MHz, CDCl3)δ 9.96(s,1H), 8.29(d,J=2.0Hz, 1H), 8.07(dd,J=8.6, 2.0Hz, 1H), 7.50(d,J=8.6Hz, 1H), 5.45-5.37(m ,2H), 5.35-5.24(m,2H), 4.33(d,J=8.4Hz, 1H), 3.69(s,3H), 2.11(s,3H), 2.06(s,3H), 2.05(s,3H).

[0511] (2S,3R,4S,5S,6S)-2-(4-(1-hydroxybuta-3-in-1-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triyltriacetate SYB-BA1-060 [ka]

[0512] To a suspension of newly activated zinc powder (HCl, H2O, MeOH, Et2O, high vacuum dried, 54 mg, 0.83 mmol, 8.0 equivalents) in dry THF (2 mL, 0.4 M), allyl bromide (123 mg, 0.825 mmol, 8.0 equivalents) was added at 0°C under a nitrogen atmosphere. The reaction mixture was warmed to room temperature and stirred for 1 hour until all zinc was dissolved. The reaction solution was cooled to -15°C, and a solution of (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (50 mg, 0.103 mmol, 1.0 equivalent) in THF (1 mL) was slowly added. The reaction mixture was stirred at -15°C for 30 minutes and quenched by adding saturated NH4Cl aqueous solution (20 mL). The aqueous solution was extracted with ELISA (3 x 20 mL). The combined organic layer was washed with H2O (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (12 g, 20 CV, CyH:ELISA:95:0~20:80) to obtain (2S,3R,4S,5S,6S)-2-(4-(1-hydroxybuta-3-in-1-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triyltriacetate (14 mg, 1.16 mmol, 53%).

[0513] 1 1H NMR (400 MHz, CDCl3) δ 7.87(ddd,J=5.5, 2.2, 0.6Hz, 1H), 7.58(dddd,J=8.6, 6.3, 2.2, 0.6Hz, 1H ), 7.37(d,J=8.6Hz, 1H), 5.41-5.25(m,3H), 5.24-5.17(m,1H), 4.91(t,J= 6.2Hz, 1H), 4.24-4.18(m,1H), 3.74(s,3H), 2.70-2.57(m,2H), 2.13(s,3H) ), 2.11(t,J=2.6Hz, 1H), 2.07(s,3H), 2.05(s,3H), 2.04(d,J=3.8Hz, 1H).

[0514] (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(1-(((4-nitrophenoxy)carbonyl)oxy)buta-3-in-1-yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate SYB-BA1-94 or SYB-BA2-23 [ka]

[0515] At 0°C, (2S,3R,4S,5S,6S)-2-(4-(1-hydroxybuta-3-in-1-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triyltriacetate SYB-BA1-060 (375 mg, 0.71 mmol, 1.0 equivalent) and 4-nitrophenyl chloroformate (361 mg, 1.79 mmol, 2.5 equivalents) were mixed in dichloromethane (8 mL, 0.1 M). Pyridine (580 μL, 7.16 mmol, 10 equivalents) was added to the mixture, and the reaction was stirred at room temperature for 4 hours. The reaction was diluted with ethyl acetate (100 mL) and washed with saturated aqueous NaHCO3 solution. The aqueous layer was extracted three times with ethyl acetate. The combined organic layers were dried, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CyH / Â 5-60%) to obtain (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(1-(((4-nitrophenoxy)carbonyl)oxy)buta-3-in-1-yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (410 mg, 0.590 mmol, 83%).

[0516] R f =0.30 product(KMnO4)

[0517] 1H NMR (400MHz, CDCl3)δ 8.35-8.27(m,2H), 7.96(d,J=2.2Hz, 1H), 7.67(dd,J=8.5, 2.3Hz, 1H), 7.47-7.37(m,3H), 5.83(t,J=6.5Hz, 1H), 5.47-5.22( m,4H), 4.26(d,J=8.8Hz, 1H), 3.76(s,3H), 3.06-2.85(m,2H), 2.15(s,3H), 2.12(t,J=2.6Hz, 1H), 2.10(s,3H), 2.06(s,3H).

[0518] (2S,3R,4S,5S,6S)-2-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoeth (L)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate SYB-BA1-029 or SYB-BA2-026 [ka]

[0519] To a solution of (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(1-(((4-nitrophenoxy)carbonyl)oxy)buta-3-in-1-yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (110 mg, 0.16 mmol, 1 equivalent), monomethyl auristatin E (115 mg, 0.160 mmol, 1.0 equivalent), DIPEA (61 μL, 0.35 mmol, 2.2 equivalents), and pyridine (0.13 μL, 0.16 mmol, 1.0 equivalent), HOBT (26 mg, 0.17 mmol, 1.05 equivalents) was added.

[0520] The reaction mixture was injected directly into a reverse-phase column and purified by reverse-phase column chromatography using 5-70% methanol in 0.1% FA containing water. (2S,3R,4S,5S,6S)-2-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl (Lu-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (216 mg, 0.170 mmol, 60%) was obtained as a white solid.

[0521] 1 H NMR (400MHz, MeOD)δ 7.98-7.57(m,2H), 7.56-7.13(m,6H), 5.89-5.73(m,1H), 5.58-5.36(m,2H), 5.23(q, J=8.6Hz, 2H), 4. 71(d,J=8.7Hz, 1H), 4.65-4.46(m,3H), 4.33-4.01(m,3H), 3.80-3.63(m,4H), 3.46-3.33(m,9H), 3.29 -3.23(m,3H), 3.16-2.75(m,6H), 2.55-2.30(m,3H), 2.30-1.99(m,11H), 1.98-1.75(m,3 H), 1.59(s,1H), 1.41(s,1H), 1.23-1.08(m,6H), 0.99-0.83(m,17H), 0.69-0.48(m,2H).

[0522] LC-MS(ESI): m / z = 1268[M+H] +

[0523] (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid SYB-BA1-34 [ka]

[0524] (2S,3R,4S,5S,6S)-2-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9 in MeOH (6 mL, 0.03 M) cooled to 0°C To a solution of 12-trioxo-2,13-dioxa-5,8,11-triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate SYB-BA1-029 or SYB-BA2-026 (200 mg, 158 μmol, 1.0 equivalent), a cold solution of LiOH·H2O in H2O (6 mL) at 0°C (58 mg, 1.4 mmol, 8.8 equivalents) was added dropwise. The mixture was stirred at 0°C for 15 minutes (followed by TLC DCM:MeOH 9:1), and the solution was neutralized with Amberlite Weakly acidic cation exchange hydrogen for 15 minutes (at the pH as specified in the document) and filtered. The aqueous layer was freeze-dried overnight to obtain (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, which contained 8% impurities. HPLC (Method ACN-High Mass-2000: RT: 3.79 min, m / z=1109[M+H] +The impurity product was purified by reverse-phase column chromatography (40 g, 15 μm) using MeOH in 0.1% FA containing water to obtain (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1 (-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (89 mg, 79 μmmol, 50%, containing less than 3% impurities) and (56 mg, 50 μmmol, 31%, containing less than 10% impurities) were obtained.

[0525] If the impurity level is less than 15%, purification at this stage is not necessary, as the impurities will be removed during the reverse-phase purification of the final click reaction.

[0526] 1 H NMR (400MHz, MeOD) δ 7.97-7.85(m,1H), 7.69(m 1H), 7.50-7.17(m,6H), 5.80(d,J=9.6Hz, 1H), 5.28-5.07(m,1H), 4.75-4.42(m,2H), 4.39-3.43(m,10H), 3.35(m, 6H), 3.30-3.22(m,5H), 3.14-2.69(m,6H), 2.62-2.07(m,5H), 2.05-1.51(m,5H), 1.41(s,2H), 1.31-0.26(m,26H).

[0527] LC-MS(ESI): m / z = 1128[M+H] + LC-MS(ESI): m / z = 1128[M+H] +

[0528] General procedure for click response To a solution of an alkyne derivative (1.0 equivalent) in DCM (0.05 M), the corresponding azide (1.3 equivalents) was added, followed by the addition of tetrakis(acetonitrile) copper(i) hexafluorophosphate (1.5 equivalents). The solution was stirred at room temperature, and the reaction product was monitored by LC-MS. The desired mass was observed. After 4 hours (LC-MS confirmed that SM had been consumed), the reaction mixture was concentrated under reduced pressure to remove the DCM. Next, the crude product was diluted with DMF, and a solution of ethylenediaminetetraacetate disodium dihydrate (EDTA.2Na.2H2O) (4 equivalents) in water was added at 0°C, and the mixture was stirred at room temperature for 15 minutes. The reaction mixture was then injected directly into a reverse-phase column (25 gm), and the crude product was purified using ACN in 0.1% FA in water. The pure fraction (confirmed by LC-MS) was lyophilized to obtain the desired compound as a white solid.

[0529] (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-15-(1-(1-bromo-2-oxo-6,9,12,15,18,21,24,27-octaoxa-3-azanonacosan-29-yl)-1H-1,2,3-triazole-4-yl)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane (N-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazapentadecane-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid SYB-BA1-111 or SPC17

[0530] [ka]

[0531] After the general procedure for the click reaction, (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,1 The title product was obtained as a white solid (9.7 mg, 5.8 μmol, 16%) by adding 3-dioxa-5,8,11-triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (40 mg, 35 μmol, 1 equivalent) and N-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)-2-bromoacetamide (25 mg, 46 μmol, 1.3 equivalents).

[0532] 1 H NMR (400MHz, DMSO)δ 8.36-8.32(m,1H), 7.92-7.84(m,1H), 7.75-7.69(m,1H), 7.40-7.20(m,6H), 7.21-7.15(m,1H), 5.4 3-5.33(m,3H), 5.26-5.18(m,2H), 4.55-4.28(m,6H), 4.10-3.89(m,2H), 3.86(s,2H), 3.76(s,3H), 3.71-3.31(m,36H), 3.27-3.21(m,10H), 3.06-2.79(m,1H), 2.78-2.72(m,1H), 2.44-1.90(m,2H), 1 .72-1.62(m,1H), 1.35-1.15(m,3H), 1.10-0.94(m,10H), 0.93-0.55(m,24H), 0.47(d,J=6.6Hz, 1H).

[0533] The LCMS purity was 96%.

[0534] LC-MS(ESI): m / z = 1687[M+H] +

[0535] Example 4 - Linker for SPC17:

[0536] N-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)-2-bromoacetamide SSA-BA1-164

[0537] [ka]

[0538] To a solution of 26-azido-3,6,9,12,15,18,21,24-octaoxahexacosan-1-amine (100 mg, 0.228 mmol, 1.0 equivalent) in dry DCM (6 mL), K2CO3 (47 mg, 0.34 mmol, 1.5 equivalents) was added, followed by 2-bromoacetyl bromide (83 mg, 0.41 mmol, 1.8 equivalents). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, it was diluted with water and extracted twice with siRNA. The combined organic layer was washed with water, filtered, and concentrated to obtain the desired compound as a pale yellow liquid (110 mg, 86%). The crude product was used without further purification.

[0539] 1 H NMR (400MHz, CDCl3)δ 3.93(s,2H), 3.89(s,13H), 3.71-3.62(m,24H).

[0540] Example 5 - Synthesis of SPC19

[0541] The synthesis steps for SPC19, including the general procedure for the click reaction, are carried out for SPC07 and SPC17 as described above in Examples 1 and 3.

[0542] (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-15-(1-(2-(2-bromoacetamidoethyl)-1H-1,2,3-triazol-4-yl)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy (-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazapentadecane-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid SYB-BA1-132SPC19 [ka]

[0543] After the general procedure for the click reaction, (2S,3S,4S,5R,6S)-6-(4-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl- The title product was obtained as a white solid (22 mg, 16 μmol, 55%) from the reaction of 6,9,12-trioxo-2,13-dioxa-5,8,11-triazaheptadeca-16-in-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (33 mg, 29 μmol, 1.0 equivalent) and N-(2-azidoethyl)-2-bromoacetamide (8 mg, 41 μmol, 1.4 equivalents).

[0544] 1H NMR (400MHz, DMSO)δ 8.48-8.42(m,1H), 8.18-8.12(m,1H), 7.91-7.87(m,1H), 7.83-7.69(m,1H), 7.42-7.23(m, 6H), 7.23-7.13(m,1H), 6.12-5.68(m,1H), 5.50-5.31(m,2H), 5.29-5.15(m,2H), 4.55-4.2 7(m,5H), 4.24-3.68(m,7H), 3.54(m,7H), 3.30-3.11(m,12H), 3.10-2.63(m,6H), 2.46-1.9 0(m,4H), 1.87-1.41(m,4H), 1.11-0.95(m,8H), 0.94-0.56(m,19H), 0.48(d,J=6.5Hz, 1H).

[0545] The LCMS purity was 99%.

[0546] LC-MS(ESI): m / z = 1335[M+H] +

[0547] Example 6 - Linker for SPC19:

[0548] 2-(2-azidoethyl)isoindoline-1,3-dione SSA-BA1-127 [ka]

[0549] To a stirred solution of 2-(2-bromoethyl)isoindoline-1,3-dione (2.5 g, 9.8 mmol, 1.0 equivalent) in DMF (49 mL, 0.2 M), sodium azide (0.96 g, 1.5 equivalents, 15 mmol) was added, and the resulting reaction mixture was stirred overnight at 70°C. After the reaction was complete, a 10% aqueous LiCl solution was added, and the mixture was extracted twice with ethyl acetate. The combined organic layer was washed with water, filtered, and concentrated to obtain the desired compound as an off-white solid (2.01 g, 94%). The crude product was used without further purification.

[0550] 1H NMR(500MHz, CDCl3)δ 7.90-7.83(m,2H), 7.76-7.71(m,2H), 3.90(t,J=6.1Hz, 2H), 3.59(t,J=6.1Hz, 2H).

[0551] 2-Azidoethane-1-amine SSA-BA1-131 [ka]

[0552] To a solution of 2-(2-azidoethyl)isoindoline-1,3-dione (2.01 g, 9.30 mmol, 1 equivalent) in THF (46.5 mL, 0.2 mol), hydrazine hydrate (18.6 g, 18.1 mL, 372 mmol, 40 equivalents) was added, and the resulting reaction mixture was stirred at room temperature for 4 hours. After the reaction was complete, the THF was removed (while maintaining a pressure of over 250 mbar), the residue was washed with 10% LiCl, and extracted with diethyl ether. The combined organic layer was dried, filtered, and concentrated (while maintaining a pressure of over 250 mbar) to obtain the desired compound as a colorless oil (800 mg, crude product, containing THF and diethyl ether as solvent impurities).

[0553] Due to the volatility of the compound, it was difficult to remove the residual solvent, which was then used in the next step.

[0554] N-(2-azidoethyl)-2-bromoacetamide SSA-BA1-134 [ka]

[0555] A solution of 2-bromoacetyl bromide (0.15 mL, 1.5 mmol, 2.2 equivalents) in dry THF (3 mL) was added dropwise to a mixture of 2-azidoethane-1-amine (65 mg, 0.75 mmol, 1.0 equivalent) and TEA (0.24 mL, 1.7 mmol, 2.5 equivalents) in dry THF (4.5 mL, 0.2 M) at 0°C. The reaction mixture was stirred at room temperature for 1.3 hours. Excess acid bromide was quenched by adding methanol (1 mL). The formed triethylammonium bromide salt was removed by filtration, and the solvent was removed under reduced pressure. The crude product was dissolved in dichloromethane and washed three times with saturated ammonium chloride solution and twice with distilled water. The combined organic layers were dried, filtered, and concentrated to obtain a light brown crude oil product. The residue was purified using MPLC (SiO2, CyH / Â 0-45%) to obtain the desired compound as a pale yellow oil (65 mg, 42%).

[0556] 1 H NMR (400MHz, CDCl3)δ 6.76(s,1H), 3.90(s,2H), 3.48(d,J=4.1Hz, 4H).

[0557] Example 7 - Large-scale conjugation of antibodies

[0558] Dilute TCEP to 5 mM: Dilute 5 µl of 0.5 M TCEP in 495 µl of SPC EDTA buffer (50 mM Tris pH 8.5, 150 mM NaCl, 20 mM EDTA)

[0559] Conjugation is performed in a 1.7 ml tube. 1. Aliquot 97 µl of SPC EDTA buffer. 2.5.9 mg / ml antibody in aliquot form, 847 µl. Mix slowly in 3.22 µl of TCEP. Place the tube in the shaker at 4,300 rpm for 1 hour at 25°C. 5. Leave it on the ice for 2 minutes. Mix slowly in the linker payload (SPC004, SPC007, or SPC008) of 6.33.3ul. Place the tube in the shaker at 7,300 rpm for 2 hours and 25°C. 8. Place it on the ice. [Table 7]

[0560] The arrangement of the heavy chain and light chain variable regions is shown in Figures 2A and 2B.

[0561] Structure of BA-143-00-MC (wild-type or non-CAB parent conjugated to MC): [ka]

[0562] Abbreviations for Examples 8-18 ADC Antibody-Drug Conjugate °C (Celsius) Af488 Alexa Fluor 488 Celsius BSA (Bovine Serum Albumin) DAR drug-antibody ratio DMEM Dulbecco's Modified Eagle Medium ECD extracellular domain ELISA enzyme-linked immunosorbent assay FACS Fluorescence-Activated Cell Sort FBS Fetal Bovine Serum g grams HCl (hydrochloric acid) hr time HRP (Horseradiol Peroxidase) huNectin 4 Human Nectin 4 Extracellular Domain cyno-nectin 4, cynomolgus nectin 4 extracellular domain Rat Nectin 4, Rat Nectin 4 Extracellular Domain IC50 50% effective inhibitory concentration Logarithm MEM (Minimum Essential Medium) Median MFI fluorescence intensity min mL (milliliter) MMAE Monomethyl Auristatin E NA Not applicable NaOH (Sodium Hydroxide) NEAA Non-essential amino acids ng nanogram nm (nanometer) OD optical density PBS (phosphate-buffered saline) PE Phycoerythrin PK (Pharmacokinetics) RCF (Relative Centrifugal Force) RLU (Relative Luminescence Unit) rpm (revolutions per minute) RT room temperature SD standard deviation TMB 3,3',5,5'-tetramethylbenzidine μg (microgram) μL (microliter) T 1 / 2 Time required for concentration to decrease by 50% C max Peak serum concentration after administration

[0563] Example 8 - PK analysis of ADCs using affinity ELISA

[0564] 1.1 Test substance Mouse serum samples were collected in an in vivo PK test at 10 minutes, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, and 96 hours after the following antibody treatments (3 mg / kg). BA-143-00-MC (Wild-type or non-CAB parent conjugated to MC) BA-143-00-SPC04 (Wild-type or non-CAB parent conjugated to SPC04) BA-143-00-SPC07 (Wild-type or non-CAB parent conjugated to SPC07) BA-143-00-SPC08 (Wild-type or non-CAB parent conjugated to SPC08) BA-143-01-MC (CAB conjugated to MC) BA-143-01-SPC04 (CAB conjugated to SPC04) BA-143-01-SPC07 (CAB conjugated to SPC07) BA-143-01-SPC08 (CAB conjugated to SPC08)

[0565] 1.2 Formulation The test substance was diluted in assay buffer (PBS containing 1% BSA, pH 6.0) at different dilution ratios depending on the time of sample collection. Antibodies from the PK test were used as standard controls for the standard curve, first diluted to 3 ug / mL with assay buffer containing 1:200 normal mouse serum, and then triple-diluted for 10 concentration points.

[0566] 1.3 pH affinity ELISA assay 1) Coat the ELISA plate with 100 μL of 2 μg / mL recombinant human nectin-4 antigen in a carbonate-bicarbonate coated buffer. 2) Cover the plate with sealing film and incubate overnight at 4°C. 3) Decant the plate and gently tap it with a bundle of paper towels to remove any remaining liquid. 4) Add 200 μL of assay buffer. Shake at 200 RPM for 5 minutes at room temperature. 5) Empty the plate and wipe off any remaining liquid with a paper towel. 6) Repeat a total of three times. 7) Add 200 μL of assay buffer to each well. Cover with sealing film and place the plate on a plate shaker set to 200 rpm at room temperature for 60 minutes. 8) Decant the plate and gently tap it with a bundle of paper towels to remove any remaining liquid. 9) Dilute the test substance and standard control antibody, and add 100 μL / well to the plate. 10) Cover with sealing film and place the plate on a plate shaker set to 200 rpm for 60 minutes at room temperature. 11) Decant the plate and gently tap it with a bundle of paper towels to remove any remaining liquid. 12) Wash the wells four times by distributing 200 μL of washing buffer (PBS containing 0.05% Tween20, pH 6.0) into each well, and aspirate the contents completely. 13) Dilute the HRP secondary antibody to 1:2500 in the assay buffer. 14) Add 100 μL of diluted HRP secondary antibody to each well. 15) Cover with sealing film and place the plate on a plate shaker set to 200 rpm for 60 minutes at room temperature. 16) Decant the plate and gently tap it with a bundle of paper towels to remove any remaining liquid. 17) Wash the wells four times by distributing 200 μL of washing buffer into each well, and completely aspirate the contents. 18) Distribute 50 μL of TMB substrate solution (3,3',5,5'-tetramethylbenzidine, Life Technologies, Cat.# 002023) per well into all wells of the plate. Incubate at room temperature for approximately 2 minutes and 30 seconds. 19) Add 50 μL of 1N HCl per well to all wells in the plate. 20) The plate was read at 450nm using a PerkinElmer, EnSpire 2300 Multilabel Reader. 21) Using optical density values ​​obtained with known antibody concentrations, a four-parameter nonlinear regression curve was generated using Graph Pad Prism software version 9.0. The antibody concentration in the serum sample was determined by extrapolation from the standard curve. Non-compartment pharmacokinetic analysis (T1 / 2 and Cmax) of serum concentrations using intravenous bolus injection was performed using PK Solver 2.0.

[0567] As shown in Figures 3A and 3B, no significant differences in pharmacokinetics were observed among the control non-CAB (BAP-143-00-MC) linker-conjugate antibody, control CAB (BAP-143-01-MC) linker-conjugate antibody, non-CAB linker compound conjugate antibodies (BA-143-00-SPC04, BA-143-00-SPC07, and BA-143-00-SPC08), and CAB linker compound conjugate antibodies (BA-143-01-SPC04, BA-143-01-SPC07, AND BA-143-01-SPC08). The notations "BA" and "BAP" are synonymous.

[0568] The results are summarized in Table 3 (Figure 3A) and Table 4 (Figure 3B) below. [Table 8] [Table 9]

[0569] Example 9 - PK analysis of ADCs using MMAE ELISA

[0570] 1.1 Test substance Mouse serum samples were collected in an in vivo PK test at 10 minutes, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, and 96 hours after the following antibody treatments (3 mg / kg). BA-143-00-MC (Wild-type or non-CAB parent conjugated to MC) BA-143-00-SPC04 (Wild-type or non-CAB parent conjugated to SPC04) BA-143-00-SPC07 (Wild-type or non-CAB parent conjugated to SPC07) BA-143-00-SPC08 (Wild-type or non-CAB parent conjugated to SPC08) BA-143-01-MC (CAB conjugated to MC) BA-143-01-SPC04 (CAB conjugated to SPC04) BA-143-01-SPC07 (CAB conjugated to SPC07) BA-143-01-SPC08 (CAB conjugated to SPC08)

[0571] 1.2 Formulation The test substance was diluted with zero calibrator buffer (Epitope Diagnostics, # KTR-782) at different dilution ratios depending on the time of sample collection. Antibodies from the PK test were used as a standard control for the standard curve, first diluted to 10 ug / mL with zero calibrator buffer containing 1:200 normal mouse serum, and then serially diluted 5-fold for six other concentration points.

[0572] 1.3 MMAE ELISA assay 1) Add 25 μL of standard and serum sample to each well (from an intact MMAE ADC ELISA kit, Epitope Diagnostics, #KTR-782). 2) Immediately add 100 μL of assay buffer (Epitope Diagnostics, #30799). 3) Seal the plate well tightly, cover with foil to protect from light, and rotate on a plate shaker at 400-450 rpm for 1 hour. 4) Wash each well five times by distributing 350 μL of diluted standard washing solution (Epitope Diagnostics, # KTR-782) into each well, and then completely aspirate the contents. 5) Add 100 μL of diluted MMAE tracer antibody (Epitope Diagnostics, #30753) to each well. Gently tap the plate. 6) Seal the plate well tightly, cover with foil to protect from light, and rotate on a plate shaker at 400-450 rpm for 30 minutes. 7) Wash each well five times by distributing 350 μL of diluted standard washing solution into each well, and then completely aspirate the contents. 8) Add 100 μL of ELISA HRP substrate to each well. 9) Cover the plate with aluminum foil to avoid exposure to light. Incubate the plate at room temperature for 20 minutes while stationary. 10) Immediately add 100 μL of ELISA stop solution (Epitope Diagnostics, #10030) to each well. 11) Using a reference filter at 620 nm, read the absorbance at 450 nm. Using optical density values ​​obtained with known antibody concentrations, a four-parameter nonlinear regression curve was generated using Graph Pad Prism software version 9.0. The antibody concentration in the serum sample was determined by extrapolation from the standard curve. Non-compartment pharmacokinetic analysis (T1 / 2 and Cmax) of serum concentrations using intravenous bolus injection was performed using PK Solver 2.0.

[0573] As shown in Figures 4A and 4B, no significant differences in pharmacokinetics were observed among the control non-CAB (BAP-143-00-MC) linker conjugate antibody, control CAB (BAP-143-01-MC) linker conjugate antibody, non-CAB linker compound conjugate antibodies (BA-143-00-SPC04, BA-143-00-SPC07, AND BA-143-00-SPC08), and CAB linker compound conjugate antibodies (BA-143-01-SPC04, BA-143-01-SPC07, AND BA-143-01-SPC08) groups.

[0574] The results are summarized in Table 5 (Figure 4A) and Table 6 (Figure 4B) below. [Table 10] [Table 11]

[0575] Example 10 - In vivo antitumor efficacy experiment

[0576] Example 10A. In vivo antitumor efficacy in a subcutaneous xxT47D CDX human breast cancer xenograft model in BABL / c nude mice

[0577] Test objectives and compliance with laws and regulations

[0578] The objective of this study was to evaluate the in vivo antitumor efficacy of the test substance in a subcutaneous xxT47D CDX human bre...

Claims

1. Conjugate of Equation I, 【Chemistry 1】 Equation I (In the formula: L, 【Chemistry 2】 is a linker represented by, R １ is a nucleophilic functional group capable of binding to an antibody, and the nucleophilic functional group is C １ -C １０ alkyl halide, C １ -C １０ alkyl alcohol, phenol, C １ -C １０ alkyl thiol, and C １ -C １０ alkyl azide selected from the group consisting of, R ２ is a substituted or unsubstituted C １ -C ２０ alkylene, or a substituted or unsubstituted (PEG) ｎ moiety represented by the following, 【Transformation 3】 In the formula, n is between 1 and 20; X, 【Chemistry 4】 A substituted nitrophenyl represented by the formula, where R ３ However, C １ ~C ２０ It is alkylene, R ４ However, it is a triazole ring having a C=C double bond in the ring; Y is a glycoside or a glycoside derivative; D is a drug; Y is bonded to X at the covalent bond site indicated by the wavy line on the nitrophenyl ring, D is bonded to X at the site of the wavy covalent bond on the carbonyl group, L is R ２ and R ４ It is joined to X via a covalent bond between the two (the wavy line).

2. R １ However, C has a halide selected from the group consisting of Br, I, Cl, and F. １ ~C ６ Alkyl halide, C １ ~C ２ Alkyl alcohol, 4-phenol, or C １ ~C ２ The conjugate according to claim 1, wherein the conjugate is an alkylthiol.

3. R １ ga-CH ２ - The conjugate according to claim 1 or 2, wherein the conjugate is Br.

4. R ２ However, non-substituted C １ ~C ２０ The conjugate according to any one of claims 1 to 3, wherein the conjugate is alkylene.

5. R ２ However, non-substituted C ２ ~C ５ The conjugate according to claim 4, wherein the conjugate is alkylene.

6. R ２ However, non-substituted (PEG) ｎ The conjugate according to any one of claims 1 to 3, wherein n is 8.

7. R ３ However, -CH ２ - The conjugate according to any one of claims 1 to 6.

8. The glycoside or glycoside derivative Y has the following structure: 【Transformation 5】 【change】 A conjugate according to any one of claims 1 to 7, selected from the above.

9. The conjugate according to any one of claims 1 to 8, wherein the glycoside or glycoside derivative Y is a glucuronide.

10. The aforementioned triazole ring, 【Transformation 6】 Linker intermediate (L) having the formula represented by ｉｎｔ ) formed together, In the formula, R １ , R ２ and n are as defined in claim 1; R ７ The conjugate according to any one of claims 1 to 9, wherein the group is an azide group or an acetylene group, and the nitrogen atom of the azide group or the carbon atom of the acetylene group becomes a ring member in the triazole ring.

11. The triazole ring is bonded to L by the following azide group or acetylene group: 【Transformation 7】 The conjugate according to claim 10, which is formed together with, wherein p is 2 to 5 in the formula.

12. The triazole ring is bonded to L by the following azide group or acetylene group: 【Transformation 8】 A conjugate according to any one of claims 1 to 10, formed together with, wherein n is 8 in the formula.

13. The aforementioned conjugate is SPC07: 【Chemistry 9】 A conjugate according to any one of claims 1 to 10, having a structure represented by [the given].

14. The aforementioned conjugate is SPC19 【Chemistry 10】 A conjugate according to any one of claims 1 to 10, having a structure represented by [the given].

15. R ２ However, substituted or non-substituted (PEG) ｎ In this case, Y is a conjugate that is not a galactoside, as described in claim 11.

16. The aforementioned conjugate is SPC17 【Chemistry 11】 A conjugate according to any one of claims 1 to 10, having a structure represented by [the given].

17. The aforementioned conjugate is SPC02 【Chemistry 12】 A conjugate according to any one of claims 1 to 10, having a structure represented by (wherein MMAE is monomethyl auristatin E).

18. The aforementioned conjugate is SPC04 【Chemistry 13】 A conjugate according to any one of claims 1 to 10, having a structure represented by (wherein MMAE is monomethyl auristatin E).

19. The aforementioned conjugate is SPC05 【Chemistry 14】 A conjugate according to any one of claims 1 to 10, having a structure represented by (wherein MMAE is monomethyl auristatin E).

20. The aforementioned conjugate is SPC06 【Chemistry 15】 A conjugate according to any one of claims 1 to 10, having a structure represented by (wherein MMAE is monomethyl auristatin E).

21. The aforementioned conjugate is SPC08 【Chemistry 16】 A conjugate according to any one of claims 1 to 10, having a structure represented by (wherein MMAE is monomethyl auristatin E).

22. The conjugate according to any one of claims 1 to 21, wherein the drug D is selected from the group consisting of auristatin, drastatin, meitansinoid, calicheamicin, pyrrolobenzodiazepine, anthracycline, ribonuclease, and DNA endonuclease.

23. The conjugate according to claim 22, wherein the drug D is an auristatin selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). 【Request Item 24】 【Chemistry 17】 (In the formula, R １ However, C １ ~C １０ Alkyl halide, C １ ~C １０ Alkyl alcohols, phenols, C １ ~C １０ Alkylthiols, and C １ ~C １０ A nucleophilic functional group capable of binding to an antibody selected from the group consisting of alkyl azides, R ２ However, C is either substituted or non-substituted. １ ~C ２０ Alkylenes, or substituted or unsubstituted (PEGs) represented by the following: ｎ It is a part: [Chemistry 18] In the formula, n is between 1 and 20. R ７ (However, it is either an azide group or an acetylene group.) Linker intermediate (L) having the formula represented by ｉｎｔ ).

25. R １ However, C has a halide selected from the group consisting of Br, I, Cl, and F. １ ~C ６ Alkyl halide, C １ ~C ２ Alkyl alcohol, 4-phenol, or C １ ~C ２ The linker intermediate (L) described in claim 24 is an alkylthiol. ｉｎｔ ).

26. R １ ga-CH ２ -Br, the linker intermediate (L) according to claim 24 or 25 ｉｎｔ ).

27. R ２ However, non-substituted C １ ~C ２０ The linker intermediate (L) described in any one of claims 24 to 26 is an alkylene. ｉｎｔ ).

28. R ２ However, non-substituted C ２ ~C ５ The linker intermediate (L) described in claim 27 is an alkylene. ｉｎｔ ).

29. R ２ However, non-substituted (PEG) ｎ The linker intermediate (L) according to any one of claims 24 to 26, wherein n is 8. ｉｎｔ ).

30. R ７ The linker intermediate (L) according to any one of claims 24 to 29, wherein is an azide group. ｉｎｔ ).

31. The aforementioned linker, 【Chemistry 19】 The linker intermediate (L) according to claim 30, having a structure represented by the formula (where p is 2 to 5) ｉｎｔ ).

32. The aforementioned linker, 【Chemistry 20】 The linker intermediate (L) according to claim 31, having a structure represented by ｉｎｔ ).

33. The aforementioned linker, 【Chemistry 21】 The linker intermediate (L) according to claim 31, having a structure represented by ｉｎｔ ).

34. The aforementioned linker, 【Chemistry 22】 A linker intermediate (L) according to any one of claims 24 to 26, having a structure represented by (wherein n is 8) ｉｎｔ ).

35. Immunoconjugate of formula II 【Chemistry 23】 Formula II (In the formula: L, 【Chemistry 24】 represented by, wherein R １ is C １ to C １０ alkyl halide, C １ to C １０ alkyl alcohol, phenol, C １ to C １０ alkyl thiol, and C １ to C １０ is a nucleophilic functional group capable of binding to an antibody selected from the group consisting of alkyl azide, R ２ is a substituted or unsubstituted C １ -C ２０ alkylene, or a substituted or unsubstituted (PEG)n moiety represented by the following, 【Chemistry 25】 In the formula, n is between 1 and 20. X, 【Chemistry 26】 A substituted nitrophenyl represented by R ３ However, C １ ~C ２０ It is alkylene; R ４ However, it is a triazole ring having a C=C double bond in the ring; Y is a glycoside or a glycoside derivative; D is a drug; The wavy lines indicate the covalent bond sites; m is between 1 and 10. The term "antibody" in Formula II refers to an antibody or an antibody fragment.

36. R １ However, C has a halide selected from the group consisting of Br, I, Cl, and F. １ ~C ６ Alkyl halide, C １ ~C ２ Alkyl alcohol, 4-phenol, or C １ ~C ２ The immunoconjugate according to claim 35, wherein the immunoconjugate is an alkylthiol.

37. R １ However, -CH ２ - An immunoconjugate according to claim 35 or 36, wherein the immunoconjugate is Br.

38. R ２ However, non-substituted C １ ~C ２０ An immunoconjugate according to any one of claims 35 to 37, wherein the immunoconjugate is alkylene.

39. R ２ However, non-substituted C ２ ~C ５ The immunoconjugate according to claim 38, wherein the immunoconjugate is alkylene.

40. R ２ However, non-substituted (PEG) ｎ The immunoconjugate according to any one of claims 35 to 37, wherein n is 8 in the formula.

41. R ３ However, -CH ２ - An immunoconjugate according to any one of claims 35 to 40.

42. The glycoside or glycoside derivative Y has the following structure: 【Chemistry 27】 An immunoconjugate according to any one of claims 35 to 41, selected from the above.

43. The immunoconjugate according to any one of claims 35 to 42, wherein the glycoside or glycoside derivative Y is a glucuronide.

44. R １ However, -CH ２ - It is Br, R ２ but, 【Chemistry 28】 An immunoconjugate according to any one of claims 35 to 43, selected from the formula (wherein p is 2 to 5).

45. R １ However, -CH ２ - Br, R ２ but, 【Chemistry 29】 An immunoconjugate according to claim 44, selected from the above.

46. R １ However, -CH ２ - Br; R ２ but, 【Transformation 30】 An immunoconjugate according to claim 45, selected from the above.

47. -CH ２ -Br and R ２ but, 【Chemistry 31】 An immunoconjugate according to any one of claims 35 to 43, selected from (wherein n is 8).

48. The immunoconjugate according to any one of claims 35 to 47, wherein the drug D is selected from the group consisting of auristatin, drastatin, meitansinoid, calicheamicin, pyrrolobenzodiazepine, anthracycline, ribonuclease, and DNA endonuclease.

49. The immunoconjugate according to claim 48, wherein the drug D is an auristatin selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). 【Request Item 50】 【Chemistry 32】 However, the aforementioned antibody 【Transformation 33】 【change】 【change】 【change】 An immunoconjugate according to any one of claims 35 to 43, which is conjugated to the antibody by reacting with a compound having a structure selected from (wherein MMAE is monomethyl auristatin E).

51. The antibody is an antibody or antibody fragment that specifically binds to nectin-4, which includes a heavy chain variable region containing three complementarity-determining regions (CDRs) having sequences H1, H2, and H3, wherein: The H1 sequence is GFTFSSYNX １ N is (Sequence ID 1); The H2 sequence is ISSSSSTIYYADSVKG (Sequence ID 2); The aforementioned H3 sequence is AYYYGX ２ DX ３ (Sequence code 3) is; Here, X １ However, it is M or D; X ２ However, it is M or D; X ３ However, the heavy chain and light chain variable regions are not in combination sequence numbers 18 and 31 or sequence numbers 18 and 56, and the light chain variable region comprises three CDRs having sequences L1, L2, and L3, where: The L1 array is X ４ ASQGISGWX ５ A (sequence number 4); The L2 sequence is AASTLQS (Sequence ID 5); The L3 array is QQANSX ６ PX ７ T (Sequence ID 6) Here, X ４ However, it is R or H; X ５ However, it is L or E; X ６ However, it is F or E; X ７ However, P or D, provided that X １ , X ２ , X ３ , X ４ , X ５ , X ６ and X ７ The immunoconjugate according to any one of claims 35 to 49, wherein each of them is not simultaneously M, M, V, R, L, F, and P.

52. The antibody or antibody fragment further comprises six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9, wherein: The L4 sequence is GFTFNTYAMN (sequence number 44), The L5 sequence is RIRSKYNNYATYYADSVKD (Sequence ID 45), The L6 array is HX １１ NFX １２ NSX １３ VSWFX １４ Y (Sequence ID 46) The L7 sequence is RSSTGAVTTSNYX １５ N (sequence number 47), The L8 sequence is GTNKRAP (sequence number 48), The L9 sequence is ALWYSNLWV (sequence number 49), Here, X １１ However, it is G or S, and X １２ However, it is G or P, and X １３ However, it is Y or K, and X １４ However, it is A or Q, and X １５ The immunoconjugate according to claim 51, wherein the immunoconjugate is A or D.

53. The immunoconjugate according to claim 51 or 52, wherein the H1 sequence is selected from GTFFSSYNMN (SEQ ID NO: 7) and GTFFSSYNDN (SEQ ID NO: 8).

54. The immunoconjugate according to any one of claims 51 to 53, wherein the H3 sequence is selected from AYYYGMDV (SEQ ID NO: 9), AYYYGDDV (SEQ ID NO: 10), and AYYYGMDK (SEQ ID NO: 11).

55. The immunoconjugate according to any one of claims 51 to 54, wherein the L1 sequence is selected from RASQGISGWLA (SEQ ID NO: 12), RASQGISGWEA (SEQ ID NO: 13), and HASQGISGWLA (SEQ ID NO: 14).

56. The immunoconjugate according to claim 51, wherein the L3 sequence is selected from QQANSFPPT (SEQ ID NO: 15), QQANSEPPT (SEQ ID NO: 16), and QQANSFPDT (SEQ ID NO: 17).

57. The immunoconjugate according to claim 51 and any one of claims 52 to 56 as dependent on claim 51, wherein the L6 sequence is selected from HGNFGNSYVSWFAY (SEQ ID NO: 50), HSNFGNSKVSWFAY (SEQ ID NO: 51), HGNFPNSKVSWFQY (SEQ ID NO: 52), and HSNFGNSKVSWFAY (SEQ ID NO: 53).

58. The immunoconjugate according to claim 51 and any one of claims 52 to 57, in the case of claim 51, wherein the L7 sequence is selected from RSSTGAVTTSNYAN (sequence number 54) and RSSTGAVTTSNYDN (sequence number 55).

59. The immunoconjugate according to claim 51, wherein the heavy chain variable region has a sequence selected from sequence numbers 18 to 30.

60. The immunoconjugate according to claim 51, wherein the light chain variable region has a sequence selected from sequence numbers 31 to 43.

61. The immunoconjugate according to claim 51, comprising a heavy chain variable region and a light chain variable region having any pair of sequences selected from sequence numbers 32 and 19, 33 and 20, 34 and 21, 35 and 22, 36 and 23, 37 and 24, 38 and 25, 39 and 26, 40 and 27, 41 and 28, and 42 and 29.

62. An immunoconjugate according to claim 51, comprising a heavy chain variable region and a light chain variable region, each of which independently has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a combination of amino acid sequences selected from one of sequence numbers 18 to 30 in combination with one of sequence numbers 31 to 43; provided that the heavy chain and light chain variable regions are not sequence numbers 18 and 31 in combination; and the antibody or antibody fragment specifically binds to human nectin-4 protein.

63. The immunoconjugate according to claim 51, comprising a heavy chain variable region and a light chain variable region, each of which independently has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with each of the pairs of amino acid sequences selected from SEQ ID NOs: 32 and 19, SEQ ID NOs: 33 and 20, SEQ ID NOs: 34 and 21, SEQ ID NOs: 35 and 22, SEQ ID NOs: 36 and 23, SEQ ID NOs: 37 and 24, SEQ ID NOs: 38 and 25, SEQ ID NOs: 39 and 26, SEQ ID NOs: 40 and 27, SEQ ID NOs: 41 and 28, and SEQ ID NOs: 42 and 29; and the antibody or antibody fragment specifically binds to human nectin-4 protein.

64. The immunoconjugate according to claim 51, wherein the heavy chain variable region has sequences selected from sequence numbers 18, 25, 27, and 29.

65. The immunoconjugate according to claim 51, wherein the light chain variable region has a sequence selected from sequence numbers 56 to 60.

66. The immunoconjugate according to claim 51, comprising a heavy chain variable region and a light chain variable region having one pair of sequences selected from sequence numbers 25 and 57, sequence numbers 27 and 58, sequence numbers 29 and 59, and sequence numbers 29 and 60.

67. The immunoconjugate according to claim 51, comprising a heavy chain variable region and a light chain variable region, each of which independently has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a combination of amino acid sequences selected from one of sequence numbers 18, 25, 27, and 29 in combination with one of sequence numbers 56 to 60, wherein the antibody or antibody fragment specifically binds to human nectin-4 protein.

68. The immunoconjugate according to claim 51, comprising a heavy chain variable region and a light chain variable region, each of which independently has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a pair of amino acid sequences selected from SEQ ID NO: 25 and SEQ ID NO: 57, SEQ ID NO: 27 and SEQ ID NO: 58, SEQ ID NO: 29 and SEQ ID NO: 59, and SEQ ID NO: 29 and SEQ ID NO: 60; and the antibody or antibody fragment specifically binds to human nectin-4 protein.

69. The immunoconjugate according to any one of claims 35 to 43, wherein the antibody or antibody fragment has a higher binding affinity to the nectin-4 protein at the value under tumor microenvironmental conditions compared to different values ​​under the same conditions occurring in a non-tumor microenvironment.

70. The immunoconjugate according to claim 69, wherein the condition is pH.

71. The immunoconjugate according to claim 70, wherein the pH in the tumor microenvironment is in the range of 5.0 to 6.8, and the pH in the non-tumor microenvironment is in the range of 7.0 to 7.

6.

72. The immunoconjugate according to claim 69, wherein the antibody or antibody fragment has at least 70% of the antigen-binding activity at pH 6.0 compared to the same antigen-binding activity of the parent antibody or antibody fragment at pH 6.

0.

73. The immunoconjugate according to claim 69, wherein the antibody or antibody fragment has an antigen-binding activity at pH 7.4 that is less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% compared to the same antigen-binding activity of the parent antibody or antibody fragment at pH 7.

4.

74. The immunoconjugate according to claim 72 or 73, wherein the antigen-binding activity is binding to the nectin-4 protein.

75. The immunoconjugate according to any one of claims 69 to 74, wherein the antigen-binding activity is measured by an ELISA assay.

76. The immunoconjugate according to any one of claims 69 to 75, wherein the antibody or antibody fragment has a ratio of binding activity to the nectin-4 protein at a value within the tumor microenvironment versus binding activity to the nectin-4 protein at a different value under the same conditions in a non-tumor microenvironment, which is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:

1.

77. The immunoconjugate according to any one of claims 69 to 76, wherein the antibody is a multispecific antibody or an antibody fragment.

78. The immunoconjugate according to any one of claims 69 to 77, wherein the antibody is a bispecific antibody or an antibody fragment.

79. The immunoconjugate according to any one of claims 69 to 78, wherein the immunoconjugate comprises at least one agent selected from chemotherapeutic agents, radioactive atoms, cell proliferation inhibitors, and cytotoxic agents.

80. The immunoconjugate according to claim 79, comprising at least two of the aforementioned agents.

81. The immunoconjugate according to claim 79 or 80, wherein the at least one agent is a radioactive agent.

82. The immunoconjugate according to claim 81, wherein the radioactive agent is selected from an α-emitter, a β-emitter, and a γ-emitter.

83. The immunoconjugate according to any one of claims 79 to 82, wherein the antibody or antibody fragment and the at least one drug are covalently bonded to a linker molecule.

84. The immunoconjugate according to claim 79 or 80, wherein the at least one drug is selected from mytansinoids, auristatin, drastatin, calicheamicin, pyrrolobenzodiazepines, and anthracyclines.

85. An immunoconjugate according to any one of claims 35 to 84; and Pharmacologically acceptable carriers A pharmaceutical composition containing the following:

86. A single dose of the pharmaceutical composition according to claim 85, comprising an amount of the immunoconjugate in the range of approximately 135 mg, 235 mg, 335 mg, 435 mg, 535 mg, 635 mg, 735 mg, 835 mg, 935 mg, 1035 mg, 1135 mg, 1235 mg, or 1387 mg.

87. A single dose of the pharmaceutical composition according to claim 85, comprising an amount of the immunoconjugate in the range of 135-235 mg, 235-335 mg, 335-435 mg, 435-535 mg, 535-635 mg, 635-735 mg, 735-835 mg, 835-935 mg, 935-1035 mg, 1035-1135 mg, 1135-1235 mg, or 1235-1387 mg.

88. A pharmaceutical composition according to any one of claims 85 to 87, further comprising an immune checkpoint inhibitor molecule.

89. The pharmaceutical composition according to claim 88, wherein the immune checkpoint inhibitor molecule is an antibody or antibody fragment against an immune checkpoint.

90. The pharmaceutical composition according to claim 88, wherein the immune checkpoint is selected from CTLA4, LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, GITR, B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS.

91. The pharmaceutical composition according to claim 90, wherein the immune checkpoint is CTLA4, PD-1, or PD-L1.

92. A pharmaceutical composition according to any one of claims 88 to 91, further comprising an antibody or antibody fragment against an antigen selected from CTLA4, PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4, and WNT proteins.

93. A method for treating cancer, comprising the step of administering an immune conjugate according to any one of claims 30 to 79 or a pharmaceutical composition according to any one of claims 85 to 92 to a patient having cancer.

94. Use of an immunoconjugate conjugate according to any one of claims 35 to 84 or a pharmaceutical composition according to any one of claims 85 to 92 for the treatment of cancer.

95. A diagnostic or therapeutic kit comprising a conjugate according to any one of claims 1 to 23, a linker according to any one of claims 24 to 34, or an immunoconjugate according to any one of claims 35 to 84, or a pharmaceutical composition according to any one of claims 85 to 92, and instructions for using the antibody or antibody fragment, the immunoconjugate and / or the pharmaceutical composition for diagnostic or therapeutic purposes.

96. Compounds represented by formula III: 【Transformation 34】 (In the formula, R ５ａ and R ５ｂ However, independently or simultaneously, hydrogen, substituted or unsubstituted C １ ~C ６ Acyl, or substituted or unsubstituted C １ ~C ６ It is alkyl; R ６ but, 【Chemistry 35】 (Selected from the group consisting of the following).

97. R ６ but 【Transformation 36】 If R ５ａ is acetyl, R ５ｂ It is hydrogen, R ６ but, 【Chemistry 37】 If R ５ａ R is an acetyl group, ５ｂ It is a methyl group, R ６ but, 【Transformation 38】 If R ５ａ and R ５ｂ The compound according to claim 96, wherein all atoms are hydrogen.

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