5T4 antibody-drug conjugates and uses thereof

Abstract

The present disclosure provides 5T4 antibody-drug conjugates and uses thereof.

Description

[0001] Cross-reference of related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 341,957, filed May 13, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a computer-readable sequence list that has been submitted with this application in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence list XML file submitted with this application is named "14529-099-228_SEQ_LISTING.xml", was created on May 9, 2023, and has a size of 125,376 bytes. Technical Field

[0005] This disclosure generally relates to antibody-drug conjugates (ADCs) that bind to 5T4 (e.g., human 5T4) and methods of using them. Background Art

[0006] 5T4 is an N-glycosylated transmembrane 72 kDa glycoprotein containing eight leucine-rich repeat sequences. Also known as Wnt activator inhibitor 1 or WAIF1, 5T4 is commonly referred to as carcinoembryonic antigen (CEA) due to its expression in the fetal trophoblast (it was first discovered in the fetal trophoblast), or trophoblastic glycoprotein (TPBG). 5T4 is found in tumors such as colorectal, ovarian, and gastric tumors. Its expression is used as an adjunct to prognosis. 5T4 expression is extremely limited in normal tissues but is widespread in malignant tumors throughout their development. Although its restricted expression seems to offer the potential to target 5T4 in cancer therapy, successful treatments with antibodies, ADCs, and vaccines targeting 5T4 have not yet been achieved.

[0007] There is still a need in the field for ADCs that can target 5T4 to treat, prevent or alleviate 5T4-mediated diseases, conditions or disorders, such as those involving tumor cells that express 5T4. Summary of the Invention

[0008] This disclosure provides an ADC (“5T4-ADC”) comprising an antibody that binds to 5T4. In some embodiments, such a 5T4-ADC binds to the same epitope of human 5T4 with an antibody comprising the heavy chain variable region (VH) and light chain variable region (VL) as described herein.

[0009] This disclosure also provides pharmaceutical compositions comprising a 5T4-ADC comprising an antibody or fragment thereof binding to 5T4 and a drug substance conjugated (directly or indirectly) to said antibody or fragment thereof. In some embodiments, such pharmaceutical compositions comprise a 5T4-ADC comprising an antibody or fragment thereof that binds to substantially the same epitope as antibodies comprising VH and VL described herein to human 5T4.

[0010] This disclosure also provides methods for treating, preventing, or alleviating 5T4-mediated diseases, conditions, or disorders using 5T4-ADCs that bind to 5T4, such as alleviating one or more symptoms of 5T4-mediated diseases, conditions, or disorders.

[0011] More specifically, this disclosure provides a 5T4-ADC comprising (a) an antibody bound to 5T4 and (b) one or more pyridazine-pyrrolo coupling moieties, including a drug conjugated to the pyridazine-pyrrolo coupling moieties via a linker. In some embodiments, the 5T4-ADC is represented by formula (IV):

[0012]

[0013] in

[0014] Ab indicates an antibody that binds to 5T4;

[0015] R 2 and R 3 Each of these groups is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic; or R 2 With R 3 Ring-linked structures are used to form 5- or 6-membered heterocyclic groups;

[0016] L is a connector and is composed of -(T) 1 V 1 ) a -(T 2 V 2 ) b -(T 3 V 3 ) c -(T 4 V 4 ) d -(T 5 V 5 ) e -express;

[0017] T 1 、T 2 、T 3 、T 4 and T 5 Each independently is C1-C 12 Alkyl, substituted C1-C 12 Alkyl group, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) h - Piperidin-4-amine (P4A-R) 12 ), m-aminobenzoic acid (MABC) group, m-aminobenzooxy (MABO) group, p-aminobenzooxy (PABO) group, p-aminobenzoic acid (PABC) group, p-aminobenzyl (PAB) group, acetal, disulfide, hydrazine, (AA) p -MABC-(AA) p (AA) p -MABO-(AA) p (AA) p -PABO-(AA) p Or (AA) p -PABC-(AA) p ;

[0018] V 1 、V 2 、V 3 、V 4 and V 5 Each is independently a covalent bond, -C(=O)-, -NR 11 -、-C(=O)NR 11 -、-NR 11 C(=O)-, -C(=O)O-, -OC(=O)-, -O-, -S-, -S(=O)-, -SO2-, -SO2NR 11 -、-NR 11 SO2- or -P(=O)OH-;

[0019] a, b, c, d, and e are each independently 0 or 1, and the sum of a, b, c, d, and e is between 1 and 5;

[0020] h is an independent integer from 1 to 12;

[0021] m is 0 or 1;

[0022] n is an independent integer from 1 to 30;

[0023] p can be zero or an integer from 1 to 20 independently;

[0024] r is an integer from 1 to 10;

[0025] w is an independent integer from 1 to 20;

[0026] X 1 、X 2 、X 3 and X 4 Each can be independently C, N, O, or S;

[0027] Y 1 、Y 2 、Y 3 and Y 4 Each of these groups is independently hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic, or Y 1 With Y 2 、Y 2 With Y 3 Or Y 3 With Y 4 Connected in a ring;

[0028] W 1 For medicine;

[0029] R 1111 Independently hydrogen, alkyl, substituted alkyl, PEG, aryl, or substituted aryl; and

[0030] R 12 Independently, it comprises hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic; and

[0031] R 13 It is independently hydrogen, alkyl, substituted alkyl, aryl or substituted aryl.

[0032] In some implementations, the drug is maytansinoid or auristatin, such as MMAE or MMAF.

[0033] In some implementations, the 5T4-ADC can be represented by equation (1IV), where R 2 and R 3 Each is an alkyl group, such as a methyl group. In some embodiments, 5T4-ADC may be represented by formula (IV), where X 1 、X 2 、X 3 and X 4 Each of them is independently C or N.

[0034] In some implementations, the 5T4-ADC can be represented by equation (V):

[0035]

[0036] where X 1 、Y 1 、Y 2 、Y 3 、Y 4 、R 2 、R 3 L and W 1 As defined in this paper, such as that defined by (IV).

[0037] In some implementations, the 5T4-ADC can be represented by equation (VI):

[0038]

[0039] where X 1 It is C or N, and L and W 1 As defined in this paper, such as that defined by (IV).

[0040] In some implementations, the 5T4-ADC can be represented by equation (IV), (V), or (VI), where:

[0041] L is a connector and is composed of -(T) 1 V 1 ) a -(T 2 V 2 ) b -(T 3 V 3 ) c -(T 4 V 4 ) d -(T 5 V 5 ) e -express;

[0042] T 1 、T 2 、T3 、T 4 and T 5 Each is independently a C1-C6 alkyl group, P4A-R 12 (AA) p (AA) p -PABC or (PEG) n ;

[0043] V 1 V 2 V 3 V 4 and V 5 Each can be independently selected from the following groups: covalent bond, -C(=O)-, -NR 11 -、-C(=O)NR 11 -、-NR 11 C(=O)-, -C(=O)O-, -OC(=O)-, -O-, -S-, -S(=O)-, -SO2-, -SO2NR 11 -、-NR 11 SO2- and -P(=O)OH-;

[0044] a, b, c, d, and e are each independently 0 or 1, and the sum of a, b, c, d, and e is between 1 and 5;

[0045] p is an integer from 1 to 20;

[0046] n is an integer from 1 to 30;

[0047] R 11 Selected from the group consisting of: hydrogen, alkyl, substituted alkyl, PEG, aryl, and substituted aryl; and

[0048] R 12 It is either polyethylene glycol or polyethylene glycol modified with carboxylic acid.

[0049] In other embodiments, the 5T4-ADC may be represented by formula (IV), (V), or (VI), wherein:

[0050] a, b, c, d, and e are each independently 0 or 1, and the sum of a, b, c, d, and e is between 1 and 5;

[0051] p is an integer from 1 to 20;

[0052] n is an integer from 1 to 30;

[0053] L is a connector and is composed of -(T) 1 V 1 ) a -(T 2 V 2 )b -(T 3 V 3 ) c -(T 4 V 4 ) d -(T 5 V 5 ) e -express;

[0054] T 1 It is a C1-C6 alkyl group;

[0055] T 2 、T 3 、T 4 and T 5 Each independently constitutes (PEG) n C1-C6 alkyl groups, (AA) p P4A-R 12 (AA) p -(PABO-R 16 )-(AA) p (AA) p -(PABC-R 16 )-(AA) p (AA) p -(PABO-R 16 ) or (AA) p -(PABC-R 16 );

[0056] V 1 、V 2 、V 3 、V 4 and V 5 Each is independently -CO- or -NR 11 ;

[0057] R 11 Independently hydrogen or alkyl;

[0058] R 12 Independently, it is a carboxylic acid-modified polyethylene glycol; and

[0059] R 16 For hydrogen.

[0060] In some implementations, the 5T4-ADC can be represented by equation (IV), (V), or (VI), where:

[0061] L stands for -(C1-C6 alkyl)-(V 1 )-(P4A-R 12 )-(V 2 )-(C1-C6 alkyl)-(V3 )-;

[0062] V 1 、V 2 and V 3 Each is independently -CO- or -NR 11 -;

[0063] R 11 Independently hydrogen or C1-C6 alkyl; and

[0064] R 12 It is the polyethylene glycol moiety modified with carboxylic acid.

[0065] In some implementations, the 5T4-ADC can be represented by equation (IV), (V), or (VI), where L is:

[0066]

[0067] in This indicates the nitrogen atom linked to the pyridazine-pyrrole coupling moiety, and * indicates the nitrogen atom linked to W. 1 The connection point.

[0068] In some implementations, the 5T4-ADC can be represented by equation (VI-1):

[0069]

[0070] in:

[0071] Ab indicates an antibody that binds to 5T4;

[0072] r is an integer from 1 to 10;

[0073] X 1 It is C or N; and

[0074] W 1 It is a drug.

[0075] In some embodiments, a 5T4-ADC may be represented by formula (VI-1), wherein the drug is a maytansine or olipattin, such as MMAE. In some embodiments, a 5T4-ADC may be represented by formula (VI-1), wherein X 1 For C and W 1 It is maytansine or deacylated maytansine. In some embodiments, 5T4-ADC can be represented by formula (VI-1), where X 1 For N and W 1 For MMAE.

[0076] In some implementations, the 5T4-ADC can be represented by equation (IV), (V), or (VI), where:

[0077] L represents (C1-C6 alkyl)-V 1 -(AA)-V 2 -(PEG) n -V 3 -((AA) p -(PABC-R 16 ))-V 4 -(C1-C6 alkyl)-V 5 ;

[0078] V 1 、V 2 、V 3 、V 4 and V 5 Each independently chooses its own -CO- and -NR. 11 -A group;

[0079] R 1111 Independently hydrogen or C1-C6 alkyl;

[0080] R 16 It is hydrogen; and

[0081] n and p are each independent integers from 1 to 20;

[0082] In some implementations, the 5T4-ADC can be represented by equation (IV), (V), or (VI), where L is:

[0083]

[0084] in This indicates nitrogen linked to the pyridazine-pyrrole coupling moiety; and * indicates linkage to W. 1 The connection point.

[0085] In some implementations, the 5T4-ADC can be represented by equation (VI-2):

[0086]

[0087] in:

[0088] Ab indicates an antibody that binds to 5T4;

[0089] r is an integer from 1 to 10;

[0090] X 1 It is C or N; and

[0091] W 1 It is a drug.

[0092] In some embodiments, a 5T4-ADC may be represented by formula (VI-2), wherein the drug is a maytansine or olipattin, such as MMAE. In some embodiments, a 5T4-ADC may be represented by formula (VI-2), wherein X 1 For C and W 1 It is maytansine or deacylated maytansine.

[0093] This disclosure also provides 5T4-ADCs of formulas (IV), (V) and (VI):

[0094]

[0095] in:

[0096] Ab indicates an antibody that binds to 5T4;

[0097] r is an integer from 1 to 10;

[0098] m is 0 or 1;

[0099] R 2 and R 3 Each of these groups is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic; or R 2 With R 3 Ring-linked structures are used to form 5- or 6-membered heterocyclic groups;

[0100] X 1 X 2 X 3 and X 4 Each can independently choose a group composed of C, N, O, and S;

[0101] Y 1 、Y 2 、Y 3 and Y 4 Each of these groups is independently hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic, or Y 1 With Y 2 、Y 2 With Y3 or Y 3 With Y 4 Connected in a ring;

[0102] L is

[0103] This indicates the connection point of nitrogen to the pyridazine-pyrrole coupling portion;

[0104] * indicates a connection to W 1 connection points;

[0105] R 5 Independently, it is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0106] R 6 Independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0107] R 7 It is a pyrolytic component;

[0108] k is an integer from 1 to 10;

[0109] L 1 Includes -(T) 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -;

[0110] L 2 Includes -(T) 5 -V 5 ) e -(T 6 -V 6 ) f -(T 7 -V 7 ) g -(T 8 -V 8 ) h -;

[0111] T 1 、T2 、T 3 、T 4 、T 5 、T 6 、T 7 and T 8 Each is independently a covalent bond, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) m -、P4A-R 12 acetals, hydrazines, disulfides, and esters;

[0112] V 1 、V 2 、V 3 、V 4 、V 5 、V 6 、V 7 and V 8 Each is independently a covalent bond, -CO-, -NR 15 -、-NR 15 (CH2) q -、-NR 15 (C6H4)-、-CONR 15 -、-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-;

[0113] a, b, c, d, e, f, g, and h are each independently 1 or 0;

[0114] m is an independent integer from 1 to 12;

[0115] n is an independent integer from 1 to 30;

[0116] p is an independent integer from 1 to 20;

[0117] q is an independent integer from 1 to 6;

[0118] w is an independent integer from 1 to 20;

[0119] R 12Independently, it is hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0120] R 13 Independently hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

[0121] R 15 Independently, it is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic.

[0122] In some implementations, the 5T4-ADC can be represented by equation (IV), where R 2 and R 3 Each is an alkyl group, such as a methyl group. In some embodiments, 5T4-ADC may be represented by formula (IV), where X 1 X 2 X 3 and X 4 Each of them is independently C or N.

[0123] In some implementations, the 5T4-ADC can be represented by equation (VI-3):

[0124]

[0125] in:

[0126] Ab indicates an antibody that binds to 5T4;

[0127] X 1 It can be C, N, O, or S;

[0128] R 5 Independently, it is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0129] R 6 Independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0130] R 7 It is a pyrolytic component;

[0131] L 1 Includes -(T) 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -;

[0132] L 2 Includes -(T) 5 -V 5 ) e -(T 6 -V 6 ) f -(T 7 -V 7 ) g -(T 8 -V 8 ) h -;

[0133] T 1 、T 2 、T 3 、T 4 、T 5 、T 6 、T 7 and T 8 Each is independently a covalent bond, C1-C 12 Alkyl, substituted C 1- C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) m -、P4A-R 12 acetals, hydrazines, disulfides or esters;

[0134] V 1 、V 2 、V 3 、V 4 、V 5 、V 6 V7 and V8 are each independently covalent, -CO-, -NR15 -、-NR 15 (CH2) q -、-NR 15 (C6H4)-、-CONR 15 -、-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- or -P(O)OH-;

[0135] a, b, c, d, e, f, g, and h are each independently 1 or 0;

[0136] k is an integer from 1 to 10;

[0137] m is an independent integer from 1 to 12;

[0138] n is an independent integer from 1 to 30;

[0139] p is an independent integer from 1 to 20;

[0140] q is an independent integer from 1 to 6;

[0141] w is an independent integer from 1 to 20;

[0142] R 12 Independently, it is hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0143] R 13 Independently hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl; and

[0144] R 15 Independently, it is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic.

[0145] In some implementations, the 5T4-ADC can be represented by equation (IV), (V), or (VI), where:

[0146] L is

[0147] R5 、R 6 , L 1 , L 2 and k as defined in this paper, such as that defined for equation (VI-3); and

[0148] R 7 for The ** indicates a connection point to the phenyl group described herein, such as formula (VI-3), (V-4), or (VI-4).

[0149] In some implementations, the 5T4-ADC can be represented by equation (V-5):

[0150] Among them, Ab and L 1 , L 2 、R 2 、R 3 、R 5 、X 1 、Y 1 、Y 2 、Y 3 、Y 4 W 1 and r as defined in this paper, such as that defined for equation (VI-3), and R 6′ and R 6" Each of them independently is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic.

[0151] In some implementations, the 5T4-ADC can be represented by equation (VI-5):

[0152] Among them, Ab and L 1 , L 2 、R 5 W 1 and r as defined in this paper, such as that defined for equation (VI-3), and R 6′ and R 6″ Each of the following is independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic, and wherein X 1 It can be C or N.

[0153] In some implementations, the 5T4-ADC can be represented by equation (VI-6):

[0154] Among them, Ab and L 1 , L2 、W 1 and r as defined herein, such as that defined for equation (VI-3) and where X 1 It can be C or N.

[0155] In some implementations, the 5T4-ADC can be represented by equations (VI-3), (V-5), (VI-5), or (VI-6), wherein:

[0156] L 1 Includes -(T) 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -;

[0157] T 1 、T 2 and T 3 Each independently chooses C1-C 12 Alkyl group, (PEG) n and (AA) p the group formed;

[0158] V 1 、V 2 and V 3 Each is independently -C (=O)- or -NR 1111 -;

[0159] a, b, and c are each 1;

[0160] n is an independent integer from 1 to 10;

[0161] p is an independent integer from 1 to 10;

[0162] R 1111 Independently hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl or substituted aryl.

[0163] In some implementations, the 5T4-ADC can be represented by formulas (VI-3), (V-5), (VI-5), or (VI-6), where L 1 for in The * indicates the connection point of nitrogen to the pyridazine-pyrrole coupling portion, and * indicates the connection point to the terminal nitrogen in the connector, as described herein, such as shown in formula (VI-6).

[0164] In some implementations, the 5T4-ADC can be represented by equation (VI-7):

[0165]

[0166] Among them, Ab and L 1 , L 2 W 1 and r as defined herein, such as that defined for equation (VI-3) and where X 1 It can be C or N.

[0167] In some implementations, the 5T4-ADC can be represented by equation (VI-7), where L 2 The radical is carbonyl. In some embodiments, 5T4-ADC can be represented by formula (VI-7), where X is a carbonyl group. 1 For N and W 1 For MMAE.

[0168] In some embodiments, the 5T4-ADC may be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), wherein the antibody (Ab) comprises: (i) VH CDR1, VH CDR2, and VH CDR3 as shown in VH of SEQ ID NO: 25 and VL CDR1, VL CDR2, and VL CDR3 as shown in VL of SEQ ID NO: 26; (ii) VH CDR1, VHCDR2, and VH CDR3 as shown in VH of SEQ ID NO: 44 and VL CDR1, VL CDR2, and VLCDR3 as shown in VL of SEQ ID NO: 45; or (iii) VH CDR1, VH CDR2, and VH CDR3 as shown in VH of SEQ ID NO: 62 and VL CDR1, VH CDR2, and VLCDR3 as shown in VL of SEQ ID NO: 65. VL CDR1, VL CDR2 and VL CDR3 are shown in NO:63.

[0169] In some embodiments, the 5T4-ADC may be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), wherein the antibody (Ab) comprises: (a) a VH region comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20; and (b) a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; and (2) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22. CDR2; and (3) VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 17 and 23.

[0170] In some embodiments, the 5T4-ADC may be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), wherein the antibody (Ab) comprises: (i) a VH region comprising VH CDR1 containing the amino acid sequence SEQ ID NO: 1, VH CDR2 containing the amino acid sequence SEQ ID NO: 2, and VH CDR3 containing the amino acid sequence SEQ ID NO: 3; and a VL region comprising VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 6; or (ii) a VH region comprising VH CDR1 containing the amino acid sequence SEQ ID NO: 7, VH CDR2 containing the amino acid sequence SEQ ID NO: 8, and VH CDR3 containing the amino acid sequence SEQ ID NO: 9; and a VL region comprising VH CDR1 containing the amino acid sequence SEQ ID NO: 4, VH CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 6; and a VL region comprising VH CDR1 containing the amino acid sequence SEQ ID NO: 7, VH CDR2 containing the amino acid sequence SEQ ID NO: 8, and VH CDR3 containing the amino acid sequence SEQ ID NO: 9; and a VL region comprising VH CDR1 containing the amino acid sequence SEQ ID NO: 6, VH CDR2 containing the amino acid sequence SEQ ID NO: 7, VH CDR2 ... VLCDR1 containing SEQ ID NO: 10, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VLCDR3 containing the amino acid sequence SEQ ID NO: 6; or (iii) the VH region, which includes VH CDR1 containing the amino acid sequence SEQ ID NO: 12, VH CDR2 containing the amino acid sequence SEQ ID NO: 2, and VH CDR3 containing the amino acid sequence SEQ ID NO: 3; and the VL region, which includes VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 6; or (iv) the VH region, which includes VH CDR1 containing the amino acid sequence SEQ ID NO: 13, VH CDR2 containing the amino acid sequence SEQ ID NO: 14, and VH CDR3 containing the amino acid sequence SEQ ID NO: 15; and the VL region, which includes VL CDR1 containing the amino acid sequence SEQ ID NO: 16, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VLCDR3 containing the amino acid sequence SEQ ID NO: 6; VLCDR2 containing SEQ ID NO: 11 and VL CDR3 containing the amino acid sequence SEQ ID NO: 17; or (v) VH region containing VH CDR1 containing the amino acid sequence SEQ ID NO: 18, VH CDR2 containing the amino acid sequence SEQ ID NO: 19 and VH CDR3 containing the amino acid sequence SEQ ID NO: 20.The VL region contains VL CDR1 containing the amino acid sequence SEQ ID NO: 21, VL CDR2 containing the amino acid sequence SEQ ID NO: 22, and VL CDR3 containing the amino acid sequence SEQ ID NO: 23; or (vi) the VH region contains VH CDR1 containing the amino acid sequence SEQ ID NO: 1, VH CDR2 containing the amino acid sequence SEQ ID NO: 24, and VH CDR3 containing the amino acid sequence SEQ ID NO: 3; and the VL region contains VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VLCDR3 containing the amino acid sequence SEQ ID NO: 6.

[0171] In some embodiments, the 5T4-ADC may be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), wherein the antibody (Ab) comprises: (a) a VH region comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 27, 31, 34, 35, and 39; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 28, 32, 36, 40, and 43; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 33, 37, and 41; and (b) a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 10, 16, and 21; and (2) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11, and 22. CDR2; and (3) VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 38 and 42.

[0172] In some embodiments, the 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), wherein the antibody (Ab) comprises: (i) a VH region comprising VH CDR1 containing the amino acid sequence SEQ ID NO: 27, VH CDR2 containing the amino acid sequence SEQ ID NO: 28, and VH CDR3 containing the amino acid sequence SEQ ID NO: 29; and a VL region comprising VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 30; or (ii) a VH region comprising VH CDR1 containing the amino acid sequence SEQ ID NO: 31, VH CDR2 containing the amino acid sequence SEQ ID NO: 32, and VH CDR3 containing the amino acid sequence SEQ ID NO: 33; and a VL region comprising VH CDR1 containing the amino acid sequence SEQ ID NO: 39, VH CDR2 containing the amino acid sequence SEQ ID NO: 2 ... VLCDR1 containing SEQ ID NO: 10, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VLCDR3 containing the amino acid sequence SEQ ID NO: 30; or (iii) the VH region, which includes VH CDR1 containing the amino acid sequence SEQ ID NO: 34, VH CDR2 containing the amino acid sequence SEQ ID NO: 28, and VH CDR3 containing the amino acid sequence SEQ ID NO: 29; and the VL region, which includes VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 30; or (iv) the VH region, which includes VHCDR1 containing the amino acid sequence SEQ ID NO: 35, VH CDR2 containing the amino acid sequence SEQ ID NO: 36, and VHCDR3 containing the amino acid sequence SEQ ID NO: 37; and the VL region, which includes VL CDR1 containing the amino acid sequence SEQ ID NO: 16, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VL CDR3 containing the amino acid sequence SEQ ID NO: 11. CDR2 and VL CDR3 containing the amino acid sequence SEQ ID NO: 38; or (v) VH region, which contains VH CDR1 containing the amino acid sequence SEQ ID NO: 39, VH CDR2 containing the amino acid sequence SEQ ID NO: 40 and VH CDR3 containing the amino acid sequence SEQ ID NO: 41;The VL region contains VL CDR1 containing the amino acid sequence SEQ ID NO: 21, VL CDR2 containing the amino acid sequence SEQ ID NO: 22, and VL CDR3 containing the amino acid sequence SEQ ID NO: 42; or (vi) the VH region contains VH CDR1 containing the amino acid sequence SEQ ID NO: 27, VH CDR2 containing the amino acid sequence SEQ ID NO: 43, and VH CDR3 containing the amino acid sequence SEQ ID NO: 29; and the VL region contains VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 30.

[0173] In some embodiments, the 5T4-ADC may be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), wherein the antibody (Ab) comprises: (a) a VH region comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 46, 50, 53, 13, and 57; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 47, 51, 54, 58, and 61; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 48, 52, 55, and 59; and (b) a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 10, 16, and 21; and (2) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11, and 22. CDR2; and (3) VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 56 and 60.

[0174] In some embodiments, the 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), wherein the antibody (Ab) comprises: (i) a VH region comprising VH CDR1 containing the amino acid sequence SEQ ID NO: 46, VH CDR2 containing the amino acid sequence SEQ ID NO: 47, and VH CDR3 containing the amino acid sequence SEQ ID NO: 48; and a VL region comprising VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 49; or (ii) a VH region comprising VH CDR1 containing the amino acid sequence SEQ ID NO: 50, VH CDR2 containing the amino acid sequence SEQ ID NO: 51, and VH CDR3 containing the amino acid sequence SEQ ID NO: 52; and a VL region comprising VH CDR1 containing the amino acid sequence SEQ ID NO: 49, VH CDR2 ...49, VH CDR2 containing the amino acid sequence SEQ ID NO: 49, VH CDR2 containing the amino acid sequence SEQ ID NO: 49, VH CDR2 containing the amino acid sequence SEQ ID NO: 49, VH CDR2 containing the amino acid sequence SEQ ID NO: 49, VH CDR2 containing the amino acid sequence SEQ ID NO: 49, VH CDR2 containing the amino acid sequence SEQ ID NO: 49, VH CDR2 VLCDR1 containing SEQ ID NO: 10, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VLCDR3 containing the amino acid sequence SEQ ID NO: 49; or (iii) the VH region, which includes VH CDR1 containing the amino acid sequence SEQ ID NO: 53, VH CDR2 containing the amino acid sequence SEQ ID NO: 47, and VH CDR3 containing the amino acid sequence SEQ ID NO: 48; and the VL region, which includes VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 49; or (iv) the VH region, which includes VHCDR1 containing the amino acid sequence SEQ ID NO: 13, VH CDR2 containing the amino acid sequence SEQ ID NO: 54, and VHCDR3 containing the amino acid sequence SEQ ID NO: 55; and the VL region, which includes VL CDR1 containing the amino acid sequence SEQ ID NO: 16, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VL CDR3 containing the amino acid sequence SEQ ID NO: 11. CDR2 and VL CDR3 containing the amino acid sequence SEQ ID NO: 56; or (v) VH region, which contains VH CDR1 containing the amino acid sequence SEQ ID NO: 57, VH CDR2 containing the amino acid sequence SEQ ID NO: 58 and VH CDR3 containing the amino acid sequence SEQ ID NO: 59;The VL region contains VL CDR1 containing the amino acid sequence SEQ ID NO: 21, VL CDR2 containing the amino acid sequence SEQ ID NO: 22, and VL CDR3 containing the amino acid sequence SEQ ID NO: 60; or (vi) the VH region contains VH CDR1 containing the amino acid sequence SEQ ID NO: 46, VH CDR2 containing the amino acid sequence SEQ ID NO: 61, and VH CDR3 containing the amino acid sequence SEQ ID NO: 48; and the VL region contains VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 49.

[0175] In some embodiments, the 5T4-ADC may be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), wherein the antibody (Ab) comprises the sequence of frame 1 (FR1), frame 2 (FR2), frame 3 (FR3), and / or frame 4 (FR4) shown in any of SEQ ID NO: 25, 26, 44, 45, 62, and 63.

[0176] In some implementations, 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), wherein the antibody (Ab) contains a human frame sequence.

[0177] In some embodiments, the 5T4-ADC may be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), wherein the antibody (Ab) comprises: (i) a VH comprising the amino acid sequence SEQ ID NO: 25 and a VL comprising the amino acid sequence SEQ ID NO: 26; (ii) a VH comprising the amino acid sequence SEQ ID NO: 44 and a VL comprising the amino acid sequence SEQ ID NO: 45; or (iii) a VH comprising the amino acid sequence SEQ ID NO: 62 and a VL comprising the amino acid sequence SEQ ID NO: 63.

[0178] This disclosure also provides a pharmaceutical composition comprising a 5T4-ADC, wherein the 5T4-ADC may be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7); and a pharmaceutically acceptable excipient, wherein the antibody (Ab) is as described in any of the embodiments described herein. In some embodiments, the drug / antibody ratio (DAR) of the 5T4-ADC in such pharmaceutical compositions may be from about 1 to about 10, for example, from about 1 to about 4, from about 1 to about 2, from about 1.5 to about 2, or from about 1.75 to about 1.9 DAR.

[0179] This disclosure also provides a method for treating a subject with cancer or tumor, comprising administering a 5T4-ADC to the subject, wherein the 5T4-ADC may be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7); or a pharmaceutical composition comprising a 5T4-ADC of formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7) and a pharmaceutically acceptable excipient, wherein the antibody (Ab) is as described in any embodiment herein. Attached Figure Description

[0180] Figure 1 Provides an exemplary synthetic scheme 1 for a connector-drug of VIA-21.

[0181] Figure 2 Provides an exemplary synthetic scheme 2 for the connector-drug of formula VIa-11.

[0182] Figure 3 Provides an exemplary synthetic scheme 3 for a connector-drug of formula VIb-11.

[0183] Figure 4 Provides an exemplary synthetic scheme 4 for a connector-drug of formula VIb-82.

[0184] Figure 5 Provides an exemplary synthetic scheme 5 for the connector-drug of formula VIb-81.

[0185] Figure 6 Report cell viability of MDA-MB-468 cells treated with 5T4-ADC of mAbA15-VIa-11 or mAbA17-VIa-11 ADC.

[0186] Figure 7Report cell viability of MCF-7 cells treated with 5T4-ADC via mAbA15-VIa-11 or mAbA17-VIa-11 ADC.

[0187] Figure 8 Report cell viability of MDA-MB-468 cells treated with 5T4-ADC of the formula mAbA15-VIb-82 or mAbA17-VIb-82.

[0188] Figure 9 Report cell viability of MCF-7 cells treated with 5T4-ADC of the formula mAbA15-VIb-82 or mAbA17-VIb-82.

[0189] Figure 10 Report cell viability of MDA-MB-231 cells treated with 5T4-ADC of the formula mAbA15-VIb-82 or mAbA17-VIb-82.

[0190] Figure 11 Report cell viability of MCI-H226 cells treated with 5T4-ADC of the formula mAbA15-VIb-82 or mAbA17-VIb-82.

[0191] Figure 12 Report cell viability of BxPC3 cells treated with 5T4-ADC of the formula mAbA15-VIb-82 or mAbA17-VIb-82.

[0192] Figure 13 The mean tumor volume of MCF-7.1 xenograft mice treated with 5T4-ADC of mAbA15-VIa-11 or mAbA17-VIa-11 is reported.

[0193] Figure 14 The mean tumor volume of MCF-7.1 xenograft mice treated with 5T4-ADC of mAbA15-VIb-82 or mAbA17-VIb-82 is reported.

[0194] Figure 15 The mean tumor volume of MCF-7.1 xenograft mice treated with a single dose of 0.5 mg / kg, 2.5 mg / kg, or 5.0 mg / kg of the 5T4-ADC of formula mAbA15-VIb-82 is reported.

[0195] Figure 16 The mean tumor volume of MCF-7.1 xenograft mice treated with two doses (1 week apart) of 0.25 mg / kg, 1.25 mg / kg, or 2.5 mg / kg of the 5T4-ADC of formula mAbA15-VIb-82 is reported.

[0196] Figure 17 The mean tumor volume of MCF-7.1 xenograft mice treated with a single dose of 0.5 mg / kg, 2.5 mg / kg, or 5.0 mg / kg of the 5T4-ADC of formula mAbA17-VIb-82 is reported.

[0197] Figure 18 The mean tumor volume of MCF-7.1 xenograft mice treated with two doses (one week apart) of 0.25 mg / kg, 1.25 mg / kg, or 2.5 mg / kg of the 5T4-ADC of formula mAbA17-VIb-82 is reported.

[0198] Figure 19 The mean tumor volume of MDA-MB-468 xenograft mice treated with 5T4-ADCs of mAbA15-VIa-11, mAbA15-VIb-81, or mAbA15-VIb-82 is reported.

[0199] Figure 20 The mean tumor volume of MDA-MB-231 xenograft mice treated with 5T4-ADC of mAbA15-VIb-82 or mAbA15-VIa-11 is reported.

[0200] Figure 21 The mean tumor volume of NCI-H226 xenograft mice treated with 5T4-ADC of mAbA15-VIb-82 or mAbA15-VIa-11 is reported.

[0201] Figure 22 The mean tumor volume of BxPC3 xenograft mice treated with 5T4-ADC of mAbA15-VIb-82 or mAbA15-VIa-11 is reported.

[0202] Figure 23 The report describes the mean tumor volume in Calu-6 xenograft mice treated with a 5T4-ADC containing mAbA15-VIb-82.

[0203] Figures 24A to 24C The results showed that in the toxicokinetics (TK) study, two ELISA methods were used to analyze the toxicokinetics of a 30 mg / kg sample. Figure 24A ), 60mg / kg Figure 24B ) and 90mg / kg ( Figure 24C The total antibody and total ADC concentration (μg / mL) of rats treated with 5T4-ADC of the formula mAbA15-VIb-82 were quantified. Detailed Implementation

[0204] This disclosure provides antibody-drug conjugates (ADCs) that bind to 5T4 and drugs conjugated thereto (directly or indirectly). Such 5T4-ADCs are suitable for use in compositions and methods for treating, preventing, or alleviating 5T4-mediated diseases, conditions, or disorders (including one or more symptoms of said diseases, conditions, or disorders). 5T4-mediated diseases, conditions, and disorders include a variety of cancers, including (but not limited to) any cancer in which tumor cells express or overexpress 5T4. Additionally, 5T4-ADCs are suitable for killing and / or removing tumor cells. The 5T4-ADCs described herein are suitable for use in compositions and methods for treating cancer.

[0205] The term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group having one to twelve carbon atoms, such as 1 to 10, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 carbon atoms. This term includes, for example, straight-chain and branched-chain hydrocarbon groups, such as methyl (-CH3), ethyl (-CH2CH3), n-propyl (-CH2CH2CH3), isopropyl (-CH(CH3)2), n-butyl (-CH2CH2CH2CH3), isobutyl (-CH2CH(CH3)2), sec-butyl (-CH(CH3)(CH3CH2)), tert-butyl (-C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), and neopentyl (-CH2C(CH3)3).

[0206] The term "substituted alkyl" refers to an alkyl group as defined herein, wherein one or more carbon atoms (other than the C1 carbon atom) in the alkyl chain have been optionally replaced with heteroatoms such as -O-, -N-, -S-, -S(O). n - (where n is 0 to 2), -NR- (where R is hydrogen or alkyl), and having one to five substituents selected from the group consisting of: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl and -NR a R b , where R a With R b They may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0207] The term "alkenyl" refers to a straight-chain or branched divalent aliphatic hydrocarbon group, preferably having one to six, more preferably one to three, carbon atoms, and optionally substituted by one or more groups selected from: -O-, -NR. 10 -、-NR 10 C(O)-、-C(O)NR 10 - and similar groups. This term includes, for example, methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-) and similar groups.

[0208] The term “substituted alkenyl” refers to an alkylene group in which one to three hydrogens are replaced by substituents for carbon as defined below for “substituted”.

[0209] The terms “alkylaryl” or “aryl” refer to the groups -alkylene-aryl and -substituted alkylene-aryl, wherein the alkylene, substituted alkylene and aryl are as defined herein.

[0210] The term "alkoxy" refers to the -O-alkyl group, where alkyl is as defined herein. Alkoxy groups include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, and similar groups. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and ynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and ynyl are as defined herein.

[0211] The term “substituted alkoxy” refers to the following groups: substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O- and substituted alkynyl-O-, wherein the substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0212] The term "alkoxyamino" refers to the group -NH-alkoxy, where the alkoxy group is as defined herein.

[0213] The term “alkylalkoxy” refers to the group -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl, wherein the alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0214] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group having two to six carbon atoms, such as two to four carbon atoms, and having at least one, preferably one to two, unsaturated double bond sites. This term includes, for example, divinyl, allyl, and but-3-en-1-yl. This term includes cis and trans isomers or mixtures of these isomers.

[0215] The term "substituted alkenyl" refers to an alkenyl group as defined herein that has one to five or one to three substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxylamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0216] The term "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group having two to six carbon atoms, preferably two to three carbon atoms, and having at least one, preferably one to two, unsaturated triple bonds. Examples of such alkynyl groups include ethynyl (-C≡CH) and propynyl (-CH2C≡CH).

[0217] The term "substituted alkynyl" refers to an alkynyl group as defined herein, having one to five substituents or one to three substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxylamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0218] The term "acylamino" refers to a group selected from the following: -NR 20 C(O)alkyl, -NR 20 C(O) substituted alkyl groups, NR 20 C(O)cycloalkyl, -NR 20C(O) substituted cycloalkyl groups, -NR 20 C(O)-cycloalkenyl, -NR 20 C(O)-substituted cycloalkenyl, -NR 20 C(O) alkenyl, -NR 20 C(O)-substituted alkenyl, -NR 20 C(O) ynyl group, -NR 20 C(O)-substituted alkynyl groups, -NR 20 C(O)aryl, -NR 20 C(O)-substituted aryl, -NR 20 C(O)-heteroaryl, -NR 20 C(O)-substituted heteroaryl, -NR 20 C(O)-heterocyclic rings, and -NR 20 C(O)-substituted heterocycles, where R 20 It is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle are as defined herein.

[0219] The term "acyl" refers to a group selected from the following: HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, etc. The terms "aryl-C(O)-", "heteroaryl-C(O)-", "substituted heteroaryl-C(O)-", "heterocyclic-C(O)-", and "substituted heterocyclic-C(O)-" refer to alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl groups include "acetyl" CH3C(O)-.

[0220] The term "aminocarbonyl" or "aminoacyl" refers to the group -C(O)NR. 21 R 22 , where R 21 and R 22 Independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle, wherein R 21 and R 22Optionally, it is combined with the nitrogen to which it is attached to form a heterocyclic group or a substituted heterocyclic group, wherein the alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic groups are as defined herein.

[0221] The term “acyloxy” means a group selected from the following groups: alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O- and heterocyclic-C(O)O-, wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl and heterocyclic are as defined herein.

[0222] The term "aminosulfonyl" refers to the group -SO2NR. 21 R 22 , where R 21 and R 22 Independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle, wherein R 21 and R 22 Optionally, it may combine with the nitrogen to which it is attached to form a heterocyclic group or a substituted heterocyclic group, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic as defined herein.

[0223] The term "sulfonylamino" refers to the group -NR 21 SO2R 22 , where R 21 and R 22 Independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle, wherein R 21 and R 22 Optionally bonded to the atoms to which it is attached, it forms a heterocyclic group or a substituted heterocyclic group, wherein the alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic groups are as defined herein.

[0224] The term "aryl" refers to a monovalent aromatic carbocyclic group having six to eighteen carbon atoms, either in the form of a single ring (such as phenyl) or in the form of a ring system with multiple fused rings (examples of such aromatic ring systems include naphthyl, anthracel, and dihydroindenyl), wherein the fused rings may or may not be aromatic, the limitation being that the connecting points are atoms via the aromatic ring. This term includes, for example, phenyl and naphthyl. Unless otherwise limited by the definition of aryl substituents, such aryl groups may optionally be substituted with one to five or one to three substituents selected from acyloxy, hydroxyl, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkylaryl, aryl, aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0225] The term “aryloxy” refers to a group -O-aryl, wherein aryl, as defined herein, includes, for example, phenoxy, naphthoxy and similar groups, including optionally substituted aryl groups as defined herein.

[0226] The term "amino" refers to the -NH2 group.

[0227] The term "substituted amino" refers to a group -NRR, wherein each R is independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic, with the restriction that at least one R is not hydrogen.

[0228] The term "azido" refers to the group -N3.

[0229] The terms "carboxyl", "carboxy", or "carboxylate" refer to -CO2H or its salt.

[0230] The terms "carboxyl ester" or "carboxy ester," or "carboxyalkyl" or "carboxylalkyl," refer to a group selected from the following: -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-ynyl, -C(O)O-substituted ynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl ... -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclic and -C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

[0231] The terms "cyano" or "nitrile" refer to the -CN group.

[0232] The term "cycloalkyl" refers to a cycloalkyl group with three to ten carbon atoms, having one or more rings, including fused, bridged, and helical ring systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and similar groups.

[0233] The term "substituted cycloalkyl" refers to a cycloalkyl group having one to five substituents or one to three substituents, wherein the substituents are independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0234] The term "cycloalkenyl" refers to a non-aromatic cycloalkyl group with three to ten carbon atoms, having one or more rings and at least one double bond, preferably one or two double bonds.

[0235] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having one to five substituents or one to three substituents, wherein the substituents are independently selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, ketone, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxylamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0236] The terms "halogen" and "halogen" refer to fluorine, chlorine, bromine, or iodine.

[0237] The terms "hydroxyl" and "hydroxyl" refer to the -OH group.

[0238] The term "heteroaryl" refers to an aromatic group having one to fifteen carbon atoms, such as one to ten carbon atoms, and one to ten heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within a ring. Such heteroaryls may have a single ring (such as pyridyl, imidazolyl, or furanyl) or multiple fused rings (e.g., groups such as indoleazinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothiophene), wherein at least one ring within the ring system is aromatic. To satisfy valence requirements, any heteroatom in such a heteroaryl ring may or may not be bonded to H or a substituent, such as an alkyl group or other substituents as described herein. In some embodiments, the nitrogen and / or sulfur ring atoms in the heteroaryl group are optionally oxidized to provide an N-oxide (N→O), sulfinyl, or sulfonyl moiety. This term includes, for example, pyridyl, pyrroleyl, indoleyl, thiophene, and furanyl. Unless otherwise limited by the definition of heteroaryl substituents, such heteroaryl groups may optionally be substituted with one to five substituents or one to three substituents, wherein said substituents are independently selected from acyloxy, hydroxyl, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkylaryl, aryl, Aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl and trihalomethyl.

[0239] The term "heteroarylalkyl" refers to a group comprising an alkylene group bonded to a heteroaryl group, wherein the alkylene group and the heteroaryl group are as defined herein. This term includes, for example, pyridylmethyl, pyridylethyl, indolylmethyl, and similar groups.

[0240] The term "heteroaryloxy" refers to -O-heteroaryl.

[0241] The terms “heterocycle,” “heterocyclic,” “heterocyclic alkyl,” and “heterocyclic group” are used interchangeably herein to refer to a saturated or unsaturated group having a single ring or multiple fused rings (including fused, bridged, and helicaled ring systems) having three to twenty ring atoms, including one to ten heteroatoms. These ring atoms are selected from nitrogen, sulfur, and oxygen, wherein in fused ring systems, one or more rings may be cycloalkyl, aryl, or heteroaryl, limited by the connection point being via a non-aromatic ring. In some embodiments, the nitrogen and / or sulfur atoms in the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety. To satisfy valence requirements, any heteroatom in such a heterocycle may or may not be bonded to one or more H atoms or one or more substituents, such as alkyl groups or other substituents as described herein.

[0242] Examples of heterocyclic and heteroaryl compounds include, but are not limited to, aza-butane, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indoleazine, isoindole, indole, dihydroindole, indazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, caroline, phenanthridine, acridine, phenanthroxaline, isothiazine, phenazine, isoxazine, phenoxazine, phenanthridine Thiazines, imidazolines, imidazolines, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazoles, thiazolines, thiophene, benzo[b]thiophene, morpholino, thiomorpholino (also known as thiomorpholino), 1,1-dioxathiomorpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl and similar groups.

[0243] Unless otherwise limited by the definition of heterocyclic substituents, such heterocyclic groups may optionally be substituted with one to five or one to three substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclicoxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and fused heterocycles.

[0244] The term "hydroxyamino" refers to the group -NHOH.

[0245] The term "nitro" refers to the group -NO2.

[0246] The term "oxo group" refers to the oxygen atom (=O) in a double bond.

[0247] The term "sulfonyl" refers to the group -SO2-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, -SO2-substituted alkenyl, -SO2-cycloalkyl, -SO2-substituted cycloalkyl, -SO2-cycloalkenyl, -SO2-substituted cycloalkenyl, -SO2-aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2-substituted heteroaryl, -SO2-heterocyclic, and -SO2-substituted heterocyclic, wherein the alkyl, substituted alkyl, alkenyl, substituted alkenyl, ynyl, substituted ynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl groups include, for example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0248] The term "thiol" refers to the -SH group.

[0249] The term "thioxo" or "thioketo" refers to the sulfur atom (=S) in a double bond.

[0250] The term "alkylthio" or "thioalkoxy" refers to the group -S-alkyl, wherein the alkyl group is as defined herein. In some embodiments, sulfur may be oxidized to -S(O)-. The sulfoxide may be present in one or more stereoisomers.

[0251] The term "substituted thioalkoxy" refers to an alkyl group that is -S-substituted.

[0252] The term “thioaryloxy” refers to the aryl group -S-, wherein the aryl group is as defined herein, including optionally substituted aryl groups as also defined herein.

[0253] The term “thioheteroaryloxy” refers to the heteroaryl-S- group, wherein the heteroaryl is as defined herein, including optionally substituted aryl groups as also defined herein.

[0254] The term “thioheterocyclic group” refers to a heterocyclic group -S-, wherein the heterocyclic group is as defined herein, including optionally substituted heterocyclic groups as also defined herein.

[0255] In addition to the disclosure herein, the term “substituted” when used to modify a specified group or radical may also mean that one or more hydrogen atoms in the specified group or radical are independently replaced by the same or different substituents as defined below.

[0256] In addition to the groups disclosed herein with respect to individual terms, the group used to replace one or more hydrogen atoms on a saturated carbon atom in a specified group or radical (any two hydrogen atoms on a single carbon may be =O, =NR). 70 =N-OR 70 Substituents (=N2 or =S substitutions) are also -R unless otherwise specified. 60 , halogen, =O, -OR 70 -SR 70 、-NR 80 R 80 Trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 -SO2O - M + -SO2OR 70 -OSO2R 70 -OSO2O - M + -OSO2OR 70 -P(O)(O) - )2(M + )2、-P(O)(OR 70 )O - M + -P(O)(OR) 70 )2、-C(O)R 70 -C(S)R 70 -C(NR) 70 )R 70 -C(O)O - M + -C(O)OR 70 -C(S)OR 70 -C(O)NR 80 R 80 -C(NR) 70 )NR 80 R 80 、-OC(O)R 70 -OC(S)R 70 -OC(O)O - M + 、-OC(O)OR 70 -OC(S)OR 70 、-NR 70 C(O)R 70 、-NR 70 C(S)R 70 、-NR 70 CO2-M + 、-NR 70 CO2R 70-NR 70 C(S)OR 70 -NR 70 C(O)NR 80 R 80 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 Choose from the group consisting of: optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl, each R 70 Independently hydrogen or R 60 Each R 80 Independently for R 70 , or alternatively two R 80′ Together with the nitrogen atom it is bonded to, it forms a 5-, 6-, or 7-membered heterocyclic alkyl group, wherein the heterocyclic alkyl group may optionally include one to four identical or different additional heteroatoms selected from the group consisting of O, N, and S, wherein N may have -H or C1-C6 alkyl substitutions; and each M + Each M is a counterion with a single net positive charge. + It can be independently, for example, an alkali metal ion, such as K. + Na + Li + Ammonium ions, such as + N(R 60 )4; or alkaline earth metal ions, such as [Ca 2+ ] 0.5 、[Mg 2+ ] 0.5 or[Ba 2+ ] 0.5 ("Subscript 0.5 means that one of the counter ions of such divalent alkaline earth metal ions can be the ionized form of the compound of the present invention, and the other is a typical counter ion (such as chloride ion), or that the two ionized compounds disclosed herein can act as counter ions of such divalent alkaline earth metal ions, or that the dual ionized compound of the present invention can act as a counter ion of such divalent alkaline earth metal ions.") As a specific example, -NR 80 R 80 The intended components include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl, and N-morpholinyl.

[0257] In addition to what is disclosed herein, the substituents on the hydrogen atom of the unsaturated carbon atom in the "substituted" alkenyl, ynyl, aryl, and heteroaryl groups are also -R unless otherwise stated.60 、halo group, -O - M + 、-OR 70 、-SR 70 、-S - M + 、-NR 80 R 80 、trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 、-SO3-M + 、-SO3R 70 、-OSO2R 70 、-OSO3-M + 、-OSO3R 70 、-PO3 2 (M + )2、-P(O)(OR 70 )O - M [[ID=...]] + 、-P(O)(OR 70 )2、-C(O)R 70 、-C(S)R 70 、-C(NR 70 )R 70 、-CO2-M + 、-CO... 70 、-C(S)OR 70 、-C(O)NR 80 R 80 、-C(NR 70 )NR 80 R 80 、-OC(O)R 70 、-OC(S)R 70 、-OCO2 - M + 、-OCO2R 70 、-OC(S)OR 70 、-NR 70 C(O)R 70 、-NR 70 C(S)R 70 、-NR 70 CO2-M + 、-NR 70 CO2R 70 、-NR {...} 70 C(S)OR 70 、-NR 70 C(O)NR 80 R 80 、-NR 70 C(NR [[ID=...]] 70 )R (注:由于原始文本中部分内容重复且格式不清晰,以上翻译仅按照指令要求进行了逐行翻译,可能存在部分内容在整体语境中含义不连贯的情况。)70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 、R 70 、R 80 and M + As defined herein, the restriction is that, in the case of a substituted alkenyl or ynyl group, the substituent is not -O. - M + 、-OR 70 -SR 70 or -SM + .

[0258] In addition to the groups disclosed herein with respect to individual terms, the substituents on the hydrogen atom of the nitrogen atom in "substituted" heteroalkyl and cyclohexaalkyl groups are also -R unless otherwise specified. 60 -OM + 、-OR 70 -SR 70 -SM + 、-NR 80 R 80 Trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 -S(O)2O - M + -S(O)2OR 70 、-OS(O)2R 70 -OS(O)2O - M + -OS(O)2OR 70 -P(O)(O)2(M + )2、-P(O)(OR 70 )O - M + -P(O)(OR) 70 )(OR 70 -C(O)R 70 -C(S)R 70 -C(NR) 70 )R 70 -C(O)OR 70 -C(S)OR 70 -C(O)NR 80 R 80 -C(NR) 70 )NR 80 R 80 、-OC(O)R 70 -OC(S)R 70 、-OC(O)OR70 -OC(S)OR 70 、-NR 70 C(O)R 70 、-NR 70 C(S)R 70 、-NR 70 C(O)OR 70 、-NR 70 C(S)OR 70 、-NR 70 C(O)NR 80 R 80 、-NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 、R 70 、R 80 and M + As defined in this article.

[0259] The substituted group may have one, two, three or four substituents, such as one, two or three substituents, or one or two substituents, or one substituent.

[0260] Unless otherwise specified, the naming of substituents not explicitly defined herein is achieved by naming the terminal portion of the functional group, followed by naming the adjacent functional group at the junction. For example, the substituent “arylalkoxycarbonyl” refers to the group (aryl)-(alkyl)-OC(O)-.

[0261] For any group containing one or more substituents disclosed herein, it should be understood that such group does not contain any substitution or substitution pattern that is sterically impractical and / or synthetically infeasible. Furthermore, the compounds disclosed herein include all stereochemical isomers arising from the substitution of these compounds.

[0262] The term "subject" refers to human and non-human subjects, especially mammalian subjects.

[0263] As used herein, the term "treating" or "treatment" means treating or treating a disease or medical condition in a subject (such as a mammal, particularly a human) including: (a) preventing the subject from developing a disease or medical condition, such as preventative treatment of the subject; (b) improving the subject's disease or medical condition, such as eliminating or promoting the remission of the subject's disease or medical condition; (c) inhibiting the subject's disease or medical condition, for example by slowing or halting the development of the subject's disease or medical condition; or (d) alleviating the symptoms of the subject's disease or medical condition. In some embodiments, the term "treating" or "treatment" excludes preventative treatment.

[0264] As used herein, the term "native amino acid sequence" refers to the polypeptide amino acid sequence before modification to include the modified amino acid residues.

[0265] The terms "amino acid analog," "non-natural amino acid," and their analogues are used interchangeably and include amino acid-like compounds that are structurally and / or have a similar overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include natural amino acids having modified side chains or backbones. Amino acid analogs also include amino acid analogs having the same stereochemical configuration as the naturally occurring D and L-forms of amino acid analogs. In some embodiments, amino acid analogs share a backbone structure and / or the side chain structure of one or more natural amino acids, differing only in one or more modified groups in the molecule. Such modifications may include, but are not limited to, substitution of an atom (such as N) for an atom (such as S), addition of a group (such as methyl or hydroxyl and similar groups) or an atom (such as Cl or Br and similar atoms), deletion of a group, substitution of a covalent bond (substitution of a single bond for a double bond, and the like), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids and α-amino acids, and analogs thereof.

[0266] The term "amino acid side chain" is used to refer to a substituent attached to the α-carbon of an amino acid residue, including native amino acids, non-native amino acids, and amino acid analogs. Amino acid side chains may also include those described in the context of modified amino acids and / or conjugates as discussed herein.

[0267] The term "carbohydrate" is used to refer to monomeric units and / or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term "sugar" is used to refer to smaller carbohydrates, such as monosaccharides and disaccharides. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemical reaction), or absent (e.g., eliminated or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and can be adapted for use in the compounds and conjugates disclosed herein.

[0268] The term "glycoside" or "glycosyl group" refers to a sugar molecule or group bound to a part via a glycosidic bond. For example, the part to which a glycoside is bound can be a cleavable linker as described herein. Glycosidic bonds can link glycosides to other parts via various types of bonds, such as (but not limited to) O-glycosidic bonds (O-glycosides), N-glycosidic bonds (glycosylamines), S-glycosidic bonds (thioglycosides), or C-glycosidic bonds (C-glycosides or C-glycosyl groups). In some cases, the glycoside can cleave from the part to which it is attached, such as through chemically mediated or enzyme-mediated hydrolysis.

[0269] Unless otherwise specified, the term "5T4" refers to a polypeptide (“polypeptide” and “protein” are used interchangeably herein) or any native 5T4 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys, cyno)), dogs, and rodents (e.g., mice and rats). 5T4, also known as “5T4 carcinoembryonic trophoblast glycoprotein” or “Wnt activation inhibitor 1” or “5T4 carcinoembryonic antigen” or “WAIF1” or “M6P1” or “5T4-AG” or “5T4AG” or “TPBG”, is a glycoprotein encoded by the TPBG gene. The term 5T4 encompasses “full-length” 5T4, as well as any form of 5T4 or any fragment thereof produced through cellular processing. In some embodiments, 5T4 includes a signal sequence. In some embodiments, 5T4 does not include a signal sequence. In some implementations, the term 5T4 refers to the segment of full-length 5T4 containing the 5T4 extracellular domain. The term 5T4 also encompasses naturally occurring variants of 5T4, such as SNP variants, splice variants, and paired gene variants. The full-length amino acid sequence of human 5T4 is provided below (illustrative signal sequence = italicized text; illustrative extracellular domain = underlined text):

[0270]

[0271] The following is the full-length amino acid sequence of 5T4 in the cynomolgus monkey (cyno) (illustrative signal sequence = italic text; illustrative extracellular domain = underlined text):

[0272]

[0273] The following is the full-length amino acid sequence of mouse 5T4 (illustrative signal sequence = italic text; illustrative extracellular domain = underlined text):

[0274]

[0275]

[0276] The terms “antibody,” “immunoglobulin,” or “Ig” are used interchangeably herein and in the broadest sense, and particularly encompass, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions having multiple or single epitope specificity, recombinant antibodies, single-domain antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human forms of antibodies having full-length heavy and / or light chains. Antibodies also include a single antibody domain retaining the 5T4 binding signature, as well as antibody fragments (and / or polypeptides containing antibody fragments). Non-limiting examples of antibody fragments include antigen-binding regions and / or effector regions of antibodies, such as Fab, Fab′, F(ab′)2, Fv, scFv, (scFv)2, single-chain antibody molecules, bivariate domain antibodies, monovariate domain antibodies, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, bifunctional antibodies, di-bifunctional antibodies, disulfide-linked Fv(dsFv), monovariate antibodies (e.g., nanobodies), or other fragments (e.g., fragments composed of variable regions of non-covalently coupled heavy and light chains). Generally, the variable (V) region can be any suitable arrangement of immunoglobulin heavy chain (VH) and / or light chain (VL) variable regions. For example, antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers. Thus, for example, the V region can be a dimer and contain VH-VH, VH-VL, or VL-VL dimers binding to 5T4. In any implementation, the VH and VL regions may be covalently coupled directly or via a linker to form a single-chain Fv (scFv). For ease of reference, the scFv protein mentioned herein is included in the category of “antibody fragments.” Another form of antibody fragment is a peptide containing one or more complementarity-determining regions (CDRs) of an antibody. CDRs (also known as “minimum recognition units” or “hypervariates”) can be obtained by constructing a polynucleotide encoding one or more CDRs of interest.Such polynucleotides are prepared as follows: for example, using mRNA from antibody-producing cells as a template, the variable region is synthesized by polymerase chain reaction (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies", in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), p. 166, Cambridge University Press (1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies", in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), p. 137, Wiley-Liss, Inc. (1995)). Antibody fragments may be incorporated, for example, into single-domain antibodies, maximal antibodies, microantibodies, intracellular antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, variable regions (v-NARs) of neoantigen receptors, and double single-chain Fv regions (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9): 1126-1136, 2005). In some embodiments, antibodies containing VH and / or VL contain light chain and / or heavy chain constant regions, such as one or more constant regions, including one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions. In some embodiments, antibodies may include epitope-binding fragments of any of the above. The antibodies described herein may belong to any class of immunoglobulin molecules (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0277] As used herein, the term “monospecific” means that an antibody has one or more binding sites, each binding to the same epitope of the same antigen.

[0278] The term "bispecific" refers to an antibody that specifically binds to at least two different antigenic determinants, for example, each consisting of two binding sites formed by an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), which bind to different antigens or to different epitopes on the same antigen. Such bispecific antibodies may have a 1+1 format (containing one binding site for a first antigen or epitope and one binding site for a second antigen or epitope). Other bispecific antibody formats may be 2+1 or 1+2 formats (containing two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 formats (containing two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific antibody contains two antigen-binding sites, each binding site can bind to different antigenic determinants. Such bispecific antibodies can bind to two different epitopes on the same antigen (e.g., epitopes on 5T4).

[0279] In the context of two or more nucleic acids or peptides, the term "consistent" or "consistency" percentage refers to the identical or having a specified percentage of identical nucleotide or amino acid residues when compared and aligned according to maximum consistency (with gaps introduced where necessary), without considering any conserved amino acid substitutions as part of sequence consistency. Consistency percentage can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are well known in the art for obtaining amino acid or nucleotide sequence alignments. These algorithms and software include (but are not limited to) the Basic Local Alignment Search Tool (BLAST), ALIGN, MegAlign, BestFit, the GCG Wisconsin suite, and variations thereof. In some embodiments, two nucleic acids or peptides are substantially consistent, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue consistency when compared and aligned according to maximum consistency, as measured using sequence comparison algorithms or by visual inspection. In some embodiments, there is consistency in amino acid sequence regions of at least about 10 residues, at least about 20 residues, at least about 40 to 60 residues, at least about 60 to 80 residues, or any integer value therebetween. In some embodiments, consistency exists in regions longer than 60 to 80 residues (e.g., at least about 80 to 100 residues), and in some embodiments, the sequences are substantially consistent across the full length of the compared sequence (e.g., the coding region of a target protein or antibody). In some embodiments, there is consistency in nucleotide sequence regions of at least about 10 bases, at least about 20 bases, at least about 40 to 60 bases, at least about 60 to 80 bases, or any integer value therebetween. In some embodiments, consistency exists in regions longer than 60 to 80 bases (e.g., at least about 80 to 1000 or more bases), and in some embodiments, the sequences are substantially consistent across the full length of the compared sequence (e.g., the nucleotide sequence encoding the protein of interest).

[0280] "Conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue with a side chain having similar chemical characteristics. Families of amino acid residues with similar side chains are generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, phenylalanine replacing tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of peptides, soluble proteins, and / or antibodies of this disclosure do not eliminate the binding of the peptide, soluble protein, or antibody containing the amino acid sequence to the target binding site. Methods for identifying conservative amino acid substitutions that do not eliminate binding are well known in the art.

[0281] The term "peptide" refers to an amino acid polymer of any length. The polymer may be linear or branched, may contain modified amino acids, and may include non-amino acids (e.g., substituted with non-amino acids). The term also covers amino acid polymers that have been naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as linking or conjugating (directly or indirectly) to a portion of a component such as a labeled component or a drug (e.g., a toxin). The definition also includes, for example, one or more analogues containing amino acids (including, for example, non-natural amino acids) and other modified peptides known in the art. It should be understood that while the peptides of this disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, the peptides may exist in single-chain or single-chain dimer form.

[0282] As used herein, "antigen" is a portion or molecule containing an epitope that an antibody can specifically bind to. Therefore, an antigen can be bound by an antibody. In some embodiments, the antigen bound by the antibody described herein is 5T4 (e.g., human 5T4), or a fragment thereof, such as a fragment containing one or more regions of 5T4.

[0283] As used herein, "epitope" is a term used in the art and refers to a localized region of an antigen that can be specifically bound by an antibody. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. For example, in the case of a polypeptide antigen, the epitope can be an adjacent amino acid of the polypeptide ("linear" epitope) or the epitope can comprise amino acids from two or more non-adjacent regions of the polypeptide ("conformational," "non-linear," or "discontinuous" epitopes), such as human 5T4. Those skilled in the art will understand that, in general, a linear epitope may or may not depend on secondary, tertiary, or quaternary structures. For example, in some embodiments, the antibody binds to a set of amino acids, regardless of whether it folds into a native three-dimensional protein structure. In other embodiments, the antibody requires the amino acid residues constituting the epitope to exhibit a specific conformation (e.g., bending, twisting, turning, or folding) in order to recognize and bind to the epitope.

[0284] An antibody binds to a reference antibody to an "epitope," "substantially identical epitope," or "identical epitope." The most widely used rapid method for determining whether two antibodies bind to the same, overlapping, or adjacent epitopes in three-dimensional space is a competition assay, which can be configured in many different formats, such as using labeled antigens or labeled antibodies. In some assays, the antigen is immobilized on a 96-well plate or expressed on a cell surface, and radioactive, fluorescent, or enzyme labeling is used to measure the ability of the unlabeled antibody to block the binding of the labeled antibody.

[0285] "Epitope grouping" is a method for grouping antibodies based on the epitopes they recognize. More specifically, epitope grouping includes methods and systems that utilize a combination of competitive analysis and computational methods to distinguish the epitope recognition characteristics of different antibodies, thereby clustering antibodies based on their epitope recognition characteristics and identifying antibodies with different binding specificities. Further details regarding epitope grouping and methods for determining antibody epitope binding are described herein, as shown in Example 5.

[0286] As used herein, the terms “specific binding,” “specific recognition,” “immune-specific binding,” “selective binding,” “immune-specific recognition,” and “immune specificity” are similar terms in the context of antibodies and refer to molecules that bind to antigens (e.g., epitopes), as understood by those skilled in the art.

[0287] In some implementations, "specific binding" means, for example, that the interaction between a peptide or molecule and an epitope, protein, or target molecule is more frequent, faster, longer-lasting, more affinity, or a combination thereof than with alternative substances (including related and unrelated proteins). For example, a molecule that specifically binds to an antigen may often bind to other peptides or peptides with lower affinity, such as through immunoassays, BIACORE, etc. TMThe binding specificity is determined using a KinExA 3000 instrument (Sapidyne Instruments, Boise, ID.), an OctetQK384 system (ForteBio, Menlo Park, CA), or other analyses known in the art. In some embodiments, the antibody or antigen-binding domain binds to or specifically binds to an antigen when its affinity for that antigen is higher than that for any cross-reactive antigen, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, the specific or selective reaction is at least twice the background signal or noise and may be more than ten times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul, ed., 2nd ed., 1989). In some embodiments, the degree to which an antibody or antigen-binding region binds to a “non-target” protein is less than about 10% of the binding of the antibody or antigen-binding region to its target antigen, as determined, for example, by fluorescence activated cell sorting (FACS) analysis or RIA. In some embodiments, the KA of a molecule specifically bound to one antigen is at least 2 log, 2.5 log, 3 log, 4 log, or greater than the KA of the molecule bound to another antigen. In some embodiments, the molecule specifically bound to the antigen does not cross-react with other proteins. In another specific embodiment, the molecule specifically bound to the antigen does not cross-react with non-5T4 proteins. In some embodiments, "specific binding" means, for example, a peptide or molecule with a KA of about 0.1 mM or less, but more typically less than about 1 μM. D Binding to a protein or target. In some embodiments, "specific binding" means that the peptide or molecule binds at a concentration of at least about 0.1 μM or less, at least about 0.01 μM or less, or at least about 1 nM or less. DTarget binding. Due to sequence similarity between homologous proteins of different species, specific binding may include peptides or molecules recognizing proteins or targets of more than one species. Similarly, due to homology in certain regions of peptide sequences of different proteins, specific binding may include peptides or molecules recognizing more than one protein or target. It should be understood that in some embodiments, an antibody or molecule that specifically binds to a first target may or may not specifically bind to a second target. Therefore, “specific binding” does not necessarily require (although it may include) exclusive binding, such as binding to a single target. Thus, in some embodiments, a peptide or molecule may specifically bind to more than one target. In some embodiments, multiple targets may be bound by the same antigen-binding site on the peptide or molecule. For example, in some embodiments, an antibody may contain two identical antigen-binding sites, each specifically binding to the same epitope on two or more proteins. In alternative embodiments, the antibody may be bispecific and contain at least two antigen-binding sites with different specificities. Generally, but not necessarily, the term “binding” means “specific binding.”

[0288] The term "binding affinity" generally refers to the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding complex (e.g., an antigen, such as 5T4). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its binding complex Y can typically be determined by the dissociation constant (K). D () indicates. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens more quickly and tend to remain bound for a longer period. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. In one embodiment, “K” D "or "K D The K value can be measured via biolayer interferometry (BLI) using a system such as the Octet QK384 (ForteBio, Menlo Park, CA). Alternatively, it can be measured in radiolabeled antigen binding assay (RIA). D For example, using the Fab plate of the antibody of interest and its antigen (Chen et al., (1999) J. Mol Biol 293: 865-881) or using surface plasmon resonance (SPR) analysis, via BIACORE TM The execution, for example, using BIACORE TM -2000 or BIACORE TM-3000 (BIAcore, Inc., Piscataway, NJ). "On-rate" or "Rate of Association" or "Association Rate" or "k on "and dissociation rate (offrate) or dissociation rate (rate of dissociation) or dissociation rate (k)" off "The same SPR or BLI technology described herein can also be used, using, for example, the Octet QK384 system (ForteBio, Menlo Park, CA) or BIACORE." TM -2000 or BIACORE TM -3000 (BIACORE) TM , Inc., Piscataway, NJ) determination.

[0289] The term "competition," when used in the context of 5T4 antibodies, describes a situation where one binder, in the presence of another binder, is at least partially inhibited from binding to an epitope or binding site due to the binding of the other binder. Competition can be determined by analysis in which the binder under investigation prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand, or a reference antigen-binding protein, such as a reference antibody) to a common antigen (e.g., 5T4). Various types of competitive binding assays can be used to determine whether a test antibody competes with a reference molecule for binding to 5T4 (e.g., human 5T4). Examples of analyses that can be used include solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, for example, Stahli et al. (1983), Methods in Enzymology 9: 242-253); direct solid-phase biotin-antibiotin EIA (see, for example, Kirkland et al. (1986), J. Immunol. 137: 3614-3619 or Cheung et al. (1990), Virology 176: 546-552); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using I-125 labeling (see, for example, Morel et al. (1988), Molec. Immunol. 25: 7-15); and direct labeling RIA. (Moldenhauer et al., (1990) Scand. J. Immunol. 32: 77-82). Typically, such analyses involve using a purified antigen (e.g., 5T4, such as human 5T4) bound to a solid surface or cell carrying an unlabeled test antigen-binding protein (e.g., a test 5T4 antibody or ADC) or a labeled reference antigen-binding protein (e.g., a reference 5T4 antibody or ADC). Competitive inhibition can be measured by determining the amount of labeling bound to the solid surface or cell in the presence of the test immunoglobulin. The test antigen-binding protein is often present in excess. Antibodies identified by competitive analysis (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and / or antibodies that bind to a neighboring epitope, which is sufficiently close to the epitope bound by the reference antibody to cause steric hindrance (e.g., similar or overlapping epitopes). Typically, when a competitive antibody is present in excess, it will inhibit the specific binding of the reference antibody to the common antigen by at least 20%, such as at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.In some implementations, the combination is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater.

[0290] The terms “constant region” and “constant domain” are used interchangeably herein and are well-known antibody terms in the art, referring to antibody moieties, such as the carboxyl-terminal portions of the light and / or heavy chains that do not directly participate in antibody binding to antigens but can exhibit various effector functions (such as interaction with Fc receptors). The terms also include portions of immunoglobulin molecules that have a generally more conserved amino acid sequence compared to the variable regions of immunoglobulins.

[0291] Antibody "effective function" refers to those biological activities attributable to the antibody's Fc region (e.g., the native Fc region or the Fc region with amino acid sequence variations) and varying from antibody isotype to antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0292] In this document, the term "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 (EU numbering system) or Pro230 (EU numbering system) to its C-terminus. The C-terminal lysine of the Fc region (residue 447 in the EU numbering system) can be removed, for example, during antibody preparation or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Illustrative Fc region sequences are provided below (CH2 domain = bold text; CH3 domain = underlined text):

[0293]

[0294] The “functional Fc region” possesses the “effective function” of the native Fc region. Exemplary “effective functions” include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR) and analogues. Such effector functions typically require the combination of the Fc region with a binding region or binding domain (e.g., antibody variable region or domain) and can be assessed using the various analyses disclosed.

[0295] The “native sequence Fc region” comprises an amino acid sequence that is identical to the naturally occurring Fc region and has not been manipulated, modified, and / or altered (e.g., isolated, purified, selected, or combined with other sequences, such as variable region sequences). The native sequence human Fc region includes the native sequence human IgG1 Fc region (non-A and A isoforms); the native sequence human IgG2 Fc region; the native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region; as well as naturally occurring variants thereof.

[0296] The “variant Fc region” comprises an amino acid sequence that differs from the amino acid sequence of the native Fc region due to at least one amino acid modification (e.g., substitution, addition, or deletion, preferably one or more amino acid substitutions). In some embodiments, the variant Fc region has at least one amino acid substitution in the native Fc region or the Fc region of the parent peptide, for example, about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, compared to the native Fc region or the Fc region of the parent peptide. The variant Fc region described herein may have at least about 80% homology with the native Fc region and / or the Fc region of the parent peptide, or at least about 90% homology with it, for example, at least about 95% homology with it. The variant Fc region described herein may have loss-of-effect function (e.g., silencing Fc). An exemplary variant Fc region (“silencing Fc”) sequence is provided below (CH2 domain = bold text, where amino acid changes are underlined; CH3 domain = underlined text):

[0297]

[0298] When used in conjunction with antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50 to 70 kDa, wherein the amino-terminal portion includes a variable region of approximately 120 to 130 or more amino acids, and the carboxyl-terminal portion includes one or more constant regions. Based on the amino acid sequence of the constant regions, the term "heavy chain" can refer to any different type, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including IgG subclasses such as IgG1, IgG2, IgG3, and IgG4.

[0299] As used herein, the term "light chain," when used in conjunction with an antibody, can refer to a polypeptide chain of approximately 25 kDa, wherein the amino-terminal portion comprises a variable region of approximately 100 to approximately 110 or more amino acids, and the carboxyl-terminal portion comprises a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant region, two distinct types exist, referred to as kappa(K) or lambda(λ). The amino acid sequences of light chains are well known in the art.

[0300] The terms “antigen-binding fragment,” “antigen-binding domain,” “antigen-binding region,” and similar terms refer to a portion of an antibody containing amino acid residues that interact with an antigen, thereby imparting specificity and affinity for the antigen to the binding fragment or region (e.g., a CDR). As used herein, “antigen-binding fragment” includes “antibody fragment” which contains a portion of an antibody, including one or more CDRs, such as an antigen-binding region or variable region of an antibody.

[0301] The antibodies described herein include (but are not limited to) synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, single-chain Fv (scFv) (e.g., including monospecific, bispecific and similar), camelified antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0302] In some embodiments, the antibodies described herein include immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, such as molecules containing one or more antigen-binding sites that bind to 5T4.

[0303] The 5T4 antibody described herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In some embodiments, the 5T4 antibody described herein is an IgG antibody (e.g., human IgG) or a class (e.g., human IgG1, IgG2, IgG3, or IgG4) or a subclass thereof.

[0304] In some embodiments, the 5T4 antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, wherein the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical. In some embodiments, the H and / or L chains contain constant regions, such as human constant regions. In some embodiments, the L chain constant region of the 5T4 antibody is a K or λ light chain constant region, such as a human K or λ light chain constant region. In some embodiments, the H chain constant region of the 5T4 antibody contains a γ heavy chain constant region, such as a human γ heavy chain constant region. In some embodiments, the 5T4 antibody contains an IgG constant region, such as a human IgG constant region (e.g., IgG1, IgG2, IgG3, and / or IgG4 constant regions).

[0305] As used herein, "5T4 antibody" and "antibody bound to 5T4" are used interchangeably and refer to an antibody that preferentially binds to 5T4. An antibody or fragment thereof may preferentially bind to 5T4, such as human 5T4, meaning that the antibody or fragment thereof binds to 5T4, such as human 5T4, with a greater affinity than it binds to an unrelated control protein. For example, the antibody or fragment thereof may specifically recognize and bind to 5T4 or a portion thereof. "Specific binding" means that the affinity of the 5T4 antibody or fragment thereof for 5T4 is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than its affinity for an unrelated control protein (e.g., hen oocyte albumin lysozyme). In some embodiments, the 5T4 antibody or fragment thereof may exclusively and substantially bind to 5T4 (e.g., capable of distinguishing 5T4 from other known peptides, for example, based on measurable differences in binding affinity). In some implementations, the 5T4 antibody can react with 5T4 sequences other than the human 5T4 sequence (e.g., the cynomolgus monkey 5T4 sequence).

[0306] The terms “variable region” and “variable domain” are used interchangeably and refer to a portion of the light or heavy chain of an antibody, typically located at the amino terminus of the light or heavy chain, having a length of approximately 120 to 130 amino acids in the heavy chain and approximately 100 to 110 amino acids in the light chain, and serving as the binding and specificity of various antibodies to their antigens. The heavy chain variable region is referred to herein as “VH”. The light chain variable region is referred to herein as “VL”. The term “variable” refers to the fact that the sequence of certain segments of the variable region varies widely among antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody to its antigen. However, variability is not uniformly distributed within the 110-amino acid span of the variable region. In practice, the V region consists of smaller variability (e.g., relatively unchanged) segments of approximately 15 to 30 amino acids (called framework regions (FRs)) separated by shorter regions of greater variability (e.g., highly variable) (called “hypervariable regions” or alternatively, “complementarity-determining regions”). The variable regions of both the heavy and light chains each contain four frames (FR1, FR2, FR3, and FR4) that are essentially β-sheets. These frames are connected by three hypervariable regions, forming loops that connect the β-sheet structure and, in some cases, part of it. The hypervariable regions in each chain are closely linked together by the frames and, together with hypervariable regions from other chains, contribute to the formation of the antibody's antigen-binding site (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). Constant regions do not directly involve antibody-antigen binding but exhibit various effector functions, such as enabling antibodies to participate in antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cell cytotoxicity (CDC). The sequences of the variable regions vary widely among different antibodies. Sequence variability is concentrated in the CDR, while smaller variable portions within the variable regions are called frame regions (FRs). The CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions. In certain implementations, the variable area is a human-variable area.

[0307] The terms “hypervariant region,” “HVR,” “HV,” “complementarity-determining region,” and “CDR,” as used herein, refer to regions within the variable region of an antibody that are sequence-hypervariant and / or form structurally defined loops. Generally, an antibody contains six hypervariant regions: three located in the VH region (H1 or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3), and three located in the VL region (L1 or VL CDR1, L2 or VL CDR2, and L3 or VL CDR3). Several hypervariant region boundaries are used and covered herein. The Kabat CDR is based on sequence variability and is the most commonly used (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Chothia actually refers to the location of the structural ring (see, for example, Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)). When numbered using the Kabat numbering scheme, the end of the Chothia CDR-H1 ring varies between H32 and H34, depending on the length of the ring (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B exists, the ring end is at 32; if only 35A exists, the ring end is at 33; if both 35A and 35B exist, the ring end is at 34). The AbM hypervariable region represents the balance between the Kabat CDR and the Chothia structural ring and is utilized by the AbM antibody modeling software of Oxford Molecular (see, for example, Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The “contact” hypervariable region is based on the analysis of the available complex crystal structures. The residues from each of these hypervariable regions or CDRs are indicated below.

[0308] v Develop and widely adopt a universal numbering system: ImMunoGene Tics Information Systems (Lafranc et al., Dev. Comp. Immunol. 27(1): 55-77 (2003)). This is an integrated information system specifically for immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complex (MHC) in humans and other vertebrates. In this paper, CDRs are referred to based on their amino acid sequence and position within the light or heavy chain. Since the “position” of CDRs within the variable region structure of immunoglobulins is conserved across species and exists in structures called loops, CDRs and framework residues can be easily identified using a numbering system that compares the variable region sequence to structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into the receptor framework, typically derived from human antibodies. Honegger and Plückthun, J. Mol. Biol. 309: 657-670 (2001), have developed another numbering system (AHon). Numbering systems (including, for example, Kabat numbering and...) Consistency among unique numbering systems is well known to those skilled in the art (see, for example, Kabat, ibid.; Chothia and Lesk, ibid.; Martin, ibid.; Lefranc et al., ibid.) and is also described below. Various systems known in the art or described herein represent different approaches to defining CDRs, and are generally considered equivalent when used to define the same antibody. The illustrative system shown herein is a combination of Kabat and Chothia.

[0309]

[0310] The high-variance region may include the following "extended high-variance regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. As used herein, the terms "high-variance region," "HVR," "HV," "complementary determination region," or "CDR" are used interchangeably.

[0311] The term "vector" refers to a substance used to carry or include, for example, a nucleic acid sequence for introducing the nucleic acid sequence into a host cell. Examples of vectors include expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include optional sequences or markers operable to stably integrate into the host cell chromosome. Additionally, a vector may include one or more optional marker genes and appropriate expression control sequences. Optional marker genes may be included, for example, to provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or to supply critical nutrients absent in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and analogues well known in the art. When two or more nucleic acid molecules are co-expressed (e.g., antibody heavy and light chains or antibody VH and VL regions), the two nucleic acid molecules may be inserted, for example, into a single expression vector or a single expression vector. For single-vector expression, the encoding nucleic acid is operatively ligated to a shared expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of the nucleic acid molecule into the host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blotting or polymerase chain reaction (PCR) amplification of mRNA; or immunoblotting of gene product expression; or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that the expression level of the nucleic acid molecule is sufficient to produce the desired product (e.g., 5T4 antibody), and further understand that the expression level can be optimized to achieve sufficient expression using methods well known in the art.

[0312] The terms “5T4-mediated disease,” “5T4-mediated symptom,” and “5T4-mediated disorder” are used interchangeably and refer to any disease, symptom, or disorder associated with or characterized by cells expressing 5T4, such as tumor cells expressing 5T4. 5T4-mediated diseases include cancers, including (but not limited to) cancers that express or overexpress 5T4.

[0313] In any implementation thereof, the term “tumor” means any proliferative cell growth or proliferation (whether malignant or benign), and all precancerous and cancerous cells and tissues.

[0314] The terms “cancer” and “cancerous” refer to or describe physiological disorders in mammals that are typically characterized by abnormalities in the regulation of cell growth.

[0315] The term "ADC" refers to an antibody-drug conjugate, and in the context of this invention, it refers to the conjugation of a 5T4 antibody with another component (including a drug as described herein).

[0316] As used herein, “drug” refers to a biologically active compound, such as a cytotoxic compound (e.g., cytotoxic small molecules, cytotoxic synthetic peptides and their analogues).

[0317] Examples of drugs include small molecule drugs, such as cancer chemotherapeutic agents. For example, in the case where the peptide is an antibody (or fragment thereof) specific to tumor cells, the antibody can be modified as described herein to include modified amino acids, and subsequently conjugated to a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptide (i.e., non-protein) compounds that reduce the proliferation of cancer cells and encompass cytotoxic agents and cell growth inhibitors. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, phytoalkaloids (catharanthus vinca), and steroid hormones. Peptide compounds may also be used.

[0318] Suitable cancer chemotherapy agents include dolastastatin and its active analogs and derivatives; and auristatin and its active analogs and derivatives (e.g., monomethylauristatin D (MMAD), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF) and their analogs). See, for example, WO 96 / 33212, WO 96 / 14856 and US6,323,315. For example, dolastastatin 10 or auristatin PE may be included in the 5T4-ADC of this disclosure. Suitable cancer chemotherapy agents also include maytansine and its active analogs and derivatives (see, for example, EP 1391213; and Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623); duocarmycins and their active analogs and derivatives (e.g., including synthetic analogs KW-2189 and CB 1-TM1); and benzodiazepines. and its active analogues and derivatives (e.g., pyrrolobenzodiazepines) (PBD)).

[0319] Agents for reducing cell proliferation are known and widely used in the art. Such agents include alkylating agents such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkyl sulfonates, and triazines, including (but not limited to) mechlorethamine and cyclophosphamide. TM), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipebroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0320] Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including (but not limited to) cytarabine. Cytosine arabinoside, fluorouracil (5-FU), fluxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-didenifolinic acid (PDDF, CB3717), 5,8-didenifolinic acid (DDATHF), formyltetrahydrofolate, fludarabine phosphate, pentostatin, and gemcitabine.

[0321] Suitable natural products and their derivatives (such as vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include (but are not limited to) Ara-C and paclitaxel. Docetaxel Deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, such as vincristine, vinblastine, vinorelbine, vindesine and their analogues; podophyllotoxins, such as etoposide, teniposide and their analogues; antibiotics, such as anthracycline, daunorubicin hydroehloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives and their analogues; phenoxizone. Biscyclopeptides, such as dactinomycin; basic glycopeptides, such as basic glycopeptides; anthraquinone glycosides, such as plicamycin (mithramycin); anthraquinones, such as mitoxantrone; azahexacyclic acrylonitrile-pyrroloindole-diones, such as mitomycin; macrocyclic immunosuppressants, such as cyclosporine, FK-506 (taerolimus, prograf), rapamycin and their analogues; and their analogues.

[0322] Other anti-cytotoxic agents include navelbene, CPT-11, anastrozole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

[0323] Agents that affect microtubules and have antiproliferative activity are also applicable and include (but are not limited to) allocolchicine (NSC 406042), squalene B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), sea haretoxin 10 (NSC 376128), maytansine (NSC 153858), rhizobium glycosides (NSC332598), and paclitaxel. Derivatives, Docetaxel Colchicine thiocarbazepine (NSC361792), triphenylmethylcysteine, vincristine sulfate, vincristine sulfate, natural and synthetic epothilones (including but not limited to epothilone A and epothilone B), discodermolide; estramustine, nocodazole, and their analogues.

[0324] Suitable hormone modulators and steroids (including synthetic analogues) include (but are not limited to) corticosteroids, such as prednisone, dexamethasone, and their analogues; estrogens and progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, and their analogues; adrenocortical inhibitors, such as aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, and dromostanolone propionate. Propionate, testolactone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estradiol, medroxyprogesterone acetate, leuprolide, and flutamide. Toremifene And Gosherrelin And its analogues. Estrogen stimulates proliferation and differentiation; therefore, compounds that bind to estrogen receptors are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0325] Other suitable chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin and its analogues; ureas, such as hydroxyurea; hydrazine, such as N-methylhydrazine; epidophyllotoxin; topoisomerase inhibitors; procarbazine; mitoxantrone; folate; tegafur and its analogues. Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); gefitinib (… ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazolin; and analogues thereof.

[0326] Taxanes are suitable for use. "Taxanes" includes paclitaxel, as well as any active taxane derivatives or prodrugs. "Paclitaxel" (as understood herein) includes analogues, formulations, and derivatives such as docetaxel, (Docetaxel formulations), 10-deacetylated analogs of paclitaxel, and 3′N-debenzoyl-3′N-tert-butoxycarbonyl analogs of paclitaxel can be readily prepared using techniques known to those skilled in the art (see also WO94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO93 / 10076; U.S. Patents 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from various commercial sources, including, for example, Sigma Chemical. Co., St. Louis, Mo. (T7402 from *Taxus brevifolia*; or T-1912 from *Taxus yannanensis*). It should be understood that paclitaxel refers not only to the chemically available common form of paclitaxel, but also to its analogues and derivatives (e.g., those noted herein). Docetaxel and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-glucan, or paclitaxel-xylose).

[0327] The term "taxane" also includes a variety of known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include (but are not limited to) galactose and mannose derivatives as described in International Patent Application WO 99 / 18113; piperazine and other derivatives as described in WO 99 / 14209; taxane derivatives as described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No. 5,869,680; 6-thio derivatives as described in WO 98 / 28288; sulfinamide derivatives as described in U.S. Patent No. 5,821,263; and paclitaxel derivatives as described in U.S. Patent No. 5,415,869. It further includes paclitaxel prodrugs, including (but not limited to) those described in WO 98 / 58927; WO 98 / 13059; and U.S. Patent No. 5,824,701.

[0328] Suitable biological response modifiers include (but are not limited to) (1) tyrosine kinase (RTK) activity inhibitors; (2) serine / threonine kinase activity inhibitors; (3) tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony-stimulating factors; and (9) angiogenesis inhibitors.

[0329] An "effective amount" is generally an amount sufficient to achieve the following effects: reduce the severity and / or frequency of symptoms, eliminate symptoms and / or underlying causes, prevent the occurrence of symptoms and / or their underlying causes, and / or improve or repair damage caused by or related to a disease, condition, or disorder. In some implementations, the effective amount is a therapeutically effective amount.

[0330] As used herein, the term "therapeuticly effective amount" means an amount of antibody or ADC described herein sufficient to reduce and / or improve the severity and / or duration of a specified disease, condition, or disorder and / or associated symptoms. A therapeutically effective amount of a drug (including a therapeutic agent) may be an amount necessary to achieve: (i) reducing or improving the progression or course of a specified disease, condition, or disorder; (ii) reducing or improving the recurrence, development, or onset of a specified disease, condition, or disorder; and / or (iii) improving or enhancing the therapeutic effect of another therapy (e.g., a therapy other than the administration of an antibody or ADC described herein). The "therapeuticly effective amount" of a substance / molecule / drug agent (e.g., a 5T4 antibody or ADC) disclosed herein may vary based on a variety of factors, such as an individual's disease state, age, sex, and weight, and the ability of the substance / molecule / drug agent to induce a desired response in the individual. A therapeutically effective amount encompasses an amount in which the therapeutically beneficial effect of the substance / molecule / drug agent exceeds any toxic or harmful effect. In some implementations, the term "therapeutic effective amount" refers to the amount of antibody or other agent (e.g., drug) that effectively "treats" a disease, condition, or ailment in a subject or mammal.

[0331] In some embodiments, the drug is a microtubule-affecting agent with antiproliferative activity, such as maytansin. In some embodiments, the drug is an antimitotic agent, such as oligristatin or an active oligristatin analog or derivative thereof. In some embodiments, the drug is a DNA alkylating agent.

[0332] As used in this article, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopoeia for use in animals, and more specifically, in humans.

[0333] "Excipients" include carriers, excipients, preservatives, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used and may be included to achieve, for example, stability, improve formulation, or impart therapeutic enhancements to the active ingredient in the final dosage form (e.g., improve absorption, reduce viscosity, improve solubility). "Excipients" may be natural or synthetic organic or inorganic components with which the active ingredient is incorporated to facilitate the utilization of the active ingredient, such as when the active ingredient is administered to a subject. Examples of excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about ten amino acid residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN. TM Polyethylene glycol (PEG) and PLURONICS TMThe term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or medium that is administered with the therapeutic agent. Such excipients can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is used as an illustrative excipient when the composition (e.g., a pharmaceutical composition) is administered intravenously. Physiological saline solutions, dextran solutions, and glycerol solutions can also be used as liquid excipients, especially for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol, propylene, glycol, water, ethanol, and the like. In any embodiment, the composition may also contain small amounts of a wetting agent or emulsifier, or a pH buffer. The composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, or similar forms. Oral compositions (such as formulations) may include standard excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or the like. Examples of suitable excipients are described in Remington: The Science and Practice of Pharmacy (2020) (Elsevier Science, Amsterdam, Netherlands), including pharmaceutical compounds that may contain an effective or therapeutically effective amount of 5T4-ADC, such as in an isolated or purified form, and an appropriate amount of excipient to provide the correct form for administration to the subject. The formulation should be suitable for the mode of administration.

[0334] 5T4 antibody

[0335] The 5T4-ADCs described herein comprise a drug and a 5T4 antibody conjugated thereto. In some embodiments, a 5T4 antibody refers to an antibody that specifically binds to 5T4, such as the 5T4 protein, 5T4 polypeptide, 5T4 polypeptide fragment, 5T4 peptide, or 5T4 epitope. In some embodiments, the 5T4 antibody is a human or humanized antibody (e.g., containing a human constant region) that binds to 5T4 expressed on the surface of mammalian (e.g., human) cells (including tumor cells expressing 5T4). In some embodiments, the 5T4 antibody binds to extracellular 5T4 epitopes (e.g., extracellular 5T4 epitopes) expressed on cells (such as tumor cells). In some embodiments, 5T4 is human 5T4. An exemplary amino acid sequence of human 5T4 is described herein.

[0336] In some embodiments, the 5T4 antibody competitively binds to 5T4 with a reference 5T4 antibody comprising the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any antibody described herein, such as the amino acid sequence of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and / or VL CDR3 described in any of Tables 1 to 3. Therefore, in some embodiments, the 5T4 antibody competitively binds to 5T4 with a reference 5T4 antibody comprising one, two, and / or three VH CDRs and / or one, two, and / or three VL CDRs from: (a) an antibody named mAbA4; or (b) an antibody named mAbA15; or (c) an antibody named mAbA17. In some embodiments, the 5T4-ADC comprises a drug (directly or indirectly) conjugated to a 5T4 antibody that competitively binds to 5T4 with a reference 5T4 antibody comprising one, two, and / or three VH CDRs and one, two, and / or three VLCDRs: (a) an antibody named mAbA4; or (b) an antibody named mAbA15; or (c) an antibody named mAbA17. In some embodiments, the 5T4 antibody competitively binds to 5T4 with a reference 5T4 antibody comprising VH and VL regions from the following antibodies: (a) an antibody named mAbA4; or (b) an antibody named mAbA15; or (c) an antibody named mAbA17. In some embodiments, the 5T4 antibody competitively binds to 5T4 with a reference 5T4 antibody comprising: (a) a VH region comprising the amino acid sequence SEQ ID NO: 25 and a VL region comprising the amino acid sequence SEQ ID NO: 26; or (b) a VH region comprising the amino acid sequence SEQ ID NO: 44 and a VL region comprising the amino acid sequence SEQ ID NO: 45; or (c) a VH region comprising the amino acid sequence SEQ ID NO: 62 and a VL region comprising the amino acid sequence SEQ ID NO: 63.

[0337] In some embodiments, the 5T4 antibody comprises the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any of the antibodies described herein, such as the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 described in any of Tables 1 to 3. Therefore, in some embodiments, the 5T4 antibody comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from the following antibodies: (a) an antibody named mAbA4; or (b) an antibody named mAbA15; or (c) an antibody named mAbA17. In some embodiments, the 5T4 antibody comprises one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs from the following antibodies: (a) an antibody named mAbA4; or (b) an antibody named mAbA15; or (c) an antibody named mAbA17.

[0338] In some embodiments, the 5T4 antibody comprises a VH region containing one or more (such as one, two, or three) of VH CDR1, VH CDR2, and VH CDR3 as described herein (such as any of Tables 1 to 3); and / or a VL region containing one or more (such as one, two, or three) of VL CDR1, VL CDR2, and VL CDR3 as described herein (such as any of Tables 1 to 3). In some embodiments, the 5T4 antibody is bispecific and comprises a first binding region containing one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs as described in any of Tables 1 to 3; and a second region containing one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs of a binder, said binder binding to a second target antigen that is not 5T4. In some embodiments, the 5T4 antibody is bispecific and includes a first binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs as described in any of Tables 1 to 3; and a second binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from a binder that binds to a second 5T4 epitope.

[0339] The antibody named mAbA4 contains the VH amino acid sequence of SEQ ID NO: 25 and the VL amino acid sequence of SEQ ID NO: 26.

[0340] The antibody named mAbA15 contains the VH amino acid sequence of SEQ ID NO: 44 and the VL amino acid sequence of SEQ ID NO: 45.

[0341] The antibody named mAbA17 contains the VH amino acid sequence of SEQ ID NO: 62 and the VL amino acid sequence of SEQ ID NO: 63.

[0342]

[0343]

[0344]

[0345] In some embodiments, the 5T4 antibody includes a VH region. In some embodiments, the 5T4 antibody includes a VL region. In some embodiments, the 5T4 antibody has a combination of (i) a VH region and (ii) a VL region.

[0346] In some embodiments, the 5T4 antibody comprises a heavy chain having a combination of: (i) a VH as described herein, such as any of those in Tables 1 to 3; and (ii) one or more heavy chain constant regions (e.g., CH1, hinge, CH2, and CH3). An exemplary IgG heavy chain may comprise any VH amino acid sequence as described herein and the following CH1, hinge, CH2, and CH3 amino acid sequences:

[0347]

[0348] Another exemplary IgG heavy chain may comprise any VH amino acid sequence as described herein and the following CH1, hinge, CH2, and CH3 amino acid sequences:

[0349]

[0350] In some embodiments, the 5T4 antibody comprises a light chain having a combination of: (i) a VL region as described herein, such as any of those in Tables 1 to 3; and (ii) a light chain constant region (CL). An exemplary light chain (e.g., paired with an IgG heavy chain) may comprise any VL amino acid sequence described herein and the following CL amino acid sequences:

[0351]

[0352] In some embodiments, the 5T4 antibody comprises (a) a heavy chain having a combination of: (i) a VH as described herein, such as any of the tables 1 to 3, and (ii) one or more heavy chain constant regions (e.g., CH1, hinge, CH2, and CH3); and (b) a light chain having a combination of: (i) a VL as described herein, such as any of the tables 1 to 3, and (ii) a light chain constant region (CL1) in IgG format.

[0353] In some embodiments, the antibody binding to 5T4 may comprise: a heavy chain having an amino acid sequence.

[0354] And light chains, which have an amino acid sequence

[0355]

[0356] In some embodiments, the antibody binding to 5T4 may be a heavy chain, which has been modified to include formylglycine residues and thus has an amino acid sequence.

[0357] Where f(Gly) is a formylglycine residue.

[0358] In some implementations, the 5T4-ADC comprises a heavy chain having an amino acid sequence.

[0359] Where n is an amino acid whose side chain is replaced by a linker-drug as disclosed herein, such as a linker-drug of formula (XIV), (XV) or (XVI).

[0360] In some implementations, the antibody binding to 5T4 comprises a heavy chain having an amino acid sequence.

[0361] And light chains, which have an amino acid sequence

[0362]

[0363] In some embodiments, the antibody binding to 5T4 may be a heavy chain, which has been modified to include formylglycine residues and thus has an amino acid sequence.

[0364] Where f(Gly) is a formylglycine residue.

[0365] In some implementations, the 5T4-ADC comprises: a heavy chain having an amino acid sequence Where n is an amino acid whose side chain is replaced by a linker-drug as disclosed herein, such as a linker-drug of formula (XIV), (XV) or (XVI).

[0366] In some implementations, the antibody binding to 5T4 comprises a heavy chain having an amino acid sequence.

[0367] And light chains, which have an amino acid sequence

[0368] In some embodiments, the antibody binding to 5T4 may be a heavy chain, which has been modified to include formylglycine residues and thus has an amino acid sequence.

[0369] Where f(Gly) is a formylglycine residue.

[0370] In some implementations, the 5T4-ADC comprises: a heavy chain having an amino acid sequence

[0371] Where n is an amino acid whose side chain is replaced by a linker-drug as disclosed herein, such as a linker-drug of formula (XIV), (XV) or (XVI).

[0372] In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, three, four, five, or six CDRs), such as VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VLCDR3 as described in Table 1. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, three, four, five, or six CDRs), such as VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VLCDR3 as described in Table 2. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, three, four, five, or six CDRs), such as VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VLCDR3 as described in Table 3.

[0373] In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, or three VH CDRs), such as VH CDR1, VH CDR2, and VH CDR3 as described in Table 1. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, or three VL CDRs), such as VL CDR1, VL CDR2, and / or VL CDR3 as described in Table 1. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, or three VH CDRs), such as VH CDR1, VH CDR2, and VH CDR3 as described in Table 1; and one or more CDRs (e.g., one, two, or three VL CDRs), such as VL CDR1, VL CDR2, and / or VL CDR3 as described in Table 1.

[0374] In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, or three VH CDRs), such as VH CDR1, VH CDR2, and VH CDR3 described in Table 2. In other embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, or three VL CDRs), such as VL CDR1, VL CDR2, and / or VL CDR3 described in Table 2. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, or three VH CDRs), such as VH CDR1, VH CDR2, and VH CDR3 described in Table 2; and one or more CDRs (e.g., one, two, or three VL CDRs), such as VL CDR1, VL CDR2, and / or VL CDR3 described in Table 2.

[0375] In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, or three VH CDRs), such as VH CDR1, VH CDR2, and VH CDR3 described in Table 3. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, or three VL CDRs), such as VL CDR1, VL CDR2, and / or VL CDR3 described in Table 3. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., one, two, or three VH CDRs), such as VH CDR1, VH CDR2, and VH CDR3 described in Table 3; and one or more CDRs (e.g., one, two, or three VL CDRs), such as VL CDR1, VL CDR2, and / or VL CDR3 described in Table 3.

[0376] In some embodiments, the 5T4 antibody comprises one or more (e.g., one, two, or three) VH CDRs as described herein, such as those listed in Tables 1 to 3. In other embodiments, the 5T4 antibody comprises one or more (e.g., one, two, or three) VL CDRs as described herein, such as those listed in Tables 1 to 3. In some embodiments, the 5T4 antibody comprises one or more (e.g., one, two, or three) VH CDRs as described herein (such as those listed in Tables 1 to 3) and one or more (e.g., one, two, or three) VL CDRs as described herein (such as those listed in Tables 1 to 3). Thus, in some embodiments, the 5T4 antibody comprises VH CDR1 whose amino acid sequence contains any one of SEQ ID NO: 1, 7, 12, 13, 18, 27, 31, 34, 35, 39, 46, 50, 53, and 57. In some embodiments, the 5T4 antibody comprises VH CDR2 containing the amino acid sequence of any one of SEQ ID NO: 2, 8, 14, 19, 24, 28, 32, 36, 40, 43, 47, 51, 54, 58, and 61. In some embodiments, the 5T4 antibody comprises VHCDR3 containing the amino acid sequence of any one of SEQ ID NO: 3, 9, 15, 20, 29, 33, 37, 41, 48, 52, 55, and 59. In some embodiments, the 5T4 antibody comprises VH CDR1 and / or VH CDR2 and / or VHCDR3 independently selected from any one of the following: VH CDR1, VH CDR2, VH CDR3 as described in Tables 1 to 3. In some embodiments, the 5T4 antibody comprises VL CDR1 containing the amino acid sequence of any one of SEQ ID NO: 4, 10, 16, and 21. In some embodiments, the 5T4 antibody comprises a VL CDR2 containing the amino acid sequence of any one of SEQ ID NO: 5, 11, and 22. In some embodiments, the 5T4 antibody comprises a VL CDR3 containing the amino acid sequence of any one of SEQ ID NO: 6, 17, 23, 30, 38, 42, 49, 56, and 60. In some embodiments, the 5T4 antibody comprises VL CDR1 and / or VLCDR2 and / or VL CDR3 independently selected from the following: VL CDR1, VL CDR2, VL CDR3 as described herein (such as any one of Tables 1 to 3).

[0377] In some embodiments, the 5T4 antibody comprises a heavy chain variable region (VH) comprising: (1) a VH CDR1 comprising any of the following amino acid sequences: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 7, 31 or 50, (iii) SEQ ID NO: 12, 34 or 53, (iv) SEQ ID NO: 13 or 53 and (v) SEQ ID NO: 18, 39 or 57; (2) a VH CDR2 comprising any of the following amino acid sequences: (i) SEQ ID NO: 2, 28 or 47, (ii) SEQ ID NO: 8, 32 or 51, (iii) SEQ ID NO: 14, 36 or 54, (iv) SEQ ID NO: 19, 40 or 58 and (v) SEQ ID NO: 24, 43 or 61; and (3) a VH CDR3 comprising any of the following amino acid sequences: (i) SEQ ID NO: 3, 29 or 48, (ii) SEQ ID NO: 12, 27 or 46, (ii) SEQ ID NO: 13 or 53, (iii) SEQ ID NO: 14, 36 or 54, (iv) SEQ ID NO: 19, 40 or 58 and (v) SEQ ID NO: 24, 43 or 61; and (3) a VH CDR3 comprising any of the following amino acid sequences: (i) SEQ ID NO: 3, 29 or 48, (ii) SEQ ID NO: 13 or 53, (iv) SEQ ID NO: 13 or 53 and (v) SEQ ID NO: 18, 39 or 57; (i) SEQ ID NO: 13 or 53 and (v) SEQ ID NO: 18, 39 or 57; ( ID NO: 9, 33 or 52, (iii) SEQ ID NO: 15, 37 or 55 and (iv) SEQ ID NO: 20, 41 or 59; and / or VL region comprising: (1) VL CDR1 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 4, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 16 and (iv) SEQ ID NO: 21; (2) VLCDR2 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11 and (iii) SEQ ID NO: 22; and (3) VL CDR3 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 6, 30 or 49, (ii) SEQ ID NO: 17, 38 or 56 and (iii) SEQ ID NO: 23, 42 or 60.

[0378] In some embodiments, the 5T4 antibody comprises a VH region comprising: (1) a VH CDR1 comprising any of the following amino acid sequences: (i) SEQ ID NO: 1, 27, or 46; (ii) SEQ ID NO: 7, 31, or 50; (iii) SEQ ID NO: 12, 34, or 53; (iv) SEQ ID NO: 53; and (v) SEQ ID NO: 18, 39, or 57; (2) a VH CDR2 comprising any of the following amino acid sequences: (i) SEQ ID NO: 2, 28, or 47; (ii) SEQ ID NO: 8, 32, or 51; (iii) SEQ ID NO: 14, 36, or 54; (iv) SEQ ID NO: 19, 40, or 58; and (v) SEQ ID NO: 24, 43, or 61; and (3) a VH CDR3 comprising any of the following amino acid sequences: (i) SEQ ID NO: 3, 29, or 48; (ii) SEQ ID NO: 12, 27, or 46; (iii) SEQ ID NO: 12, 34, or 53; (iv) SEQ ID NO: 53; and (v) SEQ ID NO: 18, 39, or 57; (iv) SEQ ID NO: 19, 40, or 58; and (v) SEQ ID NO: 24, 43, or 61; and (3) a VH CDR3 comprising any of the following amino acid sequences: (i) SEQ ID NO: 3, 29, or 48; (ii) SEQ ID NO: 12, 29, or 46; (iii) SEQ ID NO: 12, 34, or 53; (iv) SEQ ID NO: 53; and (v) SEQ ID NO (iii) SEQ ID NO: 15, 37 or 55 and (iv) SEQ ID NO: 20, 41 or 59.

[0379] In some embodiments, the 5T4 antibody comprises a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 4, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 16 and (iv) SEQ ID NO: 21; (2) a VL CDR2 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11 and (iii) SEQ ID NO: 22; and (3) a VL CDR3 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 6, 30 or 49, (ii) SEQ ID NO: 17, 38 or 56 and (iii) SEQ ID NO: 23, 42 or 60.

[0380] In some embodiments, the 5T4-ADC comprises an antibody bound to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising an amino acid sequence comprising any of the following: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 7, 31 or 50, (iii) SEQ ID NO: 12, 34 or 53, (iv) SEQ ID NO: 53 and (v) SEQ ID NO: 18, 39 or 57; (2) a VH CDR2 comprising an amino acid sequence comprising any of the following: (i) SEQ ID NO: 2, 28 or 47, (ii) SEQ ID NO: 8, 32 or 51, (iii) SEQ ID NO: 14, 36 or 54, (iv) SEQ ID NO: 19, 40 or 58 and (v) SEQ ID NO: 24, 43 or 61; and (3) a VH CDR3 comprising an amino acid sequence comprising any of the following: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 7, 31 or 50, (iii) SEQ ID NO: 12, 34 or 53, (iv) SEQ ID NO: 53 and (v) SEQ ID NO: 24, 43 or 61; and (3) a VH CDR3 comprising an amino acid sequence comprising any of the following: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 7, 31 or 50, (iii) SEQ ID NO: 12, 34 or 53, (iv) SEQ ID NO: 19, 40 or 58 and (v) SEQ ID NO: 24, 43 or 61; and (4) SEQ ID NO: 19, 40 or ID NO: 3, 29 or 48, (ii) SEQ ID NO: 9, 33 or 52, (iii) SEQ ID NO: 15, 37 or 55 and (iv) SEQ ID NO: 20, 41 or 59; and / or VL region comprising: (1) VL CDR1 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 4, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 16 and (iv) SEQ ID NO: 21; (2) VL CDR2 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11 and (iii) SEQ ID NO: 22; and (3) VL CDR3 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 6, 30 or 49, (ii) SEQ ID NO: 17, 38 or 56 and (iii) SEQ ID NO: 23, 42 or 60.

[0381] In some embodiments, the 5T4-ADC comprises an antibody bound to 5T4, wherein the antibody comprises a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 7, 31 or 50, (iii) SEQ ID NO: 12, 34 or 53, (iv) SEQ ID NO: 13, 35 or 53 and (v) SEQ ID NO: 18, 39 or 57; (2) a VH CDR2 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 2, 28 or 47, (ii) SEQ ID NO: 8, 32 or 51, (iii) SEQ ID NO: 14, 36 or 54, (iv) SEQ ID NO: 19, 40 or 58 and (v) SEQ ID NO: 24, 43 or 61; and (3) a VH CDR3 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 7, 31 or 50, (iii) SEQ ID NO: 12, 34 or 53, (iv) SEQ ID NO: 13, 35 or 53 and (v) SEQ ID NO: 18, 39 or 57; and (3) a VH CDR3 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 8, 32 or 51, (iii) SEQ ID NO: 14, 36 or 54, (iv) SEQ ID NO: 19, 40 or 58 and (v) SEQ ID NO: 24, 43 or 61; and (4) a V (ii) SEQ ID NO: 3, 29 or 48, (iii) SEQ ID NO: 9, 33 or 52, (iii) SEQ ID NO: 15, 37 or 55 and (iv) SEQ ID NO: 20, 41 or 59.

[0382] In some embodiments, the 5T4-ADC comprises an antibody bound to 5T4, wherein the antibody comprises a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 4, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 16 and (iv) SEQ ID NO: 21; (2) a VLCDR2 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11 and (iii) SEQ ID NO: 22; and (3) a VL CDR3 comprising the amino acid sequence of any of the following: (i) SEQ ID NO: 6, 30 or 49, (ii) SEQ ID NO: 17, 38 or 56 and (iii) SEQ ID NO: 23, 42 or 60.

[0383] In some embodiments, the 5T4-ADC comprises an antibody binding to 5T4, wherein the antibody comprises all three heavy chain CDRs and / or all three light chain CDRs from the following antibodies: (a) an antibody named mAbA4 comprising the VH amino acid sequence of SEQ ID NO: 25 and the VL amino acid sequence of SEQ ID NO: 26; or (b) an antibody named mAbA15 comprising the VH amino acid sequence of SEQ ID NO: 44 and the VL amino acid sequence of SEQ ID NO: 45; or (c) an antibody named mAbA17 comprising the VH amino acid sequence of SEQ ID NO: 62 and the VL amino acid sequence of SEQ ID NO: 63. In some embodiments, the 5T4-ADC comprises a 5T4 antibody, wherein the 5T4 antibody comprises all three heavy chain CDRs and / or all three light chain CDRs from the antibody named mAbA4. In some embodiments, the 5T4-ADC comprises a 5T4 antibody, wherein the 5T4 antibody comprises all three heavy chain CDRs and / or all three light chain CDRs from the antibody named mAbA15. In some embodiments, the 5T4-ADC comprises a 5T4 antibody, wherein the 5T4 antibody comprises all three heavy chain CDRs and / or all three light chain CDRs from an antibody named mAbA17.

[0384] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 as described herein (such as Tables 1 to 3); and / or (b) a VL region comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 as described herein (such as Tables 1 to 3). In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: a VH region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 as described herein (such as Tables 1 to 3). In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises a VL region comprising the amino acid sequences of VL CDR1, VLCDR2, and VL CDR3 as described herein (such as Tables 1 to 3).

[0385] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (1) VL CDR1, which comprises the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) VL CDR2, which comprises the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0386] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; and (2) a VLCDR2 comprising the amino acid sequence SEQ ID NO: 5.

[0387] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 10; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11.

[0388] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 16; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11.

[0389] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 21; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 22.

[0390] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 1; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0391] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 1; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5.

[0392] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 7; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0393] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 7; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 10; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11.

[0394] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 12; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0395] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 12; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 10; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11.

[0396] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 18; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0397] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 18; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 21; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 22.

[0398] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 27; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0399] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 27; and a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5.

[0400] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 31; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0401] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 31; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 10; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11.

[0402] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 34; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0403] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 34; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5.

[0404] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 35; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0405] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 35; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 16; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11.

[0406] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 39; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0407] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 39; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 21; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 22.

[0408] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 46; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0409] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 46; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5.

[0410] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 50; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0411] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 50; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 10; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11.

[0412] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 53; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0413] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 53; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5.

[0414] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 57; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0415] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 57; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 21; and (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 22.

[0416] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 7, 12, 13 and 18; (2) a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 2, 8, 14, 19 and 24; and (3) a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 3, 9, 15 and 20; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22; and (3) a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 6, 17 and 23.

[0417] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 1; (2) a VHCDR2 comprising the amino acid sequence SEQ ID NO: 2; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 3; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 6.

[0418] In some embodiments, the 5T4-ADC comprises an antibody bound to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 7; (2) a VHCDR2 comprising the amino acid sequence SEQ ID NO: 8; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 9; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 10; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 6.

[0419] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 12; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 2; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 3; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 6.

[0420] In some embodiments, the 5T4-ADC comprises an antibody bound to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 13; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 14; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 15; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 16; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 17.

[0421] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 18; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 19; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 20; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 21; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 22; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 23.

[0422] In some embodiments, the 5T4-ADC comprises an antibody bound to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 1; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 24; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 3; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 6.

[0423] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 27, 31, 34, 35 and 39; (2) a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 28, 32, 36, 40 and 43; and (3) a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 29, 33, 37 and 41; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22; and (3) a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 30, 38 and 42.

[0424] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 27; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 28; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 29; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 30.

[0425] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 31; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 32; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 33; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 10; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 30.

[0426] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 34; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 28; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 29; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 30.

[0427] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 35; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 36; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 37; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 16; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 38.

[0428] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 39; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 40; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 41; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 21; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 22; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 42.

[0429] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 27; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 43; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 29; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 30.

[0430] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 46, 50, 53 and 57; (2) a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 47, 51, 54, 58 and 61; and (3) a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 48, 52, 55 and 59; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22; and (3) a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 49, 56 and 60.

[0431] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 46; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 47; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 48; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 49.

[0432] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 50; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 51; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 52; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 10; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 49.

[0433] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 53; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 47; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 48; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 49.

[0434] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 13; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 54; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 55; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 16; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 11; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 56.

[0435] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 57; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 58; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 59; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 21; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 22; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 60.

[0436] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence SEQ ID NO: 46; (2) a VH CDR2 comprising the amino acid sequence SEQ ID NO: 61; and (3) a VH CDR3 comprising the amino acid sequence SEQ ID NO: 48; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence SEQ ID NO: 4; (2) a VL CDR2 comprising the amino acid sequence SEQ ID NO: 5; and (3) a VL CDR3 comprising the amino acid sequence SEQ ID NO: 49.

[0437] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises: a VH region comprising: a VH CDR1 whose amino acid sequence is selected from the group consisting of SEQ ID NO: 27, 31, 34, 35 and 39; and a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22.

[0438] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, wherein the antibody comprises the VH and / or VL regions described herein, wherein the VH and / or VL comprise human frame sequences. In some embodiments, the VH and / or VL regions comprise frame 1 (FR1), frame 2 (FR2), frame 3 (FR3), and / or frame 4 (FR4) sequences, such as human FR1, human FR2, human FR3, and / or human FR4.

[0439] In some implementations, the CDR of the 5T4 antibody can be determined using the Kabat system (Kabat et al., (1971) Ann. NY Acad. Sci. 190: 382-391 and Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Bulletin No. 91-3242).

[0440] In some implementations, the CDR of the 5T4 antibody can be determined by the Chothia system, referred to herein as “Chothia CDR” (see, for example, Chothia and Lesk, 1987, J. Mol. Biol., 196: 901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273: 927-948; Chothia et al., 1992, J. Mol. Biol., 227: 799-817; Tramontano A. et al., 1990, J. Mol. Biol. 215(1): 175-82; and U.S. Patent No. 7,709,226).

[0441] In some implementations, the CDR of the 5T4 antibody can be obtained via ImMunoGeneTics. Systematic assays, such as those described in the following literature: Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27: 209-212 (“ CDR).

[0442] In some implementations, the CDR of the 5T4 antibody can be determined by the AbM system, referred to herein as “AbMCDR”, as described, for example, in MacCallum et al., 1996, J. Mol. Biol., 262: 732-745. See also, for example, Martin, A., “Protein Sequence and Structure Analysis of Antibody Variable Domains”, in Antibody Engineering, Kontermann and Dübel eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0443] In some implementations, the CDR of the 5T4 antibody can be determined using a Contact system, which will be referred to herein as “Contact CDR” (see, for example, MacCallum RM et al., 1996, J Mol Biol 5: 732-745). Contact CDR is based on the analysis of the available complex crystal structure.

[0444] In some embodiments, the position of one, two, three, four, five, or six amino acid positions of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the 5T4 antibody may be altered, subject to maintaining (e.g., substantially maintaining, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) binding to 5T4 (e.g., human 5T4). For example, in some embodiments, the position of the delimited CDR in any of Tables 1, 2, or 3 may be altered by shifting the N-terminal and / or C-terminal boundary of the CDR relative to the current CDR position by one, two, three, four, five, or six amino acids, subject to maintaining (e.g., substantially maintaining, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) binding to 5T4 (e.g., human 5T4). Alternatively or in some embodiments, the length of one or more (e.g., one, two, or three) CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the 5T4 antibody (e.g., human 5T4 antibody) described herein may be varied (e.g., shorter or longer) by one, two, three, four, five, or more amino acids, subject to the constraint of maintaining (e.g., substantially maintaining, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) binding to 5T4 (e.g., human 5T4). For example, in some embodiments, VH and / or VL CDR1, CDR2, and / or CDR3 may be one, two, three, four, five, or more amino acids shorter than one or more CDRs described in SEQ ID NO: 1-24, 27-43, or 46-61, subject to the constraint of maintaining (e.g., substantially maintaining, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) binding to 5T4 (e.g., human 5T4). In other embodiments, VH and / or VL CDR1, CDR2, and / or CDR3 may be one, two, three, four, five, or more amino acids longer than one or more CDRs described in SEQ ID NO: 1-24, 27-43, or 46-61, subject to the constraint of maintaining (e.g., substantially maintaining, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) binding to 5T4 (e.g., human 5T4).Alternatively, in some embodiments, the amino terminus of VH and / or VL CDR1, CDR2 and / or CDR3 may be extended by one, two, three, four, five or more amino acids compared to one or more CDRs described in SEQ ID NO: 1-24, 27-43 or 46-61, subject to the limitation of maintaining (e.g. substantially maintaining, e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%) binding to 5T4 (e.g. human 5T4). Alternatively, in some embodiments, the carboxyl termini of VH and / or VL CDR1, CDR2 and / or CDR3 may be extended by one, two, three, four, five or more amino acids, compared to one or more CDRs described in SEQ ID NO: 1-24, 27-43 or 46-61, subject to maintaining (e.g. substantially maintaining, e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%) binding to 5T4 (e.g. human 5T4). Alternatively, in some embodiments, the amino termini of VH and / or VL CDR1, CDR2 and / or CDR3 may be shortened by one, two, three, four, five or more amino acids compared to one or more CDRs described in SEQ ID NO: 1-24, 27-43 or 46-61, subject to the constraint of maintaining (e.g. substantially maintaining, e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%) binding to 5T4 (e.g. human 5T4). In some embodiments, the carboxyl termini of VH and / or VL CDR1, CDR2, and / or CDR3 may be shortened by one, two, three, four, five, or more amino acids compared to one or more CDRs described in SEQ ID NO: 1-24, 27-43, or 46-61, subject to the constraint of maintaining (e.g., substantially maintaining, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) the binding to 5T4 (e.g., human 5T4). Any method known in the art can be used to determine whether the binding to 5T4 (e.g., human 5T4) is maintained, such as the binding analyses and conditions described in the “Examples” section herein. For example, Example 2 described herein describes an analysis for measuring the binding to 5T4 (e.g., human 5T4).

[0445] In some embodiments, a 5T4-ADC may comprise a 5T4 antibody containing one or more (e.g., one, two, three, four, or more) conserved sequence modifications. For a polypeptide that is a 5T4 antibody (such as a human 5T4 antibody), conserved sequence modifications include conserved amino acid substitutions, wherein an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families are disclosed herein. Therefore, in some embodiments, a predicted non-essential amino acid residue in the 5T4 antibody may be replaced by another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions that do not eliminate antigen binding and the nucleotides encoding them are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)). In some embodiments, the conserved sequence modifications described herein modify the amino acid sequence of the 5T4 antibody (e.g., human 5T4 antibody) by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, the amino acid sequence modification refers to the substitution of up to one, two, three, four, five, or six amino acids of the CDR, such as those described in any of Tables 1 to 3. Therefore, for example, each of these CDRs may contain up to five conserved amino acid substitutions, such as up to (no more than) four conserved amino acid substitutions, such as up to (no more than) three conserved amino acid substitutions, such as up to (no more than) two conserved amino acid substitutions, or no more than one conserved amino acid substitution. In some embodiments, the 5T4-ADC comprises a 5T4 antibody containing one or more CDRs (e.g., one, two, three, four, five, or six CDRs) that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% concordant with the CDRs of mAbA4, mAbA15, or mAbA17 (see, for example, Tables 1, 2, or 3). In some embodiments, the 5T4-ADC comprises a 5T4 antibody containing VH and VL concordant with the CDRs of mAbA4, mAbA15, or mAbA17 (see, for example, Tables 1, 2, or 3). In some embodiments, the amino acid sequence modification does not include any modification within the specificity-determining residue (SDR). In some embodiments, the amino acid sequence modification does not include any modification within the CDRs (such as CDR1, CDR2, CDR3, or any combination thereof). In other embodiments, the amino acid sequence modification is located in the frame or constant region.

[0446] In some embodiments, the antibody in the 5T4-ADC comprises a VH whose amino acid sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 25, and a VL whose amino acid sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 26, and maintains (e.g., substantially maintains, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) the binding of the antibody to 5T4 (e.g., human 5T4).

[0447] In some embodiments, the antibody in the 5T4-ADC comprises a VH whose amino acid sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 44, and a VL whose amino acid sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 45, and maintains (e.g., substantially maintains, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) the binding of the antibody to 5T4 (e.g., human 5T4).

[0448] In some embodiments, the antibody in the 5T4-ADC comprises a VH whose amino acid sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 62, and a VL whose amino acid sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 63, and maintains (e.g., substantially maintains, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) the binding of the antibody to 5T4 (e.g., human 5T4).

[0449] Additionally, nucleic acids encoding 5T4 binding agents (e.g., antibodies or antibody fragments) or fusion polypeptides as disclosed herein, complementary nucleic acids thereto, vectors containing nucleic acids as disclosed herein, and cells containing nucleic acids or vectors as disclosed herein are provided. In some embodiments, the cells express the 5T4 binding agent. In some embodiments, the cells replicate the nucleic acid or vector. In some embodiments, materials suitable for generating 5T4 antibodies (including human 5T4 antibodies) and for preparing ADCs are further provided. In this regard, cells (e.g., isolated cells) can generate antibodies containing VH and VL as described herein. In some embodiments, the polynucleotide may contain one or more nucleic acid sequences encoding 5T4 antibodies or antibody fragments. In some embodiments, the polynucleotide is an isolated and / or recombinant polynucleotide. In some embodiments, the isolated polynucleotide contains nucleotide sequences encoding antibody heavy chain variable regions (VH) and / or antibody light chain variable regions (VL), wherein the VH and VL contain CDRs consistent with CDRs as described herein.

[0450] In some implementations, one or more vectors (e.g., expression vectors) may contain one or more polynucleotides for expression in a suitable host cell. Using recombinant techniques, such vectors are suitable for, for example, amplifying polynucleotides in a host cell to produce a suitable quantity, and for expressing binding agents, such as antibodies or antibody fragments.

[0451] In some embodiments, one or more vectors are expression vectors, wherein one or more polynucleotides are operatively linked to one or more polynucleotides containing an expression control sequence. Self-replicating recombinant expression constructs are particularly contemplated, such as plasmids and viral DNA vectors containing one or more polynucleotides encoding an antibody sequence binding to 5T4. The expression control DNA sequence includes a promoter, an enhancer, and an operator, and is typically selected based on the expression system in which the expression construct will be used. The promoter and enhancer sequences are typically selected based on their ability to enhance gene expression, while the operator sequence is typically selected based on its ability to regulate gene expression. The expression construct may also include sequences encoding one or more optional markers that allow identification of the host cell carrying the construct. The expression construct may also include sequences that facilitate (e.g., promote) homologous recombination in the host cell. In some embodiments, the expression construct also includes sequences necessary for replication in the host cell.

[0452] In some implementations, the expression control sequence may include a promoter / enhancer sequence, such as the cell giant virus promoter / enhancer (Lehner et al., J. Clin Microbiol., 29: 2494-2502, 1991; Boshart et al., Cell, 41: 521-530, 1985); the Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4: 151, 1993); the Tie promoter (Korhonen et al., Blood, 86(5): 1828-1835, 1995); the simian virus 40 promoter; DRA (downregulated in adenomas; Allefai et al., Am. J. Physiol. Gastrointest. Liver Physiol., 293: G923-G934, 2007); MCT1 (monocarboxylic acid transporter 1; Cuff et al., Am. J. Physiol. Gastrointest. Liver Physiol., G977-G979. 2005); and Math1 (mouse unregulated homolog 1; Shroyer et al., Gastroenterology, 132: 2477-2478, 2007), for expression in mammalian cells, wherein the promoter is operatively linked upstream (e.g., 5′) of the polypeptide coding sequence. In some embodiments, the promoter may be an epithelial-specific promoter or an endothelial-specific promoter. The polynucleotide may also optionally include a suitable polyadenylated sequence (e.g., SV40 or human growth hormone gene polyadenylated sequence), which is operatively linked downstream (e.g., 3′) of the polypeptide coding sequence.

[0453] In any embodiment, one or more polynucleotides may optionally additionally comprise one or more nucleotide sequences encoding one or more secretion signal peptides fused in frame with a polypeptide sequence. The one or more secretion signal peptides may guide cells expressing the one or more polynucleotides to secrete antibody polypeptides and may be cleaved by the cell with the secreted polypeptides. In any embodiment, one or more polynucleotides may optionally further comprise one or more sequences whose predetermined function is solely to facilitate the large-scale production of the vector.

[0454] Expression vectors can be prepared using standard recombinant DNA techniques, such as those described in the following literature: Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994). Optionally, the viral vector can be made replication-deficient, for example, by deleting or interrupting the selection genes necessary for viral replication.

[0455] Cells may contain one or more polynucleotides and / or one or more vectors. For example, in any embodiment, cells may be transformed or transfected with one or more polynucleotides encoding a 5T4 antibody (e.g., human 5T4 antibody) or with one or more vectors containing one or more polynucleotides encoding a 5T4 antibody (e.g., human 5T4 antibody). In some embodiments, cells may express a 5T4 antibody (e.g., human 5T4 antibody) containing one or more (e.g., one, two, three, four, five, or six) CDRs that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to CDRs as described herein (such as mAbA4, mAbA15, and / or mAbA17 (see, for example, Tables 1, 2, and / or 3)). In some embodiments, cells may express 5T4 antibodies (e.g., human 5T4 antibodies) containing VH and VL regions with CDRs consistent with those described herein (such as CDRs of mAbA4, mAbA15, and / or mAbA17 (see, for example, Tables 1, 2, and / or 3)). Suitable cells for producing 5T4 antibodies as described herein include prokaryotic cells, such as *Escherichia coli* (see, for example, Plückthun et al., Methods Enzymol., 178:497-515, 1989); and eukaryotic cells, such as animal cells (e.g., myeloma cells, Chinese hamster ovary (CHO) cells, or fusion tumor cells), yeast (e.g., *Saccharomyces cerevisiae*), or plant cells (e.g., tobacco, corn, soybean, or rice cells). The use of mammalian host cells allows for translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) that may be necessary to confer optimal biological activity to the recombinant expression product. Similarly, in any embodiment, the peptide (e.g., a 5T4 antibody, including a human 5T4 antibody) may be glycosylated or deglycosylated and / or covalently modified to include one or more water-soluble polymers linked together, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0456] Methods for introducing DNA or RNA into host cells are well known and include (but are not limited to) transformation, transfection, electroporation, nuclear injection, and fusion with vectors such as liposomes, microcells, shadow cells, and protoplasts. Such host cells are suitable for amplifying polynucleotides and expressing polypeptides encoded by polynucleotides. In this regard, methods for generating 5T4 antibodies may include introducing RNA or DNA encoding 5T4 antibodies as described herein into host cells; culturing said host cells; and isolating the resulting 5T4 antibodies.

[0457] Various methods for generating antibodies from polynucleotides are generally well known. For example, basic molecular biology procedures are described in Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989 (see also Maniatis et al., 3rd ed., Cold Spring Harbor Laboratory, New York, 2001). In addition, many publications describe techniques suitable for antibody preparation by manipulating DNA, constructing expression vectors, and transforming and culturing appropriate cells (see, for example, Chapter 1 of Mountain and Adair, Biotechnology and Genetic Engineering Reviews, ed. Tombs, Intercept, Andover, UK, 1992); and Current Protocols in Molecular Biology, ed. Ausubel, Wiley Interscience, New York, 1999).

[0458] 5T4-ADCS

[0459] Antibodies and drugs binding to 5T4 can be directly or indirectly linked to each other via a pyridazine-pyrrole coupling portion to form a 5T4-ADC as described herein. In some embodiments, the 5T4-ADC can be represented by formula (IV):

[0460]

[0461] in:

[0462] Ab is an antibody that binds to 5T4;

[0463] r is an integer from 1 to 10;

[0464] m is 0 or 1;

[0465] R2 and R 3 Each of these groups is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic; or R 2 With R 3 Ring-linked structures are used to form 5- or 6-membered heterocyclic groups;

[0466] X 1 、X 2 、X 3 and X 4 Each can be independently C, N, O, or S;

[0467] Y 1 、Y 2 、Y 3 and Y 4 Each of these groups is independently hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic, or Y 1 With Y 2 、Y 2 With Y 3 Or Y 3 With Y 4 Connected in a ring;

[0468] L is the connector as described herein; and

[0469] W 1 It is a drug.

[0470] Formula (IV) was previously described in WO 2015 / 081282, which is incorporated herein by reference in its entirety.

[0471] In some implementations, m is 0 or 1 and R 2 and R 3 Each is an alkyl group. For example, R 2 and R 3 Each can be independently C1-C 10 Alkyl (e.g., C1-C6 alkyl). In some embodiments, R 2 and R 3Each is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl. In some embodiments, R 2 and R 3 At least one of them is methyl. In some embodiments, R 2 With R 3 All are methyl groups. In some embodiments, X 1 、X 2 、X 3 and X 4 Each of them is independently C or N. In some implementations, X 1 For C. In some implementations, X 1 For N. In some implementations, X 2 For C or N. In some implementations, X 2 For C. In some implementations, X 3 For C or N. In some implementations, X 3 For C. In some implementations, X 4 For C or N. In some implementations, X 4 It is C. X 1 、X 2 、X 3 and X 4 Various combinations of X are possible. For example, in some implementations, X 1 、X 2 、X 3 and X 4 Each of them is C. In other implementations, X 1 、X 2 、X 3 and X 4 The three are C and X 1 、X 2 、X 3 and X 4 One of them is N. In some implementations, Y 1 、Y 2 、Y 3 and Y 4 Each of them is hydrogen.

[0472] In the context of the 5T4-ADC of formula (IV), the connector-drug of formula (IV) can be represented by the following formula (XIV):

[0473]

[0474] in The linker site representing the antibody binding to 5T4 is, for example, residue n of SEQ ID NO: 93, 97, or 101. All other variables of the linker-drug of formula (XIV) may be as defined herein, such as those defined with respect to formula (IV), and / or as described herein.

[0475] In some implementations, the 5T4-ADC can be represented by equation (V):

[0476]

[0477] in:

[0478] Ab is an antibody that binds to 5T4;

[0479] r is an integer from 1 to 10;

[0480] R 2 and R 3 Each of these groups is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic; or R 2 With R 3 Ring-linked structures are used to form 5- or 6-membered heterocyclic groups;

[0481] X 1 It can be C or N;

[0482] Y 1 、Y 2 、Y 3 and Y 4 Each of these groups is independently hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic, or Y 1 With Y 2 、Y 2 With Y 3 or Y 3 With Y 4 Connected in a ring;

[0483] L is the connector as described herein; and

[0484] W 1It is a drug.

[0485] Equation (V) was previously described in WO 2015 / 081282, which is incorporated herein by reference in its entirety.

[0486] In some embodiments, R 2 and R 3 Each is an alkyl group. For example, R 2 and R 3 Each can be independently C1-C 10 Alkyl (e.g., C1-C6 alkyl). In some embodiments, R 2 and R 3 Each is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl. In some embodiments, R 2 and R 3 At least one of them is methyl. In some embodiments, R 2 With R 3 All are methyl groups. In some embodiments, Y 1 、Y 2 、Y 3 and Y 4 Each of them is hydrogen.

[0487] In the context of the 5T4-ADC of equation (V), the connector-drug of equation (V) can be represented by the following equation (XV):

[0488]

[0489] in The linker site represents the antibody binding to 5T4, such as residue n of SEQ ID NO: 93, 97, or 101. All other variables of the linker-drug of formula (XV) may be as defined herein, such as those defined with respect to formula (V), and / or as described herein.

[0490] In some implementations, the 5T4-ADC can be represented by equation (VI):

[0491]

[0492] in:

[0493] Ab is an antibody that binds to 5T4;

[0494] r is an integer from 1 to 10;

[0495] X 1 It can be C or N;

[0496] L is the connector as described herein; and

[0497] W1 It is a drug.

[0498] Formula (IV) was previously described in WO 2015 / 081282, which is incorporated herein by reference in its entirety.

[0499] In the context of the 5T4-ADC of Equation (VI), the connector-drug of Equation (VI) can be represented by the following Equation (XVI):

[0500]

[0501] in The linker site representing the antibody binding to 5T4 is, for example, residue n of SEQ ID NO: 93, 97, or 101. All other variables of the linker-drug of formula (XVI) may be as defined herein, such as those defined with respect to formula (VI), and / or as described herein.

[0502] connector

[0503] In some embodiments, the 5T4-ADC may include a connector (L) portion that links the pyridazine-pyrrolo coupling portion, as described herein, to the drug. In any embodiment, the connector L may be represented by the following structure:

[0504] -(T 1 V 1 ) a -(T 2 V 2 ) b -(T 3 V 3 ) c -(T 4 V 4 ) d -(T 5 V 5 ) e -,

[0505] Where a, b, c, d, and e are each independently 0 or 1, and the sum of a, b, c, d, and e is 1 to 5. In some embodiments, the sum of a, b, c, d, and e is 3. In some embodiments, the sum of a, b, c, d, and e is 5.

[0506] In connector L, T 1 、T 2 、T 3 、T 4 and T 5 Each independently is C1-C 12 Alkyl, substituted C1-C 12 Alkyl group, (EDA) w(PEG) n (AA) p 、-(CR 13 OH) h - Piperidin-4-amine (P4A), m-aminobenzoic acid (MABC) group, m-aminobenzooxy (MABO) group, p-aminobenzooxy (PABO) group, p-aminobenzoic acid (PABC) group, p-aminobenzyl (PAB) group, acetal, disulfide, hydrazine, protease-cleavable moiety, glucuronidase-cleavable moiety, β-lactamase-cleavable moiety, ester, (AA) p -MABC-(AA) p (AA) p -MABO-(AA) p (AA) p -PABO-(AA) p Or (AA) p -PABC-(AA) p Where w is an integer from 1 to 20, n is an integer from 1 to 30, each p is independently zero or an integer from 1 to 20, h is an integer from 1 to 12, and each R 13 It is independently hydrogen, alkyl, substituted alkyl, aryl or substituted aryl.

[0507] As used in this article, "(EDA)" w "The ethylenediamine moiety has the following structure:

[0508]

[0509] Where y is an integer from 1 to 6, r is 0 or 1, w is an integer from 1 to 20, and each R 14 Independently hydrogen, alkyl, substituted alkyl, PEG, aryl, or substituted aryl, and any two adjacent Rs are... 14 The groups can be linked in a ring to form a piperazine ring, wherein each This indicates a connection point that connects to an adjacent chemical component in any direction.

[0510] The piperidine-4-amine moiety may optionally be substituted at one or more positions with any substituent, such as alkyl, substituted alkyl, polyethylene glycol moiety, acyl, substituted acyl, aryl, or substituted aryl. Therefore, as used herein, piperidine-4-amine can be referred to by the term "P4A-R" corresponding to the following structure. 12 "describe:

[0511]

[0512] where R 12Independently, it is hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g., polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic, wherein each This indicates a connection point that connects to an adjacent chemical component in any direction.

[0513] As used in this article, (PEG) n It is polyethylene glycol with the following structure:

[0514]

[0515] Where n is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, where each This indicates a connection point that connects to an adjacent chemical component in any direction.

[0516] As used herein, “AA” refers to an amino acid as described herein, including (but not limited to) any naturally occurring amino acid, naturally occurring amino acid analogues, mirror isomers of naturally occurring amino acids, and non-naturally occurring amino acids. p In this document, each amino acid residue may be independently selected from the amino acid residues described herein and p may be an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0517] As used herein, the MABC group can be referred to by the term "MABC-R". 16 "and the following structural descriptions:

[0518]

[0519] As used herein, the MABO group can be referred to by the term "MABO-R". 16 And the following structural descriptions:

[0520]

[0521] As used herein, the PABC group can be referred to by the term "PABC-R". 16 And the following structural descriptions:

[0522]

[0523] As used herein, the PABO group can be referred to by the term "PABO-R". 16 And the following structural descriptions:

[0524]

[0525] As used herein, the p-aminobenzyl (PAB) group can be referred to by the term "PAB-R". 16 And the following structural descriptions:

[0526]

[0527] In MABC-R 16 MABO-R 16 PABC-R 16 PABO-R 16 and PAB-R 16 In the middle, each This indicates a connection point that connects to an adjacent chemical part in any direction, and R 16 Independently, it is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic.

[0528] In some implementations, T 1 、T 2 、T 3 、T 4 and T 5 One or more of them are C1-C6 alkyl groups. In some embodiments, T 1 Independently, it is a C1-C6 alkyl group. In some embodiments, T 1 It is ethyl. In some embodiments, T 1 and T 3 Each of them is a C1-C6 alkyl group. In some embodiments, T 1 and T 3 Each of them is an ethyl group.

[0529] In some implementations, T 1 、T 2 、T 3 、T 4 and T 5One or more of them are P4A-R 12 In some implementation schemes, each R 12 Independent of the polyethylene glycol portion. In some embodiments, each R... 12 Independently, it is polyethylene glycol modified with carboxylic acid. In some embodiments, T 2 For P4A-R 12 , where R 12 It is polyethylene glycol modified with carboxylic acid.

[0530] In some implementations, T 1 、T 2 、T 3 、T 4 and T 5 One or more of them are (PEG). n In some implementations, n is 2. In some implementations, T 3 For (PEG) n , where n is 2.

[0531] In some implementations, T 1 、T 2 、T 3 、T 4 and T 5 One or more of them are (AA) p In some implementations, p is 1. In some implementations, T 2 (AA) p , where p is 1. In some implementations, (AA) p In this context, AA refers to an amino acid with a polar, uncharged side chain (e.g., serine, threonine, asparagine, or glutamine). In some embodiments, (AA) p AA in the text stands for glutamine.

[0532] In some implementations, T 1 、T 2 、T 3 、T 4 and T 5 One or more of them are (AA) p -(PABC-R 16 )-(AA) p In some embodiments, R 16 It is hydrogen. In some embodiments, p is 0. In some embodiments, R 16 It is hydrogen, and p is 0. Therefore, in some embodiments, T 1 、T 2 、T 3 、T 4 and T 5One or more of them are AA-PABC as described by the following structure:

[0533]

[0534] Among them This indicates a connection point that connects to an adjacent chemical component in the connector in any direction.

[0535] In some implementations, p in AA-PABC is 2. In some implementations, T 4 It is AA-PABC, where p is 2. In some embodiments, the two amino acids in (AA)2 are valine and citrulline.

[0536] V in connector L 1 V 2 V 3 V 4 and V 5 Each group can independently be covalent, amino, carbonyl, amide, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phosphoryl, aminophosphate, or thioaminophosphate. In some embodiments, V 1 V 2 V 3 V 4 and V 5 Each is independently a covalent bond, -C(=O)-, -NR 11 -、-C(=O)NR 11 -、-NR 11 C(=O)-, -C(=O)O-, -OC(=O)-, -O-, -S-, -S(=O)-, -SO2-, -SO2NR 11 -、-NR 11 SO2- and -P(=O)OH-, where R 11 It is hydrogen, alkyl, substituted alkyl, PEG, aryl, or substituted aryl. In some embodiments, V 1 V 2 and V 3 Independently C (=O) or -NR 11 -, where R 11 It is independently hydrogen or C1-C6 alkyl.

[0537] In some implementations, the connector L can be represented by the following structure:

[0538] -(T 1 V 1 ) a -(T 2 V 2 ) b -(T3 V 3 ) c -(T 4 V 4 ) d -(T 5 V 5 ) e -,

[0539] in:

[0540] a, b, c, d, and e are each independently 0 or 1, and the sum of a, b, c, d, and e is between 1 and 5;

[0541] T 1 It is a C1-C6 alkyl group;

[0542] T 2 、T 3 、T 4 and T 5 Each independently constitutes (PEG) n C1-C6 alkyl groups, (AA) p P4A-R 12 (AA) p -(PABO-R 16 )-(AA) p (AA) p -(PABC-R 16 )-(AA) p (AA) p -(PABO-R 16 ) or (AA) p -(PABC-R 16 );

[0543] V 1 V 2 V 3 V 4 and V 5 Each is independently a covalent bond, -CO- or -NR. 11 -;

[0544] Each R 11 It is independently hydrogen, alkyl, substituted alkyl, polyethylene glycol, aryl or substituted aryl;

[0545] Each R 12 Independently, it is a hydrogen, alkyl, substituted alkyl, or modified or unmodified polyethylene glycol moiety;

[0546] R 16 It is hydrogen;

[0547] n is an integer from 1 to 30; and

[0548] p is an integer from 1 to 20.

[0549] In some implementation schemes, R 12 For (PEG) n In some implementations, R 12 It is a PEG with terminal carboxylic acid modification. n In some implementations, R 1111 It is hydrogen or C1-C 12 Alkyl group. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 1 at one position in the connector and 2 at another position in the connector. In some embodiments, n is an integer from 1 to 5. In some embodiments, n is 2.

[0550] Non-disintegrable connector

[0551] In some implementations, the connector L can be represented by the following structure:

[0552] (T 1 V 1 ) a -(T 2 V 2 ) b -(T 3 V 3 ) c -(T 4 V 4 ) d -(T 5 V 5 ) e -,

[0553] in:

[0554] a, b, c, d, and e are each independently 0 or 1, and the sum of a, b, c, d, and e is between 1 and 5;

[0555] T 1 It is a C1-C6 alkyl group;

[0556] T 2 and T 3 Each is independently a C1-C6 alkyl or P4A-R 12 ;

[0557] T 4 and T 5 Each independently constitutes (PEG) n C1-C6 alkyl groups, (AA) p P4A-R 12 (AA) p -(PABO-R 16 )-(AA)p (AA) p -(PABC-R 16 )-(AA) p (AA) p -(PABO-R 16 ) or (AA) p -(PABC-R 16 );

[0558] V 1 V 2 V 3 V 4 and V 5 Each is independently a covalent bond, -CO- or -NR. 11 -;and

[0559] Each R 11 Independently hydrogen or C1-C6 alkyl; and

[0560] Each R 12 Independent of the modified or unmodified polyethylene glycol moiety.

[0561] In some implementations, T 1 and T 3 Each of them is independently a C1-C6 alkyl group, and independently such as methyl, ethyl, or propyl. In some embodiments, T 1 and T 3 Each of them is ethyl. In some embodiments, T 2 For P4A-R 12 , where R 12 This refers to the modified or unmodified polyethylene glycol moiety. In some embodiments, d and e are 0.

[0562] In some implementations, the connector L can be represented by the following structure:

[0563] -(C1-C6 alkyl)-(V 1 )-(P4A-R 12 )-(V 2 )-(C1-C6 alkyl)-(V 3 )-,

[0564] in:

[0565] V 1 V 2 and V 3 Each is independently -CO- or -NR 11 -;

[0566] Each R 11 Independently hydrogen or C1-C6 alkyl; and

[0567] Each R 12 Independently, it is a polyethylene glycol moiety modified with carboxylic acid.

[0568] In some implementations, connector L is connector (L-1):

[0569]

[0570] in * indicates a connection point to the nitrogen of the pyridazine-pyrrole coupling portion as described herein, and * indicates a connection point to W. 1 The connection point.

[0571] Therefore, in some implementations, the 5T4-ADC can be represented by equation (VI-1):

[0572]

[0573] in:

[0574] Ab is an antibody that binds to 5T4;

[0575] r is an integer from 1 to 10; and

[0576] W 1 It is a drug.

[0577] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0578] In some implementations, the 5T4-ADC can be represented by equation (VI-1), where X 1 Let C be the value. Therefore, in some implementations, the 5T4-ADC can be represented by equation (VIa-1):

[0579]

[0580] in:

[0581] Ab is an antibody that binds to 5T4;

[0582] r is an integer from 1 to 10; and

[0583] W 1 It is a drug.

[0584] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0585] In some implementations, the 5T4-ADC can be represented by equation (VI-1), where X 1 Let N be the number of digits. Therefore, in some implementations, the 5T4-ADC can be represented by equation (VIb-1):

[0586]

[0587] in:

[0588] Ab is an antibody that binds to 5T4;

[0589] r is an integer from 1 to 10; and

[0590] W 1 It is a drug.

[0591] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0592] Disintegral connector

[0593] In some embodiments, the antibody and drug are conjugated to each other via a cleavable linker. A cleavable linker is a linker comprising one or more cleavable portions, wherein said cleavable portions include one or more bonds that can dissociate under certain conditions, thereby separating the cleavable linker into two or more separable portions. For example, the cleavable portions may include one or more covalent bonds that can dissociate or decompose under certain conditions to separate the cleavable linker into two or more portions. Therefore, a cleavable linker may be included in a 5T4-ADC such that, under certain conditions, the cleavable linker is cleaved to allow the drug to separate from or be released from the antibody at the desired target site of the drug.

[0594] In some embodiments, the cleavable linker may be an enzyme-cleavable linker. The enzyme-cleavable linker can be separated into two or more parts as described above by enzymatic action of an enzyme. The enzyme-cleavable linker may contain any cleavable moiety that can be cleaved by enzymatic action, such as (but not limited to) peptides, glycosides, and analogues. In some cases, the enzyme that cleaves the enzyme-cleavable linker is present at the desired target site, such as the desired target site of a drug to be released from the antibody-drug conjugate. In some cases, the enzyme that cleaves the enzyme-cleavable linker is not present in large quantities in other regions, such as whole blood, plasma, or serum. This allows for controllable cleavage of the enzyme-cleavable moiety, ensuring that substantial cleavage occurs at the desired site of action, while significant cleavage does not occur in other regions or before the antibody-drug conjugate reaches the desired site of action.

[0595] In some embodiments, the enzyme-cleavable linker comprises a peptide. The peptide can be any peptide suitable for the cleavable linker and capable of being cleaved by the enzymatic action of an enzyme. Non-limiting examples of peptides that can be used as the enzyme-cleavable portion include, for example, Val-Ala; Phe-Lys; and analogues thereof.

[0596] In some implementations, the connector L is detachable and can be represented by the following structure:

[0597] -(T1 V 1 ) a -(T 2 V 2 ) b -(T 3 V 3 ) c -(T 4 V 4 ) d -(T 5 V 5 ) e -,

[0598] in:

[0599] a, b, c, d, and e are each independently 0 or 1, and the sum of a, b, c, d, and e is between 1 and 5;

[0600] T 1 and T 5 Each is a C1-C6 alkyl group;

[0601] T 2 、T 3 and T 4 Each independently constitutes (PEG) n (AA) p Or (AA) p -PABC-R 16 ;

[0602] V 1 V 2 V 3 V 4 and V 5 Each is independently a covalent bond, -CO- or -NR. 11 -;

[0603] R 11 As mentioned in this article;

[0604] R 16 As mentioned in this article;

[0605] Each n is an independent integer from 1 to 30; and

[0606] Each p is an independent integer from 1 to 20.

[0607] In some implementations, n is an integer from 1 to 5. In some implementations, n is 2.

[0608] In some implementations, p is 1. In some implementations, p is 2. In some implementations, p is 1 at one location in the connector L and 2 at another location in the connector L.

[0609] In some implementations, T 2 For amino acid residues. In some embodiments, T 2 These are amino acid residues with polar, uncharged side chains. In some embodiments, T 2 It is a glutamine residue.

[0610] In some implementations, T 3 For (PEG) n , where n is an integer from 1 to 30, such as 1 to 5. In some implementations, n is 2.

[0611] In some implementations, T 4 (AA) p -PABC-R 16 Where p is an integer from 1 to 20, such as 1 to 5, and R 16 The molar is hydrogen. In some embodiments, p is 2. In some embodiments, the amino acid (AA) is valine and citrulline.

[0612] In some implementations, the connector L can be represented by the following structure:

[0613] -(C1-C6 alkyl)-V 1 -(AA)-V 2 -(PEG) n -V 3 -((AA) p -(PABC-R 16 ))-V 4 -(C1-C6 alkyl)-V 5 -,

[0614] in:

[0615] R 16 It is hydrogen;

[0616] Each of n and p is an integer from 1 to 30, 1 to 20, or 1 to 5;

[0617] V 1 V 2 V 3 V 4 and V 5 Each is independently a covalent bond, -CO- or -NR. 11 -;and

[0618] Each R 11 It is independently hydrogen or C1-C6 alkyl.

[0619] In some implementations, both n and p are 2.

[0620] In some implementations, connector L may be connector (L-2):

[0621]

[0622] in This indicates a connection to the pyridazine-pyrrole coupling moiety as described herein, and * indicates a connection to W. 1 The connection point.

[0623] In some implementations, the 5T4-ADC can be represented by equation (VI-2):

[0624]

[0625] in:

[0626] Ab is an antibody that binds to 5T4;

[0627] r is an integer from 1 to 10; and

[0628] W 1 It is a drug.

[0629] In some implementation schemes, X 1 It can be N or C.

[0630] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0631] In some implementations, the 5T4-ADC can be represented by equation (VI-2), where X 1 For C. Therefore, in some implementations, the 5T4-ADC can be represented by equation (VIa-2):

[0632]

[0633] in:

[0634] Ab is an antibody that binds to 5T4;

[0635] r is an integer from 1 to 10; and

[0636] W 1 It is a drug.

[0637] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0638] In some implementations, the 5T4-ADC can be represented by equation (VI-2), where X 1 Let N be the number of digits. Therefore, in some implementations, the 5T4-ADC can be represented by equation (VIb-2):

[0639]

[0640] in:

[0641] Ab is an antibody that binds to 5T4;

[0642] r is an integer from 1 to 10; and

[0643] W 1 It is a drug.

[0644] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0645] Dual pyrolysis connector

[0646] In some embodiments, the cleavable linker includes two cleavable portions, such as a first cleavable portion and a second cleavable portion. The cleavable portions can be configured such that cleavage of both cleavable portions is required to dissociate or release the drug from the antibody, for example, at a desired target site of action. For example, cleavage of the cleavable linker can be achieved by first cleaving one of the two cleavable portions and then cleaving the other. In some embodiments, the cleavable linker includes a first cleavable portion and a second cleavable portion that inhibits cleavage of the first cleavable portion. "Inhibits cleavage" means that the presence of an uncracked second cleavable portion reduces the likelihood of cleavage of the first cleavable portion or substantially inhibits cleavage of the first cleavable portion, thereby substantially reducing the amount of cleavage of the cleavable linker or preventing cleavage of the cleavable linker. In embodiments, the presence of an uncracked second cleavable portion can inhibit enzymatic and / or chemical cleavage of the first cleavable portion. The inhibitory effect of the second cleavable portion on the cleavage of the first cleavable portion, in turn, substantially reduces the amount of drug released from the antibody or prevents release. For example, it can substantially reduce or prevent premature release of the drug from the antibody until the 5T4-ADC is located at or near the desired target site of the drug.

[0647] In some embodiments, the second cleavable portion hinders the cleavage of the first cleavable portion, and cleavage of the cleavable connector can be achieved by first cleaving the second cleavable portion and then cleaving the first cleavable portion. Cleavage of the second cleavable portion can reduce or eliminate steric hindrance to the cleavage of the first cleavable portion, thereby allowing the first cleavable portion to cleave. Cleavage of the first cleavable portion can cause the cleavable connector to dissociate or separate into two or more portions as described herein, thereby allowing drug release from the 5T4-ADC. In some embodiments, the first cleavable portion does not substantially cleave in the presence of the uncracked second cleavable portion. The term “substantially” in this document means that, in the presence of an un-fractured second fractured portion, the first fractured portion undergoes about 10% or less of fracture, such as, in the presence of an un-fractured second fractured portion, the first fractured portion undergoes about 9% or less, or about 8% or less, or about 7% or less, or about 6% or less, or about 5% or less, or about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less, or about 0.5% or less, or about 0.1% or less of fracture.

[0648] In other words, the second cleavable portion prevents the first cleavable portion from cleaving. In embodiments, the presence of the uncracked second cleavable portion prevents the first cleavable portion from cleaving, and thus substantially reduces or prevents premature release of the drug from the 5T4 antibody until the 5T4-ADC is located at or near the desired target site of the drug. Therefore, cleavage of the second cleavable portion exposes the first cleavable portion (e.g., removes the protecting group of the first cleavable portion), thereby allowing the first cleavable portion to cleave, causing cleavage of the cleavable linker, which in turn allows the drug to separate or be released from the antibody at the desired target site of the drug, as described herein. In some embodiments, cleavage of the second cleavable portion exposes the first cleavable portion for subsequent cleavage, but cleavage of the second cleavable portion does not, and does not itself, cause cleavage of the cleavable linker (e.g., cleavage of the first cleavable portion is still required for cleavage of the cleavable linker).

[0649] Examples of suitable cleavable portions in cleavable connectors include chemically cleavable portions and enzyme-cleavable portions. For example, a first cleavable portion may be chemically cleavable and a second cleavable portion may be chemically cleavable, and the second cleavable portion may be the same as or different from the first cleavable portion. In other embodiments, the first cleavable portion may be enzyme-cleavable and the second cleavable portion may be chemically cleavable. In still other embodiments, the first cleavable portion may be chemically cleavable and the second cleavable portion may be enzyme-cleavable. In still other embodiments, the first cleavable portion may be enzyme-cleavable and the second cleavable portion may be enzyme-cleavable, and the second cleavable portion may be the same as or different from the first cleavable portion.

[0650] A chemically cleavable moiety includes a cleavable portion that can be cleaved in the presence of certain chemical conditions. In some embodiments, the chemically cleavable moiety includes one or more bonds that can dissociate in the presence of certain chemical conditions, thereby separating the cleavable moiety into two or more separable moieties. For example, the chemically cleavable moiety is cleavable in the presence of chemical conditions that can cause hydrolysis of the chemically cleavable moiety, such as acidic or alkaline conditions. In some embodiments, the chemical conditions by which the chemically cleavable moiety is cleaved may be present at the desired target site, such as the desired target site of a drug to be released from a 5T4-ADC. In some embodiments, the chemical conditions found at the desired cleavage site of the chemically cleavable moiety are not significantly present in other regions, such as whole blood, plasma, or serum. This allows for controlled cleavage of the chemically cleavable moiety, such that substantial cleavage occurs at the desired site of action, while cleavage does not significantly occur in other regions or before the 5T4-ADC reaches the desired site of action.

[0651] In some embodiments, the cleavable portion may be an enzyme-cleavable portion. An enzyme-cleavable portion is a cleavable portion that can be separated into two or more portions as described above by the enzymatic action of an enzyme. The enzyme-cleavable portion may be any cleavable portion that can be cleaved by the enzymatic action of an enzyme, such as (but not limited to) peptides, glycosides, and analogues. In some embodiments, the enzyme that cleaves the enzyme-cleavable portion is present at the desired target site, such as the desired target site of a drug to be released from the 5T4-ADC. In some embodiments, the enzyme that cleaves the enzyme-cleavable portion is not present in large quantities in other regions, such as whole blood, plasma, or serum. This allows for controlled cleavage of the enzyme-cleavable portion, such that substantial cleavage occurs at the desired site of action, while significant cleavage does not occur in other regions or before the 5T4-ADC reaches the desired site of action (e.g., cancer cells).

[0652] In some implementations, connector L may be connector (L-3):

[0653]

[0654] in:

[0655] This indicates the connection point to the pyridazine-pyrrole coupling portion as described herein (e.g., any of formulas IV, V, and VI);

[0656] * indicates a connection to W 1 connection points;

[0657] Each R 5 Independently, it is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0658] Each R 6 Independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0659] R 7 It is a pyrolytic component;

[0660] k is an integer from 1 to 10;

[0661] L 1 Includes -(T) 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -,

[0662] L 2 Includes -(T) 5 -V 5 ) e -(T 6 -V 6 ) f -(T 7 -V 7 ) g -(T 8 -V 8 ) h -;

[0663] T 1 、T 2 、T 3 、T 4 、T 5 、T 6 、T 7 and T 8 Each is independently a covalent bond, C1-C 12 Alkyl, substituted C 1- C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) m -、P4A-R 12acetals, hydrazines, disulfides or esters;

[0664] V 1 、V 2 、V 3 、V 4 、V 5 、V 6 、V 7 and V 8 Each is independently a covalent bond, -CO-, -NR 15 -、-NR 15 (CH2) q -、-NR 15 (C6H4)-、-CONR 15 -、-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- or -P(O)OH-;

[0665] Each of a, b, c, d, e, f, g, and h is independently 1 or 0;

[0666] m is an independent integer from 1 to 12;

[0667] n is an independent integer from 1 to 30;

[0668] p is an independent integer from 1 to 20;

[0669] q is an independent integer from 1 to 6;

[0670] w is an independent integer from 1 to 20;

[0671] Each R 12 Independently, it is hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g., polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0672] Each R 13 Independently hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl; and

[0673] Each R 15Independently, it is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic.

[0674] In some implementations, the 5T4-ADC of formula (V) includes a connector (L-3). Therefore, in some implementations, the 5T4-ADC can be represented by formula (V-3).

[0675]

[0676] in:

[0677] Ab is an antibody that binds to 5T4;

[0678] r is an integer from 1 to 10;

[0679] X 1 It can be C or N;

[0680] W 1 For medicine;

[0681] R 2 、R 3 、Y 1 、Y 2 、Y 3 and Y 4 As defined herein, such as with respect to equation (V); and

[0682] L 1 , L 2 、R 5 、R 6 、R 7 And k is as defined in this article, such as with respect to the connector (L-3).

[0683] In some implementations, r is an integer from 1 to 4. In some implementations, r is an integer from 1 to 2.

[0684] In some implementations, the 5T4-ADC of formula (VI) includes a connector (L-3). Therefore, in some implementations, the 5T4-ADC can be represented by formula (VI-3).

[0685]

[0686] in:

[0687] Ab is an antibody that binds to 5T4;

[0688] r is an integer from 1 to 10;

[0689] X 1 It can be C or N;

[0690] W 1 For medicine; and

[0691] L 1 L 2 、R 5 、R 6 、R 7 And k is as defined in this article, such as with respect to the connector (L-3).

[0692] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0693] In some embodiments of connector (L-3), k is 2. Therefore, connector L can be connector (L-4):

[0694]

[0695] in:

[0696] This indicates the connection point to the pyridazine-pyrrole coupling portion as described herein (e.g., any of formulas IV, V, and VI);

[0697] * indicates a connection point to W;

[0698] Each R 5 Independently, it is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0699] R 6′ and R 6″ Each of these can be independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0700] R 7 It is a divisible part; and

[0701] L 1 and L 2 As defined herein, such as with regard to connector (L-3).

[0702] In some implementations, the 5T4-ADC of formula (V) includes a connector (L-4). Therefore, in some implementations, the 5T4-ADC can be represented by formula (V-4):

[0703]

[0704] in:

[0705] Ab is an antibody that binds to 5T4;

[0706] r is an integer from 1 to 10;

[0707] X 1 It can be C or N;

[0708] W 1 For medicine;

[0709] R 2 、R 3 、Y 1 、Y 2 、Y 3 and Y 4 As defined in this article, such as with respect to equation (V);

[0710] L 1 L 2 、R 5 、R 6′ 、R 6″ and R 7 As defined herein, such as with regard to connector (L-4).

[0711] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0712] In some embodiments, the 5T4-ADC of formula (VI) includes a connector (L-4). In some embodiments, the 5T4-ADC may be represented by formula (VI-4).

[0713]

[0714] in:

[0715] Ab is an antibody that binds to 5T4;

[0716] r is an integer from 1 to 10;

[0717] X 1 It can be C or N;

[0718] W 1 For medicine;

[0719] L 1 L 2 、R 5 、R 6′ 、R 6″ and R 7 As defined herein, such as with regard to connector (L-4).

[0720] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0721] In some embodiments, R 7 The cleavable portion of the enzyme contains a sugar moiety, such as a glycoside or glycosyl group. In some embodiments, the glycoside may contribute to the enhanced hydrophilicity of the cleavable linker compared to a cleavable linker that does not contain a glycoside. The glycoside may be any glycoside suitable for the cleavable linker and capable of being cleaved by the enzymatic action of an enzyme.

[0722] R 7 The glycoside in one portion can be linked to the rest of the 5T4-ADC via a glycosidic bond. The glycosidic bond allows the glycoside to be linked to the 5T4-ADC via various types of bonds, such as (but not limited to) O-glycosidic bonds (O-glycosides), N-glycosidic bonds (glycosylamines), S-glycosidic bonds (thioglycosides), or C-glycosidic bonds (C-glycosides or C-glycosyl groups). In some embodiments, the glycosidic bond is an O-glycosidic bond (O-glycoside).

[0723] In some embodiments, R 7 for Where ** indicates a connection point to the phenyl group described herein, such as formula (VI-3), (V-4), or (VI-4). Therefore, connector L can be connector (L-5):

[0724]

[0725] in:

[0726] This indicates the connection point to the pyridazine-pyrrole coupling portion as described herein (e.g., any of formulas IV, V, and VI);

[0727] * indicates a connection to W 1 connection points;

[0728] Each R 5 Independently, it is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0729] R 6′ and R 6″ Each of these groups is independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, or substituted heterocyclic; and

[0730] L 1 and L 2As defined herein, such as with regard to connector (L-3).

[0731] In some implementations, the connector (L-5) is connected to formula (V). In some implementations, the 5T4-ADC can be represented by formula (V-5):

[0732]

[0733] in:

[0734] Ab is an antibody that binds to 5T4;

[0735] r is an integer from 1 to 10;

[0736] X 1 It can be C or N;

[0737] W 1 For medicine;

[0738] R 2 、R 3 、Y 1 、Y 2 、Y 3 and Y 4 As defined in this article, such as with respect to equation (V);

[0739] L 1 L 2 、R 5 、R 6′ and R 6″ As defined herein, such as with regard to connector (L-5).

[0740] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0741] In some embodiments, the 5T4-ADC of formula (VI) may include a connector (L-5). Therefore, in some embodiments, the 5T4-ADC may be represented by formula (VI-5):

[0742]

[0743] in:

[0744] Ab is an antibody that binds to 5T4;

[0745] r is an integer from 1 to 10;

[0746] X 1 It can be C or N;

[0747] W 1 For medicine;

[0748] L1 , L 2 、R 5 、R 6′ and R 6″ As defined herein, such as with regard to connector (L-5).

[0749] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0750] In some implementations of the connector (L-5), R 5 For hydrogen, R 6′ It is isopropyl and R 6" It is methyl. Therefore, in some embodiments, the connector L can be a connector (L-6):

[0751]

[0752] in:

[0753] This indicates the connection point to the pyridazine-pyrrole coupled portion (e.g., any of formulas IV, V, and VI);

[0754] * indicates a connection to W 1 The connection point; and

[0755] L 1 and L 2 As defined herein, such as with regard to connector (L-5).

[0756] In some implementations, the 5T4-ADC of formula (V) includes a connector (L-6). Therefore, in some implementations, the 5T4-ADC can be represented by formula (V-6):

[0757]

[0758] in:

[0759] Ab is an antibody that binds to 5T4;

[0760] r is an integer from 1 to 10;

[0761] X 1 It can be C or N;

[0762] R 2 、R 3 、Y 1 、Y 2 、Y 3 and Y 4 As defined in this article, such as with respect to equation (V);

[0763] W 1 For medicine; and

[0764] L 1 and L 2 As defined herein, such as with regard to connector (L-6).

[0765] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0766] In some implementations, the 5T4-ADC of formula (VI) includes a connector (L-6). Therefore, in some implementations, the 5T4-ADC can be represented by formula (VI-6):

[0767]

[0768] in:

[0769] Ab is an antibody that binds to 5T4;

[0770] r is an integer from 1 to 10;

[0771] X 1 It can be C or N;

[0772] W 1 For medicine; and

[0773] L 1 and L 2 As defined herein, such as with regard to connector (L-6).

[0774] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0775] In some implementations, the 5T4-ADC can be represented by formulas (V-3), (V-4), (V-5), (V-6), (VI-3), (VI-4), (VI-5), or (VI-6), where:

[0776] L 1 Includes -(T) 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -;

[0777] L 2 Includes -(T) 5 -V 5 ) e-(T 6 -V 6 ) f -(T 7 -V 7 ) g -(T 8 -V 8 ) h -;

[0778] T 1 、T 2 and T 3 Each independently is C1-C 12 Alkyl group, (PEG) n Or (AA) p ;

[0779] T 5 、T 6 、T 7 and T 8 Each is independently a covalent bond, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) m -、P4A-R 12 acetals, hydrazines, disulfides, and esters;

[0780] V 1 、V 2 、V 3 、V 4 、V 5 、V 6 、V 7 and V 8 Each is independently -C (=O)- or -NR 1111 -;

[0781] a, b, and c are each 1;

[0782] d is 0 and therefore, T 4 and V 4 does not exist;

[0783] Each of e, f, g, and h is independently 1 or 0;

[0784] n is an integer from 1 to 10;

[0785] p is an integer from 1 to 10; and

[0786] R1111 It can be hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl or substituted aryl.

[0787] In some implementations, the 5T4-ADC can be represented by formulas (V-3), (V-4), (V-5), (V-6), (VI-3), (VI-4), (VI-5), or (VI-6), where T 1 、T 2 and T 3 It is a C1-C6 alkyl group, (AA) p Or (PEG) n And p and n are independent integers from 1 to 10. In some implementations, p is 1. In some implementations, n is 2. In some implementations, (AA) p It may include non-naturally occurring amino acids, such as cysteine ​​sulfonate amino acids. In some embodiments, V 1 、V 2 and V 3 Independently -C (=O)- or -NR 15 -, where R 15 For hydrogen.

[0788] In any of the implementations described herein, L 1 It can be as follows:

[0789] -(C1-C6 alkyl)-V 1 -(AA) p -V2-(PEG) n -V 3

[0790] Where V 1 、V 2 、V 3 AA, p, and n are as described herein, such as as defined with respect to the connector (L-3). In some embodiments, V 1 、V 2 and V 3 -C (=O) or -NR 15 -, where R 15 It is hydrogen. In some embodiments, p is 1 and n is 2. In some embodiments, (AA) p It may include non-naturally occurring amino acids, such as cysteine ​​sulfonic acid amino acids.

[0791] In some implementations, L 1 for Among them each This indicates the connection point that connects to an adjacent chemical portion of the connector in any direction. Therefore, in some embodiments, connector L may be connector (L-7):

[0792]

[0793] in:

[0794] This indicates the connection point to the pyridazine-pyrrole coupling portion as described herein (e.g., any of formulas IV, V, and VI);

[0795] * indicates a connection to W 1 The connection point; and

[0796] L 2 As defined herein, such as with regard to connector (L-3).

[0797] In some implementations, the 5T4-ADC can be represented by equation (V-7):

[0798]

[0799] in:

[0800] Ab is an antibody that binds to 5T4;

[0801] r is an integer from 1 to 10;

[0802] X 1 It can be C or N;

[0803] R 2 、R 3 、Y 1 、Y 2 、Y 3 and Y 4 As defined in this article, such as with respect to equation (V);

[0804] W 1 For medicine; and

[0805] L 2 As defined herein, such as with regard to connector (L-7).

[0806] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0807] In some implementations, the 5T4-ADC can be represented by equation (VI-7):

[0808]

[0809] in:

[0810] Ab is an antibody that binds to 5T4;

[0811] r is an integer from 1 to 10;

[0812] X 1 It can be C or N;

[0813] W 1 For medicine; and

[0814] L 2 As defined herein, such as with regard to connector (L-7).

[0815] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0816] In L 2 In some implementations, e is 1, T 5 For covalent bonds, V 5 It is a carbonyl group, and each of f, g, and h is 0. In some embodiments, L 2 It is a carbonyl group (C=O). Therefore, in some embodiments, the connector L can be a connector (L-8):

[0817]

[0818] in:

[0819] This indicates the connection point to the pyridazine-pyrrolo coupling portion as described herein (e.g., any of formulas IV, V, and VI); and

[0820] * indicates a connection to W 1 The connection point.

[0821] In some implementations, the connector (L) in the 5T4-ADC of formula (V) can be connector (L-8). Therefore, in some implementations, the 5T4-ADC can be represented by formula (V-8):

[0822]

[0823] in:

[0824] Ab is an antibody that binds to 5T4;

[0825] r is an integer from 1 to 10;

[0826] X 1 It can be C or N;

[0827] R 2 、R 3 、Y 1 、Y 2 、Y 3 and Y 4 As defined herein, such as with respect to equation (V); and

[0828] W 1 It is a drug.

[0829] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0830] In some implementations, the connector (L) in the 5T4-ADC of formula (VI) can be connector (L-8). Therefore, in some implementations, the 5T4-ADC can be represented by formula (VI-8):

[0831]

[0832] in:

[0833] Ab is an antibody that binds to 5T4;

[0834] r is an integer from 1 to 10;

[0835] X 1 It is C or N; and

[0836] W 1 It is a drug.

[0837] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0838] In some implementations, the 5T4-ADC can be represented by equation (VI-8), where X 1 For C. Therefore, in some implementations, the 5T4-ADC can be represented by equation (VIa-8):

[0839]

[0840] in:

[0841] Ab is an antibody that binds to 5T4;

[0842] r is an integer from 1 to 10; and

[0843] W 1 It is a drug.

[0844] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0845] In some implementations, the 5T4-ADC can be represented by equation (VI-8), where X 1 Let N be the number of digits. Therefore, in some implementations, the 5T4-ADC can be represented by equation (VIb-8):

[0846]

[0847] in:

[0848] Ab is an antibody that binds to 5T4;

[0849] r is an integer from 1 to 10; and

[0850] W 1 It is a drug.

[0851] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0852] drug

[0853] Drugs applicable to 5T4-ADCs as described herein (represented as W in the formulas provided herein) 1 There are no particular limitations. Examples of suitable drugs include (but are not limited to) small molecule drugs, such as cancer chemotherapy agents. For example, a drug may be an agent that affects microtubules. In some embodiments, the drug is an agent that affects microtubules and has antiproliferative activity, such as levothyroxine or olistatin.

[0854] Maytansine

[0855] In some implementations, the drug is a maytansine-like substance. For example, the drug maytansine has the following structure (W-1):

[0856]

[0857] Or its derivatives, such as deacylated maytansine (W-1′):

[0858]

[0859] in The connection point of the connector in the 5T4-ADC indicates the connection of the methyl methacrylate (MTA) to the connector.

[0860] For example, in any 5T4-ADC represented by equations (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), and (VIb-8), W 1 It can be a maytansine, such as W-1 or W-1′ maytansine, in which Indicator of gentamicin and L or L 2 The connection points between them.

[0861] In some implementations, the 5T4-ADC can be represented by equation (VI-11), equation (VI-21), or equation (VI-81):

[0862]

[0863]

[0864] in:

[0865] Ab is an antibody that binds to 5T4;

[0866] r is an integer from 1 to 10; and

[0867] X 1 It can be C or N.

[0868] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0869] In some implementation schemes, X 1 Let C be the value of 5T4-ADC, and 5T4-ADC can be represented by equation (VIa-11) or equation (VIa-21):

[0870]

[0871]

[0872] in:

[0873] Ab is an antibody that binds to 5T4; and

[0874] r is an integer from 1 to 10.

[0875] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0876] In some implementation schemes, X 1 Let N be the integer part of the 5T4-ADC, and let N be the integer part of the ADC. Then N can be expressed by equation (VIb-81):

[0877]

[0878] in:

[0879] Ab is an antibody that binds to 5T4; and

[0880] r is an integer from 1 to 10.

[0881] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0882] In some implementations, the above-described maytansine may be referred to as deacylated maytansine or deacylated maytansine.

[0883] Oristatin

[0884] In some embodiments, the drug is an antimitotic agent, such as oliquistatin or an active oliquistatin analog or derivative thereof (e.g., monomethyloliquistatin D (MMAD), monomethyloliquistatin E (MMAE), monomethyloliquistatin F (MMAF), and analogs thereof). For example, in any formula representing the 5T4-ADC described herein, W 1 It can be MMAE, MMAF, or MMAD.

[0885] In some implementation schemes, W 1 For MMAE, it has the following structure (W-2):

[0886]

[0887] in Indicates the MMAE in the 5T4-ADC and the connector L (such as L disclosed herein). 2 Connection points between parts.

[0888] For example, the MMAE can be included in a 5T4-ADC that includes a connector (L-1). Therefore, in some embodiments, the 5T4-ADC can be represented by formula (VI-12):

[0889]

[0890] in:

[0891] Ab is an antibody that binds to 5T4;

[0892] r is an integer from 1 to 10; and

[0893] X 1 It can be C or N.

[0894] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0895] In some implementations, the MMAE may be included in a 5T4-ADC containing a connector (L-5). Therefore, in some implementations, the 5T4-ADC may be represented by formula (VI-52):

[0896]

[0897] in:

[0898] Ab is an antibody that binds to 5T4;

[0899] r is an integer from 1 to 10;

[0900] X 1 It is C or N; and

[0901] L 1 and L 2 As defined herein, such as with regard to connector (L-3).

[0902] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0903] In some implementations, the MMAE may be included in a 5T4-ADC that includes a connector (L-6). Therefore, in some implementations, the 5T4-ADC may be represented by formula (VI-62):

[0904]

[0905] in:

[0906] Ab is an antibody that binds to 5T4;

[0907] r is an integer from 1 to 10;

[0908] X 1 It is C or N; and

[0909] L 1 and L 2 As defined herein, such as with regard to connector (L-3).

[0910] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0911] In some implementations, the 5T4-ADC can be represented by equations (VI-12), (VI-52), and (VI-62), where X 1 For N. In some implementations, the 5T4-ADC can be represented by equation (VIb-12), equation (VIb-52), or equation (VIb-62):

[0912]

[0913] in

[0914] Ab is an antibody that binds to 5T4;

[0915] r is an integer from 1 to 10;

[0916] R 5 、R 6′ 、R 6″ L 1 and L 2 As defined herein, such as those relating to connector (L-5); and

[0917] r is an integer from 1 to 10.

[0918] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0919] In some implementations, the MMAE may be included in a 5T4-ADC containing a connector (L-7). Therefore, the 5T4-ADC can be represented by equation (VIb-72):

[0920]

[0921] in:

[0922] Ab is an antibody that binds to 5T4;

[0923] r is an integer from 1 to 10; and

[0924] L 2 As defined herein, such as with regard to connector (L-6).

[0925] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0926] In some implementations, the MMAE may be included in a 5T4-ADC containing a connector (L-8). Therefore, the 5T4-ADC can be represented by equation (VIb-82):

[0927]

[0928] in

[0929] Ab is an antibody that binds to 5T4; and

[0930] r is an integer from 1 to 10.

[0931] In some implementations, r is an integer from 1 to 4. In some implementations, r is 1 or 2.

[0932] In some implementation schemes, W 1 For MMAE, it has the following structure (W-3):

[0933]

[0934] in Indicates the connection point between the MMAF and the connector in the 5T4-ADC.

[0935] For example, MMAF can be included in a 5T4-ADC, as shown below:

[0936]

[0937] in:

[0938] Ab is an antibody that binds to 5T4;

[0939] r is an integer from 1 to 10; and

[0940] Y 1 、Y 2 、Y 3 、Y 4 、X 1 、R 2 、R 3 And L as defined in this paper, such as with respect to equation (V).

[0941] In some embodiments, L is selected from connectors (L-1), (L-2), (L-3), (L-4), (L-5), (L-6), (L-7), and (L-8). In some embodiments, X 1 It is C or N. In some implementations, Y 1 、Y 2 、Y 3 and Y 4 Each of them is hydrogen. In some implementations, R 2 and R 3 Independently, it is a C1-C6 alkyl group, such as methyl. In some embodiments, r is an integer from 1 to 10. In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0942] Summary Chapter

[0943] In some embodiments, 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-1 2), (VI-52), (VI-62), (VIb-1 2), (VIb-52), (VIb-62), (VIb-72), or (VIb-82), where Ab is the antibody that binds to 5T4 as disclosed herein. In some embodiments, the antibody comprises a VH, which contains one or more (such as one, two, or three) CDRs that are consistent with or substantially consistent with the CDRs described herein (such as any of Tables 1 to 3). Alternatively or additionally, in some embodiments, the antibody comprises a VL, which contains one or more (such as one, two, or three) CDRs that are consistent with or substantially consistent with the CDRs described herein (such as any of Tables 1 to 3).

[0944] In some embodiments, 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-1 2), (VI-52), (VI-62), (VIb-1 2), (VIb-52), (VIb-62), (VIb-72), or (VIb-82), where Ab is the antibody that binds to 5T4 as disclosed herein. In some embodiments, the antibody contains a VH that is consistent with or substantially consistent with the VH described herein (such as any of Tables 1 to 3). Alternatively or additionally, in some embodiments, the antibody contains a VL that is consistent with or substantially consistent with the VL described herein (such as any of Tables 1 to 3).

[0945] In some implementations, 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (V1-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (V1-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-1 2), (VI-52), (VI-62), (VIb-1 2), (VIb-52), (VIb-62), (VIb-72), or (VIb-82), where Ab is an antibody that binds to 5T4. In some embodiments, the antibody comprises: (i) a VH containing the amino acid sequence SEQ ID NO: 25 and a VL containing the amino acid sequence SEQ ID NO: 26; or (ii) a VH containing the amino acid sequence SEQ ID NO: 44 and a VL containing the amino acid sequence SEQ ID NO: 45; or (iii) a VH containing the amino acid sequence SEQ ID NO: 62 and a VL containing the amino acid sequence SEQ ID NO: 63.

[0946] In some embodiments, 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-1 2), (VI-52), (VI-62), (VIb-1 2), (VIb-52), (VIb-62), (VIb-72), or (VIb-82), where Ab is the antibody that binds to 5T4 as disclosed herein. In some embodiments, the antibody comprises (a) a VH region comprising: (1) a VHCDR1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 7, 12, 13 and 18; (2) a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 2, 8, 14, 19 and 24; and (3) a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 3, 9, 15 and 20; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22; and (3) a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 6, 17 and 23.

[0947] In some implementations, 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-1 2), (VI-52), (VI-62), (VIb-1 2), (VIb-52), (VIb-62), (VIb-72), or (VIb-82), where Ab is an antibody that binds to 5T4. In some embodiments, the antibody comprises (i) a VH region comprising: VH CDR1 containing the amino acid sequence SEQ ID NO: 1, VH CDR2 containing the amino acid sequence SEQ ID NO: 2, and VH CDR3 containing the amino acid sequence SEQ ID NO: 3; and a VL region comprising: VLCDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VLCDR3 containing the amino acid sequence SEQ ID NO: 6; or (ii) a VH region comprising: VH CDR1 containing the amino acid sequence SEQ ID NO: 7, VH CDR2 containing the amino acid sequence SEQ ID NO: 8, and VH CDR3 containing the amino acid sequence SEQ ID NO: 9; and a VL region comprising: VL CDR1 containing the amino acid sequence SEQ ID NO: 10, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VL CDR3 containing the amino acid sequence SEQ ID NO: 6; or (iii) a VH region comprising: VH CDR1 containing the amino acid sequence SEQ ID NO: 1, VH CDR2 containing the amino acid sequence SEQ ID NO: ... VH CDR3 containing the amino acid sequence SEQ ID NO: 3, VH CDR3 containing the amino acid sequence SEQ ID NO: 4, VH CDR2 containing the amino acid sequence SEQ ID NO: 5, and VLCDR3 containing the amino acid sequence SEQ ID NO: 6; or (iii) a VH region comprising: VH CDR1 containing the amino acid sequence SEQ ID NO: 1, VH CDR2 containing the amino acid sequence SEQ ID NO: 1, VH CDR3 containing the amino acid sequence SEQ ID NO: 6, VH CDR VHCDR1 containing SEQ ID NO: 12, VH CDR2 containing the amino acid sequence SEQ ID NO: 2, and VHCDR3 containing the amino acid sequence SEQ ID NO: 3; and VL region containing: VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 6; or (iv) VH region containing: VH CDR1 containing the amino acid sequence SEQ ID NO: 13, VH CDR2 containing the amino acid sequence SEQ ID NO: 14, and VH CDR3 containing the amino acid sequence SEQ ID NO: 15;The VL region comprises: VLCDR1 containing the amino acid sequence SEQ ID NO: 16, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VLCDR3 containing the amino acid sequence SEQ ID NO: 17; or (v) the VH region comprises: VH CDR1 containing the amino acid sequence SEQ ID NO: 18, VH CDR2 containing the amino acid sequence SEQ ID NO: 19, and VH CDR3 containing the amino acid sequence SEQ ID NO: 20; and the VL region comprises: VL CDR1 containing the amino acid sequence SEQ ID NO: 21, VL CDR2 containing the amino acid sequence SEQ ID NO: 22, and VL CDR3 containing the amino acid sequence SEQ ID NO: 23; or (vi) the VH region comprises: VHCDR1 containing the amino acid sequence SEQ ID NO: 1, VH CDR2 containing the amino acid sequence SEQ ID NO: 24, and VHCDR3 containing the amino acid sequence SEQ ID NO: 3; and the VL region comprises: VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VH CDR2 containing the amino acid sequence SEQ ID NO: 16, VH CDR2 containing the amino acid sequence SEQ ID NO: 11, and VHCDR3 containing the amino acid sequence SEQ ID NO: 3; and the VL region comprises: VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VH CDR2 containing the amino acid sequence SEQ ID NO: 16, VH CDR2 containing the amino acid sequence SEQ ID NO: 11, and VHCDR3 containing the amino acid sequence SEQ ID NO: 17; CDR1, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 6.

[0948] In some implementations, the 5T4-ADC can be derived from formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-12), (VI-52), (VI-62), (VIb-12). 2), (VIb-52), (VIb-62), (VIb-72), or (VIb-82) represent an antibody that binds to 5T4 and comprises (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 27, 31, 34, 35, and 39; (2) a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 28, 32, 36, 40, and 43; and (3) a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 29, 33, 37, and 41; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16, and 21; (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11, and 2 ...L CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 29, 33, 37, and 41; and (c) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11, and 22; and (d) a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 29, 33, 37, and 41; and NO: VL CDR3 of the amino acid sequence of any one of 30, 38 and 42.

[0949] In some implementations, 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-1 2), (VI-52), (VI-62), (VIb-1 2), (VIb-52), (VIb-62), (VIb-72), or (VIb-82), where Ab is an antibody that binds to 5T4. In some embodiments, the antibody comprises (i) a VH region comprising: VH CDR1 containing the amino acid sequence SEQ ID NO: 27, VH CDR2 containing the amino acid sequence SEQ ID NO: 28, and VH CDR3 containing the amino acid sequence SEQ ID NO: 29; and a VL region comprising: VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 30; or (ii) a VH region comprising: VH CDR1 containing the amino acid sequence SEQ ID NO: 31, VH CDR2 containing the amino acid sequence SEQ ID NO: 32, and VH CDR3 containing the amino acid sequence SEQ ID NO: 33; and a VL region comprising: VL CDR1 containing the amino acid sequence SEQ ID NO: 10, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VL CDR3 containing the amino acid sequence SEQ ID NO: 30. CDR3; or (iii) the VH region, comprising: VH CDR1 containing the amino acid sequence SEQ ID NO: 34, VH CDR2 containing the amino acid sequence SEQ ID NO: 28, and VH CDR3 containing the amino acid sequence SEQ ID NO: 29; and the VL region, comprising: VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 30; or (iv) the VH region, comprising: VH CDR1 containing the amino acid sequence SEQ ID NO: 35, VH CDR2 containing the amino acid sequence SEQ ID NO: 36, and VH CDR3 containing the amino acid sequence SEQ ID NO: 37;The VL region comprises: VL CDR1 containing the amino acid sequence SEQ ID NO: 16, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VL CDR3 containing the amino acid sequence SEQ ID NO: 38; or (v) the VH region comprises: VH CDR1 containing the amino acid sequence SEQ ID NO: 39, VH CDR2 containing the amino acid sequence SEQ ID NO: 40, and VH CDR3 containing the amino acid sequence SEQ ID NO: 41; and the VL region comprises: VL CDR1 containing the amino acid sequence SEQ ID NO: 21, VL CDR2 containing the amino acid sequence SEQ ID NO: 22, and VL CDR3 containing the amino acid sequence SEQ ID NO: 42; or (vi) the VH region comprises: VH CDR1 containing the amino acid sequence SEQ ID NO: 27, VH CDR2 containing the amino acid sequence SEQ ID NO: 43, and VH CDR3 containing the amino acid sequence SEQ ID NO: 29; and the VL region comprises: VL CDR1 containing the amino acid sequence SEQ ID NO: 16, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VL CDR3 containing the amino acid sequence SEQ ID NO: 38; and the VL region comprises: VL CDR1 containing the amino acid sequence SEQ ID NO: 16, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VL CDR3 containing the amino acid sequence SEQ ID NO: 38. VL CDR1 containing amino acid sequence SEQ ID NO: 4, VL CDR2 containing amino acid sequence SEQ ID NO: 5, and VL CDR3 containing amino acid sequence SEQ ID NO: 30.

[0950] In some implementations, 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-1 2), (VI-52), (VI-62), (VIb-1 2), (VIb-52), (VIb-62), (VIb-72), or (VIb-82), where Ab is an antibody that binds to 5T4. In some embodiments, the antibody comprises (a) a VH region comprising: (1) a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 46, 50, 53, 13 and 57; (2) a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 47, 51, 54, 58 and 61; and (3) a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 48, 52, 55 and 59; and (b) a VL region comprising: (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 10, 16 and 21; (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 11 and 22; and (3) a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 49, 56 and 60.

[0951] In some implementations, the 5T4-ADC can be derived from formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-12), (VI-52), (VI-62), (VIb-12). 2) (VIb-52), (VIb-62), (VIb-72), or (VIb-82) indicates that the Ab is an antibody that binds to 5T4 and contains (i) a VH region comprising: VH CDR1 containing the amino acid sequence SEQ ID NO: 46, VH CDR2 containing the amino acid sequence SEQ ID NO: 47, and VH CDR3 containing the amino acid sequence SEQ ID NO: 48; and a VL region comprising: VL CDR1 containing the amino acid sequence SEQ ID NO: 4, VL CDR2 containing the amino acid sequence SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence SEQ ID NO: 49; (ii) a VH region comprising: VH CDR1 containing the amino acid sequence SEQ ID NO: 50, VH CDR2 containing the amino acid sequence SEQ ID NO: 51, and VH CDR3 containing the amino acid sequence SEQ ID NO: 52; and a VL region comprising: VL CDR1 containing the amino acid sequence SEQ ID NO: 10, VH CDR2 containing the amino acid sequence SEQ ID NO: 52, and VH CDR3 containing the amino acid sequence SEQ ID NO: 52. (iii) VL CDR2 containing amino acid sequence SEQ ID NO: 11 and VL CDR3 containing amino acid sequence SEQ ID NO: 49; or (iii) VH region containing: VH CDR1 containing amino acid sequence SEQ ID NO: 53, VH CDR2 containing amino acid sequence SEQ ID NO: 47 and VH CDR3 containing amino acid sequence SEQ ID NO: 48; and VL region containing: VL CDR1 containing amino acid sequence SEQ ID NO: 4, VL CDR2 containing amino acid sequence SEQ ID NO: 5 and VL CDR3 containing amino acid sequence SEQ ID NO: 49; or (iv) VH region containing: VH CDR1 containing amino acid sequence SEQ ID NO: 13, VH CDR2 containing amino acid sequence SEQ ID NO: 54 and VH CDR3 containing amino acid sequence SEQ ID NO: 55;The VL region comprises: VL CDR1 containing the amino acid sequence SEQ ID NO: 16, VL CDR2 containing the amino acid sequence SEQ ID NO: 11, and VL CDR3 containing the amino acid sequence SEQ ID NO: 56; or (v) the VH region comprises: VH CDR1 containing the amino acid sequence SEQ ID NO: 57, VH CDR2 containing the amino acid sequence SEQ ID NO: 58, and VH CDR3 containing the amino acid sequence SEQ ID NO: 59; and the VL region comprises: VL CDR1 containing the amino acid sequence SEQ ID NO: 21, VL CDR2 containing the amino acid sequence SEQ ID NO: 22, and VL CDR3 containing the amino acid sequence SEQ ID NO: 60; or (vi) the VH region comprises: VH CDR1 containing the amino acid sequence SEQ ID NO: 46, VH CDR2 containing the amino acid sequence SEQ ID NO: 61, and VH CDR3 containing the amino acid sequence SEQ ID NO: 48; and the VL region comprises: containing the amino acid sequence SEQ ID NO: 16, VH CDR1 containing the amino acid sequence SEQ ID NO: 16, VH CDR2 containing the amino acid sequence SEQ ID NO: 11, and VH CDR3 containing the amino acid sequence SEQ ID NO: 56. VL CDR1 containing amino acid sequence SEQ ID NO: 4, VL CDR2 containing amino acid sequence SEQ ID NO: 5, and VL CDR3 containing amino acid sequence SEQ ID NO: 49.

[0952] In some implementations, 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-1 2), (VI-52), (VI-62), (VIb-1 2), (VIb-52), (VIb-62), (VIb-72), or (VIb-82), where Ab is an antibody that binds to 5T4. In some embodiments, the antibody further comprises the frame 1 (FR1), frame 2 (FR2), frame 3 (FR3) and / or frame 4 (FR4) sequences shown in any of SEQ ID NO: 25, 26, 44, 45, 62 and 63.

[0953] In some implementations, 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-1 2), (VI-52), (VI-62), (VIb-1 2), (VIb-52), (VIb-62), (VIb-72), or (VIb-82), where Ab is an antibody that binds to 5T4 and contains a human frame sequence.

[0954] In some implementations, 5T4-ADC may be represented by formulas (IV), (V), (VI), (VI-1), (VIa-1), (VIb-1), (VI-2), (VIa-2), (VIb-2), (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), (VIb-8), (VI-11), (VI-21), (VI-81), (VIa-11), (VIa-21), (VIb-81), (VI-12), (VI-52), (VI-62), (VIb-12), (VIb-52), (VIb-62), (VIb-72), or (VIb-82), where Ab is an antibody that binds to 5T4. In some embodiments, the antibody comprises: (i) a VH containing the amino acid sequence SEQ ID NO: 25 and a VL containing the amino acid sequence SEQ ID NO: 26; or (ii) a VH containing the amino acid sequence SEQ ID NO: 44 and a VL containing the amino acid sequence SEQ ID NO: 45; or (iii) a VH containing the amino acid sequence SEQ ID NO: 62 and a VL containing the amino acid sequence SEQ ID NO: 63.

[0955] Antibody-connector-drug conjugation

[0956] In some implementations, the antibody may be modified prior to adapter-drug conjugation. Modification of the antibody can produce a modified antibody containing one or more reactive groups suitable for adapter-drug conjugation.

[0957] In some embodiments, antibodies may be modified at one or more amino acid residues to provide one or more reactive groups suitable for linker-drug conjugation. For example, introducing a carbonyl group into a polypeptide can selectively react with an α-nucleophile (such as a compound carrying an aminooxy group and an acylhydrazine). Chemicals selective for carbonyl functional groups on proteins, along with enhanced kinetics, site selectivity, and conjugate stability, can improve the bioconjugate.

[0958] In some embodiments, the antibody may be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). The reactive aldehyde may be included in an “aldehyde tag” or “ald tag,” the tag referring to an amino acid sequence derived from a sulfatase motif that has been converted by formylglycinase (FGE) to contain a 2-formylglycine residue (referred to herein as “fGly”). The fGly residue generated by FGE is also referred to in the literature as “formylglycine.” In other words, the term “aldehyde tag” is used herein to refer to an amino acid sequence containing a “converted” sulfatase motif (i.e., a sulfatase motif in which a cysteine ​​or serine residue has been converted to fGly by FGE, such as L(fGly)TPSR (SEQ ID NO: 102)). The transformed sulfatase motif can be derived from an amino acid sequence containing an “untransformed” sulfatase motif (i.e., a sulfatase motif in which cysteine ​​or serine residues have not yet been converted to fGly by FGE, but are capable of transformation, such as an untransformed sulfatase motif having the sequence: L(C / S)TPSR (SEQ ID NO: 103)). In the context of the action of formylglycinase (FGE) on the sulfatase motif, “transformation” refers to the biochemical modification of a cysteine ​​or serine residue in the sulfatase motif to a formylglyine (fGly) residue (e.g., Cys modified to fGly, or Ser modified to fGly). As used herein, an aldehyde-labeled antibody refers to an antibody in which a cysteine ​​or serine residue has been modified to form a formylglyine residue. Aldehyde tags and other aspects thereof used for site-specific protein modification are described in U.S. Patent Application Publication No. 7,985,783 and U.S. Application Publication No. 2011 / 0117621, the disclosures of which are each incorporated herein by reference.

[0959] The transformation of a peptide to include fGly can be accomplished by either cell-based (in vivo) or free-method (in vitro) approaches. Similarly, modifications to a peptide to produce a peptide suitable for conjugation (e.g., modifications to produce a peptide containing a reactive group suitable for conjugation) can be accomplished by either cell-based (in vivo) or free-method (in vitro) approaches.

[0960] The amino acid sequence of the antibody may be modified to include a sulfatase motif containing serine or cysteine ​​residues, which can be converted (oxidized) to FGly residues by the action of FGE in vivo (e.g., during the translation of aldehyde-tagged proteins in cells) or in vitro (e.g., by contacting aldehyde-tagged proteins with FGE in a free system). The 5T4 antibody used to generate the 5T4-ADC includes at least one Ig constant region, such as an Ig heavy chain constant region (e.g., at least a CH1 domain; at least CH1 and CH2 domains; CH1, CH2, and CH3 domains; or CH1, CH2, CH3, and CH4 domains), or an Ig light chain constant region.

[0961] Sulfatase motifs can be introduced into any convenient site on the antibody. As noted herein, in some embodiments, modifications to the native amino acid sequence of the target polypeptide are minimized to reduce the number of inserted, deleted, substituted, and / or added (e.g., N-terminal or C-terminal) amino acid residues. Minimizing modifications to the amino acid sequence of the target antibody minimizes the potential impact of such modifications on antibody function and / or structure.

[0962] In some embodiments, the aldehyde-labeled antibody comprises an aldehyde-labeled Ig heavy chain constant region (e.g., at least a CH1 domain; at least CH1 and CH2 domains; CH1, CH2, and CH3 domains; or CH1, CH2, CH3, and CH4 domains). The aldehyde-labeled Ig heavy chain constant region may include a heavy chain constant region sequence of an IgA, IgM, IgD, IgE, IgG1, IgG2, IgG3, or IgG4 homotype heavy chain or any allotype thereof, such as a human or mouse heavy chain constant region sequence, a hybrid heavy chain constant region, a synthetic heavy chain constant region, or a common heavy chain constant region sequence or similar sequences, said sequences being modified to include at least one sulfatase motif, which can be modified by FGE to produce an fGly-modified Ig polypeptide. Allotypes of the Ig heavy chain are known in the art. See, for example, Jefferis and Lefranc (2009) MAbs1:4.

[0963] In some embodiments, the aldehyde-labeled antibody comprises an aldehyde-labeled Ig light chain constant region. The aldehyde-labeled Ig light chain constant region may include a constant region sequence of a K light chain, a λ light chain, such as a K or λ light chain constant region, a hybrid light chain constant region, a synthetic light chain constant region, or a common light chain constant region sequence and similar sequences. The constant region sequence is modified to include at least one sulfatase motif, which can be modified with FGE to produce an fGly-modified antibody.

[0964] Alternatively, the isolated unmodified polypeptide can be isolated after recombinant production in a host cell lacking a suitable enzyme or after synthesis. The isolated polypeptide can then be contacted with a suitable enzyme under conditions that achieve the desired modification to include fGly. The polypeptide can be unfolded and the denatured protein contacted with a suitable enzyme using methods known in the art (e.g., using heating, pH adjustment, dissociation agents (e.g., urea and its analogues), organic solvents (e.g., hydrocarbons: octane, benzene, chloroform) and their analogues). The modified polypeptide can then be refolded under suitable conditions.

[0965] In some embodiments, a modified antibody containing fGly residues can be conjugated to a linker-drug by reacting fGly with a coupling portion (such as the hydrazyl-pyrrolo coupling portion described herein) on the linker-drug. For example, an fGly-containing antibody can be isolated from a source of production (e.g., a source producing recombinant host cells, a synthetically produced source) and contacted with a drug or other portion (e.g., a detectable label) containing a reactive conjugate under conditions suitable for achieving conjugation of the drug or other portion to the 5T4 antibody. For example, the drug or other portion containing the reactive conjugate may include a conjugated portion (e.g., the hydrazyl-pyrrolo coupling portion described herein). The drug containing the hydrazyl-pyrrolo can be reacted with the antibody to produce a 5T4-ADC as described herein.

[0966] The general protocol for coupling the antibody with the pyridazine-pyrrole coupling portion is shown below.

[0967]

[0968] The hydrazino-pyrrolo coupling moiety, which is interchangeably referred to herein as the aza-hydrazino-iso-Pictet-Spengler (azaHIPS) coupling moiety, forms a pyridazine-pyrrolo coupling moiety upon conjugation with formyl-glycine, as shown in formula (III). A 5T4 antibody may include a 2-formylglycine residue (fGly) that reacts with the azaHIPS coupling moiety, thereby conjugating the two together. To generate a 5T4-ADC, the drug may be directly or indirectly (e.g., via a linker portion) conjugated with the azaHIPS moiety at any position on the azaHIPS moiety (e.g., R...). 16 、Y 1 、Y 2 、Y 3 Or Q 4 ) Coupling. R 2 and R 3Each can be independently any desired substituent, such as (but not limited to) hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic. R 16 、Y 1 、Y 2 、Y 3 and Q 4 It may be as described herein, such as with respect to formula (I) or formula (II).

[0969] Formula (I) below represents the hydrazine-pyrrole coupling moiety, which can be used to link antibodies bound to 5T4 with drugs in any of the 5T4-ADCs described herein.

[0970]

[0971] in:

[0972] R 16 、Y 1 、Y 2 、Y 3 and Q 4 One of them is -LW 1 ,

[0973] where Q 3 and Q 4 One of them is -(CH2). m NR 3 NHR 2 ;

[0974] m is 0 or 1;

[0975] R 2 and R 3 Each of these groups is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic; or R 2 With R 3 Ring-linked structures are used to form 5- or 6-membered heterocyclic groups;

[0976] If Q 3 Not -(CH2) mNR 3 NHR 2 Then Q 3 For Y 4 ;

[0977] If Q 4 Not for -LW 1 or -(CH2) m NR 3 NHR 2 Then Q 4 For Y 4 ;

[0978] X 1 X 2 X 3 and X 4 Each is independently C, N, O, or S, where X 1 X 2 X 3 and X 4 One of them may be absent;

[0979] Y 1 、Y 2 、Y 3 (If not -LW) 1 ) and Y 4 Each of these groups is independently hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic; or Y 1 With Y 2 、Y 2 With Y 3 or Y 4 With Y 3 (If Q) 4 For Y 4 ) Can optionally be connected in a ring;

[0980] If R 16 Not for LW 1 Then R 16 It can be hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic;

[0981] L is the connector as described herein; and

[0982] W 1It is a drug.

[0983] Equation (I) was previously described in WO 2015 / 081282, which is incorporated herein by reference in its entirety.

[0984] In some embodiments, the hydrazine-pyrrolo coupling moiety can be represented by formula (II):

[0985]

[0986] in:

[0987] Q 3 and Q 4 One of them is [-(CH2)] m NR 3 NR 2 ];

[0988] Q 3 and Q 4 The other one is Y 4 ;

[0989] m is 0 or 1; and

[0990] R 2 and R 3 Each of these groups is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic; or R 2 With R 3 Ring-linked structures are used to form 5- or 6-membered heterocyclic groups;

[0991] X 1 X 2 X 3 and X 4 Each is independently C, N, O, or S, where X 1 X 2 X 3 and X 4 One of them may be absent;

[0992] Y 1 、Y 2 、Y 3 and Y 4Each of these groups is independently hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic; or Y 1 With Y 2 、Y 2 With Y 3 Or Y 3 With Y 4 (If Q) 4 For Y 4 ) Can optionally be connected in a ring;

[0993] L is the connector as described herein; and

[0994] W 1 It is a drug.

[0995] Equation (II) has been previously described in WO 2015 / 081282 and WO 2020 / 154437, each of which is incorporated herein by reference in its entirety. In some embodiments, m is 0 or 1 and R 2 and R 3 Each is an alkyl group. For example, R 2 and R 3 Each can be independently C1-C 10 Alkyl (e.g., C1-C6 alkyl). In some embodiments, R 2 and R 3 Each is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl. In some embodiments, R 2 and R 3 At least one of them is methyl. In some embodiments, R 2 With R 3 All are methyl groups. In some embodiments, X 1 、X 2 、X 3 and X 4 Each of them is independently C or N. In some implementations, X 1 For C. In some implementations, X 1 For N. In some implementations, X 2 For C or N. In some implementations, X 2 For C. In some implementations, X 3 For C or N. In some implementations, X 3 For C. In some implementations, X4 For C or N. In some implementations, X 4 It is C. X 1 、X 2 、X 3 and X 4 Various combinations of X are possible. For example, in some implementations, X 1 、X 2 、X 3 and X 4 Each of them is C. In other implementations, X 1 、X 2 、X 3 and X 4 The three are C and X 1 、X 2 、X 3 and X 4 One of them is N. In some implementations, Y 1 、Y 2 、Y 3 and Y 4 Each of them is hydrogen.

[0996] An exemplary scheme for preparing a 5T4-ADC is shown in Scheme A:

[0997]

[0998] In scheme A, the linker-drug is conjugated to an antibody to 5T4. The 5T4 antibody has a recognition motif in a constant region of the light chain, such as the L(C / S)TPSR (SEQ ID NO: 103) recognition motif (e.g., in SEQ ID NO: 90, 94, 98). The cysteine ​​residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and subsequently conjugated to the azaHIPs moiety via a Pictet-Spengler reaction.

[0999] An exemplary scheme for preparing the 5T4-ADC is shown in Scheme B:

[1000]

[1001] In scheme B, the linker-drug is conjugated to an antibody attached to 5T4. The 5T4 antibody has a recognition motif in a constant region of the heavy chain, such as the L(C / S)TPSR (SEQ ID NO: 103) recognition motif (e.g., in SEQ ID NO: 90, 94, 98). The cysteine ​​residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and subsequently conjugated to the azaHIPs moiety via a Pictet-Spengler reaction.

[1002] In some embodiments, the 5T4-ADC includes a linker-drug conjugate connected to the C-terminus of each heavy chain constant region of the 5T4 antibody via a pyridazine-pyrrolo coupling moiety, and may be characterized by an antibody / linker-drug stoichiometric ratio of 1 to 4, 1 to 2, 1.75 to 2, or 1.9 to 2. In another embodiment, the 5T4-ADC includes a pyridazine-pyrrolo coupling moiety linked to the C-terminus of only one heavy chain constant region, wherein the antibody-drug stoichiometric ratio is 0.5 to 1, 0.75 to 1, or 0.9 to 1.

[1003] In some embodiments, the 5T4-ADC includes a linker-drug conjugated to a 5T4 antibody via a pyridazine-pyrrolo coupling moiety, wherein the linker-drug comprises maytansine conjugated to the pyridazine-pyrrolo coupling moiety via a linker (L-1), as shown in Scheme C below:

[1004]

[1005] In some embodiments, the 5T4 antibody has a recognition motif in a constant region of the heavy chain, such as the L(C / S)TPSR (SEQ ID NO: 103) recognition motif (e.g., in SEQ ID NO: 90, 94, 98). The cysteine ​​residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and subsequently conjugated to azaHIPs via a Pickett-Spengler reaction, the azaHIPs portion being conjugated to MMAE via a linker (L-1).

[1006] In some embodiments, the 5T4-ADC includes a linker-drug conjugated to a 5T4 antibody via a pyridazine-pyrrolo coupling moiety, wherein the linker-drug comprises an MMAE conjugated to the pyridazine-pyrrolo coupling moiety via a linker (L-2), as shown in scheme D below:

[1007]

[1008] In some embodiments, the 5T4 antibody has a recognition motif in the constant region of the heavy chain, such as the L(C / S)TPSR (SEQ ID NO: 103) recognition motif. The cysteine ​​residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and subsequently conjugated to azaHIPs via a Pickett-Spengler reaction, wherein the azaHIPs portion is conjugated to MMAE via a linker (L-2).

[1009] In some embodiments, the 5T4-ADC includes a linker-drug conjugated to a 5T4 antibody via a pyridazine-pyrrolo coupling moiety, wherein the linker-drug comprises an MMAE conjugated to the pyridazine-pyrrolo coupling moiety via a linker (L-7), as shown in scheme E below:

[1010]

[1011] In some embodiments, the 5T4 antibody has a recognition motif in the constant region of the heavy chain, such as the L(C / S)TPSR (SEQ ID NO: 103) recognition motif (e.g., in SEQ ID NO: 90, 94, 98). The cysteine ​​residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and subsequently conjugated to azaHIPs via a Pickett-Spengler reaction, the azaHIPs portion being conjugated to MMAE via a linker (L-7).

[1012] In some embodiments, the 5T4-ADC includes a linker-drug conjugated to a 5T4 antibody via a pyridazine-pyrrolo coupling moiety, wherein the linker-drug comprises an MMAE conjugated to the pyridazine-pyrrolo coupling moiety via a linker (L-8), as shown in the following scheme F:

[1013]

[1014] In some embodiments, the antibody binding to 5T4 has a recognition motif in the constant region of the heavy chain, such as the L(C / S)TPSR (SEQ ID NO: 103) recognition motif (e.g., in SEQ ID NO: 90, 94, 98). The cysteine ​​residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and subsequently conjugated to azaHIPs via a Pickett-Spengler reaction, the azaHIPs portion being conjugated to MMAE via a linker (L-8).

[1015] Formulations and Compositions

[1016] 5T4-ADCs may be included in pharmaceutical compositions for administration to, for example, a subject, to treat a disease, condition, or ailment. The pharmaceutical composition may contain a 5T4-ADC that, on average, exhibits a drug / antibody ratio (“DAR”) of about 1 to about 2. In other words, various antibodies are conjugated to one or two linker-drug conjugates. Methods for determining DAR are well known to those skilled in the art and include methods using reversed-phase chromatography or HPLC-MS.

[1017] For example, in any embodiment, a pharmaceutical composition comprising a 5T4-ADC may exhibit a DAR of about 1 to about 1.9, or about 1 to about 1.8, about 1 to about 1.7, about 1.5 to about 1.9, about 1.5 to about 1.8, about 1.5 to about 1.7, about 1.7 to about 1.9, about 1.7 to about 1.8, or about 1.8 to about 1.9. In some embodiments, the pharmaceutical composition comprises a 5T4-ADC comprising an antibody that binds to 5T4 as described herein, including mAbA4, mAbA15, and mAbA17 as described herein (such as any of Tables 1 to 3); and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a 5T4-ADC of formula (IV), (V), or (VI), and L is selected from linkers (L-1), (L-2), (L-3), (L-4), (L-5), (L-6), (L-7), and (L-8). In some embodiments, the pharmaceutical composition comprises a 5T4-ADC containing MMAE, MMAF, maytansine, or any derivative thereof. In some embodiments, the pharmaceutical composition comprises a 5T4-ADC of formula (VI-1), (VIa-1), or (VIb-1) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a 5T4-ADC of formula (VI-2), (VIa-2), or (VIb-2) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a 5T4-ADC of formula (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (VI-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), or (VIb-8) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a 5T4-ADC of formula (VI-11), (VI-12), (VIa-11), or (VIb-12) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a 5T4-ADC of formula (VI-21) or (VIa-21) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a 5T4-ADC of formula (VI-52), (VI-62), (VI-81), (VIb-81), (VIb-52), (VIb-62), (VIb-72), or (VIb-82) and a pharmaceutically acceptable excipient.In some embodiments, the pharmaceutical composition comprises a 5T4-ADC of mAbA4-VIa-21 (i.e., a linker-drug of formula VIa-21 conjugated with antibody mAbA4), mAbA15-VIa-21 (i.e., a linker-drug of formula VIa-21 conjugated with antibody mAbA15), mAbA15-VIb-82 (i.e., a linker-drug of formula VIb-82 conjugated with antibody mAbA15), or mAbA17-VIb-82 (i.e., a linker-drug of formula VIb-82 conjugated with antibody mAbA17) and a pharmaceutically acceptable excipient.

[1018] 5T4-ADC can be formulated using any of a variety of different methods. The 5T4-ADC of this disclosure can be provided in any suitable form, such as a pharmaceutically acceptable salt, and can be formulated according to any suitable route of administration, such as oral, topical, or parenteral. In cases where 5T4-ADC is provided in an injectable form (such as in embodiments where it can be administered intravenously or directly to tissues), 5T4-ADC can be provided in ready-to-use dosage forms or as a storage-stability-restoring powder or liquid consisting of pharmaceutically acceptable excipients and excipients.

[1019] Methods for formulating 5T4-ADC can be adapted using those methods available in the art. For example, 5T4-ADC can be provided as a pharmaceutical composition comprising an effective amount of 5T4-ADC and a pharmaceutically acceptable excipient (e.g., physiological saline). The pharmaceutical composition may optionally include other additives (e.g., buffers, stabilizers, preservatives, and the like). In some embodiments, the formulation is suitable for administration to mammals, such as those suitable for administration to humans.

[1020] This document also provides pharmaceutical compositions containing an effective amount of the 5T4-ADC described herein and a pharmaceutically acceptable excipient. In some embodiments, the 5T4-ADC comprises an antibody binding to 5T4 as described herein, including mAbA4, mAbA15, and mAbA17, as described in any of Tables 1 to 3. In some embodiments, the pharmaceutical composition comprises an effective amount of a 5T4-ADC of formula (IV), (V), or (VI), and L is selected from linkers (L-1), (L-2), (L-3), (L-4), (L-5), (L-6), (L-7), or (L-8). In some embodiments, the pharmaceutical composition comprises an effective amount of a 5T4-ADC comprising MMAE, MMAF, maytansine, or any derivative thereof. In some embodiments, the pharmaceutical composition comprises an effective amount of a 5T4-ADC of formula (VI-1), (VIa-1), or (VIb-1) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a 5T4-ADC of formula (VI-2), (VIa-2), or (VIb-2) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a 5T4-ADC of formula (V-3), (VI-3), (V-4), (VI-4), (V-5), (VI-5), (V-6), (V1-6), (V-7), (VI-7), (V-8), (VI-8), (VIa-8), or (VIb-8) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a 5T4-ADC of formula (VI-11), (VI-12), (VIa-11), or (VIb-12) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a 5T4-ADC of formula (VI-21) or (VIa-21) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a 5T4-ADC of formula (VI-52), (VI-62), (VI-81), (VIb-81), (VIb-52), (VIb-62), (VIb-72), or (VIb-82) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a 5T4-ADC of mAbA4-VIa-21, mAbA1 5-VIa-21, mAbA1 5-VIb-82, or mAbA1 7-VIb-82 and a pharmaceutically acceptable excipient.

[1021] Pharmaceutically acceptable excipients can be one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances (e.g., physiologically acceptable and / or pharmacologically acceptable) suitable for administration to human or veterinary subjects. When more than one pharmaceutically acceptable excipient is present in a pharmaceutical composition, the pharmaceutically acceptable excipients may be blended with one or more active ingredients (e.g., hybrid molecules) in a manner that avoids significantly reducing the desired efficacy of the drug. Pharmaceutically acceptable materials are generally capable of being administered to subjects without producing significant undesirable physiological effects, such as nausea, dizziness, rash, or gastrointestinal upset. For example, compositions containing pharmaceutically acceptable excipients need to be immunogenic when administered to human subjects for therapeutic purposes.

[1022] The pharmaceutical compositions of the present invention may additionally contain suitable buffers, including, for example, acetic acid in salt form, citric acid in salt form, boric acid in salt form, and phosphoric acid in salt form. The pharmaceutical compositions may also optionally contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal. The pharmaceutical compositions of the present invention can be presented in unit dosage forms and can be prepared by any suitable method, many of which are well known in pharmaceutical technology. Such methods include the step of combining the antibody or antigen-binding fragment of the present invention with an excipient constituting one or more adjunct components. Generally, pharmaceutical compositions are prepared by uniformly and tightly combining an active agent with a liquid excipient, a finely powdered solid excipient, or both, and then, if necessary, shaping the product.

[1023] Compositions suitable for parenteral administration preferably comprise sterile aqueous formulations of the composition, which are preferably isotonic to the recipient's blood. These aqueous formulations may be prepared using suitable dispersants or wetting agents and suspending agents, formulated by known methods. Sterile injectable formulations may also be sterile injectable solutions or suspensions present in parenteral acceptable non-toxic diluents or solvents, such as solutions in 1,3-butanediol. Water, Ringer's solution, and isotonic sodium chloride solutions may be used among acceptable media and solvents. Additionally, sterile non-volatile oils are routinely used as solvents or suspension media. For this purpose, any mild non-volatile oil may be used, such as synthetic monoglycerides or diglycerides. Alternatively, injectable formulations may be prepared using fatty acids (such as oleic acid). Excipient formulations suitable for oral, subcutaneous, intravenous, intramuscular, and similar administration are available at Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

[1024] The preparat...

Claims

1. An antibody-drug conjugate (ADC) of formula (IV), comprising: a. an antibody that binds to 5T4; and b. One or more drugs conjugated to one or more pyridazine-pyrrolo coupling moieties via a linker in: Ab represents the antibody that binds to 5T4; r is an integer from 1 to 10; m is 0 or 1; R 2 and R 3 each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; or R 2 With R 3 The rings are connected to form a 5-membered or 6-membered heterocyclic group; X 1 , X 2 , X 3 and X 4 Each independently selected from the group consisting of C, N, O and S; Y 1 , Y 2 , Y 3 and Y 4 each independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or Y 1 With Y 2 , Y 2 With Y 3 , or Y 3 With Y 4 Ring connection; W 1 for the drug; L represents a linker and is -(T 1 V 1 ) a -(T 2 V 2 ) b -(T 3 V 3 ) c -(T 4 V 4 ) d -(T 5 V 5 ) e -,in: a, b, c, d and e are each independently 0 or 1, wherein the sum of a, b, c, d and e is 1 to 5; T 1 , T 2 , T 3 , T 4 and T 5 Each independently contains C1-C 12 Alkyl, substituted C1-C 12 Alkyl, (EDA) w , (PEG) n 、(AA) p 、-(CR 13 OH) h -, piperidin-4-amine (P4A-R 12 ), m-aminobenzyl carbamate (MABC) group, m-aminobenzyloxy (MABO) group, p-aminobenzyloxy (PABO) group, p-aminobenzyl carbamate (PABC) group, p-aminobenzyl (PAB) group, acetal, disulfide, hydrazine, (AA) p -MABC-(AA) p 、(AA) p -MABO-(AA) p 、(AA) p -PABO-(AA) p or (AA) p -PABC-(AA) p , w is an integer from 1 to 20; n is an integer from 1 to 30; Each p is independently zero or an integer from 1 to 20; h is an integer from 1 to 12; and Each R 12 is hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; Each R 13 is hydrogen, alkyl, substituted alkyl, aryl or substituted aryl; V 1 、V 2 、V 3 、V 4 and V 5 are independently selected from the group consisting of: a covalent bond, -C(=O)-, -NR 11 -、-C(=O)NR 11 -、-NR 11 C(=O)-, -C(=O)O-, -OC(=O)-, -O-, -S-, -S(=O)-, -SO2-, -SO2NR 11 -、-NR 11 SO2- and -P(=O)OH-, where R 11 Selected from the group consisting of hydrogen, alkyl, substituted alkyl, PEG, aryl and substituted aryl.

2. The ADC of claim 1, wherein W 1 Selected from the group consisting of maytansines and auristatins.

3. The ADC of claim 1 or 2, wherein the auristatin is MMAE.

4. The ADC of any one of claims 1 to 3, wherein R 2 and R 3 Each is an alkyl group.

5. The ADC of any one of claims 1 to 4, wherein R 2 and R 3 Each is a methyl group.

6. The ADC of any one of claims 1 to 5, wherein X 1 , X 2 , X 3 and X 4 are each independently selected from C and N.

7. The ADC of any one of claims 1 to 6, wherein the ADC is represented by formula (V):

8. The ADC of any one of claims 1 to 7, wherein the ADC is represented by formula (VI): Where X 1 It is C or N.

9. The ADC of any one of claims 1 to 8, wherein T 1 , T 2 , T 3 , T 4 and T 5 Each independently selected from the group consisting of: C1-C6 alkyl, P4A-R 12 、(AA) p 、(AA) p -PABC and (PEG) n , where p is an integer from 1 to 5, and R 12 It is polyethylene glycol or polyethylene glycol modified with carboxylic acid.

10. The ADC of any one of claims 1 to 9, wherein T 1 is a C1-C6 alkyl group; T 2 , T 3 , T 4 and T 5 Each independently selected from (PEG) n 、C1-C6 alkyl、(AA) p 、P4A-R 12 、(AA) p -(PABO-R 16 )-(AA) p 、(AA) p -(PABC-R 16 )-(AA) p 、(AA) p -(PABO-R 16 ) and (AA) p -(PABC-R 16 ), where R 12 is a carboxylic acid-modified polyethylene glycol and R 16 is hydrogen; and V 1 、V 2 、V 3 、V 4 and V 5 Each independently selected from -CO- and -NR 11 -, where R 11 is selected from hydrogen and alkyl.

11. The ADC of any one of claims 1 to 10, wherein L is: -(C1-C6 alkyl)-(V 1 )-(P4A-R 12 )-(V 2 )-(C1-C6 alkyl)-(V 3 )-, in: V 1 、V 2 and V 3 Each independently selected from -CO- and -NR 1111 - The groups formed; Each R 11 are independently selected from the group consisting of hydrogen and C1-C6 alkyl; and R 12 It is a polyethylene glycol moiety modified with a carboxylic acid.

12. The ADC of any one of claims 1 to 11, wherein L is: in: represents the nitrogen attached to the pyridazine-pyrrolo coupling moiety; and *Indicates connection to W 1 .

13. An antibody-drug conjugate (ADC) of formula (VI-1), comprising: a. an antibody that binds to 5T4; and b. One or more drugs conjugated to one or more pyridazine-pyrrolo coupling moieties via a linker in: Ab represents the antibody that binds to 5T4; r is an integer from 1 to 10; X 1 is C or N; and W 1 For the drug.

14. The ADC of claim 13, wherein W 1 Selected from the group consisting of maytansines and auristatins.

15. The ADC of claim 14, wherein the auristatin is MMAE.

16. The ADC of claim 13 or 14, wherein X 1 C and W 1 It is deacylated maytansine.

17. The ADC of any one of claims 14 to 15, wherein X 1 N and W 1 It is MMAE.

18. The ADC of any one of claims 13 to 17, wherein L is: (C1-C6 alkyl)-V 1 -(AA)-V 2 -(PEG) n -V 3 -((AA) p -(PABC-R 16 ))-V 4 -(C1-C6 alkyl)-V 5 , in: R 16 is hydrogen; n and p are each independently an integer from 1 to 20; V 1 、V 2 、V 3 、V 4 and V 5 Each independently selected from -CO- and -NR 11 - a group of people; and Each R 1111 Independently selected from the group consisting of hydrogen and C1-C6 alkyl.

19. The ADC of any one of claims 13 to 17, wherein L is: in: represents the nitrogen attached to the pyridazine-pyrrolo coupling moiety; and *Indicates connection to W 1 .

20. An antibody-drug conjugate (ADC), comprising an antibody bound to 5T4, wherein the antibody is conjugated to one or more drugs via one or more pyridazine-pyrrolo coupling moieties, as shown in formula (VI-2): in: Ab represents the antibody that binds to 5T4; r is an integer from 1 to 10; X 1 is C or N; and W 1 For the drug.

21. The ADC of claim 20, wherein W 1 Selected from maytansines and auristatins.

22. The ADC of claim 21, wherein the auristatin is MMAE.

23. The ADC of claim 20 or 21, wherein X 1 C and W 1 It is deacylated maytansine.

24. An antibody-drug conjugate (ADC) of formula (IV) comprising: a. an antibody that binds to 5T4; and b. One or more drugs conjugated to one or more pyridazine-pyrrolo coupling moieties via a linker in: Ab represents the antibody that binds to 5T4; W 1 for the drug; r is an integer from 1 to 10; m is 0 or 1; R 2 and R 3 each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 With R 3 The rings are connected to form a 5-membered or 6-membered heterocyclic group; X 1 , X 2 , X 3 and X 4 Each independently selected from the group consisting of C, N, O and S; Y 1 , Y 2 , Y 3 and Y 4 each independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or Y 1 With Y 2 , Y 2 With Y 3 , or Y 3 With Y 4 Ring connection; L is in: represents the nitrogen attached to the pyridazine-pyrrolo coupling moiety; *Indicates connection to W 1 ; Each R 5 are independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; Each R 6 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R 7 is a cleavable portion; k is an integer from 1 to 10; L 1 include-(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -; L 2 include-(T 5 -V 5 ) e -(T 6 -V 6 ) f -(T 7 -V 7 ) g -(T 8 -V 8 ) h -; a, b, c, d, e, f, g and h are each independently 1 or 0; T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 and T 8 Each independently selected from the group consisting of: a covalent bond, a C1-C 12 Alkyl, substituted C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) w , (PEG) n 、(AA) p 、-(CR 13 OH) m -、P4A-R 12 , acetals, hydrazines, disulfides, and esters; Each w is an integer from 1 to 20; Each n is an integer from 1 to 30; Each p is an integer from 1 to 20; Each m is an integer from 1 to 12; V 1 、V 2 、V 3 、V 4 、V 5 、V 6 、V 7 and V 8 Each independently selected from the group consisting of: a covalent bond, -CO-, -NR 15 -、-NR 15 (CH2) q -、-NR 15 (C6H4)-、-CONR 15 -、-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-; Each q is an integer from 1 to 6; R 12 selected from the group consisting of hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; Each R 13 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

25. The ADC of claim 24, wherein W 1 Selected from maytansines and auristatins.

26. The ADC of claim 25, wherein the auristatin is MMAE.

27. The ADC of any one of claims 24 to 26, wherein R 2 and R 3 Each is an alkyl group.

28. The ADC of claim 27, wherein R 2 and R 3 Each is a methyl group.

29. The ADC of any one of claims 24 to 28, wherein X 1 , X 2 , X 3 and X 4 are each independently selected from C and N.

30. The ADC according to any one of claims 24 to 29, which is represented by formula (VI-3):

31. The ADC of claim 30, wherein R7 is wherein ** represents the point of attachment to the phenyl group in formula (VI-3).

32. The ADC of any one of claims 24 to 31, which is represented by formula (V-5): Where R 6′ and R 6″ Each is independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl.

33. The ADC according to any one of claims 24 to 32, which is represented by formula (VI-5): Where X 1 is C or N, where R 6′ and R 6″ Each is independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl.

34. The ADC according to any one of claims 24 to 33, which is represented by formula (VI-6):

35. The ADC of any one of claims 24 to 34, wherein L 1 include-(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -; a, b and c are each 1; d is 0 and therefore T 4 and V 4 does not exist; T 1 , T 2 and T 3 Each independently selected from C1-C 12 Alkyl, (PEG) n and (AA) p The group composed of n is an integer from 1 to 10; p is an integer from 1 to 10; V 1 、V 2 and V 3 Each independently selected from -C(=O)- and -NR 11 - a group of people; and R 11 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl.

36. The ADC of any one of claims 24 to 35, wherein L 1 for in represents the point of attachment to the nitrogen of the pyridazine-pyrrolo coupling moiety and wherein * represents the point of attachment to the rest of the linker in any direction.

37. The ADC according to any one of claims 24 to 36, which is represented by formula (VI-7): (VI-7), where X 1 It is C or N.

38. The ADC of any one of claims 24 to 37, wherein L 2 It is a carbonyl group.

39. The ADC of any one of claims 24 to 38, wherein X 1 N and W 1 It is MMAE.

40. The ADC of any one of claims 24 to 39, represented by formula (VIb-82):

41. The ADC of any one of claims 1 to 40, wherein the antibody comprises: (i) comprising the VH CDR1, VH CDR2 and VH CDR3 shown in VH of the amino acid sequence SEQ ID NO: 25, and the VL CDR1, VL CDR2 and VL CDR3 shown in VL of the amino acid sequence SEQ ID NO: 26; or (ii) VH CDR1, VH CDR2 and VH CDR3 shown in VH comprising the amino acid sequence of SEQ ID NO: 44, and VL CDR1, VL CDR2 and VL CDR3 shown in VL comprising the amino acid sequence of SEQ ID NO: 45; or (iii) VH CDR1, VH CDR2 and VH CDR3 shown in VH comprising the amino acid sequence of SEQ ID NO: 62, and VL CDR1, VL CDR2 and VL CDR3 shown in VL comprising the amino acid sequence of SEQ ID NO:

63.

42. The ADC of any one of claims 1 to 41, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13 and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19 and 24; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15 and 20; and (b) a VL region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16 and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) A VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17 and 23.

43. The ADC of any one of claims 1 to 42, wherein the antibody comprises: (i) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (ii) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (iii) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (iv) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or (v) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 23; or (vi) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

44. The ADC of any one of claims 1 to 41, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 31, 34, 35 and 39; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 32, 36, 40 and 43; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 33, 37 and 41; and (b) a VL region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16 and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) A VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 38 and 42.

45. The ADC of any one of claims 1 to 41 and 44, wherein the antibody comprises: (i) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30; or (ii) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30; or (iii) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30; or (iv) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 38; or (v) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 41; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (vi) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

30.

46. ​​The ADC of any one of claims 1 to 41, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 50, 53, 13, and 57; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 54, 58 and 61; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 52, 55 and 59; and (b) a VL region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16 and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) A VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 56 and 60.

47. The ADC of any one of claims 1 to 41 and 46, wherein the antibody comprises: (i) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 49; or (ii) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 52; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 49; or (iii) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 49; or (iv) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 54, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 55; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 56; or (v) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 57, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 59; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 60; or (vi) a VH region comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48; and a VL region comprising: a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

49.

48. The ADC of any one of claims 1 to 46, wherein the antibody further comprises a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and / or a framework 4 (FR4) sequence shown in any one of SEQ ID NOs: 25, 26, 44, 45, 62 and 63.

49. The ADC of any one of claims 1 to 48, wherein the antibody further comprises a human framework sequence.

50. The ADC of any one of claims 1 to 49, wherein the antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 26; or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 44 and a VL comprising the amino acid sequence of SEQ ID NO: 45; or (iii) VH comprising the amino acid sequence of SEQ ID NO:62 and VL comprising the amino acid sequence of SEQ ID NO:

63.

51. A pharmaceutical composition comprising the ADC of any one of claims 1 to 50 and a pharmaceutically acceptable excipient.

52. The pharmaceutical composition of claim 51, wherein the antibody-drug conjugate has a drug / antibody ratio of about 1 to about 4.

53. The pharmaceutical composition of claim 52, wherein the drug / antibody ratio is about 2.

54. A method for treating cancer or a tumor in a subject, comprising administering to the subject the antibody-drug conjugate of any one of claims 1-49 or the pharmaceutical composition of any one of claims 51 to 53.

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