5T4 Antibody-Drug Conjugate and Its Use

Abstract

The present disclosure provides a 5T4 antibody-drug conjugate and its use. The present disclosure provides an ADC (a "5T4-ADC") comprising an antibody that binds to 5T4. Such 5T4-ADCs, in some embodiments, bind to the same epitope of human 5T4 as the antibody comprising the heavy chain variable region (VH) and light chain variable region (VL) described herein. The present disclosure also provides a pharmaceutical composition comprising a 5T4-ADC comprising an antibody or a fragment thereof that binds to 5T4 and a drug conjugated thereto (directly or indirectly).

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 341,957, filed on May 13, 2022. The disclosure of the provisional application is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a computer - readable sequence listing submitted in XML file format together with this application, the entire content of which is incorporated herein by reference in its entirety. The sequence listing of the XML file submitted together with this application is named "14529 - 099 - 228_SEQ_LISTING.xml", was created on May 9, 2023, and is 125,376 bytes in size.

[0003] Field The present disclosure generally relates to antibody - drug conjugates (ADCs) that bind to 5T4 (e.g., human 5T4) and methods of using them.

Background Art

[0004] Background 5T4 is an N - glycosylated transmembrane 72 kDa glycoprotein containing eight leucine - rich repeats. Also known as Wnt - inhibitory factor 1 or WAIF1, 5T4 is often called a cancer - fetal antigen because it is expressed in fetal trophoblast cells (where it was first discovered) or trophoblast glycoprotein (TPBG). 5T4 is found within tumors such as colorectal tumors, ovarian tumors, and gastric tumors. Its expression has been used as a prognostic adjunct. 5T4 has very limited expression in normal tissues but is widely distributed throughout its occurrence in malignant tumors. Its limited expression would seem to give 5T4 the potential to be a target for cancer therapy, but the success of treatment with antibodies, ADCs, and vaccines targeting 5T4 has not yet been achieved.

[0005] In the art, there is still a need for an ADC that targets 5T4 and can treat, prevent, or alleviate 5T4-mediated diseases, disorders, or conditions, such as those involving tumor cells expressing 5T4. SUMMARY OF THE INVENTION

[0006] Summary The present disclosure provides an ADC (a "5T4-ADC") comprising an antibody that binds to 5T4. In some embodiments, such 5T4-ADCs bind to the same epitope of human 5T4 as the antibodies comprising the heavy chain variable region (VH) and light chain variable region (VL) described herein.

[0007] The present disclosure also provides a pharmaceutical composition comprising a 5T4-ADC comprising an antibody or a fragment thereof that binds to 5T4 and a drug conjugated (directly or indirectly) thereto. In some embodiments, such pharmaceutical compositions comprise a 5T4-ADC comprising an antibody or a fragment thereof that binds to the same epitope of human 5T4 as the antibodies comprising VH and VL described herein.

[0008] The present disclosure also provides methods of treating, preventing, or alleviating a 5T4-mediated disease, disorder, or condition, such as alleviating one or more symptoms of a 5T4-mediated disease, disorder, or condition with a 5T4-ADC that binds to 5T4.

[0009] More specifically, the present disclosure provides a 5T4-ADC comprising (a) an antibody that binds to 5T4 and (b) one or more pyridazine-pyrrole coupling moieties comprising a drug conjugated to a pyridazine-pyrrole coupling moiety via a linker. In some embodiments, the 5T4-ADC is represented by formula (IV):

Chemical formula

[0010] In some embodiments, the drug is a maytansinoid or an auristatin such as MMAE or MMAF.

[0011] In some embodiments, the 5T4-ADC may be represented by formula (IV), wherein R 2 and R 3Each is alkyl, for example, methyl. In some embodiments, 5T4-ADC can be represented by formula (IV), where X 1 , X 2 , X 3 , and X 4 are each independently C or N.

[0012] In some embodiments, 5T4-ADC is of formula (V):

Chemical formula

[0013] In some embodiments, 5T4-ADC can be represented by formula (VI):

Chemical formula

[0014] In some embodiments, 5T4-ADC can be represented by formula (IV), (V), or (VI), wherein L is a linker, - (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 - represented by T 1, T 2 , T 3 , T 4 , and T 5 are each independently C 1 ~C 6 alkyl, P4A-R 12 , (AA) p , (AA) p -PABC, or (PEG) n and V 1 , V 2 , V 3 , V 4 and V 5 are each 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)-, -SO 2 -, -SO 2 NR 11 -, -NR 11 SO 2 -, or -P(=O)OH- and are selected from the group consisting of 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, p is an integer from 1 to 20, n is an integer from 1 to 30, R 11 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, PEG, aryl, and substituted aryl, and R 12 is independently polyethylene glycol or carboxylic acid-modified polyethylene glycol.

[0015] In a further embodiment, 5T4-ADC can be represented by formula (IV), (V), or (VI), wherein 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, p is an integer from 1 to 20, n is an integer from 1 to 30, L is a linker, -(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 - is represented by T 1 is C 1 ~C 6 alkyl, T 2 , T 3 , T 4 , and T 5 are each independently (PEG) n , C 1 ~C 6 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 ) or (AA) p -(PABC-R 16 ) V 1 , V 2 , V 3 , V 4 , and V 5 are each independently -CO- or -NR 11 R 11 is independently hydrogen or alkyl, R 12 is independently carboxylic acid-modified polyethylene glycol, and R 16 is hydrogen.

[0016] ​In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), or (VI), wherein L is -(C 1 ~C 6 alkyl)-(V 1 )-(P4A-R 12 )-(V 2 )-(C 1 ~C 6 alkyl)-(V 3 )- and V 1 V 2 V 3 are each independently -CO- or -NR 11 and R 11 is independently hydrogen or C 1 ~C 6 alkyl and R 12 is a carboxylic acid-modified polyethylene glycol moiety.

[0017] In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), or (VI), wherein L is

Chemical formula

Chemical formula

[0018] In some embodiments, the 5T4-ADC can be represented by formula (VI-1):

Chemical formula

[0019] In some embodiments, the 5T4-ADC can be represented by formula (VI-1), wherein the drug is a maytansinoid or an auristatin, for example, MMAE. In some embodiments, the 5T4-ADC can be represented by formula (VI-1), wherein X 1 is C, and W 1 is maytansine or deacylated maytansine. In some embodiments, the 5T4-ADC can be represented by formula (VI-1), wherein X 1 is N, and W 1 is MMAE.

[0020] In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), or (VI), wherein L is (C 1 ~C 6 alkyl)-V 1 -(AA)-V 2 -(PEG) n -V 3 -((AA) p -(PABC-R 16 ))-V 4 -(C 1 ~C 6 alkyl)-V 5 and V 1 、V 2 、V 3 、V 4 、and V 5 are each independently selected from the group consisting of -CO- and -NR 11 -. R 11 is independently hydrogen or C 1 ~C 6 alkyl, R 16 is hydrogen, and n and p are each independently an integer from 1 to 20.

[0021] In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), or (VI), wherein L is [Chemical formula] In the formula, [Chemical formula] (where) represents the bonding point to the nitrogen of the pyridazine - pyrrolocoupling moiety, and * represents the bonding point to W 1 represents the bonding point to.

[0022] In some embodiments, the 5T4 - ADC can be represented by formula (VI - 2): [Chemical formula] In the formula, Ab represents an antibody that binds to 5T4, r is an integer from 1 to 10, X 1 is C or N, and W 1 is a drug.

[0023] In some embodiments, the 5T4 - ADC can be represented by formula (VI - 2), wherein the drug is a maytansinoid or an auristatin, for example, MMAE. In some embodiments, the 5T4 - ADC can be represented by formula (VI - 2), wherein X 1 is C, and W 1 is maytansine or deacylated maytansine.

[0024] The present disclosure also provides 5T4 - ADCs of formulas (IV), (V), and (VI): [Chemical formula] In the formula, Ab represents an antibody that binds to 5T4, r is an integer from 1 to 10, m is 0 or 1, R 2 and R 3is, independently of each other, 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 and R 3 are cyclically linked to form a 5- or 6-membered heterocyclyl, X 1 X 2 X 3 and X 4 are each independently selected from the group consisting of C, N, O, and S, Y 1 Y 2 Y 3 and Y 4 are each 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, heterocyclyl, or substituted heterocyclyl, or Y 1 and Y 2 Y 2 and Y 3 or Y 3 and Y 4 are cyclically linked, L is,

Chemical formula

Chemical formula

[0025] In some embodiments, the 5T4-ADC can be represented by formula (IV), wherein R 2 and R 3 are each alkyl, for example, methyl. In some embodiments, the 5T4-ADC can be represented by formula (IV), wherein X 1 , X 2 , X 3 , and X 4 are each independently C or N.

[0026] In some embodiments, the 5T4-ADC can be represented by formula (VI-3):

Chemical formula

[0027] In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), or (IV), wherein L is

Chemical formula

Chemical formula

[0028] In some embodiments, the 5T4-ADC can be represented by formula (V-5): [Chemical formula] In the formula, Ab, L 1 , L 2 , R 2 , R 3 , R 5 , X 1 , Y 1 , Y 2 , Y 3 , Y 4 , W 1 , and r are as defined herein, such as in formula (VI-3), and R 6’ and R 6” are each independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl.

[0029] In some embodiments, the 5T4-ADC can be represented by formula (VI-5): [Chemical formula] In the formula, Ab, L 1 , L 2 , R 5 , W 1 , and r are as defined herein, such as in formula (VI-3), and R 6’ and R 6” are each independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl, and X 1 is C or N.

[0030] In some embodiments, the 5T4-ADC can be represented by formula (VI-6): [Chemical formula] In the formula, Ab, L 1 , L2 , W 1 , and r are as defined herein, for example, in formula (VI-3), where X 1 is C or N.

[0031] In some embodiments, the 5T4-ADC can be represented by formula (VI-3), (V-5), (VI-5), or (VI-6), L 1 is -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c - and includes, T 1 , T 2 and T 3 are each independently selected from the group consisting of C 1 ~C 12 alkyl, (PEG) n and (AA) p , V 1 , V 2 , and V 3 are each independently -C(=O)- or -NR 11 -, a, b, and c are each 1, n is independently an integer from 1 to 10, p is independently an integer from 1 to 10, R 11 is independently hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety, aryl, or substituted aryl.

[0032] In some embodiments, the 5T4-ADC can be represented by formula (VI-3), (V-5), (VI-5), or (VI-6), where L 1 is

Chemical formula

Chemical formula

[0033] In some embodiments, the 5T4 - ADC can be represented by formula (VI - 7):

Chemical formula

[0034] In some embodiments, the 5T4 - ADC can be represented by formula (VI - 7), wherein L 2 is a carbonyl group. In some embodiments, the 5T4 - ADC can be represented by formula (VI - 7), wherein X 1 is N and W 1 is MMAE.

[0035] 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), and the antibody (Ab) comprises (i) VH CDR1, VH CDR2, and VH CDR3 as described in VH comprising the amino acid sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2, and VL CDR3 as described in VL comprising the amino acid sequence of SEQ ID NO: 26, (ii) VH CDR1, VH CDR2, and VH CDR3 as described in VH comprising the amino acid sequence of SEQ ID NO: 44, and VL CDR1, VL CDR2, and VL CDR3 as described in VL comprising the amino acid sequence of SEQ ID NO: 45, or (iii) VH CDR1, VH CDR2, and VH CDR3 as described in VH comprising the amino acid sequence of SEQ ID NO: 62, and VL CDR1, VL CDR2, and VL CDR3 as described in VL comprising the amino acid sequence of SEQ ID NO: 63.

[0036] 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), and the antibody (Ab) comprises (a) a VH region comprising (1) VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18, (2) VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24, and (3) 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) VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, (2) VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22, and (3) VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23.

[0037] In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), and the antibody (Ab) is (i) a VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (ii) a VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 7, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 8, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 9, and a VL region comprising a VL CDR1 containing SEQ ID NO: 10, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (iii) a VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 12, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (iv) a VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 13, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 15, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 17, or (v) a VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 18, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 20, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 21, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 23, or (vi) a VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, a VHIt includes a VH region containing CDR2 and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 3, and a VL region containing a VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0038] In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), and the antibody (Ab) includes (a) (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) (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.

[0039] In some embodiments, the 5T4-ADC can be represented by Formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), and the antibody (Ab) is (i) a VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 27, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 29, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 30, or (ii) a VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 10, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 30, or (iii) a VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 34, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 29, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 30, or (iv) a VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 35, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 38, or (v) a VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 39, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 40, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 41, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 21, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 42, or (vi) a VH comprising the amino acid sequence of SEQ ID NO: 27It includes a VH region containing CDR1, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 29, and a VL region containing a VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 30.

[0040] In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), and the antibody (Ab) has (a) (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 includes a VH region, and (b) (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, and includes a VL region.

[0041] In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), and the antibody (Ab) is (i) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 46, a VH CDR2 having the amino acid sequence of SEQ ID NO: 47, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 48, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 49, or (ii) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 50, a VH CDR2 having the amino acid sequence of SEQ ID NO: 51, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 52, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 49, or (iii) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 53, a CDR2 having the amino acid sequence of SEQ ID NO: 47, and a CDR3 having the amino acid sequence of SEQ ID NO: 48, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 49, or (iv) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 13, a CDR2 having the amino acid sequence of SEQ ID NO: 54, and a CDR3 having the amino acid sequence of SEQ ID NO: 55, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 56, or (v) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 57, a CDR2 having the amino acid sequence of SEQ ID NO: 58, and a CDR3 having the amino acid sequence of SEQ ID NO: 59, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 21, a VL CDR2 having the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 60, or (vi) a VH having the amino acid sequence of SEQ ID NO: 46A VH region comprising CDR1, a CDR2 having the amino acid sequence of SEQ ID NO: 61, and a CDR3 having the amino acid sequence of SEQ ID NO: 48, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 49.

[0042] In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), and the antibody (Ab) comprises a framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequence shown in any one of SEQ ID NOs: 25, 26, 44, 45, 62, and 63.

[0043] In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), and the antibody (Ab) comprises a human framework sequence.

[0044] In some embodiments, the 5T4-ADC can be represented by formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7), and the antibody (Ab) 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, (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) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 63.

[0045] The present disclosure also provides a pharmaceutical composition comprising a 5T4-ADC, which can be represented by Formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7) and its pharmaceutically acceptable excipients, and the antibody (Ab) is as described in any of the embodiments described herein. In some embodiments, the drug-to-antibody ratio (DAR) of the 5T4-ADC in such a pharmaceutical composition can be from about 1 to about 10, for example, the DAR can be 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.

[0046] The present disclosure also provides a method for treating cancer or tumor in a subject, the method comprising administering a 5T4-ADC to the subject, the 5T4-ADC being represented by Formula (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (V-5), (VI-5), (VI-6), or (VI-7) or 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 its pharmaceutically acceptable excipients, and the antibody (Ab) is as described in any of the embodiments herein.

Brief Description of the Drawings

[0047]

Figure 1-1

Figure 1-2

[0048]

Figure 2-1

Figure 2-2

[0049]

Figure 3

[0050]

Figure 4-1

Figure 4-2

[0051]

Figure 5-1

Figure 5-2

[0052]

Figure 6

[0053]

Figure 7

[0054]

Figure 8

[0055]

Figure 9

[0056]

Figure 10

[0057]

Figure 11

[0058]

Figure 12

[0059]

Figure 13

[0060]

Figure 14

[0061]

Figure 15

[0062]

Figure 16

[0063]

Figure 17

[0064]

Figure 18

[0065]

Figure 19

[0066]

Figure 20

[0067]

Figure 21

[0068]

Figure 22

[0069]

Figure 23

[0070]

Figure 24A

Figure 24B

Figure 24C

Mode for Carrying Out the Invention

[0071] Detailed Description The present disclosure provides an antibody-drug conjugate (ADC) that binds to 5T4 and a drug conjugated (directly or indirectly) thereto. Such 5T4-ADC is useful in compositions and methods for treating, preventing, or alleviating 5T4-mediated diseases, disorders, or conditions, including one or more symptoms of a disease, disorder, or condition. 5T4-mediated diseases, disorders, and conditions include, but are not limited to, various cancers in which tumor cells express or overexpress 5T4. Further, 5T4-ADC is useful for killing and / or removing tumor cells. The 5T4-ADC described herein is useful in compositions and methods for treating cancer.

[0072] The term "alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 12 carbon atoms, such as 1 to 10, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 carbon atoms. By way of example, this term includes straight-chain and branched-chain hydrocarbyl groups, such as methyl (-CH 3 )), ethyl (-CH 2 CH 3)、n-propyl (-CH 2 CH 2 CH 3 )、isopropyl (-CH(CH 3 ) 2 )、n-butyl (-CH 2 CH 2 CH 2 CH 3 )、isobutyl (-CH 2 CH(CH 3 ) 2 )、sec-butyl (-CH(CH 3 )(CH 3 CH 2 ))、t-butyl (-C(CH 3 ) 3 )、n-pentyl (-CH 2 CH 2 CH 2 CH 2 CH 3 )、and neopentyl (-CH 2 C(CH 3 ) 3 ) etc. are included.

[0073] The term "substituted alkyl" refers to an alkyl group as defined herein, where one or more carbon atoms (C 1 excluding carbon atoms) in the alkyl chain are optionally substituted with heteroatoms such as -O-, -N-, -S-, -S(O) n -(where n is 0 to 2), -NR- (where R is hydrogen or alkyl), etc., and are also substituted with alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketone, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO 2-Alkyl, -SO 2 -Aryl, -SO 2 -Heteroaryl, and -NR a R b having 1 to 5 substituents selected from the group consisting of, wherein R a and R b may be the same or different and are each selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocycle.

[0074] The term "alkenyl" preferably refers to a divalent aliphatic hydrocarbyl group having 1 to 6, more preferably 1 to 3 straight-chain or branched-chain carbon atoms, optionally substituted with one or more groups selected from -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, etc. This term includes, for example, methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), n-propylene (-CH 2 CH 2 CH 2 -), isopropylene (-CH 2 CH(CH 3 ))-, (-C(CH 3 )) 2 CH 2 CH 2 -), (-C(CH 3 )) 2 CH 2 C(O)-), (-C(CH 3 )) 2 CH 2 C(O)NH-), (-CH(CH 3 ))CH 2 -), etc.

[0075] The term "substituted alkenyl" refers to an alkylene group having 1 to 3 hydrogens substituted with substituents as described in the following definition of "substitution" for carbon.

[0076] The term "alkaryl" or "aralkyl" refers to an -alkylene-aryl group and a -substituted alkylene-aryl group, and alkylene, substituted alkylene and aryl are defined herein.

[0077] The term "alkoxy" refers to an -O-alkyl group, and alkyl is as defined herein. Examples of alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy and the like. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, and alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0078] The term "substituted alkoxy" refers to a substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- group, and substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0079] The term "alkoxyamino" refers to an -NH-alkoxy group, and alkoxy is defined herein.

[0080] The term "alkylalkoxy" refers to -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl, and alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0081] The term "alkenyl" refers to a straight-chain or branched hydrocarbyl group having 2 to 6 carbon atoms, for example 2 to 4 carbon atoms, and having at least one, preferably 1 to 2 sites of double bond unsaturation. This term includes, by way of example, vinyl, allyl, and but-3-en-1-yl. This term includes cis and trans isomers or mixtures of these isomers.

[0082] The term "substituted alkenyl" refers to an alkenyl group having 1 to 5 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, thioketo, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl and -SO 2 -heteroaryl, or an alkenyl group as defined herein having 1 to 3 substituents.

[0083] The term "alkynyl" refers to a straight-chain or branched-chain monovalent hydrocarbyl group having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and at least 1, preferably 1 to 2 sites of triple-bond unsaturation. Examples of such alkynyl groups include ethynyl (-C≡CH) and propargyl (-CH 2 C≡CH).

[0084] The term "substituted alkynyl" refers to one to five 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, thioketo, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl and -SO 2 -heteroaryl, and refers to an alkynyl group defined herein having one to three substituents, or one to five substituents selected therefrom.

[0085] The term "acylamino" refers to -NR 20 C(O)alkyl, -NR 20 C(O)substituted alkyl, NR 20 C(O)cycloalkyl, -NR 20 C(O)substituted cycloalkyl, -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)alkynyl, -NR 20 C(O)substituted alkynyl, -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)heterocycle, and -NR 20 C(O)substituted heterocyclic group, wherein R 20is hydrogen or alkyl, and 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.

[0086] The term "acyl" refers to a group selected from H-C(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)-, substituted aryl C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, and 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. For example, acyl includes the "acetyl" group CH 3 C(O)-.

[0087] The term "aminocarbonyl" or the term "aminoacyl" refers to a -C(O)NR 21 R 22 group, wherein R 21 and R 22 are 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, and R 21 and R 22Optionally, together with the nitrogen atom attached thereto, they form a heterocyclic or 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, heterocycle, and substituted heterocycle are as defined herein.

[0088] The term "acyloxy" refers to a group selected from 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 heterocyclyl-C(O)O-, and alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0089] The term "aminosulfonyl" refers to -SO 2 NR 21 R 22 group, and R 21 and R 22 are 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, and R 21 and R 22 optionally, together with the nitrogen atom attached thereto, form a heterocyclic or 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, heterocycle, and substituted heterocycle are as defined herein.

[0090] The term "sulfonylamino" refers to -NR 21 SO 2 R 22Refers to a group, where R 21 and R 22 are 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, and R 21 and R 22 may optionally, together with the atoms to which they are attached, form a heterocyclic or 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, heterocycle, and substituted heterocycle are as defined herein.

[0091] The term "aryl" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (such as present in a phenyl group) or a ring system having a plurality of fused rings (examples of such aromatic ring systems include naphthyl, anthryl, and indanyl), and the fused rings may or may not be aromatic as long as the point of attachment is through an atom of the aromatic ring. This term includes, for example, phenyl and naphthyl. Unless otherwise restricted by the definition of the aryl substituent, such an aryl group may optionally be acyloxy, hydroxy, 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, aralkyl, aryl, aryloxy, azide, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl, -SO 2 1 to 5 substituents selected from -heteroaryl and trihalomethyl, or can be substituted with 1 to 3 substituents.

[0092] The term "aryloxy" refers to an -O-aryl group, where aryl is as defined herein, and examples include phenoxy, naphthoxy, etc., and optionally include substituted aryl groups as defined herein.

[0093] The term "amino" refers to an -NH 2 group.

[0094] The term "substituted amino" refers to an -NRR group, where 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 heterocyclyl, provided that at least one R is not hydrogen.

[0095] The term "azide" refers to an -N 3 group.

[0096] The terms "carboxyl", "carboxy" or "carboxylate" refer to -CO 2 H or their salts.

[0097] The term "carboxyl ester" or "carboxy ester" or the term "carboxyalkyl" or "carboxylalkyl" refers to a group selected from -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, -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-heterocycle, and -C(O)O-substituted heterocycle, where 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.

[0098] The term "cyano" or "nitrile" refers to a -CN group.

[0099] The term "cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms, including monocyclic or polycyclic rings, including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like.

[0100] The term "substituted cycloalkyl" refers to an alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketone, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl and -SO 2 selected from -heteroaryl, one to five substituents, or a cycloalkyl group having one to three substituents.

[0101] The term "cycloalkenyl" refers to a non-aromatic cyclic alkyl group having a monocyclic or polycyclic structure and at least one double bond, preferably one to two double bonds, and having three to ten carbon atoms.

[0102] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having 1 to 5 substituents, or 1 to 3 substituents, and the substituents are alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl and -SO 2 -heteroaryl, and are independently selected from.

[0103] The term "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[0104] The terms "hydroxy" and "hydroxyl" refer to the -OH group.

[0105] The term "heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms, such as 1 to 10 carbon atoms, and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a monocyclic ring (e.g., pyridinyl, imidazolyl or furyl), or can have a plurality of fused rings within the ring system (as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), in which case at least one ring within the ring system is aromatic. To satisfy the valence requirements, any heteroatom in such a heteroaryl ring may or may not be bonded to an H or a substituent, such as an alkyl group or other substituent described herein. In some embodiments, the nitrogen and / or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide an N-oxide (N→O), sulfinyl, or sulfonyl moiety. The term includes, for example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise restricted by the definition of the heteroaryl substituent, such heteroaryl groups can optionally be substituted with 1 to 5 substituents, or 1 to 3 substituents, and the substituents are acyloxy, hydroxy, 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, alkaryl, aryl, aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl and -SO 2-heteroaryl and independently selected from trihalomethyl.

[0106] The term "heteroalkyl" refers to a group containing an alkylene attached to a heteroaryl, where the alkylene and heteroaryl are as defined herein. This term includes, for example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0107] The term "heteroaryloxy" refers to -O-heteroaryl.

[0108] The terms "heterocyclic", "heterocyclic ring", "heterocycloalkyl", and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings, including fused bridge and spiro ring systems, and having 3 to 20 ring atoms containing 1 to 10 heteroatoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, and in a fused ring system, one or more rings can be cycloalkyl, aryl, or heteroaryl as long as the point of attachment is through a non-aromatic ring. In some embodiments, the nitrogen and / or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO 2 - moiety. To satisfy the valence requirements, any heteroatom in such a heterocyclic group may or may not be bonded to one or more H or one or more substituents (plural possible), such as an alkyl group or other substituents described herein.

[0109] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolidine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, etc.

[0110] Unless otherwise restricted by the definition of the heterocyclic substituent, such a heterocyclic group can optionally be substituted with 1 to 5, or 1 to 3 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, thioketone, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl, -SO 2 -heteroaryl, and fused heterocycles.

[0111] The term "hydroxyamino" refers to the -NHOH group.

[0112] The term "nitro" refers to the -NO 2 group.

[0113] The term "oxo" refers to a double-bonded oxygen atom (=O).

[0114] The term "sulfonyl" refers to -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -alkenyl, -SO 2 -substituted alkenyl, -SO 2 -cycloalkyl, -SO 2 -substituted cycloalkyl, -SO 2 -cycloalkenyl, -SO 2 -substituted cycloalkenyl, -SO 2 -aryl, -SO 2 -substituted aryl, -SO 2 -heteroaryl, -SO 2 -substituted heteroaryl, -SO 2 -heterocyclic ring, and -SO 2 -substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein. Sulfonyl includes, for example, methyl-SO 2 -, phenyl-SO 2 -, and 4-methylphenyl-SO 2 -.

[0115] The term "thiol" refers to the -SH group.

[0116] The term "thioxo" or the term "thioket" refers to the atom (=S).

[0117] The term "alkylthio" or the term "thioalkoxy" refers to an -S-alkyl group, where alkyl is as defined herein. In some embodiments, sulfur can be oxidized to -S(O)-. Sulfoxides can exist as one or more stereoisomers.

[0118] The term "substituted thioalkoxy" refers to an -S-substituted alkyl group.

[0119] The term "thioaryloxy" refers to an aryl-S-group, where the aryl group is as defined herein and includes optionally substituted aryl groups, which are also as defined herein.

[0120] The term "thioheteroaryloxy" refers to a heteroaryl-S-group, where the heteroaryl group is as defined herein and includes optionally substituted aryl groups, which are also as defined herein.

[0121] The term "thioheterocyclooxy" refers to a heterocyclyl-S-group, where the heterocyclyl group is as defined herein and includes optionally substituted heterocyclyl groups, which are also as defined herein.

[0122] In addition to the disclosure herein, when the term "substituted" is used to modify a particular group or radical, it can also mean substituting one or more hydrogen atoms of the particular group or radical, each independently of one another, with the same or different substituents as defined below.

[0123] In addition to the groups disclosed herein with respect to the individual terms herein, substituents for substituting one or more hydrogens on a saturated carbon atom in a particular group or radical (any two hydrogens on a single carbon can be substituted with =O, =NR 70 , =N-OR 70 , =N 2 or =S) are, unless otherwise specified, -R 60, halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO 2 , =N 2 , -N 3 , -SO 2 R 70 , -SO 2 O - M + , -SO 2 OR 70 , -OSO 2 R 70 , -OSO 2 O - M + , -OSO 2 OR 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 CO 2 - M+ 、 -NR 70 CO 2 R 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 wherein, R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R 70 is independently hydrogen or R 60 , and each R 80 is independently R 70 , or two R 80 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocycloalkyl which may optionally have 1 to 4 additional heteroatoms the same or different selected from the group consisting of O, N and S (wherein N is -H or C 1 -C 6 may optionally have an alkyl substitution); each M + is a counterion having a positive charge. Each M + is independently, for example, an alkali ion such as K + , Na + , Li + ; an ammonium ion such as + N(R 60 ) 4 ; or [Ca 2 0.5 , [Mg 2+ 0.5 , or [Ba 2+ 0.5 ​​​It may also be an alkaline earth ion such as (the subscript "0.5" means whether one of the counterions of such divalent alkaline earth ions can be the ionized form of the compound of the present invention and the other can be a typical counterion such as chloride, or whether the two ionized compounds disclosed in the present specification can act as counterions for such divalent alkaline earth ions, or whether the double-ionized compound of the present invention can act as a counterion for such divalent alkaline earth ions). As a specific example, -NR 80 R 80 is, -NH 2 , -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N-morpholinyl.

[0124] In addition to the disclosure in the present specification, the substituents of hydrogen on the unsaturated carbon atoms in the "substituted" alkenyl, alkynyl, aryl and heteroaryl groups are, unless otherwise specified, -R 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , -N 3 , -SO 2 R 70 , -SO 3 - M + , -SO 3 R 70 , -OSO 2 R 70 , -OSO 3 - M + , -OSO 3 R 70 , -PO 3 -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 ,-CO 2 - M + ,-CO 2 R 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 ,-OCO 2 - M + ,-OCO 2 R 70 ,-OC(S)OR 70 ,-NR 70 C(O)R 70 ,-NR 70 C(S)R 70 ,-NR 70 CO 2 - M + ,-NR 70 CO 2 R 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 wherein, R 60 , R 70 , R 80 and M + are as defined herein, provided that in the case of substituted alkenyl or alkynyl, the substituents are -O - M + , -OR 70 , -SR 70 , or -S- M + is not.

[0125] In addition to the disclosure bases for the individual terms in this specification, the substituents for the hydrogen on the nitrogen atom in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -NO, -NO 2 , -S(O) 2 R 70 , -S(O) 2 O - M + , -S(O) 2 OR 70 , -OS(O) 2 R 70 , -OS(O) 2 O - M + , -OS(O) 2 OR 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)OR 70 , -OC(S)OR70 、 -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 wherein R 60 、 R 70 、 R 80 and M + are as previously defined.

[0126] The substituted group may have 1, 2, 3 or 4 substituents, for example, 1, 2, or 3 substituents, or 1 or 2 substituents, or 1 substituent.

[0127] Unless otherwise specified, the nomenclature of substituents not explicitly defined in this specification is obtained by naming the functional group adjacent to the point of attachment towards the end of the functional group. For example, the substituent "arylalkyloxycarbonyl" refers to the (aryl)-(alkyl)-O-C(O)- group.

[0128] With respect to any of the groups disclosed herein that contain one or more substituents, it is understood that such groups do not include any substitutions or substitution patterns that are sterically unrealistic and / or synthetically infeasible. Furthermore, the compounds of the present invention include all stereochemical isomers resulting from the substitution of these compounds.

[0129] The term "subject" refers to human and non-human subjects, particularly mammalian subjects.

[0130] As used herein, the term "treat" or "treatment" means treating or ameliorating a disease or condition in a subject, such as a mammal (particularly a human), and includes (a) preventing a disease or condition, such as prophylactic treatment of a subject, (b) ameliorating a disease or condition in a subject, such as eliminating or regressing the disease or condition, (c) suppressing a disease or condition, such as delaying or halting the onset of a disease or condition in a subject, or (d) reducing the symptoms of a disease or condition in a subject. In some embodiments, the terms "treating" or "treatment" exclude prophylactic treatment.

[0131] As used herein, the term "native amino acid sequence" refers to the amino acid sequence of a polypeptide prior to modification, including modified amino acid residues.

[0132] Terms such as "amino acid analog" and "non-natural amino acid" are used interchangeably and include amino acid-like compounds that are structurally and / or overall shaped similar to one or more amino acids commonly found in natural 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 stereochemistry as the naturally occurring D- and L-forms of amino acid analogs. In some embodiments, an amino acid analog shares the backbone structure, and / or side chain structure, of one or more natural amino acids, and the difference(s) are one or more modifying groups within the molecule. Such modifications can include, but are not limited to, substitution of an atom (such as N) with a related atom (such as S), addition of a group (such as methyl, or hydroxyl) or atom (such as Cl or Br), deletion of a group, substitution of a covalent bond (such as a double bond to a single bond), or combinations thereof. For example, amino acid analogs can include α-hydroxy acids, and α-amino acids, among others.

[0133] The term "amino acid side chain" is used to refer to a substituent attached to the α-carbon of an amino acid residue, including natural amino acids, non-natural amino acids, and amino acid analogs. Amino acid side chains can also include amino acid side chains described in the context of the modified amino acids and / or conjugates described herein.

[0134] The term "carbohydrate" is used to refer to monomer 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 action), or absent (e.g., removed or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and can be adapted for use in the compounds and conjugates of interest.

[0135] The term "glycoside" or "glycosyl" refers to a sugar molecule or group attached to a moiety via a glycosidic bond. For example, the moiety to which the glycoside is attached can be a cleavable linker as described herein. The glycosidic bond can be an O-glycosidic bond (O-glycoside), an N-glycosidic bond (glycosylamine), an S-glycosidic bond (thioglycoside), or a C-glycosidic bond (C-glycoside or C-glycosyl), among other types of bonds through which the glycoside can be linked to another moiety, although not limited thereto. In some cases, the glycosides can be cleaved from the moieties to which they are attached, for example, by chemical-mediated hydrolysis or enzyme-mediated hydrolysis.

[0136] Unless otherwise indicated, the term "5T4" refers to any native 5T4 from any vertebrate source, including a polypeptide (the terms "polypeptide" and "protein" are used interchangeably herein), or a mammal such as a primate (e.g., human, cynomolgus monkey (cyno)), dog, and rodent (e.g., mouse and rat). 5T4, also known as "5T4 cancer fetal trophoblast glycoprotein" or "Wnt activation inhibitory factor 1" or "5T4 tumor trophoblast glycoprotein" or "5T4 cancer fetal 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 or any fragment of 5T4 resulting from intracellular processing. In some embodiments, 5T4 includes a signal sequence. In some embodiments, 5T4 does not include a signal sequence. In some embodiments, the term "5T4" refers to a fragment of full-length 5T4 that includes the 5T4 extracellular domain. The term "5T4" also encompasses naturally occurring variants of 5T4, such as SNP variants, splice variants, and allelic variants. The full-length amino acid sequence of human 5T4 is shown below (exemplary signal sequence = italicized, exemplary extracellular domain = underlined).

Chemical Structure

Chemical Structure

Chemical Structure

[0137] The terms "antibody", "immunoglobulin", or "Ig" are used interchangeably herein and in the broadest sense, and specifically include, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length monoclonal antibodies), antibody compositions having polyepitope or monospecificity, recombinantly produced antibodies, single domain antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies having full-length heavy and / or light chains. Antibodies include single antibody domains, as well as antibody fragments (and / or polypeptides containing antibody fragments) that retain 5T4 binding properties. Non-limiting examples of antibody fragments include the antigen-binding region and / or effector region of an antibody, for example, Fab, Fab’, F(ab’) 2 , Fv, scFv, (scFv) 2 , single-chain antibody molecules, diabody antibodies, single variable domain antibodies, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab) 2, Fd, Fc, diabody, bispecific diabody, disulfide-bonded Fv (dsFv), single-domain antibody (e.g., nanobody) or other fragment (e.g., fragment consisting of non-covalently associated heavy and light chain variable regions). Generally speaking, the variable (V) region can be any suitable arrangement of the variable regions of the heavy chain (VH) and / or light chain (VL) of an immunoglobulin. For example, antibodies include tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers. Thus, for example, the V region may be a dimer and may include VH-VH, VH-VL, or VL-VL dimers that bind to 5T4. In any embodiment, the VH region and the VL region can be covalently linked either directly or via a linker to form a single-chain Fv (scFv). For ease of reference, scFv proteins are considered herein to be 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 referred to as "minimal recognition units" or "hypervariable regions") can be obtained by constructing a polynucleotide encoding one or more CDRs of interest.Such polynucleotides can be synthesized, for example, in the variable region using polymerase chain reaction with mRNA of antibody-producing cells as a template (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.), page 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.), page 137, Wiley-Liss, Inc. (1995)). Antibody fragments can be incorporated, for example, into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable regions of novel antigen receptors (v-NAR), and bis single-chain Fv regions (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). In some embodiments, antibodies comprising VH and / or VL contain one or more constant regions, such as one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions, including light chain and / or heavy chain constant regions. In some embodiments, the antibody can comprise any of the epitope-binding fragments described above. The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules.

[0138] As used herein, the term "monospecific" refers to an antigen-binding protein having one or more binding sites, each of which binds to the same epitope of the same antigen.

[0139] The term "bispecific" means that an antibody can specifically bind to two binding sites formed by pairs of antibody heavy chain variable regions (VH) and antibody light chain variable regions (VL), respectively, where the antibody binds to at least two distinct antigenic determinants, e.g., different antigens or different epitopes on the same antigen. Such bispecific antibodies can have a 1+1 format (comprising one binding site for a first antigen or epitope and one binding site for a second antigen or epitope). Other bispecific antibody formats can be 2+1 or 1+2 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope), or a 2+2 format (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific antibody comprises two antigen-binding sites, each can bind to a different antigenic determinant. Such bispecific antibodies can bind to two different epitopes on the same antigen (e.g., epitopes on 5T4).

[0140] The terms "identical" or "percent identity" in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned to obtain a maximum correspondence (introducing gaps if necessary) without considering any conservative amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain an alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, basic local alignment search tool (BLAST), ALIGN, MegAlign, BestFit, GCG Wisconsin Package, and their variants. In some embodiments, two nucleic acids or polypeptides are substantially identical if, when measured using a sequence comparison algorithm or by visual inspection and compared and aligned for maximum correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity. In some embodiments, identity exists over a region of an amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40 - 60 residues, at least about 60 - 80 residues in length, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60 - 80 residues, for example, at least about 80 - 100 residues, and in some embodiments, the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or antibody. In some embodiments, identity exists over a region of a nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40 - 60 bases, at least about 60 - 80 bases in length, or any integer value therebetween.In some embodiments, the identity exists over a region longer than 60 - 80 bases, for example, over at least about 80 - 1000 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as the nucleotide sequence encoding the protein of interest.

[0141] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art, and these families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar 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, substituting phenylalanine with tyrosine is a conservative substitution. In general, conservative substitutions in the sequences of the polypeptides, soluble proteins, and / or antibodies of the present disclosure do not inhibit binding to the target binding site of the polypeptide, soluble protein, or antibody containing the amino acid sequence. Methods for identifying amino acid conservative substitutions that do not eliminate binding are well known in the art.

[0142] The term "polypeptide" refers to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may contain non-amino acids (e.g., which may be substituted thereby). These terms also embrace amino acid polymers that have been modified either naturally or 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 moiety (e.g., a toxin) such as a label component or a drug. Also included in this definition are, for example, polypeptides containing one or more analogs of amino acids (including, e.g., non-natural amino acids, etc.), and other modifications known in the art. Since the polypeptides of the present disclosure can be based on antibodies or other immunoglobulin superfamilies, in some embodiments, it is understood that the polypeptides can exist as single chains or linked chains.

[0143] As used herein, an "antigen" is a moiety or molecule that contains an epitope to which an antibody can bind. Thus, an antigen can be bound by an antibody. In some embodiments, the antigen to which the antibodies described herein bind is 5T4 (e.g., human 5T4), or a fragment thereof, e.g., comprising one or more regions of 5T4.

[0144] As used herein, "epitope" is a term in the art and refers to the localized region of an antigen to which an antibody can bind. An epitope can be a linear epitope or a higher order, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, the epitope can be a continuous amino acid of the polypeptide ("linear" epitope), or the epitope can be amino acids of two or more non-contiguous regions of the polypeptide ("higher order structure", "non-linear" or "discontinuous" epitope), for example including human 5T4. In general, it will be understood by those skilled in the art that linear epitopes may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, the antibody binds to a group of amino acids regardless of whether it is folded into a native three-dimensional protein structure. In other embodiments, the antibody requires that the amino acid residues constituting the epitope exhibit a specific higher order structure (e.g., bend, twist, rotation or fold) in order to recognize and bind the epitope.

[0145] An antibody binds to an "epitope", "substantially the same epitope", or "the same epitope" as a reference antibody. 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 various different formats using, for example, either a labeled antigen or a labeled antibody. In some assays, the antigen is immobilized on a 96-well plate or expressed on the cell surface, and the ability of an unlabeled antibody to block the binding of a labeled antibody is measured using a radioactive, fluorescent, or enzyme label.

[0146] "Epitope binning" is a process of classifying antibodies based on the epitopes recognized by the antibodies. More specifically, epitope binning involves clustering antibodies based on their epitope recognition characteristics and using a competitive assay combined with a computational process to identify antibodies with different binding specificities, and includes methods and systems for discriminating the epitope recognition characteristics of different antibodies. Further details regarding the determination methods of antibody epitope binning and epitope binding are described herein, as shown in Example 5.

[0147] As used herein, the terms "specifically binds", "specifically recognizes", "immunologically specifically binds", "selectively binds", "immunologically specifically recognizes", and "immunologically specific" are similar terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope), as understood by those skilled in the art.

[0148] In some embodiments, "specifically binds" means that, for example, a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for a longer period of time, with higher affinity, or in any combination of the above, compared to alternative substances including related and unrelated proteins. For example, a molecule that specifically binds an antigen generally binds other peptides or polypeptides with lower affinity, as determined by, for example, immunoassays, BIACORE™, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), OctetQK384 system (ForteBio, Menlo Park, CA), or other assays known in the art. In some embodiments, an antibody or antigen-binding region binds or specifically binds an antigen when it binds the antigen with higher affinity than any cross-reactive antigen, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective reaction is at least two-fold the background signal or noise, and may exceed ten-fold the background. For considerations regarding binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In some embodiments, the degree of binding of an antibody or antigen-binding region 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, a molecule that specifically binds an antigen has a K A that is at least 2 log, 2.5 log, 3 log, 4 log, or greater than that of K AIt binds to the antigen. In some embodiments, the molecule that specifically binds to the antigen does not cross-react with other proteins. In another specific embodiment, the molecule that specifically binds to the antigen does not cross-react with other non-5T4 proteins. In some embodiments, "specifically binds" means, for example, that a polypeptide or molecule binds to a protein or target with a K D of about 0.1 mM or less, but more typically less than about 1 μM. In some embodiments, "specifically binds" means that a polypeptide or molecule binds to a target with a K D 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. Due to sequence identity between homologous proteins in different species, specific binding can include polypeptides or molecules that recognize a protein or target in multiple species. Similarly, due to homology within a certain region of the polypeptide sequences of different proteins, specific binding can include polypeptides or molecules that recognize multiple proteins or targets. In some embodiments, it is understood that a polypeptide or molecule that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require exclusive binding, e.g., binding to a single target (although it can include that). Thus, in some embodiments, a polypeptide or molecule can specifically bind to multiple targets. In some embodiments, multiple targets can be bound by the same antigen-binding site on the polypeptide or molecule. For example, in some embodiments, an antibody can include two identical antigen-binding sites, each of which specifically binds to the same epitope on two or more proteins. In another embodiment, the antibody is bispecific and can include at least two antigen-binding sites with different specificities. Generally, although not necessarily, reference to "binding" means "specific binding".

[0149] The term "binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (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., an antibody and an antigen). The affinity of a binding molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including the methods 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 longer. Various methods for measuring binding affinity are known in the art, and any of these can be used for the purposes of the present disclosure. In one embodiment, "K D " or "K D value" can be measured, for example, by biolayer interferometry (BLI) using an OctetQK384 system (ForteBio, Menlo Park, CA). As another method, K D can also be measured, for example, by a radiolabeled antigen binding assay (RIA) performed with the Fab version of the antibody of interest and its antigen (Chen et al., (1999) J. Mol Biol 293:865-881), or, for example, using a BIACORE™ surface plasmon resonance (SPR) assay using a BIACORE™-2000 or BIACORE™-3000 (BIAcore, Inc., Piscataway, NJ). "On-rate" or "rate of binding" or "binding rate" or "k on ", as well as "off-rate" or "rate of dissociation" or "dissociation rate" or "k off」 can also be determined, for example, by the same SPR or BLI techniques described herein using, respectively, an OctetQK384 system (ForteBio, Menlo Park, CA) or a BIACORE™-2000 or BIACORE™-3000 (BIACORE™, Inc., Piscataway, NJ).

[0150] The term "competitive", when used in the context of a 5T4 antibody, refers to a binder whose binding to an epitope or binding site is at least partially inhibited for binding by another binder in the presence of the other binder. Competition can be determined by an assay in which the binder being tested prevents or inhibits specific binding of a reference molecule (e.g., a reference antigen-binding protein such as a reference ligand or a reference antibody) to a common antigen (e.g., 5T4). To determine whether a test binder competes with a reference molecule for binding to 5T4 (e.g., human 5T4), a number of different types of competitive binding assays can be used. Examples of assays that can be used include solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol. 137:3614-3619, or Cheung et al., (1990) Virology 176:546-552), solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct labeled RIA using I-125 labeling (e.g., Morel et al., (1988) Molec. Immunol. 25:7-15), and direct labeled RIA (see Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of a purified antigen (e.g., 5T4 such as human 5T4) bound to a solid-phase surface or cell having either an unlabeled test antigen-binding protein (e.g., a test 5T4 antibody or ADC) or a labeled antigen-binding protein (e.g., a reference 5T4 antibody or ADC). Competitive inhibition can be measured by determining the amount of label that binds to the solid-phase surface or cell in the presence of the test antigen-binding protein.Typically, the test antigen-binding protein is present in excess. Competitive antibodies identified by a competitive assay include antibodies that bind to the same epitope as the reference antibody and / or antibodies that bind to an adjacent epitope that is close enough to the epitope bound by reference to cause steric hindrance of the antibody (e.g., epitopes with overlapping similar epitopes). Typically, when the competitive antibody is present in excess, specific binding of the reference antibody to the common antigen is inhibited by at least 20%, e.g., at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some embodiments, the binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

[0151] The terms "constant region" or "constant domain" are used interchangeably herein and are well-known terms in the art for antibodies, referring to the carboxy-terminal portions of the light and heavy chains that are part of the antibody but not directly involved in antigen binding, such as the antibody, but exhibit various effector functions such as interaction with Fc receptors. This term includes portions of immunoglobulin molecules that generally have more conserved amino acid sequences compared to the immunoglobulin variable regions.

[0152] The "effector function" of an antibody refers to the biological activity attributed to the Fc region of the antibody (e.g., the native sequence Fc region or an amino acid sequence variant Fc region) and varies depending on the isotype of the 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.

[0153] The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. The boundaries of the Fc region of an immunoglobulin heavy chain can vary widely, but the human IgG heavy chain Fc region is often defined as extending from the amino acid residue at position Cys226 (according to the EU numbering system) or Pro230 (according to the EU numbering system) to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinant manipulation of the nucleic acid encoding the antibody heavy chain. Exemplary Fc region sequences are provided below (CH2 domain = bold, CH3 domain = underlined). [Chemical formula] (SEQ ID NO: 67).

[0154] A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector 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 the like. Such effector functions generally require the binding of the Fc region to a binding region or domain (e.g., an antibody variable region or domain), and can be evaluated using the various assays disclosed herein.

[0155] A "native sequence Fc region" is identical to the amino acid sequence of an Fc region found in nature and includes an amino acid sequence in which no artificial manipulation, modification, and / or alteration (e.g., isolation, purification, selection, inclusion or combination with other sequences such as variable region sequences) has been performed. Native sequence human Fc regions include the Fc region of native sequence human IgG1 (non-A and A allotypes), the Fc region of native sequence human IgG2, the Fc region of native sequence human IgG3, and the Fc region of native sequence human IgG4, as well as their natural variants.

[0156] A "variant Fc region" includes an amino acid sequence that is different from that of the native sequence Fc region by 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, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc region or the Fc region of the parental polypeptide, as compared to the native sequence Fc region or the Fc region of the parental polypeptide. The variant Fc region herein can have at least about 80% homology, or at least about 90% homology, e.g., at least about 95% homology, with the native sequence Fc region and / or the Fc region of the parental polypeptide. The variant Fc region described herein can have a loss of effector function (e.g., silent Fc). Exemplary variant Fc region ("silent Fc") sequences are provided below (CH2 domain = amino acid changes in bold underlined, CH3 domain = underlined text).

Chemical formula

[0157] The term "heavy chain," when used in reference to an antibody, refers to a polypeptide chain of about 50 - 70 kDa, the amino-terminal portion of which includes a variable region of about 120 - 130 or more amino acids, and the carboxy-terminal portion of which includes a constant region. "Heavy chain" can refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), for example, based on the amino acid sequence of the heavy chain. These give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, and include subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0158] As used herein, the term "light chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 25 kDa, the amino-terminal portion of which contains a variable region of approximately 100 to approximately 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of the light chain is 211 to 217 amino acids. There are two different types of light chains, called kappa (κ) or lambda (λ) based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art.

[0159] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to the portion of an antibody that contains the amino acid residues that interact with an antigen to confer specificity and affinity for the antigen on the binding fragment or region (e.g., CDRs). As used herein, "antigen-binding fragment" includes "antibody fragments" that contain portions of an antibody that include one or more CDRs, such as the antigen-binding or variable region of an antibody.

[0160] Antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab’) fragments, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0161] In some embodiments, the antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules containing one or more antigen-binding sites that bind to 5T4.

[0162] The antibodies described herein can be of any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b). In some embodiments, the 5T4 antibodies described herein are IgG antibodies (e.g., human IgG), or of that class (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclass thereof.

[0163] In some embodiments, the 5T4 antibody is a four-chain antibody unit comprising two heavy (H)-chain / light (L)-chain pairs, 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 chain and / or the L chain comprises a constant region, e.g., a human constant region. In some embodiments, the L-chain constant region of the 5T4 antibody is a kappa or lambda light-chain constant region, e.g., a human kappa or lambda light-chain constant region. In some embodiments, the H-chain constant region of the 5T4 antibody comprises a gamma heavy-chain constant region, e.g., a human gamma heavy-chain constant region. In some embodiments, the 5T4 antibody comprises an IgG constant region, e.g., a human IgG constant region (e.g., IgG1, IgG2, IgG3, and / or IgG4 constant region).

[0164] As used herein, the terms "5T4 antibody" and "antibody that binds to 5T4" are used interchangeably and refer to an antibody that preferentially binds to 5T4. The antibody or fragment thereof can preferentially bind to 5T4, such as human 5T4, which means that the antibody or fragment thereof binds to 5T4, such as human 5T4, with a higher affinity than it binds to an irrelevant control protein. For example, the antibody or fragment thereof can specifically recognize and bind to 5T4 or a portion thereof. "Specific binding" means that the 5T4 antibody or fragment thereof binds to 5T4 with an affinity that is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times higher than its affinity for an irrelevant control protein (e.g., chicken egg white lysozyme). In some embodiments, the 5T4 antibody or fragment thereof can bind substantially exclusively to 5T4 (e.g., distinguishable from other known polypeptides by a measurable difference in binding affinity). In some embodiments, the 5T4 antibody can react with 5T4 sequences other than the human 5T4 sequence (e.g., cynomolgus monkey 5T4 sequence).

[0165] The terms "variable region" and "variable domain" are used interchangeably and generally refer to the portions of the light and heavy chains of an antibody that are located at the amino termini of the chains, have lengths of approximately 120-130 amino acids for the heavy chain and approximately 100-110 amino acids for the light chain, and are used for the binding and specificity of the antibody to its antigen. The variable region of the heavy chain may be referred to herein as "VH". The variable region of the light chain may be referred to herein as "VL". The term "variable" refers to the fact that the sequences of certain segments of the variable regions vary widely between antibodies. The V regions mediate antigen binding and define the specificity of a particular antibody for its antigen. However, the variability is not uniformly distributed throughout the 110 amino acid span of the variable regions. Rather, the V regions consist of stretches of relatively low (e.g., relatively invariant) variability, called framework regions (FRs), of about 15-30 amino acids, separated by shorter regions of greater (e.g., extreme) variability, called "hypervariable regions" or "complementarity determining regions". The variable regions of the heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4), which predominantly adopt a β-sheet conformation and are connected by three hypervariable regions, which form loops connecting the β-sheet structures and, in some cases, form part of the β-sheet structures. The hypervariable regions in each chain are held together in close proximity by the framework and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions such as involvement in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of the antibody. The variable regions vary widely in sequence between different antibodies. The sequence variability is concentrated in the CDRs, while the less variable portions of the variable regions are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen.In a specific embodiment, the variable region is a human variable region.

[0166] As used herein, the terms "hypervariable region", "HVR", "HV", "complementarity determining region", and "CDR" refer to regions of an antibody variable domain that are hypervariable and / or form structurally defined loops. Generally, an antibody contains six hypervariable regions, three in VH (H1 or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3) and three in VL (L1 or VL CDR1, L2 or VL CDR2, and L3 or VL CDR3). Several descriptions of hypervariable regions are used and are included herein. Kabat CDRs are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia, in contrast, refers to the positions of structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). When numbered using the Kabat numbering rules, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places an insertion at H35A and H35B, and if neither 35A nor 35B is present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering Vol. 2, Chapter 3, Springer Verlag). "Contact" hypervariable regions are based on the analysis of available complex crystal structures. Residues from each of these hypervariable regions are shown below.

[0167] The Universal Numbering System has been developed and widely adopted (ImMunoGeneTics (IMGT®) Information System (Lefranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT® is an integrated information system specialized for human and other vertebrate immunoglobulins (IG), T cell receptors (TR), and major histocompatibility gene complexes (MHC). In this specification, CDRs are referred to both for the amino acid sequence and the position within the light or heavy chain. The "position" of the CDRs within the structure of the immunoglobulin variable domain is conserved across species and exists in a structure called a loop, so that by using a numbering system that aligns the variable domain sequences of the structural features, CDRs and framework residues can be readily identified. This information can be used when grafting and substituting CDR residues derived from a certain immunoglobulin, typically into an acceptor framework derived from a human antibody. A further numbering system (AHon) has been developed by Honegger and Pluckthun, J. Mol. Biol. 309:657-670 (2001). For example, the correspondence between numbering systems, including the Kabat numbering and the IMGT® proprietary numbering system, is well known to those skilled in the art and is also described below (see, for example, Kabat (supra); Chothia and Lesk (supra); Martin (supra); Lefranc et al. (supra)). The various systems known in the art or described herein represent different ways of defining CDRs and are often considered equivalent when they are used to define the same antibody. The exemplary system shown herein is a combination of Kabat and Chothia.

Table 15

[0168] The hypervariable regions may include the following "extended hypervariable regions": 24 - 36 or 24 - 34 (L1), 46 - 56 or 50 - 56 (L2), and 89 - 97 or 89 - 96 (L3) for VL, and 26 - 35 or 26 - 35A (H1), 50 - 65 or 49 - 65 (H2), and 93 - 102, 94 - 102, or 95 - 102 (H3) for VH. The terms "hypervariable region", "HVR", "HV", "complementary determining region", or "CDR" as used herein are used interchangeably.

[0169] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, for example, to introduce the nucleic acid sequence into a host cell. Examples of vectors include expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, and may include selectable sequences or selectable markers for stable integration into the chromosome of the host cell. Further, the vector may include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included confer resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply essential nutrients not present in the medium. Expression control sequences can include constitutive and inducible promoters, transcriptional enhancers, transcriptional terminators, etc., well known in the art. When two or more nucleic acid molecules (e.g., both the antibody heavy chain and light chain or the antibody VH and VL regions) are co-expressed, both nucleic acid molecules can be inserted, for example, into a single expression vector or separate expression vectors. In the case of single vector expression, the coding nucleic acids can be operably linked to one common expression control sequence or to different expression control sequences such as one inducible promoter and one constitutive promoter. 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 blot of mRNA or polymerase chain reaction (PCR) amplification, immunoblotting for the expression of gene products, or other analytical methods suitable for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art understand that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product (e.g., 5T4), and further understand that the expression level can be optimized to obtain sufficient expression using methods well known in the art.

[0170] The terms "5T4-mediated disease", "5T4-mediated disorder", and "5T4-mediated condition" are used interchangeably and refer to any disease, disorder or condition associated with or characterized by 5T4-expressing cells, such as 5T4-expressing tumor cells. Examples of 5T4-mediated diseases include, but are not limited to, cancers that express or overexpress 5T4.

[0171] As used in any embodiment herein, the term "tumor" refers to the growth and proliferation of any tumor cells, as well as all precancerous and cancerous cells and tissues, whether malignant or benign.

[0172] The terms "cancer" and "cancerous" refer to or describe a physiological state in mammals typically characterized by uncontrolled cell growth.

[0173] The term "ADC" refers to an antibody-drug conjugate, which in the context of the present invention refers to a 5T4 antibody conjugated to another moiety comprising a drug as described herein.

[0174] As used herein, "drug" refers to a compound having biological activity, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).

[0175] Examples of drugs include small molecule drugs such as cancer chemotherapeutic agents. For example, if the polypeptide is an antibody (or a fragment thereof) having specificity for tumor cells, the antibody can be modified as described herein to contain modified amino acids, which can then be conjugated to a cancer chemotherapeutic drug. Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinaceous) compounds that reduce the growth of cancer cells and include cytotoxic agents and cell division inhibitors. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds can also be used.

[0176] Suitable cancer chemotherapeutic agents include dolastatin and its active analogs and derivatives, and auristatin and its active analogs and derivatives (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, for example, WO96 / 33212, WO96 / 14856, and US6,323,315. For example, dolastatin 10 or auristatin PE may be included in the 5T4-ADC of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansinoid and its active analogs and derivatives (see, for example, EP1391213 and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), duocarmycin and its active analogs and derivatives (including synthetic analogs, KW-2189 and CB1-TM1); and benzodiazepine and its active analogs and derivatives (e.g., pyrrolobenzodiazepine (PBD)).

[0177] Agents that act to reduce cell proliferation are known in the art and are widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (CYNOTAN™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chloromethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramide, busulfan, dacarbazine, and temozolomide.

[0178] Examples of metabolic antagonists include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U®), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolic acid (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0179] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include Ara-C, paclitaxel (TAXOL®), docetaxel (TAXOTERE®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine, brequinar, alkaloids such as vincristine, vinblastine, vinorelbine, vindesine, etc., podophyllotoxins such as etoposide, teniposide, etc., antibiotics such as anthracyclines, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin, and morpholino derivatives; phenoxazone bicyclic peptides such as dactinomycin, basic glycopeptides such as bleomycin, anthraquinone glycosides such as pirarubicin (mitiromycin), anthraquinones such as mitoxantrone; aziridinopyrrolindiones such as mitomycin; macrocyclic immunosuppressants such as cyclosporine, FK-506 (tacrolimus, Prograf), rapamycin, etc., but are not limited thereto.

[0180] Other antiproliferative cytotoxic agents are navelbine, CPT-11, anastrozole, letrozole, capecitabine, raloxifene, cyclophosphamide, ifosfamide, and droloxifene.

[0181] Microtubule-affecting agents having antiproliferative activity are also suitable for use, including but not limited to allocolchicine (NSC406042), halicondrin B (NSC609395), colchicine (NSC757), colchicine derivatives (e.g., NSC33410), dolastatin 10 (NSC376128), maytansine (NSC153858), lysocine (NSC332598), paclitaxel (TAXOL®), TAXOL® derivatives, docetaxel (TAXOTERE®), thiocolchicine (NSC361792), tritylcysteine, vinblastine sulfate, vincristine sulfate, epothilone A, epothilone B, natural and synthetic epothilones including but not limited to discodermolide, estramustine, and noconazole.

[0182] Suitable hormone modulators and steroids (including synthetic analogs) for use include corticosteroids, such as prednisone, dexamethasone, estrogens and progestins, such as caproxyprogesterone, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, etc., adrenal cortex inhibitors, such as aminoglutethimide; 17α-ethinyl estradiol, diethylstilbestrol, testosterone, fluoxymesterone, drostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (DROGENIL®), toremifene (FARESTON®), and goserelin (ZOLADEX®), etc., but are not limited thereto. Estrogens stimulate proliferation and differentiation. Therefore, compounds that bind to estrogen receptors are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0183] Other suitable chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, etc., ureas such as hydroxyurea, hydrazines such as N-methylhydrazine, epipodophyllotoxins, topoisomerase inhibitors, procarbazine, mitoxantrone, leucovorin, tegafur, etc. Other anti-proliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, deoxyspergualin, azathioprine, leflunomide, mizoribine, azaspiracid (SKF105685), gefitinib (IRESSA (registered trademark), ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline), etc.

[0184] Taxanes are suitable for use. "Taxanes" include paclitaxel, as well as any active taxane derivative or prodrug. "Paclitaxel" (as used herein, analogs, formulations, and derivatives, such as docetaxel, TAXOL®, TAXOTERE® (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel, and 3'N-desbenzoyl-3'Nt-butoxycarbonyl analogs of paclitaxel) can be readily prepared using techniques known to those of skill in the art (see also WO94 / 07882, WO94 / 07881, WO94 / 07880, WO94 / 07876, WO93 / 23555, WO93 / 10076, U.S. Patent Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534, 5,229,529, and EP590,267), or can be obtained from various commercial sources, such as Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia, or T-1912 from Taxus yannanensis). Paclitaxel is understood to refer not only to the commonly available chemical forms of paclitaxel, but also to analogs and derivatives (such as TAXOTERE® docetaxel described herein) and paclitaxel conjugates (such as paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0185] A variety of known derivatives, including both hydrophilic and hydrophobic derivatives, are also included within the scope of the term "taxane". Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. WO99 / 18113, the piperazino and other derivatives described in WO99 / 14209, the taxane derivatives described in WO99 / 09021, WO98 / 22451, and U.S. Patent No. 5,869,680, the 6-thio derivatives described in WO98 / 28288, the sulfenamide derivatives described in U.S. Patent No. 5,821,263, and the taxol derivatives described in U.S. Patent No. 5,415,869. This further includes prodrugs of paclitaxel, including, but not limited to, those described in WO98 / 58927, WO98 / 13059, and U.S. Patent No. 5,824,701.

[0186] Biological response modifiers suitable for use include, but are not limited to: (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) tumor-related 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) inhibitors of angiogenesis.

[0187] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate the symptoms and / or underlying cause, prevent the occurrence of the symptoms and / or its underlying cause, and / or improve or repair damage resulting from or associated with a disease, disorder, or condition. In some embodiments, an effective amount is a therapeutically effective amount.

[0188] As used herein, the term "therapeutically effective amount" refers to an amount of an ADC that is sufficient to reduce and / or ameliorate a given disease, disorder or condition, and / or the severity and / or duration of the symptoms associated therewith. The therapeutically effective amount of an agent that includes a therapeutic agent can be an amount necessary for (i) reducing or ameliorating the progression or abhorrence of a given disease, disorder, or condition, (ii) reducing or ameliorating the recurrence, manifestation or onset of a given disease, disorder or condition, and / or (iii) enhancing or augmenting the therapeutic effect of another therapy (e.g., a therapy other than the administration of an antibody or ADC described herein). The "therapeutically effective amount" of a substance / molecule / agent of the present disclosure (e.g., a 5T4 antibody or ADC) can vary based on several factors such as the individual's pathology, age, gender, and body weight, and the ability of the substance / molecule / agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount where any toxic or adverse effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects. In some embodiments, the term "therapeutically effective amount" refers to an amount of an antibody or other agent (e.g., or drug) that is effective to "treat" a disease, disorder, or condition in a subject or mammal.

[0189] In some embodiments, the drug is a microtubule-affecting agent having anti-proliferative activity, such as a maytansinoid. In some embodiments, the drug is a mitotic inhibitor, such as an auristatin or an active auristatin analog or derivative thereof. In some embodiments, the drug is a DNA alkylating agent.

[0190] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, more specifically in humans.

[0191] "Excipients" include carriers, excipients, preservatives, or stabilizers that are non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations used, and can be included, for example, to affect stability, bulk up the formulation, or impart therapeutic enhancement of the active ingredient in the final dosage form (e.g., enhanced absorption, reduced viscosity, enhanced solubility). "Excipients" are organic or inorganic components, which can be natural or synthetic, that are combined with the active ingredient to facilitate its use, e.g., administration of the active ingredient to a subject. Examples of excipients include buffers such as phosphoric acid, citric acid, and other organic acids, antioxidants such as ascorbic acid, low molecular weight (e.g., less than about 10 amino acid residues) polypeptides, 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™, polyethylene glycol (PEG), and PLURONICS™. The term "excipient" can also refer to diluents, adjuvants (e.g., Freund's adjuvant (complete or incomplete)), excipients, or vehicles with which the therapeutic agent is administered. Such excipients can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Aqueous solutions of sodium chloride as well as aqueous solutions of dextrose and glycerol can also be used as liquid excipients, particularly for injection solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.The composition in any embodiment may also contain trace amounts of wetting or emulsifying agents, or pH buffers. The composition can take forms such as solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral compositions such as formulations can include standard excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable excipients are described in Remington: The Science and Practice of Pharmacy (2020) (Elsevier Science, Amsterdam, Netherlands), and can include a pharmaceutical compound, for example, 5T4-ADC in isolated or purified form, in an effective amount or therapeutically effective amount together with an appropriate amount of excipients, and provide a form for appropriate administration to a subject. The formulation needs to be suitable for the mode of administration.

[0192] 5T4 antibody

[0193] The 5T4-ADC described herein includes a drug and a 5T4 antibody conjugated thereto. In some embodiments, the 5T4 antibody refers to an antibody that specifically binds to 5T4, such as a 5T4 protein, 5T4 polypeptide, 5T4 polypeptide fragment, 5T4 peptide, or 5T4 epitope. In some embodiments, the 5T4 antibody is a human or humanized antibody that binds to 5T4 (e.g., including a human constant region). In some embodiments, the 5T4 antibody can bind to 5T4 expressed on the surface of mammalian (e.g., human) cells including 5T4-expressing tumor cells. In some embodiments, the 5T4 antibody binds to an extracellular 5T4 epitope (e.g., extracellular 5T4 epitope) expressed in cells such as tumor cells. In some embodiments, 5T4 is human 5T4. Exemplary amino acid sequences of human 5T4 are described herein.

[0194] In some embodiments, the 5T4 antibody competes for binding to 5T4 with, for example, any antibody described herein, such as the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 amino acid sequences described in any one of Tables 1-3, 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. Thus, in some embodiments, the 5T4 antibody competes for binding to 5T4 with a reference 5T4 antibody comprising 1, 2, and / or 3 of the VH CDRs and / or 1, 2, and / or 3 of the VL CDRs of (a) the antibody identified as mAbA4, or (b) the antibody identified as mAbA15, or (c) the antibody identified as mAbA17. In some embodiments, the 5T4-ADC comprises an agent conjugated (directly or indirectly) to a 5T4 antibody that competes for binding to the 5T4-ADC with a reference 5T4 antibody comprising 1, 2, and / or 3 of the VH CDRs and 1, 2, and / or 3 of the VL CDRs of (a) the antibody identified as mAbA4, or (b) the antibody identified as mAbA15, or (c) the antibody identified as mAbA17. In some embodiments, the 5T4 antibody competes for binding to 5T4 with a reference 5T4 antibody comprising the VH region and VL region from (a) the antibody identified as mAbA4, or (b) the antibody identified as mAbA15, or (c) the antibody identified as mAbA17. In some embodiments, the 5T4 antibody competes for binding to 5T4 with a reference 5T4 comprising a VH region and competing for binding with a reference 5T4 comprising a VH region that includes (a) a VH region comprising the amino acid sequence of SEQ ID NO: 25 and a VL region comprising the amino acid sequence of SEQ ID NO: 26, or (b) a VH region comprising the amino acid sequence of SEQ ID NO: 44 and a VL region comprising the amino acid sequence of SEQ ID NO: 45, or (c) a VH region comprising the amino acid sequence of SEQ ID NO: 62 and a VL region comprising the amino acid sequence of SEQ ID NO: 63.

[0195] 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, e.g., the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 set forth in any one of Tables 1-3. Thus, in some embodiments, the 5T4 antibody comprises 1, 2, and / or 3 heavy chain CDRs and / or 1, 2, and / or 3 light chain CDRs from (a) the antibody identified as mAbA4, or (b) the antibody identified as mAbA15, or (c) the antibody identified as mAbA17. In some embodiments, the 5T4 antibody comprises 1, 2, and / or 3 heavy chain CDRs and / or 1, 2, and / or 3 light chain CDRs from (a) the antibody identified as mAbA4, or (b) the antibody identified as mAbA15, or (c) the antibody identified as mAbA17.

[0196] In some embodiments, the 5T4 antibody comprises a VH region comprising one or more (such as one, two, or three, etc.) of VH CDR1, VH CDR2, and VH CDR3 described herein, such as any one of Tables 1-3, and / or a VL region comprising one or more (such as one, two, or three, etc.) of VL CDR1, VL CDR2, and VL CDR3 described herein, such as any one of Tables 1-3. In some embodiments, the 5T4 antibody is bispecific and comprises a first binding region comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs described in any one of Tables 1-3, and a second region comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs derived from a binder that binds to a second target antigen other than 5T4. In some embodiments, the 5T4 antibody is bispecific and comprises a first binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs described in any one of Tables 1-3, and a second binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs derived from a binder that binds to a second 5T4 epitope.

[0197] The antibody identified as mAbA4 comprises the VH amino acid sequence of SEQ ID NO: 25 and the VL amino acid sequence of SEQ ID NO: 26.

[0198] The antibody identified as mAbA15 comprises the VH amino acid sequence of SEQ ID NO: 44 and the VL amino acid sequence of SEQ ID NO: 45.

[0199] The antibody identified as mAbA17 comprises the VH amino acid sequence of SEQ ID NO: 62 and the VL amino acid sequence of SEQ ID NO: 63.

Table 1

Table 2

Table 3

[0200] In some embodiments, the 5T4 antibody comprises a VH region. In some embodiments, the 5T4 antibody comprises a VL region. In some embodiments, the 5T4 antibody comprises a combination of (i) a VH region and (ii) a VL region.

[0201] In some embodiments, the 5T4 antibody comprises a heavy chain having a combination of (i) a VH as described herein as set forth in any one of Tables 1-3 and (ii) one or more heavy chain constant regions (e.g., CH1, hinge, CH2, and CH3). An exemplary IgG heavy chain can comprise any VH amino acid sequence described herein as well as the following CH1, hinge, CH2, and CH3: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 69). Another exemplary IgG heavy chain can comprise any VH amino acid sequence described herein as well as the following CH1, hinge, CH2, and CH3: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 70).

[0202] In some embodiments, the 5T4 antibody comprises a light chain having a combination of (i) a VL region described herein as set forth in any one of Tables 1-3, and (ii) a light chain constant region (CL). Exemplary light chains (e.g., light chains that pair with IgG heavy chains) can comprise any VL amino acid sequence described herein and the following CL amino acid sequence: RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 71).

[0203] In some embodiments, the 5T4 antibody comprises a heavy chain having a combination of (a) (i) a VH described herein as set forth in any one of Tables 1-3, and (ii) one or more heavy chain constant regions (e.g., CH1, hinge, CH2, and CH3), and (b) (i) a VL described herein as set forth in any one of Tables 1-3, and (ii) a light chain having a light chain constant region (CL1) of the IgG format.

[0204] In some embodiments, an antibody that binds to 5T4 has a heavy chain having the following amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYYIHWVRQAPGKGLEWVAFISPGGSYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREYYSMGVLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLCTPSRGS (SEQ ID NO: 90), and a light chain having the following amino acid sequence may include DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQAYESPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 91).

[0205] In some embodiments, the antibody that binds to 5T4 may include a heavy chain modified to include formylglycine residues, and thus, the following amino acid sequence It may contain EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYYIHWVRQAPGKGLEWVAFISPGGSYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREYYSMGVLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLCTPSRGS (SEQ ID NO: 92), where f(Gly) is a formylglycine residue.

[0206] In some embodiments, the 5T4-ADC has a heavy chain having the following amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYYIHWVRQAPGKGLEWVAFISPGGSYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREYYSMGVLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLnTPSRGS (SEQ ID NO: 93), wherein n is an amino acid whose side chain is replaced by a linker drug (e.g., a linker drug of formula (XIV), (XV), or (XVI)) as disclosed herein.

[0207] In some embodiments, the antibody that binds to 5T4 has a heavy chain having the following amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYIHWVRQAPGKGLEWVAYITSTGSYTEYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYDRSTRYSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLCTPSRGS (SEQ ID NO: 94), and a light chain comprising the following amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQAYATPVTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95).

[0208] In some embodiments, the antibody that binds to 5T4 can comprise a heavy chain modified to contain a formylglycine residue, and thus, the following amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYIHWVRQAPGKGLEWVAYITSTGSYTEYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYDRSTRYSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSL(fGly)TPSRGS(SEQ ID NO: 96), wherein f(Gly) is a formylglycine residue.

[0209] In some embodiments, the 5T4-ADC has a heavy chain having the following amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYYIHWVRQAPGKGLEWVAFISPGGSYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREYYSMGVLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLnTPSRGS (SEQ ID NO: 97), wherein n is an amino acid whose side chain is replaced with a linker drug (e.g., a linker drug of formula (XIV), (XV), or (XVI)) as disclosed herein.

[0210] In some embodiments, the antibody that binds to 5T4 has a heavy chain having the following amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWIHWVRQAPGKGLEWVAAIFPYSGSTLYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSPSRSILPSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLCTPSRGS (SEQ ID NO: 98), and a light chain having the following amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGDGSPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 99).

[0211] In some embodiments, the antibody that binds to 5T4 may comprise a heavy chain modified to contain a formylglycine residue, and thus, the following amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWIHWVRQAPGKGLEWVAAIFPYSGSTLYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSPSRSILPSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSL(fGly)TPSRGS (SEQ ID NO: 100), where f(Gly) is a formylglycine residue.

[0212] In some embodiments, the 5T4-ADC has a heavy chain having the following amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYYIHWVRQAPGKGLEWVAFISPGGSYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREYYSMGVLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLnTPSRGS (SEQ ID NO: 101), where n is an amino acid whose side chain is replaced with a linker-drug (e.g., a linker-drug of formula (XIV), (XV), or (XVI)) as disclosed herein.

[0213] In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs), e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 as set forth in Table 1. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs), e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 as set forth in Table 2. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs), e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 as set forth in Table 3.

[0214] 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, VH CDR3 as set forth 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 set forth in Table 1. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., 1, 2, or 3 VH CDRs), such as VH CDR1, VH CDR2, VH CDR3 as set forth in Table 1, and one or more CDRs (e.g., 1, 2, or 3 VL CDRs), such as VL CDR1, VL CDR2, and / or VL CDR3 as set forth in Table 1.

[0215] In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., 1, 2, or 3 VH CDRs), such as VH CDR1, VH CDR2, VH CDR3 as set forth in Table 2. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., 1, 2, or 3 VL CDRs), such as VL CDR1, VL CDR2, and / or VL CDR3 as set forth in Table 2. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., 1, 2, or 3 VH CDRs), such as VH CDR1, VH CDR2, VH CDR3 as set forth in Table 2, and one or more CDRs (e.g., 1, 2, or 3 VL CDRs), such as VL CDR1, VL CDR2, and / or VL CDR3 as set forth in Table 2.

[0216] In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., 1, 2, or 3 VH CDRs), such as VH CDR1, VH CDR2, VH CDR3 as set forth in Table 3. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., 1, 2, or 3 VL CDRs), such as VL CDR1, VL CDR2, and / or VL CDR3 as set forth in Table 3. In some embodiments, the 5T4 antibody comprises one or more CDRs (e.g., 1, 2, or 3 VH CDRs), such as VH CDR1, VH CDR2, VH CDR3 and one or more CDRs (e.g., 1, 2, or 3 VL CDRs), such as VL CDR1, VL CDR2, and / or VL CDR3 as set forth in Table 3.

[0217] In some embodiments, the 5T4 antibody comprises one or more (e.g., one, two, or three) VH CDRs as described herein in Tables 1-3 and the like. In other embodiments, the 5T4 antibody comprises one or more (e.g., one, two, or three) VL CDRs as described herein in Tables 1-3 and the like. In some embodiments, the 5T4 antibody comprises one or more (e.g., one, two, or three) VH CDRs as described herein in Tables 1-3 and the like and one or more (e.g., one, two, or three) VL CDRs as described herein in Tables 1-3 and the like. Thus, in some embodiments, the 5T4 antibody comprises a VH CDR1 comprising an amino acid sequence comprising any one of SEQ ID NOs: 1, 7, 12, 13, 18, 27, 31, 34, 35, 39, 46, 50, 53, and 57. In some embodiments, the 5T4 antibody comprises a VH CDR2 comprising an amino acid sequence comprising any one of SEQ ID NOs: 2, 8, 14, 19, 24, 28, 32, 36, 40, 43, 47, 51, 54, 58, and 61. In some embodiments, the 5T4 antibody comprises a VH CDR3 comprising an amino acid sequence comprising any one of SEQ ID NOs: 3, 9, 15, 20, 29, 33, 37, 41, 48, 52, 55, and 59. In some embodiments, the 5T4 antibody comprises a VH CDR1 and / or a VH CDR2 and / or a VH CDR3 independently selected from the VH CDR1, VH CDR2, VH CDR3 described in any one of Tables 1-3. In some embodiments, the 5T4 antibody comprises a VL CDR1 comprising an amino acid sequence comprising any one of SEQ ID NOs: 4, 10, 16, and 21. In some embodiments, the 5T4 antibody comprises a VL CDR2 comprising an amino acid sequence comprising any one of SEQ ID NOs: 5, 11, and 22. In some embodiments, the 5T4 antibody comprises a VL CDR3 comprising an amino acid sequence comprising any one of SEQ ID NOs: 6, 17, 23, 30, 38, 42, 49, 56, and 60. In some embodiments, the 5T4 antibody comprises a VL CDR1 and / or a VL CDR2 and / or a VL CDR3 independently selected from the VL CDR1, VL CDR2, VL CDR3 described in any one of Tables 1-3.

[0218] In some embodiments, the 5T4 antibody comprises a heavy chain variable region (VH) comprising: (1) a VH CDR1 comprising any one amino acid sequence of (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 one amino acid sequence of (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 one amino acid sequence of (i) SEQ 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 a VL region comprising: (1) a VL CDR1 comprising any one amino acid sequence of (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 any one amino acid sequence of (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11, and (iii) SEQ ID NO: 22; and (3) a VL CDR3 comprising any one amino acid sequence of (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.

[0219] In some embodiments, the 5T4 antibody comprises a VH region comprising: (1) a VH CDR1 comprising any one amino acid sequence of (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 one amino acid sequence of (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 one amino acid sequence of (i) SEQ 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.

[0220] In some embodiments, the 5T4 antibody comprises a VL region comprising: (1) a VL CDR1 comprising any one amino acid sequence of (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 any one amino acid sequence of (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11, and (iii) SEQ ID NO: 22; and (3) a VL CDR3 comprising any one amino acid sequence of (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.

[0221] In some embodiments, the 5T4 antibody comprises an antibody that binds to 5T4, and the antibody comprises a VH region comprising (a) a VH CDR1 comprising any one amino acid sequence of (1)(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 one amino acid sequence of (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 one amino acid sequence of (i) SEQ 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 a VL region comprising (1) a VL CDR1 comprising any one amino acid sequence of (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 any one amino acid sequence of (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11, (iii) SEQ ID NO: 22, and (3) a VL CDR3 comprising any one amino acid sequence of (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.

[0222] In some embodiments, the 5T4 antibody comprises an antibody that binds to 5T4, and the antibody comprises a VH region comprising (1) a VH CDR1 comprising any one amino acid sequence of (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 any one amino acid sequence of (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 one amino acid sequence of (i) SEQ 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.

[0223] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VL region comprising (1) a VL CDR1 comprising any one amino acid sequence of (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 any one amino acid sequence of (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11, and (iii) SEQ ID NO: 22, and (3) a VL CDR3 comprising any one amino acid sequence of (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.

[0224] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises all three heavy chain CDRs and / or all three light chain CDRs from (a) an antibody identified as 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 identified as 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 identified as 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 that comprises all three heavy chain CDRs and / or all three light chain CDRs from the antibody identified as mAbA4. In some embodiments, the 5T4-ADC comprises a 5T4 antibody that comprises all three heavy chain CDRs and / or all three light chain CDRs from the antibody identified as mAbA15. In some embodiments, the 5T4-ADC comprises a 5T4 antibody that comprises all three heavy chain CDRs and / or all three light chain CDRs from the antibody identified as mAbA17.

[0225] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences described herein, such as in Tables 1-3, and / or a VL region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences described herein, such as in Tables 1-3. In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences described herein, such as in Tables 1-3. In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VL region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences described herein, such as in Tables 1-3.

[0226] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 4, 10, 16, and 21, and (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 5, 11, and 22.

[0227] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5.

[0228] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11.

[0229] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11.

[0230] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22.

[0231] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, and (b) a VL region comprising (1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 4, 10, 16, and 21, and (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 5, 11, and 22.

[0232] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5.

[0233] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, and (b) a VL region comprising (1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 4, 10, 16, and 21, and (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 11, and 22.

[0234] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11.

[0235] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and (b) a VL region comprising (1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 4, 10, 16, and 21, and (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 11, and 22.

[0236] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11.

[0237] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, and (b) a VL region comprising (1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 4, 10, 16, and 21, and (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 5, 11, and 22.

[0238] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22.

[0239] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, and (b) a VL region comprising (1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 4, 10, 16, and 21, and (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 5, 11, and 22.

[0240] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5.

[0241] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, and (b) a VL region comprising (1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 4, 10, 16, and 21, and (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 5, 11, and 22.

[0242] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11.

[0243] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 34, 4, 10, 16, and 21, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 10, 16, and 21, and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 5, 11, and 22.

[0244] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5.

[0245] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 35, 4, 10, 16, and 21, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 10, 16, and 21, and (2) a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 5, 11, and 22.

[0246] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 35, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11.

[0247] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 39, and a VL region comprising (a) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 4, 10, 16, and 21, and (b) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 5, 11, and 22.

[0248] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 39, and a VL region comprising (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, and (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22.

[0249] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, and a VL region comprising (a) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 4, 10, 16, and 21, and (b) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 5, 11, and 22.

[0250] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, and a VL region comprising (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, and (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5.

[0251] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 50, and a VL region comprising (a) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 4, 10, 16, and 21, and (b) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 5, 11, and 22.

[0252] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 50, and a VL region comprising (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, and (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11.

[0253] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 53, and a VL region comprising (a) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 4, 10, 16, and 21, and (b) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 5, 11, and 22.

[0254] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 53, and a VL region comprising (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, and (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5.

[0255] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 57, and a VL region comprising (a) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 4, 10, 16, and 21, and (b) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 5, 11, and 22.

[0256] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region that comprises (a)(1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 57, and a VL region that comprises (b)(1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, and (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22.

[0257] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region that comprises (a)(1) a VH CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 1, 7, 12, 13, and 18, (2) a VH CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 2, 8, 14, 19, and 24, and (3) a VH CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 3, 9, 15, and 20, and a VL region that comprises (b)(1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 4, 10, 16, and 21, (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 11, and 22, and (3) a VL CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 6, 17, and 23.

[0258] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region that comprises (a)(1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL region that comprises (b)(1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0259] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0260] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0261] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 15, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0262] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 23.

[0263] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0264] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising (1) a VH CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 27, 31, 34, 35, and 39, (2) a VH CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 28, 32, 36, 40, and 43, and (3) a VH CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 29, 33, 37, and 41, and (b) a VL region comprising (1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 4, 10, 16, and 21, (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 11, and 22, and (3) a VL CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 30, 38, and 42.

[0265] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30.

[0266] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30.

[0267] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30.

[0268] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 35, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 37, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 38.

[0269] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 39, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 41, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 42.

[0270] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30.

[0271] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising: (a) a VH region comprising (1) a VH CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 13, 46, 50, 53, and 57, (2) a VH CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 47, 51, 54, 58, and 61, and (3) a VH CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 48, 52, 55, and 59; and (b) a VL region comprising (1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 4, 10, 16, and 21, (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 11, and 22, and (3) a VL CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 49, 56, and 60.

[0272] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising: (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48; and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 49.

[0273] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising: (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 50, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 51, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 52; and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 49.

[0274] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 53, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 49.

[0275] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 54, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 55, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 56.

[0276] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, the antibody comprising (a) a VH region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 57, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 59, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 60.

[0277] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region comprising (a) (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, and (b) a VL region comprising (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 49.

[0278] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region comprising a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 31, 34, 35, and 39, and a VL region comprising (1) a VL CDR1 comprising SEQ ID NOs: 4, 10, 16, and 21, and (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 11, and 22.

[0279] In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4, and the antibody comprises a VH region and / or a VL region described herein, and the VH and / or VL comprises a human framework sequence. In some embodiments, the VH region and / or VL region comprises a framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequence, e.g., a human FR1, human FR2, human FR3, and / or human FR4.

[0280] In some embodiments, the CDRs of the 5T4 antibody can be determined by 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, Fifth Edition, U.S. Department of Health and Human Services, NIH Pulication No. 91-3242).

[0281] In some embodiments, the CDRs of the 5T4 antibody can be determined by the Chothia system, which is referred to herein as the "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).

[0282] In some embodiments, the CDRs of the 5T4 antibody can be determined by the ImMunoGeneTics (IMGT®) system as described in, for example, Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212 ("IMGT® CDRs").

[0283] In some embodiments, the CDRs of the 5T4 antibody can be determined by the AbM system, which is referred to herein as the "AbM CDR" and is 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 Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0284] In some embodiments, the CDRs of the 5T4 antibody can be determined by the Contact system, which is referred to herein as "Contact CDR" (see, for example, MacCallum RM et al., 1996, J Mol Biol 5:732-745). The Contact CDR is based on the analysis of available complex crystal structures.

[0285] In some embodiments, the position of one or more (e.g., 1, 2, or 3) CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the 5T4 antibody can differ by 1, 2, 3, 4, 5, or 6 amino acid positions, provided that binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the positions defining any of the CDRs in Tables 1, 2, or 3 can differ from the current CDR positions by shifting the N-terminal and / or C-terminal boundaries of the CDR by 1, 2, 3, 4, 5, or 6 amino acids, provided that binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the length of one or more (e.g., 1, 2, or 3) CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) described herein can differ by 1, 2, 3, 4, 5, or more amino acids (e.g., be shorter or longer), provided that binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, CDR1, CDR2, and / or CDR3 of VH and / or VL can be 1, 2, 3, 4, 5, or more amino acids shorter than one or more of the CDRs set forth in SEQ ID NOs: 1-24, 27-43, or 46-61, provided that binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).In other embodiments, the CDR1, CDR2, and / or CDR3 of VH and / or VL may be 1, 2, 3, 4, 5, or more amino acids longer, provided that binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the amino acid termini of the CDR1, CDR2, or CDR3 of VH and / or VL may be extended by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs set forth in SEQ ID NOs: 1-24, 27-43, or 46-61, provided that binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the carboxy termini of the CDR1, CDR2, or CDR3 of VH and / or VL may be extended by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs set forth in SEQ ID NOs: 1-24, 27-43, or 46-61, provided that binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the amino acid termini of the CDR1, CDR2, or CDR3 of VH and / or VL may be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs set forth in SEQ ID NOs: 1-24, 27-43, or 46-61, provided that binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).In some embodiments, the carboxy terminus of CDR1, CDR2, or CDR3 of VH and / or VL can be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs set forth in SEQ ID NOs: 1-24, 27-43, or 46-61, provided that binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). To confirm whether binding to 5T4 (e.g., human 5T4) is maintained, any method known in the art can be used, such as the binding assays and conditions described in the "Examples" section herein. For example, in Example 2 herein, an assay for measuring binding to 5T4 (e.g., human 5T4) is described.

[0286] In some embodiments, the 5T4-ADC can comprise a 5T4 antibody that includes one or more (e.g., 1, 2, 3, 4, or more) conservative sequence modifications. For polypeptides that are 5T4 antibodies, such as human 5T4 antibodies, conservative sequence modifications include conservative amino acid substitutions in which an amino acid residue is replaced with 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. Thus, in some embodiments, predicted non-essential amino acid residues in the 5T4 antibody can be replaced with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids that do not eliminate antigen binding and its nucleotide coding are well known in the art (see, e.g., 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 conservative 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 up to 1, 2, 3, 4, 5, or 6 amino acid substitutions to the CDR, such as those described in any one of Tables 1-3. Thus, for example, each such CDR can include up to 5 conservative amino acid substitutions, such as up to 4 (or fewer) conservative amino acid substitutions, such as up to 3 (or fewer) conservative amino acid substitutions, such as up to 2 (or fewer) conservative amino acid substitutions, or 1 or fewer conservative amino acid substitutions. In some embodiments, the 5T4-ADC comprises a 5T4 antibody having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the CDR of mAbA4, mAbA15, or mAbA17 (see, e.g., Tables 1, 2, or 3).In some embodiments, the 5T4-ADC comprises a 5T4 antibody containing VH and VL that contain CDRs identical to those of mAbA4, mAbA15, or mAbA17 (see, e.g., Tables 1, 2, or 3). In some embodiments, the amino acid sequence modifications do not include any modifications within the specificity-determining residues (SDRs). In some embodiments, the amino acid sequence modifications do not include any modifications within the CDRs (such as CDR1, CDR2, CDR3, or any combination thereof). In further embodiments, the amino acid sequence modifications are within the framework or constant regions.

[0287] In some embodiments, the antibody in the 5T4-ADC comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25, and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26, and binding of the antibody to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0288] In some embodiments, the antibody in the 5T4-ADC comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 44, and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 45, and binding of the antibody to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0289] In some embodiments, the antibody in the 5T4-ADC comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 62, and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 63, and the binding of the antibody to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0290] In addition, provided are nucleic acids encoding a 5T4 binder (e.g., an antibody or antibody fragment) or a fusion polypeptide disclosed herein, nucleic acids complementary thereto, vectors comprising the nucleic acids disclosed herein, and cells or vectors comprising the nucleic acids disclosed herein. In some embodiments, the cell expresses a 5T4 binder. In some embodiments, the cell replicates the nucleic acid or vector. In some embodiments, further provided are materials for generating a 5T4 antibody, including a human 5T4 antibody, useful for the preparation of an ADC. In this regard, a cell (e.g., an isolated cell) can produce an antibody comprising the VH and VL described herein. In some embodiments, the polynucleotide can comprise one or more nucleic acid sequences encoding a 5T4 antibody or antibody fragment. In some embodiments, the polynucleotide is an isolated and / or recombinant polynucleotide. In some embodiments, the isolated polynucleotide comprises a nucleotide sequence encoding an antibody heavy chain variable region (VH) and / or an antibody light chain variable region (VL), and the VH and VL comprise CDRs identical to the CDRs described herein.

[0291] In some embodiments, one or more vectors (e.g., expression vectors) can contain one or more polynucleotides for expressing one or more polynucleotides in a suitable host cell. Such vectors are useful, for example, to amplify polynucleotides within a host cell to create useful amounts and to express binding agents, such as antibodies or antibody fragments, using recombinant techniques.

[0292] In some embodiments, one or more vectors are expression vectors in which one or more polynucleotides are operably linked to one or more polynucleotides containing expression control sequences. Specifically contemplated are self-replicating recombinant expression constructs such as plasmids and viral DNA vectors incorporating one or more polynucleotides encoding antibody sequences that bind to 5T4. Expression control DNA sequences include promoters, enhancers, and operators and are generally selected based on the expression system in which the expression construct is utilized. Promoter and enhancer sequences are generally selected for their ability to increase gene expression, while operator sequences are generally selected for their ability to regulate gene expression. Expression constructs can also include sequences encoding one or more selectable markers that enable the identification of host cells carrying the construct. Expression constructs can 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.

[0293] In some embodiments, the expression control sequence is a promoter / enhancer sequence, such as the cytomegalovirus promoter / enhancer (Lehner et al., J. Clin. Microbiol., 29:2494 - 2502, 1991; Boshhart et al., Cell, 41: 521 - 530, 1985); Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4:151, 1993), type promoter (Korhonen et al., Blood, 86(5):1828 - 1835, 1995), simian virus 40 promoter, DRA (downregulated in adenoma; Alrefai et al., Am. J. J. Physiol. Gastrointest. Liver Physiol., 293: G923 - G934, 2007); MCT1 (monocarboxylate transporter 1, Cuff et al., Am. J. Physiol. Gastrointest. Liver Physiol., G977 - G979, 2005), and Math1 (mouse atonal homolog 1, Shroyer et al., Gastroenterology, 132:2477 - 2478, 2007). For expression in mammalian cells, the promoter is operably linked upstream (e.g., 5') of the polypeptide coding sequence. In some embodiments, the promoter can be an epithelial - specific promoter or an endothelial - specific promoter. The polynucleotide can also optionally include a suitable polyadenylation sequence (e.g., the SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (e.g., 3') of the polypeptide coding sequence.

[0294] In any embodiment, one or more polynucleotides can optionally further 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 can direct the secretion of the antibody polypeptide by a cell expressing the one or more polynucleotides and can be cleaved by the cell from the secreted polypeptide. In any embodiment, the one or more polynucleotides can further optionally comprise one or more sequences that have only the function of facilitating large-scale production of the vector.

[0295] Expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, 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, viral vectors are rendered replication-defective, for example, by deletion or disruption of a selectable gene required for viral replication.

[0296] A cell can contain one or more polynucleotides and / or one or more vectors. For example, in any embodiment, the cell can be transformed or transfected with one or more polynucleotides encoding a 5T4 antibody (e.g., a human 5T4 antibody), or one or more vectors containing one or more polynucleotides encoding a 5T4 antibody (e.g., a human 5T4 antibody). In some embodiments, the cell can express a 5T4 antibody comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the CDRs of, for example, mAbA4, mAbA15, and / or mAbA17 described herein (see, e.g., Tables 1, 2, and / or 3). In some embodiments, the cell can express a 5T4 antibody (e.g., a human 5T4 antibody) comprising a VH region and a VL region comprising mAbA4, mAbA15, and / or mAbA17 (e.g., Tables 1, 2, and / or 3). Cells suitable for generating the 5T4 antibodies described herein include prokaryotic cells, e.g., Escherichia coli (see, e.g., Pluckthun et al., Methods Enzymol., 178:497-515, 1989), and eukaryotic cells, e.g., animal cells (e.g., myeloma cells, Chinese hamster ovary (CHO) cells, or hybridoma cells), yeast (e.g., Saccharomyces cerevisiae), or plant cells (e.g., tobacco, corn, soybean, or rice cells). The use of mammalian host cells can provide translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) that may be desirable to confer optimal biological activity on the recombinant expression product. Similarly, in any embodiment, the polypeptide (e.g., a 5T4 antibody including a human 5T4 antibody) may be glycosylated or non-glycosylated and / or may be covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0297] Methods for introducing DNA or RNA into host cells are well known and include, but are not limited to, transformation, transduction, electroporation, nuclear injection, and fusion with carriers such as liposomes, micelles, shadow cells, and protoplasts. Such host cells are useful for amplifying polynucleotides and expressing polypeptides encoded by the polynucleotides. In this regard, a process for generating a 5T4 antibody can include introducing RNA or DNA encoding the 5T4 antibody into a host cell as described herein, culturing the host cell, and isolating the thus-generated 5T4 antibody.

[0298] A variety of methods for generating antibodies from polynucleotides are generally well known. For example, basic molecular biology procedures are described by 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). Additionally, numerous publications describe techniques suitable for the manipulation of DNA, the construction of expression vectors, and the preparation of antibodies by transformation and culture of appropriate cells (e.g., Mountain and Adair, Chapter 1 in Biotechnology and Genetic Engineering Reviews, Tombs ed., Intercept, Andov, UK, 1992), and Current Protocols in Molecular Biology, Ausubel ed., Wiley Interscience, New York, 1999).

[0299] 5T4-ADC Antibodies that bind to 5T4 and drugs can be directly or indirectly linked to each other via a pyridazine-pyrrolocoupling moiety to form the 5T4-ADCs described herein. In some embodiments, the 5T4-ADC can be represented by formula (IV):

Chemical formula

[0300] Formula (IV) has been previously described in WO2015 / 081282, which is hereby incorporated by reference in its entirety.

[0301] In some embodiments, m is 0 or 1, and R 2 and R 3 are each alkyl. For example, R 2 and R 3 can each independently be C 1 ~C 10 alkyl (e.g., C 1 ~C 6 alkyl). In some embodiments, R 2 and R 3 are each independently methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, or t-butyl. In some embodiments, at least one of R 2 and R 3 is methyl. In some embodiments, both R 2 and R 3 are methyl. In some embodiments, X 1 , X 2, X 3 , and X 4 each is, independently, C or N. In some embodiments, X 1 is C. In some embodiments, X 1 is N. In some embodiments, X 2 is C or N. In some embodiments, X 2 is C. In some embodiments, X 3 is C or N. In some embodiments, X 3 is C. In some embodiments, X 4 is C or N. In some embodiments, X 4 is C. X 1 , X 2 , X 3 , and X 4 various combinations are possible. For example, in some embodiments, X 1 , X 2 , X 3 , and X 4 each is C. In other embodiments, three of X 1 , X 2 , X 3 , and X 4 are C and one of X 1 , X 2 , X 3 , and X 4 is N. In some embodiments, Y 1 , Y 2 , Y 3 , and Y 4 each is hydrogen.

[0302] In the context of the 5T4-ADC of formula (IV), the linker-drug of formula (IV) can be represented by the following formula (XIV):

Chemical formula

Chemical formula

[0303] In some embodiments, the 5T4-ADC can be represented by formula (V):

Chemical formula

[0304] Formula (V) has been previously described in WO2015 / 081282, which is hereby incorporated by reference in its entirety.

[0305] In some embodiments, R 2 and R 3 are each alkyl. For example, R 2 and R 3 can each independently be C 1 ~C 10 alkyl (e.g., C 1 ~C 6 alkyl). In some embodiments, R 2 and R 3 are each independently methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, or t-butyl. In some embodiments, at least one of R 2 and R 3 is methyl. In some embodiments, both R 2 and R 3 are methyl. In some embodiments, Y 1 , Y 2 , Y 3, and Y 4 Each of them is hydrogen.

[0306] In the context of the 5T4-ADC of formula (V), the linker-drug of formula (V) is of the following formula (XV): [Chemical formula] It may be represented by, wherein [Chemical formula] represents a binding point to an antibody that binds to 5T4 (for example, residue n of SEQ ID NO: 93, 97, or 101). All other variable elements of the linker-drug of formula (XV) may be as defined herein, such as with respect to formula (V), and / or as described herein.

[0307] In some embodiments, the 5T4-ADC may be represented by formula (VI): [Chemical formula] wherein Ab is an antibody that binds to 5T4, r is an integer from 1 to 10, X 1 is C or N, L is a linker as described herein, and W 1 is a drug.

[0308] Formula (IV) has been previously described in WO2015 / 081282, which is hereby incorporated by reference in its entirety.

[0309] In the context of the 5T4-ADC of formula (VI), the linker-drug of formula (VI) may be represented by the following formula (XVI): [Chemical formula] wherein [Chemical formula] represents the binding site to an antibody that binds to 5T4 (e.g., residue n of SEQ ID NO: 93, 97, or 101). All other variable elements of the linker-drug of formula (XVI) can be as defined herein, such as with respect to formula (VI), and / or as described herein.

[0310] Linker In some embodiments, the 5T4-ADC may include a linker (L) moiety that links a pyridazine-pyrrolocoupling moiety, such as described herein, to the drug. In any embodiment, the linker L may be represented by the following structure: -(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 - wherein a, b, c, d, and e are each independently 0 or 1, and the sum of a, b, c, d, and e is from 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.

[0311] In the linker, L, T 1 , T 2 , T 3 , T 4 , and T 5 are each independently C 1 ~C 12 alkyl, substituted C 1 ~C 12 alkyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) h-, piperidin-4-amine (P4A), meta-aminobenzyl carbamate (MABC) group, meta-aminobenzyloxy (MABO) group, para-aminobenzyloxy (PABO) group, para-aminobenzyl carbamate (PABC) group, para-aminobenzyl (PAB) group, acetal, disulfide, hydrazine, protease-cleavable moiety, glucuronidase-cleavable moiety, beta-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 wherein 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 is independently hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl.

[0312] As used herein, “(EDA) w ” is an ethylenediamine moiety having the following structure:

Chemical formula

Chemical formula

[0313] The piperidin-4-amine moiety may be optionally substituted at one or more positions with any substituent, such as alkyl, substituted alkyl, polyethylene glycol moiety, acyl, substituted acyl, aryl, or substituted aryl. Thus, as used herein, the piperidin-4-amine used herein can be described by the term "P4A-R" corresponding to the following structure: 12 which can be described by the term:

Chemical formula

Chemical formula

[0314] As used herein, (PEG) n is polyethylene glycol having the following structure:

Chemical formula

Chemical formula

[0315] As used herein, "AA" refers to an amino acid described herein, including but not limited to any naturally occurring amino acid, a naturally occurring amino acid analog, an enantiomer of a naturally occurring amino acid, and a non-naturally occurring amino acid. (AA) p in which each amino acid residue can be independently selected from those described herein, and p can 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, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0316] As used herein, the MABC group can be described by the term "MABC-R 16 " and by the following structure:

Chemical formula

[0317] As used herein, the MABO group can be described by the term "MABO-R 16 " and by the following structure:

Chemical formula

[0318] As used herein, the PABC group can be described by the term "PABC-R 16 " and by the following structure:

Chemical formula

[0319] As used herein, the PABO group can be described by the term "PABO-R 16 " and by the following structure: [Chemistry]

[0320] As used herein, the para-aminobenzyl (PAB) group can be described by the term "PAB-R" 16 and by the following structure: [Chemistry]

[0321] MABC-R 16 , MABO-R 16 , PABC-R 16 , PABO-R 16 , and PAB-R 16 wherein each [Chemistry] represents a point of attachment in any direction to an adjacent chemical moiety, and R 16 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, heterocyclyl, or substituted heterocyclyl.

[0322] In some embodiments, one or more of T 1 , T 2 , T 3 , T 4 , and T 5 are C 1 ~C 6 alkyl. In some embodiments, T 1 is independently C 1 ~C 6 alkyl. In some embodiments, T 1is ethyl. In some embodiments, T 1 and T 3 each is C 1 ~C 6 alkyl. In some embodiments, T 1 and T 3 each is ethyl.

[0323] In some embodiments, one or more of T 1 , T 2 , T 3 , T 4 , and T 5 is P4A-R 12 . In some embodiments, each R 12 is independently a polyethylene glycol moiety. In some embodiments, each R 12 is independently a carboxylic acid-modified polyethylene glycol. In some embodiments, T 2 is P4A-R 12 , wherein R 12 is a carboxylic acid-modified polyethylene glycol.

[0324] In some embodiments, one or more of T 1 , T 2 , T 3 , T 4 , and T 5 is (PEG) n . In some embodiments, n is 2. In some embodiments, T 3 is (PEG) n , wherein n is 2.

[0325] In some embodiments, one or more of T 1 , T 2 , T 3 , T 4 , and T 5 is (AA) p . In some embodiments, p is 1. In some embodiments, T 2 is (AA) p , wherein p is 1. In some embodiments, (AA) pThe AA is an amino acid having a polar uncharged side chain (e.g., serine, threonine, asparagine, or glutamine). In some embodiments, (AA) p The AA is glutamine.

[0326] In some embodiments, T 1 , T 2 , T 3 , T 4 , and T 5 One or more of are (AA) p -(P4BC-R 16 )-(AA) p In some embodiments where is, R 16 is hydrogen. In some embodiments, p is 0. In some embodiments, R 16 is hydrogen and p is 0. Thus, in some embodiments, T 1 , T 2 , T 3 , T 4 , and T 5 One or more of are AA-PABCs described by the following structure:

Chemical formula

Chemical formula

[0327] In some embodiments, the p of AA-PABC is 2. In some embodiments, T 4 is AA-PABC, where p is 2. In some embodiments, the two amino acids of (AA) 2 are valine and citrulline.

[0328] The V of linker L 1 , V 2 , V 3 , V 4 , and V 5is, independently of each other, a covalent bond, amino, carbonyl, amide, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, or thiophosphoridate. In some embodiments, V 1 V 2 V 3 V 4 and V 5 are, independently of each other, a covalent bond, -C(=O)-, -NR 11 -, -C(=O)NR 11 -, NR 11 C(=O)-, -C(=O)O-, -OC(=O)-, -O-, -S-, -S(=O)-, -SO 2 -, -SO 2 NR 11 -, -NR 11 SO 2- and -P(=O)OH-, wherein R 11 is hydrogen, alkyl, substituted alkyl, PEG, aryl, or substituted aryl. In some embodiments, V 1 V 2 and V 3 are independently C(=O) or -NR 11 -, and R 11 are independently hydrogen or C 1 ~C 6 alkyl.

[0329] In some embodiments, the linker L can be represented by the following structure: -(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 -, wherein a, b, c, d, and e are each independently 0 or 1, and the sum of a, b, c, d, and e is from 1 to 5. T 1 is C 1 to C 6 alkyl. T 2 , T 3 , T 4 , and T 5 are each independently (PEG) n , C 1 to C 6 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 ), or (AA) p -(PABC-R 16 ). V 1 , V 2 , V 3 , V 4 , and V 5 are each independently a covalent bond, -CO-, or -NR 11 -. Each R 11 is independently hydrogen, alkyl, substituted alkyl, polyethylene glycol, aryl, or substituted aryl. Each R 12 is independently hydrogen, alkyl, substituted alkyl, or a modified or unmodified polyethylene glycol moiety. R 16 is hydrogen. n is an integer from 1 to 30. p is an integer from 1 to 20.

[0330] In some embodiments, R 12 is (PEG) n . In some embodiments, R 12is (PEG) terminated with a carboxylic acid n In some embodiments, R 11 is hydrogen or C 1 to C 12 alkyl. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 1 at one position within the linker and p is 2 at another position within the linker. In some embodiments, n is an integer from 1 to 5. In some embodiments, n is 2.

[0331] Non-cleavable linker

[0332] In some embodiments, the linker L can be represented by the following structure: -(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 - wherein a, b, c, d, and e are each independently 0 or 1, and the sum of a, b, c, d, and e is from 1 to 5, T 1 is C 1 to C 6 and T 2 and T 3 are each independently C 1 to C 6 alkyl or P4A-R 12 and T 4 and T 5 are each independently (PEG)n, C 1 to C 6 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 )、 or (AA) p -(PABC-R 16 )、 V 1 、V 2 、V 3 、V 4 、 and V 5 are each independently a covalent bond, -CO-, or -NR 11 -, and each R 11 is independently hydrogen or C 1 ~C 6 alkyl, and each R 12 is independently a modified or unmodified polyethylene glycol moiety.

[0333] In some embodiments, each of T 1 and T 3 is independently C 1 ~C 6 alkyl, for example, independently methyl, ethyl, or propyl. In some embodiments, each of T 1 and T 3 is ethyl. In some embodiments, T 2 is P4A-R 12 wherein R 12 is a modified or unmodified polyethylene glycol moiety. In some embodiments, d and e are 0.

[0334] In some embodiments, the linker L can be represented by the following structure: -(C 1 ~C 6 alkyl)-(V 1 )-(P4A-R 12 )-(V 2 )-(C 1 ~C6 -(alkyl)-(V 3 )- and wherein V 1 , V 2 , V 3 is each independently -CO- or -NR 11 and each R 11 is independently hydrogen or C 1 ~C 6 alkyl, and each R 12 is independently a carboxylic acid-modified polyethylene glycol moiety.

[0335] In some embodiments, the linker L is linker (L-1):

Chemical formula

Chemical formula

[0336] Thus, in some embodiments, the 5T4-ADC can be represented by formula (VI-1):

Chemical formula

[0337] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0338] In some embodiments, the 5T4-ADC can be represented by formula (VI-1), wherein X 1is C. Thus, in some embodiments, the 5T4-ADC can be represented by formula (VIa-1): [Chemical formula] wherein Ab is an antibody that binds to 5T4, r is an integer from 1 to 10, W 1 is a drug. In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0339] In some embodiments, the 5T4-ADC can be represented by formula (VI-1), wherein X 1 is N. Thus, in some embodiments, the 5T4-ADC can be represented by formula (VIb-1): [Chemical formula] wherein Ab is an antibody that binds to 5T4, r is an integer from 1 to 10, W 1 is a drug.

[0340] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0341] Cleavable linker

[0342] In some embodiments, the antibody and the drug are conjugated to each other via a cleavable linker. A cleavable linker is a linker that includes one or more cleavable moieties, and the cleavable moieties are dissociable under some conditions, and thus include one or more bonds that can separate the cleavable linker into two or more separable parts. For example, the cleavable moiety can include one or more covalent bonds, which are dissociable or decomposable under some conditions and can separate the cleavable linker into two or more parts. Thus, the cleavable linker can be included in the 5T4-ADC, and as a result, under certain conditions, the cleavable linker separates or releases the drug from the antibody at the desired target site of action of the drug.

[0343] In some embodiments, the cleavable linker can be an enzymatically cleavable linker. An enzymatically cleavable linker can include any cleavable moiety that can be cleaved via the enzymatic action of an enzyme into two or more parts as described above, and the enzymatically cleavable linker can be, but is not limited to, a peptide, a glycoside, etc. In some cases, the enzyme that cleaves the enzymatically cleavable linker is present at the desired target site of action, such as the desired target site of action of the drug released from the antibody-drug conjugate. In some cases, the enzyme that cleaves the enzymatically cleavable linker is not present in significant amounts in other regions such as whole blood, plasma, or serum. Thus, the cleavage of the enzymatically cleavable moiety can be controlled such that substantial cleavage occurs at the desired site of action, while on the other hand, cleavage does not occur significantly in other regions or before the antibody-drug conjugate reaches the desired site of action.

[0344] In some embodiments, the enzymatically cleavable linker includes a peptide. The peptide can be any peptide that is suitable for use in a cleavable linker and can be cleaved via the enzymatic action of an enzyme. Non-limiting examples of peptides that can be used as enzymatically cleavable moieties include, for example, Val-Ala, Phe-Lys, and the like.

[0345] In some embodiments, the linker L is cleavable and can be represented by the following structure: -(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 - wherein 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, T 1 and T 5 are each C 1 ~C 6 alkyl, T 2 , T 3 , and T 4 are each independently (PEG) n , (AA) p , or (AA) p -PABC-R 16 , V 1 , V 2 , V 3 , V 4 , and V 5 are each independently a covalent bond, -CO-, or -NR 11 -, R 11 is as described herein, R 16 is as described herein, each n is independently an integer from 1 to 30, and each p is independently an integer from 1 to 20.

[0346] In some embodiments, n is an integer from 1 to 5. In some embodiments, n is 2.

[0347] In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 1 at one position within linker L and 2 at another position within linker L.

[0348] In some embodiments, T 2 is an amino acid residue. In some embodiments, T 2 is an amino acid residue having a polar uncharged side chain. In some embodiments, T 2 is a glutamine residue.

[0349] In some embodiments, T 3 is (PEG) n wherein n is an integer from 1 to 30, such as 1 to 5. In some embodiments, n is 2.

[0350] In some embodiments, T 4 is (AA) p -PABC-R 16 wherein p is an integer from 1 to 20, such as 1 to 5, and R 16 is hydrogen. In some embodiments, p is 2. In some embodiments, the amino acid (AA) is valine and citrulline.

[0351] In some embodiments, linker L can be represented by the following structure: -(C 1 ~C 6 alkyl)-V 1 -(AA)-V 2 -(PEG) n -V 3 -((AA) p -(PABC-R 16 ))-V 4 -(C 1 -C 6 alkyl)-V 5 ‐, wherein, R 16 is hydrogen, Each of n and p is, independently, an integer from 1 to 30, from 1 to 20, or from 1 to 5, V 1 , V 2 , V 3 , V 4 , and V 5 is, independently, a covalent bond, -CO-, or -NR 11 -, and each R 11 is independently hydrogen or C 1 ~C 6 alkyl.

[0352] In some embodiments, n and p are both 2.

[0353] In some embodiments, the linker L can be the linker (L-2):

Chemical formula

Chemical formula

[0354] In some embodiments, the 5T4-ADC can be represented by formula (VI-2):

Chemical formula

[0355] In some embodiments, X 1 is N or C. In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0356] In some embodiments, the 5T4-ADC can be represented by formula (VI-2), where X 1 is C. Thus, in some embodiments, the 5T4-ADC can be represented by formula (VIa-2):

Chemical formula

[0357] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0358] In some embodiments, the 5T4-ADC can be represented by formula (VI-2), where X 1 is N. Thus, in some embodiments, the 5T4-ADC can be represented by formula (VIb-2):

Chemical formula

[0359] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0360] Double cleavage linker

[0361] In some embodiments, the cleavable linker comprises two cleavable moieties such as a first cleavable moiety and a second cleavable moiety. The cleavable moieties can be configured such that cleavage of both cleavable moieties is required, for example, at a desired site of action of the drug, to separate or release the drug from the antibody. For example, cleavage of the cleavable linker can be achieved by first cleaving one of the two cleavable moieties and then cleaving the other of the two cleavable moieties. In some embodiments, the cleavable linker comprises a first cleavable moiety and a second cleavable moiety that inhibits cleavage of the first cleavable moiety. "Inhibits cleavage" means that the presence of the uncleaved second cleavable moiety reduces the likelihood of cleavage of the first cleavable moiety, or substantially inhibits cleavage of the first cleavable moiety, and thus substantially reduces its amount, or prevents cleavage of the cleavable linker. In an embodiment, the presence of the uncleaved second cleavable moiety can prevent enzymatic and / or chemical cleavage of the first cleavable moiety. The hindrance of cleavage of the first cleavable moiety by the presence of the second cleavable moiety then substantially reduces its amount or prevents release of the drug from the antibody. For example, premature release of the drug from the antibody can be substantially reduced or prevented until the 5T4-ADC reaches the desired target site of action of the drug or in the vicinity thereof.

[0362] In some embodiments, the second cleavable moiety prevents cleavage of the first cleavable moiety, and cleavage of the cleavable linker can be achieved by first cleaving the second cleavable moiety and then cleaving the first cleavable moiety. Cleavage of the second cleavable moiety reduces or eliminates the hindrance in the cleavage of the first cleavable moiety, thus enabling cleavage of the first cleavable moiety. Cleavage of the first cleavable moiety results in dissociation or separation of the cleavable linker into two or more parts, allowing the release of the drug from the 5T4-ADC, as described herein. In some embodiments, cleavage of the first cleavable moiety does not substantially occur in the presence of an uncleaved second cleavable moiety. The term "substantially" in this context means that in the presence of an uncleaved second cleavable moiety, cleavage of the first cleavable moiety is about 10% or less, for example, about 9% or less, or about 8% or less, or 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, and cleavage of the first cleavable moiety occurs in the presence of an uncleaved second cleavable moiety.

[0363] Stated another way, the second cleavable moiety can protect the first cleavable moiety from cleavage. In embodiments, the presence of the non-cleaved second cleavable moiety protects the first cleavable moiety from cleavage and thus can substantially reduce or prevent premature release of the drug from the 5T4 antibody until the 5T4-ADC reaches the desired site of action or in the vicinity thereof. Thus, cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety) and thus enables the first cleavable moiety to cleave, which results in cleavage of the cleavable linker and, thus, separation or release of the drug from the antibody at the desired target site of action of the drug, as described herein. In some embodiments, cleavage of the second cleavable moiety exposes the first cleavable moiety to subsequent cleavage, but cleavage of the second cleavable moiety does not, in and of itself, result in cleavage of the cleavable linker (e.g., cleavage of the first cleavable moiety is still required to cleave the cleavable linker).

[0364] Examples of cleavable moieties suitable for use in cleavable linkers include chemically cleavable moieties and enzymatically cleavable moieties. For example, the first cleavable moiety can be a chemically cleavable moiety and the second cleavable moiety can be a chemically cleavable moiety, which can be the same as or different from the first cleavable moiety. In other embodiments, the first cleavable moiety can be an enzymatically cleavable moiety and the second cleavable moiety can be a chemically cleavable moiety. In yet other embodiments, the first cleavable moiety can be a chemically cleavable moiety and the second cleavable moiety can be an enzymatically cleavable moiety. In still further embodiments, the first cleavable moiety can be an enzymatically cleavable moiety and the second cleavable moiety can be the same as or different from the first cleavable moiety and can be an enzymatically cleavable moiety.

[0365] Chemically cleavable moieties include cleavable moieties that can cleave in the presence of certain chemical conditions. In some embodiments, the chemically cleavable moiety includes one or more linkages that 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 can cleave in the presence of chemical conditions such as acidic or alkaline conditions that can lead to hydrolysis of the chemically cleavable moiety. In some embodiments, the chemical conditions under which the chemically cleavable moiety cleaves can be present at a desired target site of action, such as the target site of the desired action of the drug released from the 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. Thus, cleavage of the enzymatically cleavable moiety can be controlled such that substantial cleavage occurs at the desired site of action, while on the other hand, cleavage does not occur significantly in other regions or before the 5T4-ADC reaches the desired site of action.

[0366] In some embodiments, the cleavable moiety can be an enzymatically cleavable moiety. An enzymatically cleavable moiety is a cleavable moiety that can be separated into two or more moieties via the enzymatic action of an enzyme, as described herein. The enzymatically cleavable moiety can be any cleavable moiety that can cleave via the enzymatic action of an enzyme, such as, but not limited to, a peptide, a glycoside, etc. In some embodiments, the enzyme that cleaves the enzymatically cleavable moiety is present at the desired target site of action, such as the target site of the desired action of the drug released from the 5T4-ADC. In some embodiments, the enzyme that cleaves the enzymatically cleavable moiety is not present in significant amounts in other regions such as whole blood, plasma, or serum. Thus, cleavage of the enzymatically cleavable moiety can be controlled such that substantial cleavage occurs at the desired site of action, while on the other hand, cleavage does not occur significantly in other regions or before the 5T4-ADC reaches the desired site of action, such as a cancer cell.

[0367] In some embodiments, the linker L can be linker (L-3): [Chemical formula] In the formula, [Chemical formula] represents the bonding point to the pyridazine - pyrrolocoupling moiety (for example, any of Formula IV, V, and VI) described in this specification, * represents the bonding point to W 1 and each R 5 is 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 is independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl, R 7 is a cleavable moiety, k is an integer from 1 to 10, L 1 is - (T 1 - V 1 ) a - (T 2 - V 2 ) b - (T 3 - V 3 ) c - (T 4 - V 4 ) d and includes -, L 2 is - (T 5 - V 5 ) e - (T 6 - V 6 ) f - (T 7 - V 7 ) g - (T 8 - V8 ) h- comprising, T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , and T 8 are each independently a covalent bond, C 1 ~C 12 alkyl, substituted C 1 ~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 , an acetal, hydrazine, disulfide, or ester, V 1 , V 2 , V 3 , V 4 , V 5 , V 6 , V 7 , and V 8 are each independently a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 -, or -P(O)OH-, a, b, c, d, e, f, g, and h are each independently 1 or 0, m is independently an integer from 1 to 12, n is independently an integer from 1 to 30, p is independently an integer from 1 to 20, q is independently an integer from 1 to 6, w is independently an integer from 1 to 20, Each R 12 is independently 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, heterocyclyl, or substituted heterocyclyl, Each R 13 is independently hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, and Each R 15 is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl.

[0368] In some embodiments, the 5T4-ADC of formula (V) comprises a linker (L-3). Thus, in some embodiments, the 5T4-ADC can be represented by formula (V-3):

Chemical formula

[0369] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is an integer from 1 to 2.

[0370] In some embodiments, the 5T4-ADC of formula (VI) comprises a linker (L-3). Thus, in some embodiments, the 5T4-ADC can be represented by formula (VI-3),

Chemical Formula

[0371] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0372] In some embodiments of the linker (L-3), k is 2. Thus, linker L can be linker (L-4):

Chemical Formula

Chem.

[0373] In some embodiments, the 5T4-ADC of Formula (V) includes a linker (L-4). Thus, in some embodiments, the 5T4-ADC can be represented by Formula (V-4):

Chem.

[0374] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0375] In some embodiments, the 5T4-ADC of formula (VI) comprises a linker (L-4). In some embodiments, the 5T4-ADC can be represented by formula (VI-4):

Chemical formula

[0376] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0377] In some embodiments, R 7is an enzymatically cleavable moiety that includes a sugar moiety such as a glycoside or a glycosyl. In some embodiments, the glycoside can promote an increase in the hydrophilicity of the cleavable linker as compared to a cleavable linker that does not include a glycoside. The glycoside can be any glycoside that is suitable for use in a cleavable linker and that can be cleaved via the enzymatic action of an enzyme.

[0378] R 7 Some of the glycosides can be attached to the remainder of the 5T4-ADC via a glycosidic bond. The glycosidic bond can be any of a variety of types of bonds, such as an O-glycosidic bond (O-glycoside), an N-glycosidic bond (glycosylamine), an S-glycosidic bond (thioglycoside), or a C-glycosidic bond (C-glycoside or C-glycosyl), etc., without being limited thereto, to link the glycoside to the 5T4-ADC. In some embodiments, the glycosidic bond is an O-glycosidic bond (O-glycoside).

[0379] In some embodiments, R 7 is

Chemical formula

Chemical formula

Chemical formula

[0380] In some embodiments, linker (L-5) is attached to formula (V). In some embodiments, 5T4-ADC may be represented by formula (V-5):

Chemical Formula

[0381] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0382] In some embodiments, the 5T4-ADC of formula (VI) comprises a linker (L-5). Thus, in some embodiments, the 5T4-ADC can be represented by formula (VI-5):

Chemical formula

[0383] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0384] In some embodiments of linker (L-5), R 5 is hydrogen, R 6’ is isopropyl, and R 6” is methyl. Thus, in some embodiments, linker L can be linker (L-6):

Chemical formula

Chemical formula

[0385] In some embodiments, the 5T4-ADC of formula (V) comprises a linker (L-6). Thus, in some embodiments, the 5T4-ADC can be represented by formula (V-6):

Chemical formula

[0386] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0387] In some embodiments, the 5T4-ADC of formula (VI) comprises a linker (L-6). Thus, in some embodiments, the 5T4-ADC can be represented by formula (VI-6):

Chemical formula

[0388] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0389] In some embodiments, 5T4-ADC can be represented by formula (V-3), (V-4), (V-5), (V-6), (VI-3), (VI-4), (VI-5) or (VI-6), L 1 is -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d and includes L 2 is -(T 5 -V 5 ) e -(T 6 -V 6 ) f -(T 7 -V 7 ) g -(T 8 -V 8 ) h and includes T 1 T 2 and T 3 are each independently C 1 ~C 12 alkyl, (PEG) n or (AA) p and T 5 T 6 T 7 and T 8 are each independently a covalent bond, C 1 ~C 12 alkyl, substituted C 1 ~C 12alkyl, 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 , acetal, hydrazine, disulfide, and ester, V 1 , V 2 , V 3 , V 4 , V 5 , V 6 , V 7 , and V 8 are each independently, -C(=O)- or -NR 11 -, a, b, and c are each 1, d is 0, and thus, T 4 and V 4 do not exist, e, f, g, and h are each independently, 1 or 0, n is an integer from 1 to 10, p is an integer from 1 to 10, R 11 is hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, or substituted aryl.

[0390] In some embodiments, 5T4-ADC is of formula (V-3), (V-4), (V-5), (V-6), (VI-3), (VI-4), (VI-5), or (VI-6), wherein T 1 , T 2 , and T 3 are C 1 ~C 6 alkyl, (AA) p , or (PEG) n , and p and n are independently integers from 1 to 10. In some embodiments, p is 1. In some embodiments, n is 2. In some embodiments, (AA) pmay include non-natural amino acids such as cysteine sulfonic acid amino acids. In some embodiments, V 1 , V 2 , and V 3 are independently -C(=O)- or -NR 15 -, wherein R 15 is hydrogen.

[0391] In any of the embodiments described herein, L 1 may be as follows: -(C 1 ~C 6 alkyl)-V 1 -(AA) p -V 2 -(PEG) n -V 3 - wherein V 1 , V 2 , V 3 , AA, p, and n are as described herein for linker (L-3) and the like. In some embodiments, V 1 , V 2 , and V 3 are -C(=O) or -NR 15 -, wherein R 15 is hydrogen. In some embodiments, p is 1 and n is 2. In some embodiments, (AA) p may include non-natural amino acids such as cysteine sulfonic acid amino acids.

[0392] In some embodiments, L 1 is

Chemical formula

Chemical formula

Chemical formula

Chem.

[0393] In some embodiments, the 5T4 - ADC can be represented by Formula (V - 7):

Chem.

[0394] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0395] In some embodiments, the 5T4 - ADC can be represented by Formula (VI - 7):

Chem.

[0396] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0397] L 2 In some embodiments of, e is 1, T 5 is a covalent bond, V 5 is a carbonyl, and each of f, g, and h is 0. In some embodiments, L 2 is a carbonyl (C=O). Thus, in some embodiments, linker L can be linker (L-8):

Chemical formula

Chemical formula

[0398] In some embodiments, the linker (L) in the 5T4-ADC of formula (V) can be linker (L-8). Thus, in some embodiments, the 5T4-ADC can be represented by formula (V-8):

Chemical formula

[0399] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0400] In some embodiments, the linker (L) in the 5T4-ADC of formula (VI) can be linker (L-8). Thus, in some embodiments, the 5T4-ADC can be represented by formula (VI-8): [Chemical formula] wherein Ab is an antibody that binds to 5T4, r is an integer from 1 to 10, , X 1 is C or N, and , W 1 is a drug.

[0401] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0402] In some embodiments, the 5T4-ADC can be represented by formula (VI-8), wherein X 1 is C. Thus, in some embodiments, the 5T4-ADC can be represented by formula (VIa-8): [Chemical formula] wherein Ab is an antibody that binds to 5T4, r is an integer from 1 to 10, , W 1 is a drug.

[0403] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0404] In some embodiments, the 5T4-ADC can be represented by formula (VI-8), where X 1 is N. Thus, in some embodiments, the 5T4-ADC can be represented by formula (VIb-8):

Chemical formula

[0405] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0406] Drug

[0407] In each of the formulas provided herein, the drug useful for use in the 5T4-ADC described herein, represented as W 1 is not particularly limited. Examples of suitable drugs include, but are not limited to, small molecule drugs such as cancer chemotherapeutic agents. For example, the drug can be an agent that affects microtubules. In some embodiments, the drug is a microtubule-affecting agent having anti-proliferative activity such as a maytansinoid or an auristatin.

[0408] Maytansine

[0409] In some embodiments, the drug is a maytansinoid. For example, the drug can be a maytansine having the following structure (W-1):

Chemical formula

[0410] For example, in any 5T4-ADC represented by formula (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 is W-1 or W-1 ’ and may be a maytansinoid such as a maytansinoid, where [Chemistry] indicates the binding point between the maytansinoid and L or L 2 and.

[0411] In some embodiments, the 5T4-ADC may be represented by formula (VI-11), formula (VI-21), or formula (VI-81): [Chemistry] [Chemistry] [Chemistry] In the formula, Ab is an antibody that binds to 5T4, r is an integer from 1 to 10, X 1 is C or N.

[0412] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0413] In some embodiments, X 1 is C, and the 5T4-ADC can be represented by formula (VIa-11) or formula (VIa-21): [Chemical formula] [Chemical formula] wherein, Ab is an antibody that binds to 5T4, and r is an integer from 1 to 10.

[0414] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0415] In some embodiments, X 1 is N, and the 5T4-ADC can be represented by formula (VIb-81): [Chemical formula] wherein, Ab is an antibody that binds to 5T4, and r is an integer from 1 to 10.

[0416] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0417] In some embodiments, the maytansinoid of the above structure may be referred to as deacylated maytansine or deacyl maytansine.

[0418] Auristatin

[0419] In some embodiments, the drug is a mitotic inhibitor, such as an auristatin or an active auristatin analog or derivative thereof (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). For example, in any of the formulas representing the 5T4-ADC described herein, W 1 can be MMAE, MMAF, or MMAD.

[0420] In some embodiments, W 1 is MMAE, which has the following structure (W-2):

Chemical formula

Chemical formula

[0421] For example, MMAE can be included in a 5T4-ADC that includes a linker (L-1). Thus, in some embodiments, the 5T4-ADC can be represented by formula (VI-12):

Chemical formula

[0422] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0423] In some embodiments, MMAE can be included in a 5T4-ADC that includes a linker (L-5). Thus, in some embodiments, the 5T4-ADC can be represented by formula (VI-52): [Chemical formula] In the formula, Ab is an antibody that binds to 5T4, r is an integer from 1 to 10, X 1 is C or N, and L 1 and L2 are as defined herein for linker (L-3) and the like.

[0424] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0425] In some embodiments, MMAE may be included in a 5T4-ADC comprising a linker (L-6). Thus, in some embodiments, the 5T4-ADC may be represented by formula (VI-62): [Chemical formula] In the formula, Ab is an antibody that binds to 5T4, r is an integer from 1 to 10, X 1 is C or N, and L 1 and L 2 are as defined herein for linker (L-3) and the like. In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0426] In some embodiments, the 5T4-ADC may be represented by formulas (VI-12), (VI-52), and (VI-62), wherein X 1 is N. In some embodiments, the 5T4-ADC may be represented by formula (VIb-12), formula (VIb-52), or formula (VIb-62): [Chemical formula] In the formula, Ab is an antibody that binds to 5T4, r is an integer from 1 to 10, R 5 、R 6’ 、R 6” 、L 1 、and L 2 are as defined herein for linker (L-5) etc., and r is an integer from 1 to 10.

[0427] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0428] In some embodiments, MMAE may be included in a 5T4-ADC comprising linker (L-7). Accordingly, the 5T4-ADC may be represented by formula (VIb-72):

Chemical formula

[0429] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0430] In some embodiments, MMAE may be included in a 5T4-ADC comprising linker (L-8). Accordingly, the 5T4-ADC may be represented by formula (VIb-82):

Chemical formula

[0431] In some embodiments, r is an integer from 1 to 4. In some embodiments, r is 1 or 2.

[0432] In some embodiments, W 1 is MMAE, which has the following structure (W-3):

Chemical formula

Chemical formula

[0433] For example, MMAF can be included in 5T4-ADC as follows, for example:

Chemical formula

[0434] In some embodiments, L is selected from linker (L-1), (L-2), (L-3), (L-4), (L-5), (L-6), (L-7), and (L-8). In some embodiments, X 1 is C or N. In some embodiments, each of Y 1 、Y 2 、Y 3 、and Y 4 is hydrogen. In some embodiments, R 2 and R 3 are independently C 1 ~C 6It is alkyl. 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.

[0435] Summary section

[0436] In some embodiments, the 5T4-ADC can be represented by Formula (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), and Ab is an antibody that binds to 5T4 disclosed herein. In some embodiments, the antibody comprises a VH comprising one or more (such as 1, 2, or 3) CDRs that are identical or substantially identical to one of the CDRs described herein, such as any one of Tables 1-3. Additionally or alternatively, in some embodiments, the antibody comprises a VL comprising one or more (such as 1, 2, or 3) CDRs that are identical or substantially identical to one of the CDRs described herein, such as any one of Tables 1-3.

[0437] In some embodiments, the 5T4-ADC can be represented by Formula (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), wherein Ab is an antibody that binds to 5T4 disclosed herein. In some embodiments, the antibody comprises a VH that is identical or substantially identical to a VH described herein, such as those described in any one of Tables 1-3. Additionally or alternatively, in some embodiments, the antibody comprises a VL that is identical or substantially identical to a VL described herein, such as those described in any one of Tables 1-3.

[0438] In some embodiments, the 5T4-ADC can be represented by Formula (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), wherein Ab is an antibody that binds to 5T4. In some embodiments, 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) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 63.

[0439] In some embodiments, the 5T4-ADC can be represented by Formula (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), and Ab is an antibody that binds to 5T4 disclosed herein. In some embodiments, the antibody comprises a VH region comprising (a) (1) a VH CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 1, 7, 12, 13, and 18, (2) a VH CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 2, 8, 14, 19, and 24, and (3) a VH CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 3, 9, 15, and 20, and a VL region comprising (b) (1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 4, 10, 16, and 21, (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 11, and 22, and (3) a VL CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 6, 17, and 23.

[0440] In some embodiments, the 5T4-ADC can be represented by Formula (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 region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 1, a VH CDR2 having the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 6, or (ii) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 7, a VH CDR2 having the amino acid sequence of SEQ ID NO: 8, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 9, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 6, or (iii) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 12, a VH CDR2 having the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 6, or (iv) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 13, a VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 15, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and a VLA VL region containing a CDR3, or (v) a VH region containing a VH CDR1 having the amino acid sequence of SEQ ID NO: 18, a VH CDR2 having the amino acid sequence of SEQ ID NO: 19, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 20, and a VL region containing a VL CDR1 having the amino acid sequence of SEQ ID NO: 21, a VL CDR2 having the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 23, or (vi) a VH region containing a VH CDR1 having the amino acid sequence of SEQ ID NO: 1, a VH CDR2 having the amino acid sequence of SEQ ID NO: 24, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 3, and a VL region containing a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0441] In some embodiments, the 5T4-ADC can be represented by formula (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, and includes (a) a VH region comprising (1) a VH CDR1 comprising any one amino acid sequence of SEQ ID NOs: 27, 31, 34, 35, and 39, (2) a VH CDR2 comprising any one amino acid sequence of SEQ ID NOs: 28, 32, 36, 40, and 43, and (3) a VH CDR3 comprising any one amino acid sequence of SEQ ID NOs: 29, 33, 37, and 41, and (b) a VL region comprising (1) a VL CDR1 comprising any one amino acid sequence of SEQ ID NOs: 4, 10, 16, and 21, (2) a VL CDR2 comprising any one amino acid sequence of SEQ ID NOs: 5, 11, and 22, and (3) a VL CDR3 comprising any one amino acid sequence of SEQ ID NOs: 30, 38, and 42.

[0442] In some embodiments, the 5T4-ADC can be represented by Formula (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), and Ab is an antibody that binds to 5T4. In some embodiments, the antibody comprises (i) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 27, a VH CDR2 having the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 29, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 30, or (ii) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 31, a VH CDR2 having the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 33, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 30, or (iii) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 34, a VH CDR2 having the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 29, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 30, or (iv) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 35, a VH CDR2 having the amino acid sequence of SEQ ID NO: 36, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 37, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and a VLA VL region comprising CDR2 and a VL CDR3 having the amino acid sequence of SEQ ID NO: 38, or (v) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 39, a VH CDR2 having the amino acid sequence of SEQ ID NO: 40, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 41, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 21, a VL CDR2 having the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 42, or (vi) a VH region comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 27, a VH CDR2 having the amino acid sequence of SEQ ID NO: 43, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 29, and a VL region comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 30.

[0443] In some embodiments, the 5T4-ADC can be represented by Formula (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 (a) a VH region comprising (1) a VH CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 46, 50, 53, 13, and 57, (2) a VH CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 47, 51, 54, 58, and 61, and (3) a VH CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 48, 52, 55, and 59, and (b) a VL region comprising (1) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 4, 10, 16, and 21, (2) a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 11, and 22, and (3) a VL CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 49, 56, and 60.

[0444] In some embodiments, the 5T4-ADC can be represented by Formula (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, and (i) a VH region comprising a VH CDR1 comprising the amino acid sequence comprising SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence comprising SEQ ID NO: 47, and a VH CDR3 comprising the amino acid sequence comprising SEQ ID NO: 48, and a VL region comprising a VL CDR1 comprising the amino acid sequence comprising SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence comprising SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence comprising SEQ ID NO: 49, (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence comprising SEQ ID NO: 50, a VH CDR2 comprising the amino acid sequence comprising SEQ ID NO: 51, a VH CDR3 comprising the amino acid sequence comprising SEQ ID NO: 52, and a VL region comprising a VL CDR1 comprising the amino acid sequence comprising SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence comprising SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence comprising SEQ ID NO: 49, or (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence comprising SEQ ID NO: 53, a VH CDR2 comprising the amino acid sequence comprising SEQ ID NO: 47, and a VH CDR3 comprising the amino acid sequence comprising SEQ ID NO: 48, and a VL region comprising a VL CDR1 comprising the amino acid sequence comprising SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence comprising SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence comprising SEQ ID NO: 49, or (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence comprising SEQ ID NO: 13, and a VH CDR2 comprising the amino acid sequence comprising SEQ ID NO: 54, and a VH CDR3 comprising the amino acid sequence comprising SEQ ID NO: 55, and a VLA VL region comprising a CDR1 having the amino acid sequence including SEQ ID NO: 11, a CDR2 having the amino acid sequence including SEQ ID NO: 56, and a CDR3 having the amino acid sequence including SEQ ID NO: 56, or (v) a VH region comprising a CDR1 having the amino acid sequence including SEQ ID NO: 57, a CDR2 having the amino acid sequence including SEQ ID NO: 58, and a CDR3 having the amino acid sequence including SEQ ID NO: 59, and a VL region comprising a CDR1 having the amino acid sequence including SEQ ID NO: 21, a CDR2 having the amino acid sequence including SEQ ID NO: 22, and a CDR3 having the amino acid sequence including SEQ ID NO: 60, or (vi) a VH region comprising a CDR1 having the amino acid sequence including SEQ ID NO: 46, a CDR2 having the amino acid sequence including SEQ ID NO: 61, and a CDR3 having the amino acid sequence including SEQ ID NO: 48, and a VL region comprising a CDR1 having the amino acid sequence including SEQ ID NO: 4, a CDR2 having the amino acid sequence including SEQ ID NO: 5, and a CDR3 having the amino acid sequence including SEQ ID NO: 49.

[0445] In some embodiments, the 5T4-ADC can be represented by formula (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 further comprises a framework 1 (FR1), framework 2 (FR2), framework 3 (FR3) and / or framework 4 (FR4) sequence shown in any one of SEQ ID NOs: 25, 26, 44, 45, 62, and 63.

[0446] In some embodiments, the 5T4-ADC can be represented by formula (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), wherein Ab is an antibody that binds to 5T4 as disclosed herein and comprises a human framework sequence.

[0447] In some embodiments, the 5T4-ADC can be represented by formula (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), wherein Ab is an antibody that binds to 5T4. In some embodiments, 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) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 63.

[0448] Antibody-Linker-Drug Conjugation In some embodiments, the antibody can be modified prior to conjugation to the linker-drug. Modification of the antibody can generate a modified antibody that contains one or more reactive groups suitable for conjugation to the linker-drug.

[0449] In some embodiments, the antibody is modified at one or more amino acid residues to provide one or more reactive groups suitable for conjugation to the linker-drug. For example, a carbonyl group introduced into the polypeptide can be selectively reacted with an α-nucleophile such as an aminooxy and hydrazide-containing compound. Selective chemical action on the carbonyl functional group on the protein with enhanced kinetics, site selectivity, and conjugate stability can result in an improved bioconjugate.

[0450] In some embodiments, the antibody can be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). The reactive aldehyde can be included in an “aldehyde tag” or “ald-tag,” where the “aldehyde tag” or “ald-tag” refers to an amino acid sequence derived from a sulfatase motif that has been converted by the action of formylglycine-generating enzyme (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.” Put another way, the term “aldehyde tag” as used herein refers to an amino acid sequence that includes a “converted” sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue has been converted to fGly by the action of FGE, e.g., L(fGly)TPSR (SEQ ID NO: 102)). The converted sulfatase motif can be derived from an amino acid sequence that includes an “unconverted” sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue has not been converted to fGly by FGE but can be converted, e.g., an unconverted sulfatase motif having the sequence: L(C / S)TPSR (SEQ ID NO: 103)). As used in the context of the action of formylglycine-generating enzyme (FGE) on a sulfatase motif, “conversion” refers to the biochemical modification of a cysteine or serine residue in the sulfatase motif to a formylglycine (fGly) residue (e.g., from Cys to fGly, or from Ser to fGly). As used herein, an aldehyde-tagged antibody refers to an antibody in which a cysteine or serine residue has been modified to form a formylglycine residue. Additional aspects of the addition of aldehyde tags in site-specific protein modification and their use are described in US 7,985,783 and US Patent Application Publication No. 2011 / 0117621, the disclosures of which are incorporated herein by reference in their entireties.

[0451] The conversion of polypeptides to include fGly can be achieved by cell-based methods (in vivo) or cell-free methods (in vitro). Similarly, the modification of polypeptides to produce polypeptides suitable for conjugation (e.g., modification to produce a polypeptide containing a reactive group suitable for conjugation) can be achieved by cell-based methods (in vivo) or cell-free methods (in vitro).

[0452] The amino acid sequence of the antibody can be modified to include a sulfatase motif containing serine or cysteine residues that can be converted (oxidized) to FGly residues by the action of FGE, either in vivo (e.g., during translation of an aldehyde tag-containing protein within a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with FGE in a cell-free system). The 5T4 antibody used for the production of 5T4-ADC includes at least an Ig constant region, e.g., an Ig heavy chain constant region (e.g., at least the CH1 domain, at least the CH1 and CH2 domains, the CH1, CH2, and CH3 domains, or the CH1, CH2, CH3, and CH4 domains), or an Ig light chain constant region.

[0453] The sulfatase motif can be introduced at any convenient site within the antibody. As described herein, in some embodiments, to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), and / or added (e.g., at the N-terminus or C-terminus), the degree of modification of the native amino acid sequence of the target polypeptide is minimized. By minimizing the degree of amino acid sequence modification of the target antibody, the impact that such modification can have on the function and / or structure of the antibody can be minimized.

[0454] In some embodiments, the aldehyde-tagged antibody comprises an aldehyde-tagged Ig heavy chain constant region (e.g., at least the CH1 domain, at least the CH1 and CH2 domains, the CH1, CH2, and CH3 domains, or the CH1, CH2, CH3, and CH4 domains). The aldehyde-tagged Ig heavy chain constant region comprises an IgA, IgM, IgD, IgE, IgG1, IgG2, IgG3, or IgG4 isotype heavy chain or any allotype variant thereof, e.g., a heavy chain constant region sequence of a human heavy chain constant region or a mouse heavy chain constant region sequence, a hybrid heavy chain constant region, a synthetic heavy chain constant region, or a consensus heavy chain constant region sequence, and is modified by FGE to comprise at least one sulfatase motif so as to generate an fGly-modified Ig polypeptide. Allotype variants of Ig heavy chains are known in the art. See, e.g., Jefferis and Lefranc (2009) MAbs 1:4.

[0455] In some embodiments, the aldehyde-tagged antibody comprises an aldehyde-tagged Ig light chain constant region. The aldehyde-tagged Ig light chain constant region can comprise a kappa light chain, a lambda light chain, e.g., a kappa or lambda light chain constant region, a hybrid light chain constant region, a synthetic light chain constant region, or a consensus light chain constant region sequence modified by FGE to comprise at least one sulfatase motif so as to generate an fGly-modified antibody.

[0456] Alternatively, an isolated unmodified polypeptide can be isolated after recombinant production in a host cell lacking the appropriate enzyme or by synthetic production. The isolated polypeptide can then be contacted with an appropriate enzyme under conditions that provide the desired modification of the polypeptide to include fGly. The polypeptide can be unfolded using methods known in the art (e.g., heat, pH adjustment, chaotropic agents (e.g., urea, etc.), organic solvents (e.g., hydrocarbons: octane, benzene, chloroform), etc.), and the denatured protein can be contacted with an appropriate enzyme. The modified polypeptide can then be refolded under suitable conditions.

[0457] In some embodiments, a modified antibody containing an fGly residue can be conjugated to a linker-drug by reacting the fGly with a coupling moiety on the linker-drug, such as the hydrazinyl-pyrrolo coupling moiety described herein. For example, an fGly-containing antibody can be isolated from a source of production (e.g., recombinant host cell production, synthetic production) and contacted with a drug or other moiety containing a reactive partner (e.g., a detectable label) under conditions suitable to provide conjugation of the drug or other moiety to the 5T4 antibody. For example, the drug or other moiety containing a reactive partner can include a conjugation moiety (e.g., the hydrazinyl-pyrrolo coupling moiety described herein). A hydrazinyl-pyrrolo-containing drug can be reacted with an antibody to produce a 5T4-ADC as described herein.

[0458] A general scheme for coupling an antibody to a pyridazine-pyrrolo coupling moiety is shown below.

Chemical formula

[0459] In this specification, the hydrazinyl-pyrrolo coupling moiety, which can be equivalently referred to as an aza-hydrazino-iso-pictet-spengler (azaHIPS) coupling moiety, forms a pyridazine-pyrrolo coupling moiety as shown in formula (III) upon conjugation to formyl-glycine. The 5T4 antibody can react with the azaHIPS coupling moiety and thus may contain a 2-formylglycine residue (fGly) that conjugates the two together. To generate a 5T4-ADC, the drug can be coupled directly or indirectly to the azaHIPS moiety at any position of the azaHIPS moiety (e.g., R 16 Y 1 Y 2 Y 3 or Q 4 ). R 2 and R 3 are independently, for example, but not limited to, a desired substituent such as 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. R 16 Y 1 Y 2 Y 3 and Q 4 can be as described herein for formula (I) or formula (II), etc.

[0460] The following formula (I) represents a hydrazinyl-pyrrolo coupling moiety that can be used to link an antibody that binds to 5T4 to a drug in any of the 5T4-ADCs described herein.

Chemical formula

[0461] Formula (I) has been previously described in WO2015 / 081282, which is hereby incorporated by reference in its entirety.

[0462] In some embodiments, the hydrazinyl-pyrrolo coupling moiety may be represented by formula (II):

Chemical formula

[0463] Formula (II) has been previously described in WO2015 / 081282 and WO2020 / 154437, each of which is hereby incorporated by reference in its entirety. In some embodiments, m is 0 or 1, and R 2 and R 3 are each alkyl. For example, R 2 and R 3 may each independently be C 1 ~C 10 alkyl (e.g., C 1 ~C 6 alkyl). In some embodiments, R 2 and R 3 are each independently methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, or t-butyl. In some embodiments, at least one of R 2 and R 3 is methyl. In some embodiments, R 2 and R 3Both are methyl. In some embodiments, X 1 , X 2 , X 3 , and X 4 each is, independently, C or N. In some embodiments, X 1 is C. In some embodiments, X 1 is N. In some embodiments, X 2 is C or N. In some embodiments, X 2 is C. In some embodiments, X 3 is C or N. In some embodiments, X 3 is C. In some embodiments, X 4 is C or N. In some embodiments, X 4 is C. X 1 , X 2 , X 3 , and X 4 various combinations are possible. For example, in some embodiments, X 1 , X 2 , X 3 , and X 4 each is C. In other embodiments, X 1 , X 2 , X 3 , and X 4 three of them are C and X 1 , X 2 , X 3 , and X 4 one of them is N. In some embodiments, Y 1 , Y 2 , Y 3 , and Y 4 each is hydrogen.

[0464] An exemplary scheme for preparing 5T4-ADC is shown in Scheme A.

Chemical formula

[0465] In Scheme A, the linker-drug is conjugated to an antibody that binds to 5T4. The 5T4 antibody has a recognition motif, such as the L(C / S)TPSR (SEQ ID NO: 103) recognition motif (e.g., SEQ ID NO: 90, 94, 98, etc.), within the constant region of the light chain. The cysteine residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and then conjugated to the azaHIPS moiety through a Pictet-Spengler reaction.

[0466] An exemplary scheme for preparing 5T4-ADC is shown in Scheme B.

Chemical formula

[0467] In Scheme B, the linker-drug is conjugated to an antibody that binds to 5T4. The 5T4 antibody has a recognition motif, such as the L(C / S)TPSR (SEQ ID NO: 103) recognition motif (e.g., SEQ ID NO: 90, 94, 98), within the constant region of the heavy chain. The cysteine residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and then conjugated to the azaHIPS moiety through a Pictet-Spengler reaction.

[0468] In some embodiments, the 5T4-ADC comprises a linker-drug conjugated to the C-terminus of each heavy chain constant region of the 5T4 antibody via a pyridazine-pyrrole coupling moiety and can be characterized by a stoichiometric ratio of 1 to 4, 1 to 2, 1.75 to 2, or 1.9 to 2 of antibody to linker-drug. In another embodiment, the 5T4-ADC comprises a pyridazine-pyrrole coupling moiety linked only to the C-terminus of one heavy chain constant region and has a stoichiometric ratio between antibody and drug of 0.5 to 1, 0.75 to 1, or 0.9 to 1.

[0469] In some embodiments, the 5T4-ADC comprises a linker-drug conjugated to the 5T4 antibody via a pyridazine-pyrrolocoupling moiety, and the linker-drug comprises a maytansine conjugated to the pyridazine-pyrrolocoupling moiety via a linker (L-1) as shown in Scheme C below.

Chem.

[0470] In some embodiments, the 5T4 antibody has a recognition motif, such as an L(C / S)TPSR (SEQ ID NO: 103) recognition motif (e.g., SEQ ID NO: 90, 94, 98), within the constant region of the heavy chain. The cysteine residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and then conjugated to an azaHIPS moiety conjugated to an MMAE linker (L-1) through a Pictet-Spengler reaction.

[0471] In some embodiments, the 5T4-ADC comprises a linker-drug conjugated to the 5T4 antibody via a pyridazine-pyrrolocoupling moiety, and the linker-drug comprises an MMAE conjugated to the pyridazine-pyrrolocoupling moiety via a linker (L-2) as shown in Scheme D below.

Chem.

[0472] In some embodiments, the 5T4 antibody has a recognition motif, such as an L(C / S)TPSR (SEQ ID NO: 103) recognition motif, within the constant region of the heavy chain. The cysteine residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and then conjugated to an azaHIPS moiety conjugated to MMAE through a Pictet-Spengler reaction via a linker (L-2).

[0473] In some embodiments, the 5T4-ADC comprises a linker-drug conjugated to the 5T4 antibody via a pyridazine-pyrrolocoupling moiety, and the linker-drug comprises MMAE conjugated to the pyridazine-pyrrolocoupling moiety via a linker (L-7) as shown in Scheme E below.

Chemical Structure

[0474] In some embodiments, the 5T4 antibody has a recognition motif, such as an L(C / S)TPSR (SEQ ID NO: 103) recognition motif (e.g., SEQ ID NO: 90, 94, 98), within the constant region of the heavy chain. The cysteine residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and then conjugated to an azaHIPS moiety conjugated to MMAE through a Pictet-Spengler reaction via a linker (L-7).

[0475] In some embodiments, the 5T4-ADC comprises a linker-drug conjugated to the 5T4 antibody via a pyridazine-pyrrolocoupling moiety, and the linker-drug comprises MMAE conjugated to the pyridazine-pyrrolocoupling moiety via a linker (L-8) as shown in Scheme F below.

Chemical Structure

[0476] In some embodiments, the antibody that binds to 5T4 has a recognition motif, such as an L(C / S)TPSR (SEQ ID NO: 103) recognition motif (e.g., SEQ ID NO: 90, 94, 98), within the constant region of the heavy chain. The cysteine residue of the L(C / S)TPSR (SEQ ID NO: 103) recognition motif can be converted to formylglycine (fGly) and then conjugated to an azaHIPS moiety conjugated to MMAE through a Pictet-Spengler reaction via a linker (L-8).

[0477] Formulations and Compositions 5T4-ADC may be included in a pharmaceutical composition for administration to a subject, for example, to treat a disease, disorder, or condition. The pharmaceutical composition may include a 5T4-ADC that may exhibit an average drug-to-antibody ratio (“DAR”) of from about 1 to about 2. In other words, each antibody is conjugated to one or two linker-drug conjugates. Methods for determining the DAR are well known to those of skill in the art and include methods using reverse phase chromatography, i.e., HPLC-MS.

[0478] For example, in any embodiment, a pharmaceutical composition comprising 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 described herein, including mAbA4, mAbA15, and mAbA17 described herein such as any one of Tables 1-3, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a 5T4-ADC of formula (IV), (V), or (VI), wherein L is selected from linker (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 comprising 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 to the antibody mAbA4), mAbA15-VIa-21 (i.e., a linker-drug of formula VIa-21 conjugated to the antibody mAbA15), mAbA15-VIb-82 (i.e., a linker-drug of formula VIb-82 conjugated to the antibody mAbA15), or mAbA17-VIb-82 (i.e., a linker-drug antibody of formula VIb-82 conjugated to the antibody mAbA17) and a pharmaceutically acceptable excipient.

[0479] The 5T4-ADC can be formulated in any of a variety of different ways. The 5T4-ADC of the present disclosure can be provided in any suitable form, for example, in the form of a pharmaceutically acceptable salt, and can be formulated for any suitable route of administration, for example, oral, topical, or parenteral administration. When the 5T4-ADC is provided as a liquid injection (such as in embodiments where they can be administered intravenously or directly into tissue), the 5T4-ADC can be provided as a ready-to-use dosage form, or as a storage-stable powder for reconstitution or as a liquid composed of pharmaceutically acceptable excipients and excipients.

[0480] Methods for formulating the 5T4-ADC can be adapted from those available in the art. For example, the 5T4-ADC can be provided as a pharmaceutical composition comprising an effective amount of the 5T4-ADC and a pharmaceutically acceptable excipient (such as saline). The pharmaceutical composition can optionally include other additives (such as buffers, stabilizers, preservatives, etc.). In some embodiments, the formulation is suitable for administration to mammals, such as those suitable for administration to humans.

[0481] Also provided herein are pharmaceutical compositions comprising an effective amount of a 5T4-ADC described herein and a pharmaceutically acceptable excipient. In some embodiments, the 5T4-ADC comprises an antibody that binds to 5T4 described herein, including mAbA4, mAbA15, and mAbA17 described in any one of Tables 1-3. In some embodiments, the pharmaceutical composition comprises an effective amount of a 5T4-ADC of formula (IV), (V), or (VI), wherein L is selected from linker (L-1), (L-2), (L-3), (L-4), (L-5), (L-6), (L-7), and (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), (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 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, mAbA15-VIb-21, mAbA15-VIb-82, or mAbA17-VIb-82 and a pharmaceutically acceptable excipient.

[0482] A pharmaceutically acceptable excipient can be one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances suitable for administration to human or veterinary subjects (e.g., physiologically acceptable and / or pharmacologically acceptable). When a plurality of pharmaceutically acceptable excipients are present in a pharmaceutical composition, the pharmaceutically acceptable excipients can be mixed with one or more of the active ingredients, such as a hybrid molecule, and can be mixed with each other so as not to substantially impair the desired pharmaceutical effectiveness. Pharmaceutically acceptable materials can generally be administered to a subject without causing significant undesirable physiological effects such as nausea, dizziness, rash, or stomach ache. For example, it is desirable that a composition containing a pharmaceutically acceptable excipient is non-immunogenic when administered to a human subject for therapeutic purposes.

[0483] The pharmaceutical composition of the present invention can further contain a suitable buffer, including, for example, acetates, citrates, borates, and phosphates. The pharmaceutical composition can also optionally contain suitable preservatives such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal. The pharmaceutical composition of the present invention can be provided in unit dosage form and can be prepared by any suitable method, many of which are well known in the pharmaceutical art. Such methods include the step of associating an antibody or antibody binding fragment of the present invention with an excipient that constitutes one or more accessory components. Generally, the formulation is prepared by uniformly and closely associating the active agent with a liquid excipient, a finely divided solid excipient, or both, and then, if necessary, shaping the product.

[0484] Compositions suitable for parenteral administration conveniently include sterile aqueous preparations of the composition, which are preferably isotonic with the recipient's blood. This aqueous preparation can be formulated by known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be utilized are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any brand of fixed oil such as synthetic mono- or di-glycerides can be used. Additionally, fatty acids such as oleic acid can be used in the preparation of injectables. Excipient formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

[0485] The preparation of the pharmaceutical compositions of the present invention and their various routes of administration can be carried out according to methods well known in the art. Delivery systems useful in the context of the present invention include sustained release, delayed release, and extended release delivery systems, whereby the delivery of the composition occurs before and for a sufficient time to cause sensitization of the site to be treated. The compositions can be used in combination with other therapeutic agents or therapies. Such systems can avoid repeated administration of the composition, thereby enhancing the convenience for the subject and the physician, and can be particularly suitable for some of the compositions of the present invention.

[0486] Many types of release delivery systems are available and are known to those of skill in the art. Suitable release delivery systems include polymeric bases such as poly(lactide-glycolide), copolyesters, polycaprolactone, polyesteramides, polyorthoesters, polyhydroxybutyrate, and polyanhydrides. Microcapsules of the aforementioned polymers containing a drug are described, for example, in U.S. Patent No. 5,075,109. Delivery systems also include non-polymeric systems that are lipids, such as sterols like cholesterol and cholesterol esters, and fatty acids or neutral fats such as monoglycerides and triglycerides, hydrogel release systems, thyrotic systems, peptide-based systems, wax coatings, compressed tablets using conventional binders and excipients, and partially fused implants. Specific examples include, but are not limited to, (a) erosion systems in which the active composition is contained in matrix form, such as those described in U.S. Patents 4,452,775, 4,667,014, 4,748,034, and 5,239,660, and (b) pharmaceutical compositions in which the active ingredient permeates at a controlled rate from polymers as described in U.S. Patents Nos. 3,832,253 and 3,854,480. Additionally, pump-based hardware delivery systems can be used, some of which are implant compatible.

[0487] Generally, the 5T4-ADC or pharmaceutical composition is suitably packaged, for example, in vials, pouches, ampules, and / or any container appropriate for the method of treatment. The components can be provided as concentrates (including lyophilized compositions) that can ...

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 wherein: 【Chemical 201】 Ab represents said antibody that binds to 5T4; r is an integer from 1 to 10; m is 0 or 1; a, b, c, d, and e are each independently 0 or 1, and the sum of a, b, c, d, and e is from 1 to 5; R ２ and R ３ are 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 and R ３ are linked cyclically to form a 5- or 6-membered heterocyclyl, X １ , X ２ , X ３ and X ４ are each independently selected from the group consisting of C, N, O, and S, Y １ 、Y ２ 、Y ３ and Y ４ are 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 and Y ２ 、Y ２ and Y ３ 、or Y ３ and Y ４ are linked cyclically, W １ is the drug, L represents the linker, -(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 - and w is an integer from 1 to 20; T １ 、T ２ 、T ３ 、T ４ 、and T ５ are each independently C １ ~C １２ alkyl, substituted C １ ~C １２ alkyl, (EDA) ｗ , (PEG) ｎ , (AA) ｐ , -(CR １３ OH) ｈ -, piperidin-4-amine (P4A-R １２ ), meta-aminobenzylcarbamate (MABC) group, meta-aminobenzyloxy (MABO) group, para-aminobenzyloxy (PABO) group, para-aminobenzylcarbamate (PABC) group, para-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 and contain 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;

2. Each R １２ is independently 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, heterocyclyl, or substituted heterocyclyl, Each R １３ is independently hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, V １ , V ２ , V ３ , V ４ , and V ５ is, independently of one another, a covalent bond, -C(=O)-, -NR １１ -, -C(=O)NR １１ -, -NR １１ C(=O)-, -C(=O)O-, -OC(=O)-, -O-, -S-, -S(=O)-, -SO ２ -, -SO ２ NR １１ -, -NR １１ SO ２ -, and -P(=O)OH- and is selected from the group consisting of, wherein R １１ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, PEG, aryl, and substituted aryl, said antibody-drug conjugate (ADC).

3. W １ The ADC according to claim 1, wherein W is selected from the group consisting of maytansinoid and auristatin. The ADC according to claim 1 or 2, wherein said auristatin is MMAE.

4.

5. R ２ and R ３ are each alkyl, the ADC according to any one of claims 1 to 3.

6. R ２ and R ３ is each methyl, the ADC according to any one of claims 1 to 4.

7. X １ , X ２ , X ３ , and X ４ where each of them is independently selected from C and N, the ADC according to any one of claims 1 to 5. The ADC according to any one of claims 1 to 6, wherein said ADC is represented by formula (V):

8. 【Chemical 202】 The ADC according to any one of claims 1 to 6, wherein said ADC is represented by formula (VI):

9. 【Chemical 203】 wherein X １ is C or N, the ADC according to any one of claims 1 to 7.

10. T １ 、T ２ 、T ３ 、T ４ 、and T ５ are each independently selected from the group consisting of C １ to C ６ alkyl, P4A-R １２ , (AA) ｐ , (AA) ｐ -PABC, and (PEG) ｎ wherein p is an integer from 1 to 5, and R １２ is polyethylene glycol or carboxylic acid-modified polyethylene glycol, the ADC according to any one of claims 1 to 8.

11. T １ is C １ to C ６ alkyl, and T ２ 、T ３ 、T ４ 、and T ５ are each independently (PEG) ｎ 、C １ ~C ６ 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 ) selected from, wherein R １２ is carboxylic acid-modified polyethylene glycol, and R １６ is hydrogen, V １ , V ２ , V ３ , V ４ and V ５ are each independently selected from -CO- and -NR １１ -, and R １１ is selected from hydrogen and alkyl. The ADC according to any one of claims 1 to 9 L is wherein: -(C １ ~C ６ alkyl)-(V １ )-(P4A-R １２ )-(V ２ )-(C １ ~C ６ alkyl)-(V ３ )-wherein,

12. V １ , V ２ and V ３ each independently selected from the group consisting of -CO- and -NR １１ -. R １１ each independently represents hydrogen or C １ -C ６ -C alkyl, and is independently selected from the group consisting of, and R １２ is the carboxylic acid-modified polyethylene glycol moiety, the ADC according to any one of claims 1 to 10. L is wherein represents the bond to nitrogen of said pyridazine-pyrrolo coupling moiety. 【Chemical 204】

13. 【Chemical 205】 An antibody-drug conjugate (ADC) of formula (VI-1), comprising: * is W １ The ADC according to any one of claims 1 to 11, wherein * represents the binding to W. 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 wherein: Ab represents said antibody that binds to 5T4; r is an integer from 1 to 10; 【Chemical 206】

14.

15. The ADC according to claim 14, wherein said auristatin is MMAE. X １ is C or N, and W １ is the antibody-drug conjugate (ADC) which is the said drug.

16. W １ The ADC according to claim 13, wherein W is selected from the group consisting of maytansinoid and auristatin.

17.

18.

19. X １ is C, and W １ is de-cyclized maytansine, the ADC according to claim 13 or 14. L is X １ is N, and W １ is MMAE, the ADC according to any one of claims 14 to 15. wherein: n and p are each independently an integer from 1 to 20; (C １ ~C ６ alkyl)-V １ -(AA)-V ２ -(PEG) ｎ -V ３ -((AA) ｐ -(PABC-R １６ ))-V ４ -(C １ -C ６ alkyl)-V ５ wherein

20. R １６ is hydrogen, and L is V １ , V ２ , V ３ , V ４ , and V ５ are each independently selected from the group consisting of -CO- and -NR １１ -. Each R １１ is independently selected from the group consisting of hydrogen and C １ to C ６ alkyl, and is the ADC according to any one of claims 13 to 17. wherein represents the bond to nitrogen of said pyridazine-pyrrolo coupling moiety.

21. 【Chemical 207】 An antibody-drug conjugate (ADC) comprising an antibody that binds to 5T4 conjugated to one or more drugs via one or more pyridazine-pyrrolo coupling moieties and represented by formula (VI-2), wherein: 【Chemical 208】 Ab represents said antibody that binds to 5T4; * is coupled to W １ The ADC according to any one of claims 13 to 17, wherein * represents a connection to W. r is an integer from 1 to 10;

22. 【Chemical 209】

23. The ADC according to claim 21, wherein said auristatin is MMAE.

24. X １ is C or N, and W １ The antibody-drug conjugate (ADC) wherein W is the drug.

25. W １ The ADC according to claim 20, wherein W is selected from maytansinoid and auristatin.

26.

27. An antibody-drug conjugate (ADC) of formula (IV), comprising: X １ is C, and W １ is decyclized maytansine, the ADC according to claim 20 or 21. a. an antibody that binds to 5T4, and ​ ​ b. One or more drugs conjugated to one or more pyridazine-pyrrolocoupling moieties via a linker comprising 【Chemical 210】 wherein Ab represents said antibody that binds to 5T4 W １ is the drug, r is an integer from 1 to 10 m is 0 or 1 R ２ and R ３ are 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 and R ３ are linked cyclically to form a 5- or 6-membered heterocyclyl, X １ 、 X ２ 、 X ３ 、 and X ４ are each independently selected from the group consisting of C, N, O, and S, Y １ 、Y ２ 、Y ３ and Y ４ are 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 and Y ２ 、Y ２ and Y ３ 、or Y ３ and Y ４ are linked cyclically, L is 【Chemical 211】 where wherein 【Chemical 212】 represents the bond to nitrogen of said pyridazine-pyrrolocoupling moiety * represents the bond to W １ represents the bond to, Each R ５ is 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 is 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 part, k is an integer from 1 to 10 L １ is -(T １ -V １ ) ａ -(T ２ -V ２ ) ｂ -(T ３ -V ３ ) ｃ -(T ４ - Ｖ４ ) ｄ - and includes L ２ is -(T ５ -V ５ ) ｅ -(T ６ -V ６ ) ｆ -(T ７ -V ７ ) ｇ -(T ８ -V ８ ) ｈ including - 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 are each independently a covalent bond, C １ -C １２ alkyl, substituted C １ -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 selected from the group consisting of acetal, hydrazine, disulfide, and ester 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 represents a covalent bond, —CO—, or —NR １５ --, --NR １５ (CH ２ ) ｑ --, --NR １５ (C ６ H ４ ) -, -CONR １５ --, --NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ N.R. １５ --, --NR １５ SO ２ -, -P(O)OH-, and -P(O)OH-; each q is an integer from 1 to 6 R １２ is independently selected from the group consisting of 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, heterocyclyl, and substituted heterocyclyl, Each R １３ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, and Each R １５ is 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, said antibody-drug conjugate (ADC).

25. W １ The ADC according to claim 24, wherein W is selected from maytansinoid and auristatin.

26. The ADC according to claim 25, wherein said auristatin is MMAE.

27. R ２ and R ３ is alkyl, respectively, the ADC according to any one of claims 24 to 26.

28. R ２ and R ３ are each methyl, the ADC according to claim 27.

29. X １ , X ２ , X ３ , and X ４ where each of which is independently selected from C and N, the ADC according to any one of claims 24 to 28.

30. The ADC according to any one of claims 24 to 29, represented by formula (VI-3): 【Chemical 213】

31. R ７ is 【Chemical 214】 wherein ** represents the point of attachment to the phenyl group in formula (VI-3), the ADC according to claim 30.

32. Represented by formula (V-5): 【Chemical 215】 In the formula, R ６’ and R ６” each independently is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl, the ADC according to any one of claims 24 to 31.

33. Represented by formula (VI-5): 【Chemical 216】 In the formula, X １ is C or N, and R ６’ and R ６” are each independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. The ADC according to any one of claims 24 to 32.

34. The ADC according to any one of claims 24 to 33, represented by formula (VI-6): 【Chemical 217】

35. L １ is -(T １ - V １ ) ａ -(T2 - V ２ ) ｂ -(T ３ - V ３ ) ｃ -(T ４ - V ４ ) ｄ and includes - a, b, and c are each 1 d is zero, and thus, T ４ and V ４ do not exist. T １ 、 T ２ and T ３ are each independently selected from the group consisting of C １ to C １２ alkyl, (PEG) ｎ and (AA) ｐ and n is an integer from 1 to 10 p is an integer from 1 to 10 V １ , V ２ , and V ３ each is independently selected from the group consisting of -C(=O)- and -NR １１ -, R １１ is selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, or substituted aryl, the ADC according to any one of claims 24 to 34.

36. L １ is 【Chemical 218】 wherein 【Chemical 219】 represents the point of attachment to nitrogen of the pyridazine-pyrrolocoupling moiety, and * represents the point of attachment in any direction to the remainder of the linker, the ADC according to any one of claims 24 to 35.

37. Represented by formula (VI-7), 【Chemical 220】 In the formula, X １ is C or N, and the ADC according to any one of claims 24 to 36.

38. L ２ The ADC according to any one of claims 24 to 37, wherein L is a carbonyl group.

39. X １ is N, and W １ is MMAE, the ADC according to any one of claims 24 to 38.

40. The ADC according to any of claims 24 to 39, represented by formula (VIb-82): 【Chemical 221】

41. Said antibody is (i) VH CDR1, VH CDR2, and VH CDR3 as described in VH comprising the amino acid sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2, and VL CDR3 as described in VL comprising the amino acid sequence of SEQ ID NO: 26, or (ii) VH CDR1, VH CDR2, and VH CDR3 as described in VH comprising the amino acid sequence of SEQ ID NO: 44, and VL CDR1, VL CDR2, and VL CDR3 as described in VL comprising the amino acid sequence of SEQ ID NO: 45, or The ADC according to any one of claims 1 to 40, comprising VH CDR1, VH CDR2, and VH CDR3 described in VH comprising the amino acid sequence of SEQ ID NO: 62, and VL CDR1, VL CDR2, and VL CDR3 described in VL comprising the amino acid sequence of SEQ ID NO:

63.

42. The antibody is (a) (1)VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18, (2)VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24, and (3)VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20, and a VH region, and (b) (1)VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, (2)VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22, and (3)VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23, and a VL region, and the ADC according to any one of claims 1 to 41.

43. The antibody is (i)a VH region comprising VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL region comprising VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or (ii)a VH region comprising VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, and a VL region comprising VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or (iii)a VH region comprising VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL region comprising VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and 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, the ADC according to any one of claims 1 to 42.

44. The antibody is (a)(1) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 31, 34, 35, and 39, (2)a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 32, 36, 40, and 43, and (3)a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 33, 37, and 41, a VH region, and (b)(1) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, (2)a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22, and (3)a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 38, and 42, a VL region, the ADC according to any one of claims 1 to 41.

45. The antibody is (i)A VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 27, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 29, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 30, or (ii)A VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 10, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 30, or (iii)A VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 34, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 29, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 30, or (iv)A VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 35, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 38, or (v)A VH region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 39, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 40, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 41, and a VL region comprising a VL CDR1 containing the amino acid sequence of SEQ ID NO: 21, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 42, or The ADC according to any one of claims 1 to 41 and 44, comprising 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. **Claim 46** wherein the antibody is (a) (1) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 50, 53, 13, and 57, (2) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 54, 58, and 61, and (3) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 52, 55, and 59, a VH region, and (b) (1) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, (2) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22, (3) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 56, and 60, a VL region, the ADC according to any one of claims 1 to 41. **Claim 47** wherein the antibody is (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, comprising the ADC according to any one of claims 1 to 41 and 46.

48. The ADC according to any one of claims 1 to 46, wherein the antibody further comprises a framework 1 (FR1), framework 2 (FR2), framework 3 (FR3) and / or framework 4 (FR4) sequence described in any one of SEQ ID NOs: 25, 26, 44, 45, 62, and 63.

49. The ADC according to any one of claims 1 to 48, wherein the antibody further comprises a human framework sequence.

50. The antibody is (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)a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 63, the ADC according to any one of claims 1 to 49. **Claim 51** A pharmaceutical composition comprising the ADC according to any one of claims 1 to 50 and a pharmaceutically acceptable excipient. **Claim 52** The pharmaceutical composition according to claim 51, wherein the drug-to-antibody ratio of the antibody-drug conjugate is from about 1 to about 4. **Claim 53** The pharmaceutical composition according to claim 52, wherein the drug-to-antibody ratio is about 2. **Claim 54** A method for treating cancer or a tumor in a subject, the method comprising administering to the subject the antibody-drug conjugate according to any one of claims 1 to 49 or the pharmaceutical composition according to any one of claims 51 to 53.

Images