Antibody drug conjugate (ADC) targeting nectin 4 and comprising exatecan payload

Abstract

To provide an antibody-exatecan conjugate for treating cancer, a pharmaceutical composition comprising the conjugate, and a method for producing the conjugate.SOLUTION: Provided is an exatecan antibody conjugate, which is a conjugate of a compound illustrated by the following formula and a nectin-4 antibody.SELECTED DRAWING: None

Description

[Technical field]

[0001] (Reference to sequence listing) This application has been submitted with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file entitled "txt_P30825_FR", created on September 27, 2023, and is 7,935 bytes in size. The Sequence Listing information in ST.26 XML format is incorporated herein by reference in its entirety. [Background technology]

[0002] The present disclosure relates to the field of medicine. More particularly, the present disclosure relates to Nectin-4 antibody drug conjugates and pharmaceutical compositions thereof, and their use in the treatment of cancer.

[0003] Nectin-4 is a member of the nectin family, which is a Ca2+-independent immunoglobulin-like cell adhesion molecule. Unlike other members of the nectin family, nectin-4 expression in healthy tissues is mainly placental or embryonic, but nectin-4 is overexpressed in several tumor types, including urothelial, breast, lung, gastric, esophageal, colorectal, pancreatic, and ovarian cancer. Studies have linked high nectin-4 expression in several cancer types to tumor development.

[0004] Antibody drug conjugates (ADCs) containing anti-Nectin-4 antibodies have been generated, including ADCs containing a monomethyl auristatin E (MMAE) payload (WO201247724), ADCs with camptothecin analog payloads (WO2022112356 and WO2021151984), and ADCs containing an exatecan payload (WO2022207825).

[0005] ADCs for use in oncology are very difficult compounds to design because they must balance multiple aspects of the molecule, such as sufficient specificity for tumor targeting over healthy cells, maintaining the desired activity against bystander tumor cells while at the same time being acceptable toxicity, and controlling the instability of the payload to allow intracellular delivery while maintaining good physical and chemical stability.Specifically, one of the challenges in developing ADCs is to select a suitable linker for conjugating the payload to the antibody of the ADC.The chemical structure of the linker affects various properties of the ADC, including toxicity, specificity, stability, and efficacy.

[0006] Currently, there is one FDA-approved anti-nectin-4 ADC, enfortumumab vedotin-ejfv, which is marketed under the trade name PADCEV®. Enfortumumab vedotin is an ADC that contains a monoclonal anti-nectin-4 antibody and monomethyl auristatin-E (MMAE) as a cytotoxic payload. Unfortunately, enfortumumab vedotin exhibits some undesirable skin toxicity that may be at least partially related to the indiscriminate binding of enfortumumab to skin cells as opposed to targeted tumor cells. In addition, chronic administration of enfortumumab vedotin may result in the development of drug-resistant cancer in human patients. Cabaud et al. (Mol Cancer Ther 21(2022):1227-1235) found upregulation of ABCB1 expression, which encodes the multidrug resistance protein MDR 1 / P-glycoprotein (P-gp), in resistant tumors in a preclinical mouse model. Sensitivity of a resistant mouse model to anti-Nectin-4 antibody-MMAE conjugates could be restored in vitro and in vivo by the P-gp inhibitor tariquidar.

[0007] In particular, there remains a need for anti-nectin-4 ADCs that include a topoisomerase I payload. In particular, there remains a need for anti-nectin-4 ADCs that avoid or allow better management of dermatological events seen in certain anti-nectin-4 ADCs. In particular, there remains a need for anti-nectin-4 ADCs that avoid optic neuropathy and / or peripheral neuropathy signals seen in certain anti-nectin-4 ADCs. In particular, there remains a need for anti-nectin-4 ADCs that have low immunogenicity, stable in vivo pharmacokinetics, and suitable chemical and physical stability. Additionally, there remains a need for anti-nectin-4 ADCs that have one or more of the following characteristics: improved anti-tumor activity measured in certain tumor models, enhanced bystander activity against nectin-4-low tumors, lower immunogenicity, no measurable antibody effector function, and / or better physical and chemical stability.

[0008] Here, we disclose novel anti-nectin-4 ADCs that address one or more of these needs. The disclosed ADCs comprise an anti-nectin-4 antibody that exhibits selectivity for tumor-expressed nectin-4, and a novel linker-payload that comprises exatecan. We show that the disclosed anti-nectin-4 ADCs exhibit one or more of these improved properties related to aggregation, toxicity, specificity, stability, and efficacy. We also show that the novel anti-nectin-4 ADCs can be used to treat auristatin-resistant cancers, including cancers that are resistant to treatment with enfortumumab vedotin. Summary of the Invention

[0009] Provided herein are anti-Nectin-4 ADCs and compositions comprising anti-Nectin-4 ADCs. Also provided herein are methods of using anti-Nectin-4 ADCs or compositions comprising anti-Nectin-4 ADCs to treat cancer in a patient, e.g., a cancer that expresses Nectin-4.

[0010] In one aspect, provided herein is an ADC comprising an antibody that binds to human nectin-4 and a payload. In one aspect, provided herein is an ADC prepared by conjugating an antibody that binds to human nectin-4 to a payload. In the ADC, the antibody and the payload are covalently linked or conjugated, either directly or via a linker. In one aspect of the disclosed ADC, the antibody and the payload are covalently linked or conjugated via a linker. In one aspect, the linker provides a suitable stable bond or conjugation between the antibody and the payload when the ADC is administered systemically, and the linker provides suitable instability when the ADC is internalized by a target cell that expresses nectin-4, such that the payload is released from the ADC.

[0011] In one aspect, provided herein is an ADC comprising or prepared from an antibody that binds to human Nectin-4, wherein the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and wherein the LCVR comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, (a) HCDR1 comprises SEQ ID NO:9 (NYGMA), HCDR2 comprises SEQ ID NO:10 (FISNLAYGINYADTVTG), HCDR3 comprises SEQ ID NO:11 (GARATGWFAY), LCDR1 comprises SEQ ID NO:12 (KASQNVDTHVA), LCDR2 comprises SEQ ID NO:13 (SASYRYS), and LCDR3 comprises SEQ ID NO:14 (QQYNSYPLT) (all Kabat numbering); or (b) HCDR1 comprises SEQ ID NO: 15 (GFTFSNYG), HCDR2 comprises SEQ ID NO: 16 (ISNLAYGI), HCDR3 comprises SEQ ID NO: 17 (ARGARATGWFAY), LCDR1 comprises SEQ ID NO: 18 (QNVDTH), LCDR2 comprises SEQ ID NO: 19 (SAS), and LCDR3 comprises SEQ ID NO: 20 (QQYNSYPLT) (all IMGT numbering).

[0012] In a further aspect, provided herein is an ADC comprising or prepared from an antibody that binds to human Nectin-4, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the HCVR comprises SEQ ID NO:7 and the LCVR comprises SEQ ID NO:8.

[0013] In another aspect, provided herein is an ADC comprising or prepared from an antibody that binds to human nectin-4, wherein the antibody comprises one or more mutations in the antibody constant region that alter Fc effector function as disclosed herein, e.g., a mutation in the constant region that results in reduced binding of the antibody to an Fc receptor. In some embodiments, the antibody comprises an IgG1 heavy chain (HC) that comprises one or more mutations selected from L234F, L235E, and P331S (EU numbering), or one or more mutations selected from L247F, L248E, and P350S (Kabat numbering). In some embodiments, the antibody comprises an IgG1 heavy chain (HC) that comprises each of the mutations L234F, L235E, and P331S (EU numbering), or each of the mutations L247F, L248E, and P350S (Kabat numbering). In some embodiments, the antibody comprises a heavy chain (HC) that comprises one or more mutations selected from L234F, L235E, and P331S relative to the wild-type sequence provided in SEQ ID NO: 2. In some embodiments, the antibody comprises a heavy chain that comprises each of the mutations L234F, L235E, and P331S relative to the wild-type sequence provided in SEQ ID NO: 2. In some embodiments, the antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 3.

[0014] In another aspect, provided herein is an ADC comprising or prepared from an antibody that binds to human Nectin-4, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO:5 and the LC comprises SEQ ID NO:6.

[0015] In another aspect, provided herein is an ADC comprising a Nectin-4 antibody conjugated directly or via a linker to a payload, which may comprise a cytotoxic agent such as exatecan or an analog thereof. In some embodiments, the Nectin-4 antibody is conjugated to the payload via the thiol group of one or more cysteine ​​residues.

[0016] In some embodiments, the ADC comprises or is prepared from an anti-Nectin-4 antibody conjugated via a thiol group of one or more cysteine ​​residues present in the HC of the antibody, the cysteine ​​residues optionally being present in the constant region of the HC. In some embodiments, the cysteine ​​residues are present in the HC at one or more positions selected from C220, C226, C229 (EU index numbering) and combinations thereof, or at a position selected from C233, C239, C242 and combinations thereof (Kabat numbering). In some embodiments, the anti-Nectin-4 antibody is conjugated via each of the thiol groups of the cysteine ​​residues present in the HC at positions C220, C226, C229 (EU index numbering), or C233, C239, C242 (Kabat numbering). In some embodiments, the anti-Nectin-4 antibody is conjugated via a thiol group of one or more cysteine ​​residues present in the HC of the antibody, the HC having the amino acid sequence of SEQ ID NO: 5, the cysteine ​​residues present in the HC at positions selected from C222, C228, C231, and combinations thereof, and optionally, the anti-Nectin-4 antibody is conjugated via a thiol group of each of the cysteine ​​residues present at positions C222, C228, and C231. In some embodiments, the anti-Nectin-4 antibody is conjugated via a thiol group of a cysteine ​​residue present in the LC. In some embodiments, the anti-Nectin-4 antibody is conjugated via a thiol group of a cysteine ​​residue present at the C-terminus of the LC, and optionally, the LC is a kappa light chain and the cysteine ​​residue is present at position C214. In some embodiments, the anti-Nectin-4 antibody is conjugated via a thiol group of one or more cysteine ​​residues present in the LC of the antibody, the LC having the amino acid sequence of SEQ ID NO: 6, and the cysteine ​​residue is at position C214 in the LC.In some embodiments, the anti-Nectin-4 antibody is conjugated through the thiol group of a cysteine ​​residue present at positions C220, C226, C229 (EU index numbering) or C233, C239, C242 (Kabat numbering) in the HC, and through the thiol group of a cysteine ​​residue present at position C214 in the LC. In some embodiments, the anti-Nectin-4 antibody is conjugated through the thiol group of a cysteine ​​residue present in the HC, the HC having the amino acid sequence of SEQ ID NO: 5, and the cysteines present at positions C222, C228, and C231, and the Nectin-4 antibody is conjugated through the thiol group of a cysteine ​​residue present in the LC, the LC having the amino acid sequence of SEQ ID NO: 6, and the cysteine ​​present at position C214.

[0017] In a further aspect, provided herein is an ADC comprising or prepared from (i) an antibody that binds to human Nectin-4, (ii) optionally a linker, and (iii) a payload. In a further aspect, provided herein is an ADC comprising an antibody that binds to human Nectin-4, wherein the antibody is conjugated to the payload directly or via a linker. In a further aspect, provided herein is an ADC prepared by conjugating an antibody that binds to human Nectin-4 to a payload, either directly or indirectly via a linker.

[0018] In a further aspect, provided herein is an ADC comprising or prepared from (i) an antibody that binds to human nectin-4, (ii) optionally a linker, and (iii) exatecan or an analogue thereof. In a further aspect, provided herein is an ADC comprising an antibody that binds to human nectin-4, wherein the antibody is conjugated to exatecan or an analogue thereof directly or via a linker. In a further aspect, provided herein is an ADC prepared by conjugating an antibody that binds to human nectin-4 and exatecan or an analogue thereof, either directly or indirectly via a linker.

[0019] In a further aspect, provided herein is an ADC comprising an antibody that binds to human Nectin-4, optionally a linker, and a compound of Formula (P), which in some aspects may be referred to as a "payload."

[0020] [ka]

[0021] The compound of formula (P) may be referred to as exatecan.

[0022] In a further aspect, provided herein is an ADC comprising an antibody that binds to human Nectin-4 conjugated directly or via a linker to a payload of formula (P). In a further aspect, provided herein is an ADC prepared by conjugating an antibody that binds to human Nectin-4, either directly or indirectly via a linker, and a payload of formula (P).

[0023] In further aspects, provided herein are compounds that, in some aspects, may be utilized as linker-payloads for ADCs. In one aspect, disclosed herein are compounds of formula (LP) that may be utilized as linker-payloads for the ADCs disclosed herein:

[0024] [ka] In the formula, m is selected from 1 to 6, n is selected from 1 to 6, and p is selected from 1 to 20.

[0025] In some embodiments of the linker-payload of formula (LP), m is 2.

[0026] In some embodiments of the linker-payload of formula (LP), n is 2.

[0027] In some embodiments of the linker-payload of formula (LP), p is 10.

[0028] In a further aspect, provided herein is an ADC comprising an antibody that binds to human Nectin-4 and a compound of Formula (LP), or an ADC prepared by conjugating an antibody that binds to human Nectin-4 and a compound of Formula (LP).

[0029] In another aspect, provided herein are compounds of formula (L-P') that can be used as linker-payloads for the ADCs disclosed herein.

[0030] [ka]

[0031] In another embodiment, the compound: (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)acetamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)- Provided herein is 11,14,17,20,23,26,29,32,35,38-decamethyl-1,6,10,13,16,19,22,25,28,31,34,37,40-tridecaoxo-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecaazatetracontan-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, which in some embodiments may be utilized as a linker-payload for the ADCs disclosed herein.

[0032] In a further aspect, provided herein is an ADC comprising an antibody that binds to human Nectin-4 conjugated to a linker-payload of formula (LP). In a further aspect, provided herein is an ADC prepared by conjugating an antibody that binds to human Nectin-4 and a linker-payload of formula (LP).

[0033] In one aspect, provided herein is an antibody-drug conjugate (ADC) of the following formula (ADC):

[0034] [ka] In the formula, Ab is an antibody that binds to Nectin-4 disclosed herein, m is selected from 1 to 6, n is selected from 1 to 6, p is selected from 1 to 20, and DAR is a value in the range of 1 to 8 (inclusive) and represents the drug-antibody ratio (DAR) of the ADC.

[0035] In some embodiments of the ADC of formula (ADC), m is 2.

[0036] In some embodiments of the ADC of formula (ADC), n is 2.

[0037] In some embodiments of the ADC of formula (ADC), p is 10.

[0038] In some embodiments of the ADC of formula (ADC), the DAR is 8.

[0039] In some embodiments of an ADC of formula (ADC), at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC have a DAR of 8. In some embodiments of an ADC of formula (ADC), the ADC is present in a composition and at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC in the composition have a DAR of 8.

[0040] In some embodiments of the ADC of formula (ADC), the Ab is conjugated to the linker-payload via a thiol group of one or more cysteine ​​residues. In some embodiments, the Ab is conjugated via a thiol group of one or more cysteine ​​residues in the HC of the Ab, optionally where the HC has the amino acid sequence of SEQ ID NO:5, and the cysteine ​​is selected from positions C222, C228, C231, and combinations thereof. In some embodiments, the Ab is conjugated via a thiol group of one or more cysteine ​​residues in the LC of the Ab, optionally where the LC has the amino acid sequence of SEQ ID NO:6, and the cysteine ​​is at position C214. In some embodiments, the Ab is conjugated via all of C222, C228, and C231 of the HC of SEQ ID NO:5, and C214 of the LC of SEQ ID NO:6.

[0041] In a further aspect, provided herein is an ADC comprising an antibody that binds to human nectin-4 conjugated to a linker-payload of formula (L-P'). In a further aspect, provided herein is an ADC prepared by conjugating an antibody that binds to human nectin-4 and a linker-payload of formula (L-P').

[0042] In one aspect, provided herein is an antibody-drug conjugate (ADC) of the following formula (ADC'):

[0043] [ka] In the formula, DAR is a value ranging from 1 to 8 (inclusive) and represents the drug-to-antibody ratio (DAR) of the ADC.

[0044] In some embodiments of the ADC of formula (ADC'), the DAR is 8.

[0045] In some embodiments of an ADC of formula (ADC'), at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC have a DAR of 8. In some embodiments of an ADC of formula (ADC'), the ADC is present in a composition and at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC in the composition have a DAR of 8.

[0046] In some embodiments of the ADC of formula (ADC'), the Ab is conjugated to the linker-payload via a thiol group of one or more cysteine ​​residues. In some embodiments, the Ab is conjugated via a thiol group of one or more cysteine ​​residues in the HC of the Ab, optionally where the HC has the amino acid sequence of SEQ ID NO:5, and the cysteine ​​is selected from positions C222, C228, C231, and combinations thereof. In some embodiments, the Ab is conjugated via a thiol group of one or more cysteine ​​residues in the LC of the Ab, optionally where the LC has the amino acid sequence of SEQ ID NO:6, and the cysteine ​​is at position C214. In some embodiments, the Ab is conjugated via all of C222, C228, and C231 of the HC of SEQ ID NO:5, and C214 of the LC of SEQ ID NO:6.

[0047] In another aspect, provided herein is an antibody-drug conjugate (ADC) of the following formula (ETx-22):

[0048] [ka] where Ab is an antibody that binds to Nectin-4 as disclosed herein. The Ab comprises a heavy chain (HC) and a light chain (LC), where the HC comprises SEQ ID NO:5 and the LC comprises SEQ ID NO:6. The Ab is conjugated to the linker-payload via the thiol groups of cysteine ​​residues including C222, C228, and C231 of the HC of SEQ ID NO:5 and C214 of the LC of SEQ ID NO:6.

[0049] In another aspect, provided herein is a composition comprising an anti-Nectin-4 ADC disclosed herein. In one aspect of the disclosed composition comprising an anti-Nectin-4 ADC, at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADCs in the composition have a DAR of 8.

[0050] In another aspect, provided herein is a pharmaceutical composition comprising an anti-Nectin-4 antibody disclosed herein and one or more pharma- ceutically acceptable carriers, diluents, or excipients. In another aspect, provided herein is a pharmaceutical composition comprising an anti-Nectin-4 ADC disclosed herein and one or more pharma- ceutically acceptable carriers, diluents, or excipients. In another aspect, provided herein is a pharmaceutical composition comprising an anti-Nectin-4 ADC disclosed herein, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADCs in the composition have a DAR of 8.

[0051] In another aspect, provided herein is a method of treating cancer, comprising administering to a patient in need of cancer treatment, such as a Nectin-4 expressing cancer, an effective amount of an anti-Nectin-4 ADC disclosed herein. In a further aspect, provided herein is a method of treating cancer, comprising administering to a patient in need of cancer treatment, an effective amount of an anti-Nectin-4 ADC disclosed herein, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer. In another aspect, provided herein is a method of treating cancer, comprising administering to a patient in need of cancer treatment, an effective amount of an anti-Nectin-4 ADC disclosed herein, wherein optionally the cancer is resistant to treatment with an ADC comprising a Nectin-4 binding agent and an auristatin, e.g., monomethylauristatin E (MMAE). In another aspect, provided herein is a method of treating cancer comprising administering to a patient in need of such treatment an effective amount of an anti-Nectin-4 ADC disclosed herein, wherein the patient has previously received enfortumab vedotin, optionally enfortumab vedotin-ejfv, which is commercially available under the trade name Padcev®.

[0052] In another aspect, provided herein is an ADC comprising a nectin-4 binding agent and exatecan for use in a method comprising administering the ADC to a patient suffering from a cancer disclosed herein, such as a nectin-4 expressing cancer. Optionally, the cancer is resistant to treatment with an ADC comprising a nectin-4 binding agent and an auristatin, e.g., monomethyl auristatin E (MMAE). In another aspect, provided herein is an ADC comprising a nectin-4 binding agent and exatecan for use in a method comprising administering the ADC to a patient suffering from a nectin-4 expressing cancer, wherein the patient has previously been administered enfortumab vedotin, optionally enfortumab vedotin-ejfv, which is commercially available under the trade name Padcev®.

[0053] In another aspect, provided herein is a pharmaceutical composition comprising a nectin-4 binding agent and exatecan for use in treating cancer, such as a nectin-4 expressing cancer. Optionally, the cancer is resistant to treatment with an ADC comprising a nectin-4 binding agent and an auristatin, e.g., monomethyl auristatin E (MMAE). In another aspect, provided herein is a pharmaceutical composition comprising a nectin-4 binding agent and exatecan for use in treating cancer, wherein the patient has previously received enfortumab vedotin, optionally enfortumab vedotin-ejfv, which is commercially available under the trade name Padcev®.

[0054] In another aspect, provided herein is the use of an ADC comprising a nectin-4 binding agent and exatecan for the manufacture of a medicament for the treatment of a patient suffering from a nectin-4 expressing cancer. Optionally, the cancer is resistant to treatment with an ADC comprising a nectin-4 binding agent and an auristatin, e.g., monomethyl auristatin E (MMAE). In another aspect, provided herein is the use of an ADC comprising a nectin-4 binding agent and exatecan for the manufacture of a medicament for the treatment of a patient suffering from a nectin-4 expressing cancer, the patient having previously been administered enfortumab vedotin, optionally enfortumab vedotin-ejfv, which is commercially available under the trade name Padcev®. [Brief description of the drawings]

[0055] [Figure 1] Schematic diagram of ETx-22. [Diagram 2] Comparative HIC of naked humanized 15A7.5 and ETx-22. [Diagram 3] Ex vivo stability evaluation. ETx-22 was incubated in mouse, cynomolgus monkey, or human serum. At the indicated times, ETx-22 was affinity captured via anti-human LC-kappa (mouse) or Fc-nectin-4 fragment (cynomolgus monkey and human) and drug-antibody ratios were measured by LC / MS. [Figure 4]ETx-22 in vitro cytotoxic activity. A dose range of ETx-22 (closed triangles) or an IgG1 isotype control coupled to the same linker payload (open diamonds) was incubated with HCT-116-2G10 clones expressing human Nectin-4 for 8 days. Viability was monitored by mitochondrial oxidation (cell titer blue reagent) using a BMG Labtech fluorometer. [Diagram 5] PK / PD characterization of ETx-22 ADC. NSG mice and TNBC PDX400-grafted NSG mice (n=3 per time point) were injected intravenously with 10 mg / kg ETx-22 when tumors averaged 150 mm3 (T0). At the indicated time points, terminal blood sampling was performed and plasma was prepared from naïve and PDX 400-grafted NSG mice. A, Pharmacokinetic analysis of ETx-22 (ADC + total antibody) by mesoscale discovery technology. B, In vivo stability measurements by LC / MS after affinity capture of ETx-22 with anti-human LC-kappa. C, Determination of circulating free exatecan concentrations in plasma by LC / MS. [Figure 6]ETx-22 mechanism of action. TNBC PDX400-implanted NSG mice (n=3 per time point) were injected intravenously with 10 mg / kg ETx-22 when tumors averaged 150 mm3 (T0). At the indicated time points, animals were euthanized and their tumors were collected, measured, and weighed. A, IHC analysis of ETx-22 invasion and pharmacodynamics in PDX400 tumors. Tumors were fixed and embedded in paraffin. ETx-22 (upper panel) was detected with rabbit anti-human IgG, which was revealed with secondary anti-rabbit IgG coupled to horseradish peroxidase and the ChromoMap DAB kit. Phosphorylated H2A.X (lower panel), a marker of topoisomerase I inhibitor activity, was detected with mouse anti-phospho-histone H2A.X, which was revealed with secondary rabbit anti-mouse IgG and tertiary anti-rabbit IgG coupled to horseradish peroxidase and the ChromoMap DAB kit. B, Western blot analysis of tumor lysates. ETx-22 was detected using goat anti-human IgG conjugated to horseradish peroxidase. C, LC / MS determination of the amount of exatecan per gram of tumor. D, Quantification of phosphorylated H2A.X positive cells. Slides from A were counted on a Hamamatsu scanner and the percentage of phosphorylated H2A.X positive cells was quantified using Tribun Calopix software. E, Tumor volume measured with a caliper (V=(L×W×H)×π / 6). [Figure 7] In vivo efficacy of ETx-22 in an MMAE-resistant TNBC cell line (SUM190). NSG mice (n=5 / group) were orthotopically xenografted bilaterally with MMAE-resistant SUM190 cells embedded in Matrigel. At the indicated times (black arrows), three different ADCs were injected intravenously: isotype control (8 mg / kg), ETx-22 at 2, 4, and 8 mg / kg, and enfortumumab vedotin at 4 and 8 mg / kg. The control group received ADC diluent. Tumor growth was monitored twice weekly (V(mm3)=L×W2×π / 6). [Figure 8]In vivo efficacy of ETx-22 in a PDX model (ES0201, esophageal cancer). Tumor fragments were implanted subcutaneously in BALB / c Nu mice. The indicated ADCs were injected intravenously twice at the indicated times (black arrows). Quick scores for Nectin-4 expression were determined by immunohistochemistry. Tumor growth was monitored twice weekly (V(mm3)=L×W2×π / 6). [Figure 9] In vivo efficacy of ETx-22 in a PDX model (BLCU003, bladder cancer). Tumor fragments were implanted subcutaneously in NMRI nude mice. The indicated ADCs were injected intravenously twice at the indicated times (black arrows). Quick scores for Nectin-4 expression were determined by immunohistochemical staining and are shown here. Tumor growth was monitored twice weekly (V(mm3)=L×W2×π / 6). [Figure 10] In vivo efficacy of ETx-22 in a PDX model (B521, bladder cancer). The bladder cancer PDX model B521 exhibits relatively high Nectin-4 expression and harbors a homozygous mutation in FGFR3 conferring resistance to erdafitinib. Cisplatin / gemcitabine was administered: 4mg / kg Q3W×2 / 60mg / kg QW×4. [Figure 11] In vivo efficacy of ETx-22 in a PDX model (HN2579, head and neck cancer). The head and neck PDX model HN2579 shows moderate to high Nectin-4 expression. NOD / SCID mice were subcutaneously implanted with tumor fragments. At the indicated times (black arrows), the indicated ADCs were injected intravenously twice. A quick score for Nectin-4 expression was determined by immunohistochemical staining and is shown here. Tumor growth was monitored twice weekly (V(mm3)=L×W2×π / 6). [Figure 12] In vivo efficacy of ETx-22 in PDX model (CV3035, cervical cancer). Tumor fragments were implanted subcutaneously in BALB / c Nu mice. The indicated ADCs were injected intravenously twice at the indicated times (black arrows). Quick scores for Nectin-4 expression were determined by immunohistochemical staining and are shown here. Tumor growth was monitored twice weekly (V(mm3)=L×W2×π / 6). [Figure 13]In vivo efficacy of ETx-22 in a PDX model (CV3560, cervical cancer). The cervical cancer PDX model CV3560 shows moderate to high Nectin-4 expression. [Figure 14] In vivo efficacy of ETx-22 in PDX model (OV2423, ovarian cancer). Tumor fragments were implanted subcutaneously in NOD / SCID mice. The indicated ADC was injected intravenously twice at the indicated times (black arrows). In each PDX model, a quick score for Nectin-4 expression was determined by immunohistochemical staining and is shown here. Tumor growth was monitored twice weekly (V(mm3)=L×W2×π / 6). [Figure 15] In vivo efficacy of ETx-22 in the TNBC PDX400 model. NSG mice were orthotopically implanted with tumor fragments bilaterally. At the indicated times (black arrows), the indicated ADCs were injected intravenously. Quick scores of Nectin-4 expression determined by immunohistochemical staining are shown. Bar = 500 μm. [Figure 16] In vivo efficacy of ETx-22 in the TNBC PDX317 model. NSG mice were orthotopically implanted with tumor fragments bilaterally. At the indicated times (black arrows), the indicated ADCs were injected intravenously. Quick scores of Nectin-4 expression determined by immunohistochemical staining are shown. Bar = 500 μm. [Figure 17] In vivo efficacy of ETx-22 in the TNBC PDX348 model. NSG mice were orthotopically implanted with tumor fragments bilaterally. At the indicated times (black arrows), the indicated ADCs were injected intravenously. Quick scores of Nectin-4 expression determined by immunohistochemical staining are shown. Bar = 500 μm. [Figure 18] In vivo efficacy of ETx-22 in the TNBC PDX434 model. The TNBC PDX434 model shows low Nectin-4 expression. NSG mice were orthotopically implanted with tumor fragments bilaterally. At the indicated times (black arrows), the indicated ADCs were injected intravenously. Quick scores of Nectin-4 expression determined by immunohistochemical staining are shown. Bar = 500 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] Nectin-4 As used herein, "human nectin-4" refers to the human nectin-4 protein or polypeptide, also known as poliovirus receptor-related 4, Ig superfamily receptor LNIR, or poliovirus receptor-related protein 4 (PVRL4). The amino acid sequence of human nectin-4 can be found in NP_112178.2 and is provided in SEQ ID NO:1.

[0057] antibody The disclosed ADCs include antibodies. As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0058] An exemplary antibody of the present disclosure is an immunoglobulin G (IgG) type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino terminal portion of each of the four polypeptide chains contains a variable region of about 100-125 or more amino acids that is primarily responsible for antigen recognition. The carboxy terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The IgG isotype can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0059] The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). The CDRs are exposed on the surface of the protein and are the regions of the antibody that are important for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3" and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3". The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs can be performed using the methods described in Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987), Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999;27:209-212). The CDRs of the present disclosure are determined according to North.

[0060] Certain antibodies of the ADCs described herein contain an IgG1 Fc region or an Fc region derived from human IgG1, e.g., a modified IgG1 Fc region with altered Fc effector functions. IgG1 is known to induce antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Some antibodies of the disclosed ADCs include amino acid substitutions introduced into the IgG1 Fc region that alter Fc effector functions. In some embodiments of the antibodies of the disclosed ADCs, mutations are introduced in the Fc region at positions 234 and 235 (EU index numbering) or 247 and 248 (Kabat numbering). In some embodiments of the antibodies disclosed herein, mutations are introduced in the Fc region at one or more positions selected from 234, 235, and 331 (EU index numbering) or 247, 248, and 350 (Kabat numbering). In some aspects, the anti-Nectin-4 antibodies of the present disclosure comprise a modified human IgG1 Fc region comprising one or more mutations selected from L234E, L235E, P331S, and combinations thereof (EU index numbering), or selected from L247F, L248E, P350S, and combinations thereof (Kabat numbering). In further aspects, these amino acid substitutions introduced into the IgG1 Fc region reduced or eliminated a measurable antibody effector function, including but not limited to, reduced binding to Fc receptors.

[0061] Expression of an antibody in a mammal from the IgG subclass may result in truncation of the C-terminal amino acids from one or both heavy chains, for example, in the case of an IgG1 antibody, one or two C-terminal amino acids may be removed. In the case of an IgG1 antibody, the C-terminal lysine, if present, may be truncated or trimmed from the heavy chain during expression. Additionally, the penultimate glycine may be truncated or trimmed from the heavy chain as well. The anti-Nectin-4 antibodies disclosed herein may accordingly include one or more truncations and truncations.

[0062] Expression of the antibody in a mammal may also result in modification of the N-terminal amino acid. For example, if the most N-terminal amino acid of the heavy or light chain is glutamine or glutamic acid, it may be changed to pyroglutamic acid. For example, if the most C-terminal amino acid of the heavy or light chain is lysine or glycine, it may be removed. The anti-Nectin-4 antibodies disclosed herein may include one or more modifications or removals accordingly.

[0063] The terms "nucleic acid" or "polynucleotide," as used interchangeably herein, refer to a polymer of nucleotides, including single- and / or double-stranded nucleotide-containing molecules, such as DNA, cDNA, and RNA molecules, that incorporate naturally occurring nucleotides, modified nucleotides, and / or nucleotide analogs. A polynucleotide of the present disclosure can also include substrates incorporated therein, for example, by a DNA or RNA polymerase or a synthetic reaction.

[0064] The polynucleotides of the present disclosure can be expressed in a host cell, for example, after the polynucleotide is operably linked to an expression control sequence. Expression control sequences capable of expressing an operably linked polynucleotide are well known in the art. For example, the expression vector can include a sequence encoding one or more signal peptides that facilitate secretion of the polypeptide from the host cell. The signal peptide can be, for example, an immunoglobulin signal peptide or a heterologous signal peptide. The expression vector containing the polynucleotide of interest (e.g., a polynucleotide encoding an antibody polypeptide) can be transferred into the host cell by well-known methods. Additionally, the expression vector can include one or more selection markers, such as, for example, tetracycline, neomycin, and dihydrofolate reductase, to aid in detection of host cells transformed with the desired polynucleotide sequence.

[0065] Host cells include cells stably or transiently transfected, transformed, transduced or infected with one or more expression vectors expressing all or a portion of the antibody of the present disclosure. According to some embodiments, host cells can be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing the HC polypeptide and an expression vector expressing the LC polypeptide of the antibody of the present disclosure. In some embodiments, host cells can be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing the HC and LC polypeptides of the antibody of the present disclosure. The antibodies of the present disclosure can be produced in mammalian cells, such as CHO, NS0, HEK293 or COS cells, according to techniques well known in the art.

[0066] The medium into which the antibody of the present disclosure is secreted can be purified by conventional techniques, such as mixed-mode methods of ion exchange and hydrophobic interaction chromatography. For example, the medium can be applied to and eluted from a Protein A or Protein G column using conventional methods, and mixed-mode methods of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, refrigerated, or lyophilized, for example at -70°C. Various methods of protein purification can be employed, such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0067] Anti-Nectin-4 antibody The antibodies for use in the disclosed ADCs exhibit specific binding to Nectin-4 relative to other proteins of the human Nectin family, such as Nectin-1. As used herein, the terms "binding" and "specific binding" refer to the binding of an antibody to an epitope of the Nectin-4 antigen. A measure of the binding strength of an antibody is called affinity. Methods for determining such binding and / or affinity using in vitro assays are known to those skilled in the art. According to the present invention, detection by flow cytometry, immunohistochemistry, and / or fluorescence are described and are particularly preferred herein. The affinity of binding of an antibody to an antigen is measured by the K a (rate constant for the association of the antibody from the antibody / antigen complex), K D (dissociation constant), and K dis (K D / K a ) can be defined by the term.

[0068] In some embodiments, the disclosed ADCs may comprise or be prepared from an anti-nectin-4 antibody that is a tumor-selective anti-nectin-4 antibody. A tumor-selective anti-nectin-4 antibody may be characterized as an anti-nectin-4 antibody that binds to nectin-4 expressed by tumors with higher affinity compared to nectin-4 expressed by normal cells. For comparison, in some embodiments, the binding affinity may be detected by flow cytometry, immunohistochemistry, and / or fluorescence. The normal cells may comprise normal human endothelial keratinocytes (NHEKs). The normal cells may comprise nectin-4-expressing human endothelial keratinocytes differentiated in vitro. In one embodiment, the tumor-selective anti-nectin-4 antibody has a dissociation constant K for binding to nectin-4 expressed on keratinocytes that is greater than the dissociation constant for binding to nectin-4 expressed on tumor cells. DIn one aspect, the tumor-selective anti-Nectin-4 antibody exhibits lower internalization and / or cytotoxic activity against keratinocytes compared to tumor cells. In one aspect, the tumor-selective anti-Nectin-4 antibody exhibits lower binding affinity, lower internalization, and lower cytotoxic activity against keratinocytes compared to tumor cells. In one aspect, the anti-Nectin-4 antibody of the disclosed ADC exhibits lower binding affinity, internalization, or cytotoxic activity against keratinocytes compared to tumor cells compared to the reference antibody HA22 mAb (Enfortumab).

[0069] Tumor-selective anti-Nectin-4 antibodies for use in the disclosed ADCs may include the antibodies disclosed in WO 2022 / 207822 and WO 2022 / 207825. In one aspect, the ADCs disclosed herein include a monoclonal antibody designated 15A7.5, or a humanized variant thereof designated H1L2_15A7.5, comprising the back mutations disclosed in WO 2022 / 207822 and WO 2022 / 207825, and having a HC comprising the amino acid sequence of SEQ ID NO:5 and a LC comprising the amino acid sequence of SEQ ID NO:6.

[0070] In one aspect, provided herein is an ADC comprising or prepared from an antibody that binds to human Nectin-4, wherein the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and wherein the LCVR comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 9 (NYGMA), HCDR2 comprises SEQ ID NO: 10 (FISNLAYGINYADTVTG), HCDR3 comprises SEQ ID NO: 11 (GARATGWFAY), LCDR1 comprises SEQ ID NO: 12 (KASQNVDTHVA), LCDR2 comprises SEQ ID NO: 13 (SASYRYS), and LCDR3 comprises SEQ ID NO: 14 (QQYNSYPLT) (all Kabat numbering). In another aspect, provided herein is an antibody that binds to human Nectin-4, wherein HCDR1 comprises SEQ ID NO: 15 (GFTFSNYG), HCDR2 comprises SEQ ID NO: 16 (ISNLAYGI), HCDR3 comprises SEQ ID NO: 17 (ARGARATGWFAY), LCDR1 comprises SEQ ID NO: 18 (QNVDTH), LCDR2 comprises SEQ ID NO: 19 (SAS), and LCDR3 comprises SEQ ID NO: 20 (QQYNSYPLT) (all IMGT numbering).

[0071] In some aspects, the ADCs disclosed herein comprise or are prepared from an anti-Nectin-4 antibody, wherein the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the antibody comprises a HCVR comprising SEQ ID NO:7 and a LCVR comprising SEQ ID NO:8.

[0072] In a further aspect, the ADC disclosed herein comprises or is prepared from an anti-Nectin-4 antibody, the antibody comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), the antibody having a human IgG1 or IgG4 isotype. In a further aspect, the anti-Nectin-4 antibody has a human IgG1 isotype. In a further aspect, the anti-Nectin-4 antibody has a human IgG1 isotype, the HC of the anti-Nectin-4 antibody comprises one or more mutations at positions L234, L235, and P331 (EU index numbering) or L247, L248, P350 (Kabat numbering). In a further aspect, the anti-Nectin-4 antibody comprises one or more mutations selected from L234F, L235E, and P331S (EU index numbering) or L247F, L248E, P350S (Kabat numbering).

[0073] In one aspect, provided herein is an ADC comprising or prepared from an antibody that binds to human Nectin-4, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO:5 and the LC comprises SEQ ID NO:6.

[0074] In a further aspect, provided herein is an ADC comprising or prepared from an antibody that binds to human Nectin-4, wherein the HC consists of SEQ ID NO:5 and the LC consists of SEQ ID NO:6.

[0075] In another aspect, provided herein is a mammalian cell comprising a DNA molecule comprising a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:5, the amino acid sequence of SEQ ID NO:6, or both the amino acid sequence of SEQ ID NO:5 and the amino acid sequence of SEQ ID NO:6. In some aspects, the cell is capable of expressing a Nectin-4 antibody disclosed herein.

[0076] In another aspect, provided herein is a mammalian cell comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule comprises a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:5, and the second DNA molecule comprises a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6.

[0077] In another aspect, provided herein is a process for producing a Nectin-4 antibody, comprising culturing one of the mammalian cells disclosed herein under conditions such that the antibody is expressed, and recovering the expressed antibody.

[0078] In another aspect, provided herein is an antibody produced by culturing a mammalian cell containing a DNA molecule comprising a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or both SEQ ID NO:5 and SEQ ID NO:6 under conditions such that the antibody is expressed, and recovering the expressed antibody.

[0079] In another aspect, provided herein is an antibody produced by culturing a mammalian cell comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule comprises a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:5, and the second DNA molecule comprises a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6, under conditions such that the antibody is expressed, and recovering the expressed antibody.

[0080] As used herein, the term "enfortumab" refers to a fully human anti-nectin-4 IgG1 kappa monoclonal antibody having the sequence disclosed in WO2012047724 (Figures 3A and 3B), expressed and purified using standard conditions. Enfortumab may alternatively be referred to herein as HA22 mAb. The term enfortumab-vedotin-ejfv refers to an antibody-drug conjugate sold under the trade name PADCEV®, which comprises enfortumab conjugated to a monomethyl auristatin E (MMAE) payload via a peptide linker, the linker-payload having the formula represented as maleimidocaproyl (MC)-valine-citrulline-PABC-MMAE.

[0081] payload The disclosed Nectin-4 antibodies can be conjugated to various payloads (including pharma- ceutically acceptable salts thereof) to form antibody drug conjugates (ADCs). In one aspect, the disclosed ADCs comprise or are prepared from (i) an antibody that binds to human Nectin-4, (ii) optionally a linker, and (iii) a payload. In a further aspect, the disclosed ADCs comprise an antibody that binds to human Nectin-4 conjugated directly or via a linker to a payload. In a further aspect, the disclosed ADCs are prepared by conjugating an antibody that binds to human Nectin-4 to a payload, either directly or indirectly via a linker. In a further aspect, the disclosed ADCs release the payload after the ADC binds to a target cell expressing Nectin-4 and the ADC is internalized by the target cell.

[0082] Moieties suitable for conjugation to the Nectin-4 antibodies disclosed herein include cytotoxic agents (e.g., chemotherapeutic agents), prodrug-converting enzymes, radioisotopes or compounds, toxins, and other payloads known in the art.

[0083] Exemplary ADCs herein utilize an exatecan-based payload (e.g., exatecan or an exatecan analog). Exatecan is a topoisomerase I (TOPO1) inhibitor that has been shown to have anticancer activity. Exatecan and its analogs bind to the TOPO1 / DNA complex, preventing the reannealing of partially cleaved DNA, the accumulation of which leads to cell death.

[0084] In a further aspect, the disclosed ADCs comprise an antibody that binds to human nectin-4, optionally a linker, and exatecan or an analog thereof. In a further aspect, the disclosed ADCs comprise an antibody that binds to human nectin-4 that is conjugated directly or via a linker to exatecan or an analog thereof. In a further aspect, the disclosed ADCs are prepared by conjugating an antibody that binds to human nectin-4 and exatecan or an analog thereof, either directly or indirectly via a linker. In the disclosed ADCs, exatecan is conjugated to its free NH 2 via a linker through a group (e.g., the NH 2 The Nectin-4 antibody may be conjugated to the Nectin-4 antibody either directly or indirectly (through a group).

[0085] In a further aspect, the disclosed ADCs comprise or are prepared from (i) an antibody that binds to human Nectin-4, (ii) optionally a linker, and a compound of formula (P), which may be referred to as a "payload."

[0086] [ka]

[0087] The compound of formula (P) may be referred to as exatecan.

[0088] In further aspects, the disclosed ADCs comprise an antibody that binds to human Nectin-4 conjugated directly or via a linker to a payload of formula (P). In further aspects, the disclosed ADCs are prepared by conjugating, either directly or indirectly via a linker, an antibody that binds to human Nectin-4 and a payload of formula (P).

[0089] Linker As disclosed herein, the payload can be conjugated to the Nectin-4 antibody by methods understood by those skilled in the art to form the Nectin-4 ADCs described herein. One example of such a conjugate includes connecting the payload described herein to the Nectin-4 antibody described herein via a linker.

[0090] The linker used in ADC is designed for stability in plasma to allow time for the ADC to localize to target cells. If the payload is released too early, it will reduce the therapeutic index of the ADC by damaging all kinds of non-target tissues. Once the ADC is internalized in the target cells, the linker should provide a mechanism for payload release so that the payload can function as designed.

[0091] Linkers known to those skilled in the art include, for example, cleavable and non-cleavable moieties. Thus, provided herein are ADCs in which a payload, for example, exatecan or an exatecan analog, is conjugated to an anti-Nectin-4 antibody via a linker having a cleavable moiety or is conjugated to the antibody via a linker having a non-cleavable moiety.

[0092] Any suitable linker known in the art can be used in preparing the ADC of the present disclosure.In certain embodiments, the linker comprises a reactive group that can be conjugated to both the antibody of the present disclosure and to a payload.Examples include, but are not limited to, the linker that comprises a maleimide group that can be conjugated to the thiol of the cysteine ​​residue of the antibody, and a succinimide ester group that can be conjugated to a payload, such as 3-(maleimido)propionic acid N-hydroxysuccinimide ester.

[0093] In certain aspects of the disclosure, the ADCs include a linker having a cleavable moiety. The cleavable moiety can include a beta-glucuronide that is cleavable by intracellular beta-glucuronidase.

[0094] In some aspects of the disclosure, the ADCs comprise a linker comprising a spacer unit, referred to herein as spacer unit A, that connects a cysteine ​​of an antibody disclosed herein to a payload as described herein. Some of the chemistries used in the art to conjugate a payload to a thiol group of a cysteine ​​include maleimide or succinimide chemistries and can be utilized in the ADCs of the disclosure. In further aspects of the disclosure, a maleimide-type spacer, such as maleimidopropionyl, is utilized in the linkers disclosed herein.

[0095] In some aspects of the disclosure, provided herein are ADCs having a spacer unit A of the formula:

[0096] [ka] where z is 1-5. Spacer unit A is conjugated to the anti-Nectin-4 antibody via a reduced maleimidyl group. Spacer unit A may be conjugated to the payload either directly or indirectly via the linker through a methylene moiety (i.e., (CH2)z) moiety. In some embodiments of spacer unit A, z is 2. In some embodiments, spacer unit A "spaces" or "positions" the remainder of the linker and the payload distally from the antibody of the ADC.

[0097] Linkers including Polysarcosine (PSAR) and Polyethylene Glycol (PEG). In some embodiments, the disclosed ADCs include a linker that includes a polysarcosine moiety. ADCs that include a polysarcosine moiety are known in the art. (See WO 2019 / 081455 and WO 2022 / 207699, and Conilh et al. "Exatecan antibody drug conjugates based on a hydrophilic polysarcosine drug-linker platform" (Pharmaceuticals 14 (2021), 247).

[0098] In some embodiments, the disclosed linkers include at least one ethylene glycol unit, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more ethylene glycol units. Further preferred linkers include highly polar spacers, such as acyl groups, carbamoyl groups, and / or sulfamide groups, appended to at least one ethylene glycol unit, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more ethylene glycol units.

[0099] In certain embodiments, the linker is a hydrophilic polysarcosine linker, e.g., comprising up to 20 sarcosine units and at least one ethylene glycol unit, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more ethylene glycol units, and the linker is subject to cleavage by glycosidases, in particular, by glucuronidase.

[0100] In certain embodiments, the linker is a hydrophilic polysarcosine linker, e.g., comprising about 8-12 sarcosine units and at least one ethylene glycol unit, e.g., up to 10 ethylene glycol units, and the linker is subject to cleavage by a glycosidase, in particular, by a glucuronidase.

[0101] In certain embodiments, the linker is a hydrophilic polysarcosine linker comprising 10 sarcosine units and 2 ethylene glycol units, and the linker is subject to cleavage by glucuronidase.

[0102] A further aspect of the invention relates to a linker-drug conjugate comprising: (i) a hydrophilic polysarcosine linker comprising, for example, about 8-12 sarcosine units and at least one ethylene glycol unit, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more ethylene glycol units, where the linker is subject to cleavage by a glycosidase, in particular to cleavage by a glucuronidase, and the linker comprises a thiol-reactive group, for example a maleimide group, capable of reacting with a cysteine ​​residue on an antibody or antigen-binding fragment thereof; and (ii) exatecan covalently attached to the linker.

[0103] In certain embodiments, the linker-drug conjugate comprises (i) a hydrophilic polysarcosine group (e.g., a polysarcosine group consisting of 10 sarcosine units) and a polyethylene group (e.g., a polyethylene glycol group consisting of two ethylene glycol units), where the linker is subject to cleavage by glycosidases, in particular glucuronidases, and the linker comprises a maleimide group capable of reacting with a cysteine ​​residue on an antibody or antigen-binding fragment thereof, and (ii) exatecan covalently attached to the linker. In some embodiments, the polysarcosine group is located at a position in the linker that is characterized as "orthogonal" to the polyethylene glycol group and the exatecan.

[0104] Self-Immolative Unit Self-immolation, or self-removal, of some ADCs can be designed into the structure of the ADC, for example, into the linker of the ADC. Self-immolation typically involves a self-immolative unit that is activated after removal of a trigger group that is conjugated to the self-immolative unit. In some embodiments of the self-immolative unit, the trigger group is enzymatically cleaved from the self-immolative unit via cellular enzymes after the ADC is internalized by the target cell. Suitable trigger groups can include beta-glucuronides that are cleaved from the self-immolative unit via intracellular beta-glucuronidase. Further biological or chemical reactions then result in the spontaneous elimination of the self-immolative unit itself from the linker. The self-immolative unit can provide favorable properties to the ADC, such as providing space to reduce steric hindrance of cellular proteases reaching peptide cleavage sites within the ADC.

[0105] In some embodiments of the present disclosure, the ADCs described herein contain a self-immolative unit. When present, the self-immolative unit may be located within a linker and may be activated after enzymatic cleavage of a trigger group from the self-immolative unit. In some embodiments, the self-immolative unit comprises a 3-nitro-octopamine group conjugated to a beta-glucuronide via its 4-hydroxyl group, the beta-glucuronide being cleavable via intracellular beta-glucuronidase expressed by tumors.

[0106] Conjugation In the disclosed ADCs, the payload, particularly exatecan or exatecan analog, is covalently conjugated to the anti-nectin-4 antibody either directly or indirectly via a linker. Methods for conjugating the antibodies disclosed herein to the payloads disclosed herein are known in the art. In some methods, the antibody is conjugated to the linker in a first reaction, and then the antibody and linker are conjugated to the payload in a second reaction. In some methods, the antibody is conjugated to the payload or payload / linker in one reaction.

[0107] The payload can be conjugated to any suitable position of the anti-Nectin-4 antibody that does not abolish the binding of the antibody to Nectin-4. For example, the payload can be conjugated to a reactive amino acid residue on the antibody, such as a cysteine ​​residue bearing a thiol group.

[0108] In certain embodiments, the ADC has a drug-antibody / antibody fragment molar ratio (DAR) greater than 1, i.e., two or more drug molecules are attached to the antibody / antibody fragment. Typically, the conjugate has a DAR of about 2:1 to about 16:1, specifically about 4:1 to about 10:1, more specifically about 6:1 to about 8:1. The DAR can be calculated from a statistical distribution according to known methods. In some embodiments, the ADC has a DAR of 8:1. In some embodiments, the ADC has a DAR of 8, or the ADC has a substantially uniform DAR of 8, e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the ADC have a DAR of 8.

[0109] In some embodiments, it may be desirable to obtain a composition comprising an ADC (or population of ADCs) with a substantially uniform DAR, where at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADCs have a selected DAR between 1 and 8. In some embodiments of the disclosed ADCs and compositions comprising disclosed ADCs, the composition comprising the ADC has a substantially uniform DAR, where at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADCs have a DAR selected from 2, 4, 6, or 8. In some embodiments of the disclosed ADCs and compositions comprising disclosed ADCs, the composition comprising the ADC has a substantially uniform DAR, where at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADCs have a DAR of 8.

[0110] A composition comprising a disclosed ADC (or population of ADCs) can include ADCs with an average DAR. In some embodiments, a composition comprising a disclosed ADC (or population of ADCs) includes ADCs with an average DAR that is a value within the range of 1 to 8, inclusive. In some embodiments, the average DAR is greater than about 1, 2, 3, 4, 5, 6, or 7.

[0111] Anti-Nectin-4 ADCs with Exatecan Payloads In a further aspect, provided herein is an ADC comprising an antibody that binds to human nectin-4 and an exatecan payload. In some aspects, the ADC comprises or is prepared from a compound of the following formula (LP), which may be utilized as a linker-payload:

[0112] [ka] In the formula, m is selected from 1 to 6, n is selected from 1 to 6, and p is selected from 1 to 20.

[0113] In some embodiments of the linker-payload of formula (LP), m is 2.

[0114] In some embodiments of the linker-payload of formula (LP), n is 2.

[0115] In some embodiments of the linker-payload of formula (LP), p is 10.

[0116] In some embodiments, the ADC comprises or is prepared from a compound of the following formula (L-P'), which can be utilized as a linker-payload:

[0117] [ka]

[0118] In some embodiments, the disclosed ADCs are selected from the group consisting of the compound: (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)acetamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b ]quinolin-1-yl)amino)-11,14,17,20,23,26,29,32,35,38-decamethyl-1,6,10,13,16,19,22,25,28,31,34,37,40-tridecaoxo-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecaazatetracontan-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, which may be utilized as a linker-payload.

[0119] In one aspect, the ADC disclosed herein is of the formula (ADC):

[0120] [ka] In the formula, Ab is an antibody that binds to Nectin-4 disclosed herein, m is selected from 1 to 6, n is selected from 1 to 6, p is selected from 1 to 20, and DAR is 1 to 8, representing the drug-to-antibody ratio (DAR) of the ADC.

[0121] In some embodiments of the ADC of formula (ADC), m is a value large enough that the linker-payload is sufficiently spaced from the Ab portion of the ADC so as not to impair the activity of the Ab, which may include binding and binding affinity to tumor-specific Nectin-4. In some embodiments of the ADC of formula (ADC), m is greater than 1, preferably m is 2.

[0122] In some embodiments of the ADC of formula (ADC), m is small enough so as not to impair the stability of the linker-payload. In some embodiments of the ADC of formula (ADC), m is less than 6, 5, 4, or 3, preferably m is 2.

[0123] In some embodiments of the ADC of formula (ADC), n is a value large enough to provide sufficient hydrophilicity and reduce aggregation of the ADC. In some embodiments of the ADC of formula (ADC), n is greater than 1, preferably n is 2.

[0124] In some embodiments of the ADC of formula (ADC), n is small enough so as not to impair the stability of the linker-payload. In some embodiments of the ADC of formula (ADC), n is less than 6, 5, 4, or 3, preferably n is 2.

[0125] In some embodiments of the ADC of formula (ADC), p is a value large enough to provide sufficient hydrophilicity and reduce aggregation of the ADC. In some embodiments of the ADC of formula (ADC), p is greater than 1, 2, 3, 4, 5, 6, 7, 8, or 9, preferably p is 10.

[0126] In some embodiments of the ADC of formula (ADC), p is small enough so as not to impair the stability of the linker-payload. In some embodiments of the ADC of formula (ADC), p is less than 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11, and preferably p is 10.

[0127] In some embodiments of the ADC of formula (ADC), the DAR is sufficiently large to provide the ADC with sufficient activity, e.g., sufficient toxicity. In some embodiments of the ADC of formula (ADC), the DAR is greater than 1, 2, 3, 4, 5, 6, 7, and preferably the DAR is 8.

[0128] In some embodiments of an ADC of Formula (ADC), at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC have a DAR of 8. In some embodiments, an ADC of Formula (ADC) is present in a composition and at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC in the composition have a DAR of 8. In some embodiments, an ADC of Formula (ADC) is present in a composition and the ADC present in the composition has an average DAR of greater than about 1, 2, 3, 4, 5, 6, or 7.

[0129] In some embodiments of the ADC of formula (ADC), the Ab is conjugated to the linker-payload via the thiol group of one or more cysteine ​​residues. In some embodiments, the Ab is conjugated via the thiol group of one or more cysteine ​​residues in the HC of the Ab, where the HC optionally has the amino acid sequence of SEQ ID NO:5, and the cysteine ​​is selected from C222, C228, C231, and combinations thereof. In some embodiments, the Ab is conjugated via the thiol group of one or more cysteine ​​residues in the LC of the Ab, where the LC optionally has the amino acid sequence of SEQ ID NO:6, and the cysteine ​​is C214. In some embodiments of the ADC of formula (ADC), the Ab comprises a HC comprising the amino acid sequence of SEQ ID NO:5 and a LC comprising the amino acid sequence of SEQ ID NO:6, and the Ab is conjugated to the linker-payload via the thiol group of cysteine ​​residues including all of C222, C228, C231 of the HC, and C214 of the LC.

[0130] In some embodiments, the ADC disclosed herein is of the following formula (ADC'):

[0131] [ka] In the formula, DAR ranges from 1 to 8 and represents the drug-to-antibody ratio (DAR) of the ADC.

[0132] In some embodiments of the ADC of formula (ADC'), the DAR is large enough to provide sufficient toxicity to the DAR. In some embodiments of the ADC of formula (ADC), the DAR is greater than 1, 2, 3, 4, 5, 6, 7, and preferably the DAR is 8.

[0133] In some embodiments of an ADC of formula (ADC'), at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC have a DAR of 8. In some embodiments of an ADC of formula (ADC'), the ADC is present in a composition and at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC in the composition have a DAR of 8. In some embodiments, an ADC of formula (ADC') is present in a composition and the ADC present in the composition has an average DAR of greater than about 1, 2, 3, 4, 5, 6, or 7.

[0134] In some embodiments of the ADC of formula (ADC'), the Ab is conjugated to the linker-payload via a thiol group of one or more cysteine ​​residues. In some embodiments, the Ab is conjugated via a thiol group of one or more cysteine ​​residues in the HC of the Ab, where the HC optionally has the amino acid sequence of SEQ ID NO:5, and the cysteine ​​is selected from C222, C228, C231, and combinations thereof. In some embodiments, the Ab is conjugated via a thiol group of one or more cysteine ​​residues in the LC of the Ab, where the LC optionally has the amino acid sequence of SEQ ID NO:6, and the cysteine ​​is C214. In some embodiments of the ADC of formula (ADC), the Ab comprises a HC comprising the amino acid sequence of SEQ ID NO:5 and a LC comprising the amino acid sequence of SEQ ID NO:6, and the Ab is conjugated to the linker-payload via a thiol group of cysteine ​​residues comprising all of C222, C228, C231 of the HC, and C214 of the LC.

[0135] In another aspect, provided herein is an ADC of formula (ETx-22):

[0136] [ka] where Ab is an antibody that binds to Nectin-4 as disclosed herein. The Ab of formula (ETx-22) comprises two HCs comprising the amino acid sequence of SEQ ID NO:5 and two LCs comprising the amino acid sequence of SEQ ID NO:6, and the Ab is conjugated to the linker payload in formula (ETx-22) via thiol groups in C222, C228, and C231 of both HCs of the Ab and via thiol groups in C214 of both chains of the LC, providing a DAR of 8 (i.e., 4 cysteine-maleimide linkages for each arm of the Ab).

[0137] As shown, the ADC of formula (ETx-22) comprises a 3-maleimido-propionyl spacer, a PEG2 spacer (i.e., -CH 2 -CH 2 -O-CH 2 -CH 2 -O-), a PSAR10 group located at a position in the linker that is characterized as orthogonal to the payload (i.e., -(N(CH3)-CH2-C(O)) 10 -), and a linker comprising a self-immolative unit comprising 4-beta-glucuronide-3-nitro-octopamine.

[0138] In some aspects, the Ab of formula (ETx-22) comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, where (a) HCDR1 comprises SEQ ID NO:9, HCDR2 comprises SEQ ID NO:10, HCDR3 comprises SEQ ID NO:11, LCDR1 comprises SEQ ID NO:12, LCDR2 comprises SEQ ID NO:13, and LCDR3 comprises SEQ ID NO:14 (all Kabat numbering), or (b) HCDR1 comprises SEQ ID NO:15, HCDR2 comprises SEQ ID NO:16, HCDR3 comprises SEQ ID NO:17, LCDR1 comprises SEQ ID NO:18, LCDR2 comprises SEQ ID NO:19, and LCDR3 comprises SEQ ID NO:20 (all IMGT numbering).

[0139] In some aspects, the Ab of an ADC of formula (ETx-22) comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the HCVR comprises SEQ ID NO:7 and the LCVR comprises SEQ ID NO:8.

[0140] In some embodiments, the Ab of an ADC of formula (ETx-22) comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO:5 and the LC comprises SEQ ID NO:6.

[0141] In some embodiments, the ADC of formula (ETx-22) comprises the linker-payload: (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)acetamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6 ,7]indolizino[1,2-b]quinolin-1-yl)amino)-11,14,17,20,23,26,29,32,35,38-decamethyl-1,6,10,13,16,19,22,25,28,31,34,37,40-tridecaoxo-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecaazatetracontan-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid.

[0142] In some embodiments, the ADC of formula (ETx-22) comprises: (i) a mAb; and (ii) a linker-payload: (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)acetamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyra (i) and (ii) are prepared by conjugating (i) and (ii) with (ii) and (ii) are prepared by conjugating (ii) and (iii) with (iii) and (iv) with (iii) and (iv) with (iii) and (iv) with (iv ...

[0143] In some embodiments, an ADC of Formula (ETx-22) is prepared by (a) reducing a mAb and (b) reacting the reduced mAb with a linker-payload: (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)acetamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[a]pyrrol-1-yl]propanamido)ethoxy)ethoxy)acetamido). and conjugating zo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-11,14,17,20,23,26,29,32,35,38-decamethyl-1,6,10,13,16,19,22,25,28,31,34,37,40-tridecaoxo-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecaazatetracontan-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid. In some embodiments, the disclosed ADC prepared by steps (a) and (b) has a DAR of 8.

[0144] In some aspects, the disclosed ADCs comprise (i) a mAb comprising an HC comprising the amino acid sequence of SEQ ID NO:5 and an LC comprising the LC of SEQ ID NO:6; and (ii) (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)acetamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H -benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-11,14,17,20,23,26,29,32,35,38-decamethyl-1,6,10,13,16,19,22,25,28,31,34,37,40-tridecaoxo-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecaazatetracontan-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid. In some embodiments, the disclosed ADCs are prepared by first reducing the mAb before performing the conjugation and conjugating the reduced mAb to a linker payload. In some embodiments, the disclosed ADCs prepared by conjugating the reduced mAb and a linker-payload have a DAR of 8.

[0145] Structural Features and Activity of ADC of Formula (ETx-22) The ADC, designated ETx-22, has the formula:

[0146] [ka] where Ab is an antibody that binds to Nectin-4 as disclosed herein, comprising two HCs comprising the amino acid sequence of SEQ ID NO:5 and two LCs comprising the amino acid sequence of SEQ ID NO:6. The Ab is conjugated to the linker payload in formula (ETx-22) via thiol groups in C222, C228, and C231 of both HCs of the Ab, and via thiol groups in C214 of both chains of the LC, providing a DAR of 8 (i.e., ETx-22 comprises four cysteine-maleimide linkages for each arm of the Ab).

[0147] As shown, the ADC of formula (ETx-22) has distinct structural features related to the anti-Nectin-4 antibody and the linker-payload of the ADC of formula (ETx-22). In some embodiments, these distinct structural features of the ADC of formula (ETx-22), or a combination thereof, impart desirable activity to the ADC of formula (ETx-22).

[0148] In one aspect, an ADC of formula (ETx-22) comprises an H1L2_15A7.5 anti-nectin-4 mAb comprising an HC of SEQ ID NO:5 and an LC of SEQ ID NO:6, which exhibits desirable selectivity. In one aspect, the H1L2_15A7.5 anti-nectin-4 mAb exhibits desirable selectivity for human nectin-4 over other nectin proteins, such as human nectin-1, nectin-2, or nectin-3. In one aspect, the H1L2_15A7.5 anti-nectin-4 mAb exhibits desirable selectivity for nectin-4 expressed by tumors compared to nectin-4 expressed by normal cells, such as normal human endothelial keratinocytes.

[0149] In one embodiment, the ADC of formula (ETx-22) has a DAR of 8. In one embodiment, the ADC of formula (ETx-22) has a DAR of 8 that provides desirable toxicity and efficacy. In one embodiment, the ADC of formula (ETx-22) has a DAR of 8, and the ADC exhibits minimal aggregation. In one embodiment, the ADC of formula (ETx-22) has a DAR of 8, and the linker of ETx-22 minimizes aggregation. In one embodiment, the linker is of sufficient length to minimize aggregation. In one embodiment, the linker is sufficiently hydrophilic to minimize aggregation.

[0150] As shown, the ADC of formula (ETx-22) includes a linker, which is a 3-maleimido-propionyl spacer, a PEG2 spacer (i.e., -CH 2 -CH 2 -O-CH 2 -CH 2 -O-), PSAR10 groups that may be in a position that is characterized as orthogonal to the payload (i.e., -(N(CH3)-CH2-C(O)) 10 -), and a self-immolative unit comprising a 4-beta-glucuronide-3-nitro-octopamine group. In one aspect, one or more of these features of the linker minimize aggregation of the ADC of formula (ETx-22). In one aspect, one or more of these structural features of the linker provide sufficient length to minimize aggregation of the ADC of formula (ETx-22). In one aspect, one or more of these structural features of the linker provide sufficient hydrophilicity to the ADC of formula (ETx-22) to minimize aggregation of the ADC.

[0151] In one embodiment, an ADC of formula (ETx-22) comprises a linker that includes a spacer having a length sufficient to space the linker-payload from the anti-nectin-4 antibody such that the conjugated linker-payload does not substantially interfere with the activity of the anti-nectin-4 antibody, such as Nectin-4 binding and affinity activity. In one embodiment, an ADC of formula (ETx-22) comprises a linker that includes a spacer having a length sufficiently short so as not to impair the stability of the linker-payload.

[0152] As shown, the ADC of formula (ETx-22) includes a linker, which is a 3-maleimido-propionyl spacer, a PEG2 spacer (i.e., -CH 2 -CH 2 -O-CH 2 -CH 2 -O-), PSAR10 groups that may be in a position that is characterized as orthogonal to the payload (i.e., -(N(CH3)-CH2-C(O)) 10 -), and a self-immolative unit comprising a 4-beta-glucuronide-3-nitro-octopamine group. In one embodiment, one or more of these structural features can provide a desired linker-payload spacing from the anti-Nectin-4 antibody.

[0153] In one embodiment, the ADC of formula (ETx-22) comprises a linker that exhibits desirable stability. In one embodiment, the ADC of formula (ETx-22) comprises a linker that exhibits desirable stability in plasma. In one embodiment, stability in plasma can be measured by determining the DAR of the ADC versus time after the ADC is placed in plasma.

[0154] As shown, the ADC of formula (ETx-22) includes a linker, which is a 3-maleimido-propionyl spacer, a PEG2 spacer (i.e., -CH 2 -CH 2 -O-CH 2 -CH 2 -O-), PSAR10 groups that may be in a position that is characterized as orthogonal to the payload (i.e., -(N(CH3)-CH2-C(O)) 10 -), and a self-immolative unit comprising a 4-beta-glucuronide-3-nitro-octopamine group. In one aspect, one or more of these structural features may provide desirable stability of the linker-payload in the bloodstream.

[0155] In one embodiment, the ADC of formula (ETx-22) comprises a linker that exhibits selective cleavage. In one embodiment, the ADC of formula (ETx-22) comprises a linker that remains intact while the ADC is in the bloodstream and is cleaved only after the ADC is delivered to the tumor site. In some embodiments, the tumor targeted by the ADC of formula (ETx-22) expresses an enzyme that cleaves a component of the linker. In some embodiments, the tumor expresses beta-glucuronidase that cleaves the beta-glucuronide of the linker.

[0156] As shown, the ADC of formula (ETx-22) comprises a beta-glucuronide group that can function as a trigger group, and a self-immolative unit that comprises or is prepared from a 3-nitro-octopamine group that undergoes rearrangement and self-immolative cleavage. In one embodiment, the self-immolative unit provides selective cleavage, and cleavage is not triggered until the beta-glucuronide is cleaved by beta-glucuronidase in targeted tumor cells. In one embodiment, the ADC of ETx-22 is delivered to tumors that express intracellular beta-glucuronidase, preferably at relatively high levels. In one embodiment, the beta-glucuronide group is cleaved from the self-immolative unit, and the self-immolative group then undergoes rearrangement and self-immolative cleavage, thereby releasing the exatecan payload.

[0157] therapeutic use In another aspect, provided herein is a method of treating cancer comprising administering to a patient in need of such treatment an effective amount of a Nectin-4 ADC or pharmaceutical composition described herein. In a further aspect, provided herein is a method of treating cancer comprising administering to a patient in need of such treatment an effective amount of an ADC or pharmaceutical composition described herein, wherein the cancer is bladder cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0158] In another aspect, provided herein is an ADC comprising a nectin-4 binding agent and exatecan for use in a method comprising administering the ADC to a patient suffering from a nectin-4-expressing cancer, optionally wherein the cancer is resistant to treatment with the ADC comprising a nectin-4 binding agent and an auristatin, e.g., monomethylauristatin E (MMAE).In another aspect, provided herein is use of an ADC comprising a nectin-4 binding agent and exatecan for the manufacture of a medicament for the treatment of a patient suffering from a nectin-4-expressing cancer, optionally wherein the cancer is resistant to treatment with the ADC comprising a nectin-4 binding agent and an auristatin, e.g., monomethylauristatin E (MMAE). In yet another aspect, provided herein is a method for treating a Nectin-4-expressing cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a conjugate comprising a Nectin-4 binding agent and exatecan, optionally wherein the disorder is resistant to treatment with an ADC comprising a Nectin-4 binding agent and an auristatin, e.g., monomethylauristatin E (MMAE).

[0159] In a further aspect, a method of treating cancer is provided, wherein the cancer is bladder cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is urothelial cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is breast cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is lung cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is gastric cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is esophageal cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is colorectal cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is pancreatic cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is head and neck cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is ovarian cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is prostate cancer.

[0160] In a further aspect, the patient has relapsed after receiving enfortumab vedotin, or the patient has become refractory to enfortumab vedotin or standard of care. In a further aspect, the patient being treated with an ADC or pharmaceutical composition described herein is ineligible for treatment with enfortumab vedotin. In a further aspect, the patient has a cancer that is resistant to treatment with an auristatin, such as monomethylauristatin E (MMAE).

[0161] In a further aspect, a patient being treated with an ADC or pharmaceutical composition described herein has previously received a programmed death receptor-1 (PD-1) or programmed death-ligand 1 (PD-L1) inhibitor in the neoadjuvant / adjuvant, locally advanced or metastatic setting, and a platinum-containing chemotherapy. In a further aspect, a patient being treated with a combination of an ADC or pharmaceutical composition described herein and a PD-1 inhibitor or PD-L1 inhibitor is ineligible for treatment with a cisplatin-containing chemotherapy.

[0162] In a further aspect, a method is provided that comprises administering an effective amount of an ADC or pharmaceutical composition described herein in simultaneous, separate or sequential combination with one or more anti-tumor agents. In a further aspect, a method is provided that comprises administering an effective amount of an ADC or pharmaceutical composition described herein in simultaneous, separate or sequential combination with a PD-1 inhibitor or a PD-L1 inhibitor. In one aspect, the patient is subjected to a diagnostic test to measure expression of PD-1 or PD-L1 by the patient's cancer prior to the simultaneous, separate or sequential administration of a PD-1 inhibitor or a PD-L1 inhibitor.

[0163] In another aspect, methods are provided herein that include administering an effective amount of a Nectin-4 ADC or pharmaceutical composition described herein in combination with an FGFR compound, either simultaneously, separately, or sequentially. In a further aspect, the cancer is urothelial carcinoma. In a further aspect, the FGFR compound is erdafitinib, LOXO-435, futibatinib, vofatamab, bemarituzumab, derazantinib, infigratinib, pemigatinib, rogaratinib, FGF401, or pemigatinib. In some embodiments, the Nectin-4 ADC is administered to a patient with a cancer that is resistant to treatment with erdafitinib.

[0164] In another aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein for use in therapy. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in treating cancer. In a further aspect, the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0165] In a further aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein for use in the treatment of bladder cancer. In a further aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein for use in the treatment of urothelial cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of breast cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of lung cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of gastric cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of esophageal cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of colorectal cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of pancreatic cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of head and neck cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of ovarian cancer.In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of prostate cancer.

[0166] In a further aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein for use in treating cancer, wherein the cancer has relapsed after treatment with enfortumab vedotin or the cancer has become refractory to enfortumab vedotin. In a further aspect, provided herein is a Nectin-4 ADC of a pharmaceutical composition described herein for use in treating cancer, wherein the cancer is resistant to treatment with an auristatin, such as monomethyl aurastatin E (MMAE). In a further aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein for use in treating cancer, wherein the cancer has relapsed after treatment with enfortumab vedotin or the cancer has become refractory to standard treatment. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in treating cancer, wherein the prior use of enfortumab vedotin was contraindicated.

[0167] In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of cancer, where the prior use occurs to a PD-1 or PD-L1 inhibitor and a platinum-containing chemotherapy in the neoadjuvant / adjuvant, locally advanced or metastatic setting. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein in simultaneous, separate or sequential combination with a PD-1 inhibitor or PD-L1 inhibitor for use in the treatment of cancer, where the cancer cannot be treated with a cisplatin-containing chemotherapy.

[0168] In a further aspect, provided herein is an ADC or pharmaceutical composition described herein in simultaneous, separate or sequential combination with one or more anti-tumor agents for use in the treatment of cancer. In a further aspect, the anti-tumor agent is a PD-1 inhibitor or a PD-L1 inhibitor.

[0169] In another aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein, combined simultaneously, separately, or sequentially with an FGFR compound for use in treating cancer. In a further aspect, the cancer is urothelial cancer. In a further aspect, the FGFR compound is erdafitinib, LOXO-435, futibatinib, vofatamab, bemarituzumab, derazantinib, infigratinib, pemigatinib, rogaratinib, FGF401, or pemigatinib. In another aspect, provided herein is a Nectin-4 ADC of a pharmaceutical composition for use in treating cancer resistant to erdafitinib.

[0170] In another aspect, provided herein is the use of a Nectin-4 ADC or pharmaceutical composition described herein for the manufacture of a medicament for the treatment of cancer. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein for the manufacture of a medicament for the treatment of cancer, wherein the cancer is bladder cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0171] In a further aspect, provided herein is the use of a Nectin-4 ADC or pharmaceutical composition described herein for the manufacture of a medicament for the treatment of cancer, wherein the cancer has relapsed following treatment with enfortumab vedotin or the cancer has become refractory to enfortumab vedotin or standard of care. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, wherein prior use of enfortumab vedotin was contraindicated. In a further aspect, provided herein is the use of a Nectin-4 ADC or pharmaceutical composition described herein for the manufacture of a medicament for the treatment of cancer, wherein the cancer is resistant to treatment with an auristatin, such as monomethylauristatin E (MMAE).

[0172] In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, which is prior use of a PD-1 or PD-L1 inhibitor and a platinum-containing chemotherapy in the neoadjuvant / adjuvant, locally advanced or metastatic setting. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, which is not treatable with a cisplatin-containing chemotherapy, and which is administered simultaneously, separately, or sequentially with a PD-1 inhibitor or PD-L1 inhibitor.

[0173] In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, where the medicament is administered simultaneously, separately, or sequentially with one or more anti-tumor agents. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, where the medicament is administered simultaneously, separately, or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor.

[0174] In another aspect, provided herein is the use of a Nectin-4 ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, wherein the medicament is administered simultaneously, separately, or sequentially with an FGFR compound. In a further aspect, the cancer is urothelial carcinoma. In a further aspect, the FGFR compound is erdafitinib, LOXO-435, futibatinib, vofatamab, bemarituzumab, derazantinib, infigratinib, pemigatinib, rogaratinib, FGF401, or pemigatinib. In another aspect, provided herein is the use of a Nectin-4 ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer that is resistant to treatment with erdafitinib.

[0175] In a further aspect, the bladder cancer is urothelial carcinoma, squamous cell carcinoma, or adenocarcinoma. In a further aspect, the bladder cancer is non-invasive, non-muscle invasive, or muscle invasive. In a further aspect, the bladder cancer is of the bladder, renal pelvis, ureter, or urethra. In a further aspect, the breast cancer is HR positive, HER2 negative, or triple negative breast cancer (TNBC). In a further aspect, the breast cancer is ductal or lobular. In a further aspect, the lung cancer is squamous non-small cell lung cancer (NSCLC) or non-squamous NSCLC. In a further aspect, the lung cancer is squamous, adenocarcinoma, or small cell carcinoma. In a further aspect, the prostate cancer is metastatic castration-resistant prostate cancer. In a further aspect, the gastric cancer is stomach cancer. In a further aspect, the gastric or esophageal cancer is gastroesophageal junction cancer. In a further aspect, the ovarian cancer is serous or mucinous. In a further aspect, the ovarian cancer is fallopian tube or peritoneal.

[0176] In further embodiments, the anti-tumor agent may be a chemotherapy treatment agent, including platinum-containing chemotherapy, and / or may include cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel, paclitaxel protein-bound particle injectable suspension, ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed, and cetuximab. In a further aspect, the anti-tumor agent may be a tumor immunotherapeutic agent, including those selected from the group consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat, and durvalumab.

[0177] Pharmaceutical Compositions and Methods of Administration The ADCs described herein can be formulated as pharmaceutical compositions, which are administered by any route that renders the antibody or ADC bioavailable, such as, for example, oral, topical, or subcutaneous administration.

[0178] Also provided herein are pharmaceutical compositions comprising an ADC provided herein and one or more agents selected from the group consisting of physiologically acceptable carriers, diluents, excipients, and adjuvants.

[0179] The ADC of the present disclosure, or a pharmaceutical composition comprising the same, may be administered by parenteral routes (e.g., subcutaneous and intravenous). The ADC of the present disclosure may be administered alone to a patient in a single dose or multiple doses together with a pharma- ceutically acceptable carrier, diluent, or excipient. The pharmaceutical compositions described herein can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press), and comprise an antibody or ADC disclosed herein and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0180] Disclosed herein in certain aspects are pharmaceutical compositions comprising an antibody disclosed herein and one or more pharma- ceutically acceptable carriers, diluents, or excipients. Disclosed herein in certain aspects are pharmaceutical compositions comprising an ADC disclosed herein and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0181] Dosage, regimen, and cycle The disclosed Nectin-4 ADCs can be administered in methods of treating cancer, comprising administering to a patient in need of such treatment an effective amount of a Nectin-4 ADC or pharmaceutical composition described herein. The patient can be administered an effective amount by adjusting the dose, dosing regimen, or dosing cycle of the Nectin-4 ADC.

[0182] In one aspect, the ADCs of the disclosure, or pharmaceutical compositions comprising same, may be administered by parenteral routes (e.g., subcutaneous and intravenous).

[0183] definition As used herein, the terms "a," "an," "the," and similar terms as used in the context of this disclosure (particularly in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context.

[0184] The term "bind," as used herein, unless otherwise specified, is intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, resulting in the proximity of the two proteins or molecules, as determined by common methods known in the art.

[0185] As used herein, the term "effective amount" refers to the amount (duration and means of administration) necessary to achieve the desired therapeutic result. The effective amount of a protein or conjugate may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein or conjugate to induce a desired response in the individual. An effective amount is also an amount in which any toxic or harmful effects of the protein or conjugate are outweighed by the therapeutically beneficial effects.

[0186] As used herein, the terms "treat," "treatment," or "treating" refer to any process that may slow, control, retard, or halt the progression of a disorder or disease disclosed herein, or that may ameliorate the symptoms of a disorder or disease, but does not necessarily indicate the complete disappearance of all symptoms of the disorder or disease.

[0187] As used herein, the term "patient" or "subject" refers to a human patient or human subject, respectively. The terms "patient" and "subject" may be used interchangeably herein.

[0188] Certain abbreviations are defined as follows: "ACN" refers to acetonitrile and "Boc2O" refers to di-tert-butyl decarbonate. "DCM" refers to dichloromethane, "DIPEA" refers to N,N-diisopropylethylamine, "DBU" refers to 1,8-diazabicyclo[5.4.0]undec-7-ene, "DMTMM" refers to (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride, "DMAC" refers to dimethylacetamide, "DMF" refers to N,N-dimethylformamide, "DTT" refers to dithiothreitol, "EtOAc" refers to ethyl acetate, "EDTA" refers to ethylenediaminetetraacetic acid, "FA" refers to formic acid, "HMPA" refers to hexamethylphosphoramide, "h" refers to hours, "HEPES" refers to (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), and "HMT "TA" refers to hexamethyltriethylenetetramine, "HATU" refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, "HOBt" refers to hydroxybenzotriazole, "Pyr" refers to pyridine, "NMM" refers to N-methylmorpholine, "NMP" refers to (N-methyl-2-pyrrolidone), "Su" refers to succinimide, "PPTS" refers to pyridinium p-toluenesulfonate, "THF" refers to tetrahydrofuran, "TsOH" refers to p-toluenesulfonic acid, and "TCEP" refers to (tris(2-carboxyethyl)phosphine). "TFA" refers to trifluoroacetic acid, and "TEA" refers to triethylamine.

[0189] Embodiment The following embodiments are illustrative and should not be construed as limiting the scope of the present invention.

[0190] Embodiment 1. An antibody-drug conjugate (ADC) of the following formula:

[0191] [ka] wherein Ab is an antibody that binds to human Nectin-4, Ab comprises a heavy chain (HC) comprising a variable region (HCVR) and a light chain (LC) comprising a variable region (LCVR), HCVR comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and LCVR comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, (i) HCDR1 comprises SEQ ID NO: 9 (NYGMA), HCDR2 comprises SEQ ID NO: 10 (FISNLAYGINYADTVTG), HCDR3 comprises SEQ ID NO: 11 (GARATGWFAY), and LCDR1 comprises SEQ ID NO: 12 (KASQN (VDTHVA), HCDR2 comprises SEQ ID NO: 13 (SASYRYS), and LCDR3 comprises SEQ ID NO: 14 (QQYNSYPLT) (all Kabat numbering); or (ii) HCDR1 comprises SEQ ID NO: 15 (GFTFSNYG), HCDR2 comprises SEQ ID NO: 16 (ISNLAYGI), HCDR3 comprises SEQ ID NO: 17 (ARGARATGWFAY), LCDR1 comprises SEQ ID NO: 18 (QNVDTH), LCDR2 comprises SEQ ID NO: 19 (SAS), and LCDR3 comprises SEQ ID NO: 20 (QQYNSYPLT) (all IMGT numbering), and DAR is 1 to 8.

[0192] Embodiment 2. The ADC of embodiment 1, wherein the HCVR comprises SEQ ID NO:7 and the LCVR comprises SEQ ID NO:8.

[0193] Embodiment 3. The ADC of embodiment 1 or 2, wherein the HC comprises SEQ ID NO:5 and the LC comprises SEQ ID NO:6.

[0194] Embodiment 4. The ADC of any one of embodiments 1-3, wherein the Ab has a human IgG1 or IgG4 isotype.

[0195] Embodiment 5. The ADC of any one of embodiments 1-4, wherein the Ab has a human IgG1 isotype.

[0196] Embodiment 6. An ADC according to any one of embodiments 1-5, wherein the Ab is conjugated via one or more thiol groups of cysteine ​​residues present in the HC or LC.

[0197] Embodiment 7. The ADC of any one of embodiments 1-6, wherein the Ab comprises a HC having the amino acid sequence of SEQ ID NO:5 and a LC having the amino acid sequence of SEQ ID NO:6, and the Ab is conjugated via a thiol group at C222, C228, or C231 of the HC, a thiol group at C214 of the LC, or a combination thereof.

[0198] Embodiment 8. The ADC of embodiment 7, wherein the ADC has a DAR of 8 and the Ab is conjugated via the thiol groups at C222, C228, and C231 of the HC and the thiol group at C214 of the LC.

[0199] Embodiment 9. The ADC of any one of embodiments 1-8, wherein at least 95% of the ADC has a DAR of 8.

[0200] Embodiment 10. The formula:

[0201] [ka] The ADC of any one of embodiments 1 to 8, comprising:

[0202] Embodiment 11. The ADC of embodiment 10, wherein the Ab comprises a HC of SEQ ID NO:5 and a LC of SEQ ID NO:6.

[0203] Embodiment 12. The ADC of embodiment 11, wherein the Ab is conjugated via cysteines at positions C222, C228, C231 of the HC and via a cysteine ​​at C214 of the LC.

[0204] Embodiment 13. A composition comprising an ADC according to any one of embodiments 1 to 8, wherein at least about 95% of the ADC in the composition has a DAR of 8.

[0205] Embodiment 14. A pharmaceutical composition comprising an ADC according to any one of embodiments 1 to 12 or a composition according to embodiment 13, and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0206] Embodiment 15. A method of treating cancer, comprising administering to a patient in need thereof an effective amount of an ADC described in any one of embodiments 1 to 11, a composition described in embodiment 13, or a pharmaceutical composition described in embodiment 14.

[0207] Embodiment 16. The method of embodiment 15, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0208] Embodiment 17 The method of embodiment 15 or 16, wherein the cancer is urothelial carcinoma.

[0209] Embodiment 18. The method of any one of embodiments 15-17, wherein the cancer has recurred after administration of enfortumab vedotin or the cancer has become refractory to treatment with enfortumab vedotin.

[0210] Embodiment 19. The method of any one of embodiments 15 to 18, wherein enfortumab vedotin has been administered to the patient as a first line, second line, or third line treatment prior to administering to the patient an effective amount of an ADC of any one of embodiments 1 to 11, a composition of embodiment 13, or a pharmaceutical composition of embodiment 14.

[0211] Embodiment 20. The method of any one of embodiments 15-17, wherein the patient is ineligible for treatment with enfortumab vedotin.

[0212] Embodiment 21. The method of any one of embodiments 15-17, wherein the cancer is resistant to treatment with monomethyl auristatin E (MMAE).

[0213] Embodiment 22. The method according to any one of embodiments 15 to 21, wherein the cancer is characterized by primary tumor growth, the occurrence and / or recurrence of tumor metastases, and / or an increase in at least one tumor marker, and / or upregulation of ABCB1 expression.

[0214] Embodiment 23. The method of any one of embodiments 15 to 22, further comprising simultaneously, separately or sequentially administering to the patient a PD-1 inhibitor or a PD-L1 inhibitor.

[0215] Embodiment 24. The method of embodiment 23, wherein the patient is administered a diagnostic test that measures expression of PD-1 or PD-L1 by the cancer prior to administration of a PD-1 inhibitor or a PD-L1 inhibitor.

[0216] Embodiment 25. An ADC according to any one of embodiments 1 to 12 for use in therapy.

[0217] Embodiment 26. An ADC according to any one of embodiments 1 to 12 for use in the treatment of cancer.

[0218] Embodiment 27. The ADC for use according to embodiment 26, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0219] Embodiment 28. The ADC for use according to embodiment 26, wherein the cancer is urothelial carcinoma.

[0220] Embodiment 29. The ADC for use according to any one of embodiments 26 to 28, wherein the cancer has recurred after treatment with enfortumab vedotin or the cancer has become refractory to treatment with enfortumab vedotin.

[0221] Embodiment 30. The ADC for use according to any one of embodiments 26 to 29, wherein the patient has previously been administered enfortumab vedotin as a first, second or third line treatment prior to administering to the patient an effective amount of the ADC according to any one of embodiments 1 to 11, the composition according to embodiment 13, or the pharmaceutical composition according to embodiment 14.

[0222] Embodiment 31. An ADC for use according to any one of embodiments 26 to 28, wherein prior use of enfortumab vedotin is contraindicated.

[0223] Embodiment 32. An ADC for use according to any one of embodiments 26 to 28, wherein the cancer is resistant to treatment with monomethylauristatin E (MMAE).

[0224] Embodiment 33. An ADC for use according to any one of embodiments 26 to 32, wherein the cancer is characterized by primary tumor growth, the occurrence and / or recurrence of tumor metastases, and / or an increase in at least one tumor marker, and / or upregulation of ABCB1 expression.

[0225] Embodiment 34. An ADC for use according to any one of embodiments 24 to 33, which is combined simultaneously, separately or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor.

[0226] Embodiment 35. The ADC for use according to embodiment 34, wherein the ADC is combined simultaneously, separately or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor after the cancer has been identified as a PD-1 or PD-L1 expressing cancer.

[0227] Embodiment 36. A pharmaceutical composition comprising an effective amount of an ADC according to any one of embodiments 1 to 12, or an effective amount of the composition according to embodiment 13, for use in the treatment of cancer.

[0228] Embodiment 37. The composition for use according to embodiment 36, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0229] Embodiment 38. The composition of embodiment 36 or 37, which is administered in combination simultaneously, separately or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor.

[0230] Embodiment 39. The composition of embodiment 36, wherein the composition is administered in simultaneous, separate or sequential combination with a PD-1 inhibitor or a PD-L1 inhibitor after the cancer has been identified as a PD-1 or PD-L1 expressing cancer.

[0231] Embodiment 40. Use of an ADC according to any one of embodiments 1 to 12 for the manufacture of a medicament for the treatment of cancer.

[0232] Embodiment 41. The use according to embodiment 40, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0233] Embodiment 42. The use according to claim 40 or 41, wherein the medicament further comprises a PD-1 inhibitor or a PD-L1 inhibitor.

[0234] Embodiment 43. A method of producing an ADC, comprising: (i) an antibody (Ab) that binds to human nectin-4, wherein the Ab comprises a heavy chain (HC) comprising a variable region (HCVR) and a light chain (LC) comprising a variable region (LCVR), wherein the HCVR comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, (a) HCDR1 comprises SEQ ID NO: 9 (NYGMA), HCDR2 comprises SEQ ID NO: 10 (FISNLAYGINYADTVTG), HCDR3 comprises SEQ ID NO: 11 (GARATGWFAY), and LCDR1 comprises SEQ ID NO: 12 (K HCDR1 comprises SEQ ID NO: 15 (GFTFSNYG), HCDR2 comprises SEQ ID NO: 16 (ISNLAYGI), HCDR3 comprises SEQ ID NO: 17 (ARGARATGWFAY), LCDR1 comprises SEQ ID NO: 18 (QNVDTH), LCDR2 comprises SEQ ID NO: 19 (SAS) and LCDR3 comprises SEQ ID NO: 20 (QQYNSYPLT) (all IMGT numbering); and (ii) a compound of the formula:

[0235] [ka]

[0236] Embodiment 44 The method of embodiment 43, wherein the Ab and compound are conjugated under conditions sufficient to conjugate the Ab and compound and provide an ADC with a DAR of 8.

[0237] Embodiment 45 The method of embodiment 43 or 44, further comprising reducing the Ab with a reducing agent prior to conjugation to produce a reduced Ab.

[0238] Embodiment 46 The method of embodiment 45, wherein the reducing agent is DTT or TCEP.

[0239] Embodiment 47 The method of any one of embodiments 43-46, wherein the compound is present in at least about 8 molar excess relative to Ab during conjugation.

[0240] Embodiment 48 The method of any one of embodiments 43-46, wherein the compound is present in at least about a 12 molar excess relative to Ab during conjugation. EXAMPLES

[0241] Example 1: Synthesis of linker-payload of formula (L-P'): (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)acetamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyra No[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-11,14,17,20,23,26,29,32,35,38-decamethyl-1,6,10,13,16,19,22,25,28,31,34,37,40-tridecaoxo-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecaazatetracontan-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0242] [ka]

[0243] The linker-payloads of formula (L-P') were synthesized from precursors or intermediates including polysarcosine (PSAR) compounds, 4-beta-glucuronide-3-nitro-octapamine compounds, exatecan compounds, and maleimido-proprionyl compounds. The linker-payloads were synthesized using the methods disclosed in WO2019081455 and WO2022 / 207699.

[0244] PSAR intermediate: FmocNH-PEG2-Glu(Su)-PSAR10-NH 2

[0245] [ka]

[0246] The PSAR intermediate can be prepared using Scheme 1.

[0247] [ka]

[0248] The on-resin synthesis of polysarcosine is carried out using a submonomer synthesis iterative procedure for Rink amide with the commercially available Fmoc-Sar-Sar-OH dipeptoid building block. All reactions are carried out at room temperature unless otherwise specified.

[0249] Rink amide preloaded with the first Fmoc-sarcosine residue is used as the starting material. Rink amide preloaded with Fmoc-sarcosine is treated twice for 15 min at room temperature with 20% piperidine in DMF (1 mL per 100 mg of resin). The resin is then washed with DMF (4 times) and DCM (4 times). To the resin was added a solution of Fmoc-Sar-Sar-OH (3 eq.), HATU (2.9 eq.), and DIPEA (6 eq.) in DMF (1 mL per 100 mg of resin). The reaction vessel is agitated for 2 h and the resin is washed with DMF (4 times) and DCM (4 times). The resin is treated twice for 15 min at room temperature with 20% piperidine in DMF (1 mL per 100 mg of resin). The resin is then washed with DMF (4x) and DCM (4x) to obtain Rink resin with n=3 polysarcosine oligomer.

[0250] The elongation of the n=3 polysarcosine oligomer is carried out using a submonomer synthesis procedure by alternating bromoacetylation and amine displacement steps until the desired length is obtained. The bromoacetylation step is carried out by adding 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL per 100 mg of resin). The mixture is stirred for 30 min, drained, and washed with DMF (4 times). For the amine displacement step, 40% (wt) methylamine in water (1.5 mL per 100 mg of resin) is added, the vessel is shaken for 30 min, drained, and washed with DMF (4 times) and DCM (4 times).

[0251] Once the desired polysarcosine oligomer length is reached (e.g., PSAR 10-mer), orthogonal chemical functionalization is performed. Orthogonal chemical functionalization may be followed by final capping with an Fmoc-protected amino acid group or other group. The Fmoc protecting group may be removed before or after resin cleavage.

[0252] Polysarcosine can be functionalized with glutamic acid and amino-3,6-dioxaoctanoic acid as follows: Fmoc-Glu(OAll)-OH (3 eq.), HATU (2.9 eq.), and DIPEA (6 eq.) in DMF (1 mL per 100 mg of resin) are added to the Rink resin. The reaction vessel is agitated for 90 min and the resin is washed with DMF (4 times) and DCM (4 times). The resin is then treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 min at room temperature. The resin is washed with DMF (4 times) and DCM (4 times) and subsequently coupled with Fmoc-amino-3,6-dioxaoctanoic acid (3 eq.), HATU (2.9 eq.), DIPEA (6 eq.) in DMF (1 mL per 100 mg of resin) for 1 h. The resin was washed with DMF (4 times) and DCM (4 times). 0.25 equivalents of Pd(PPh 3 ) 4 The alloc protecting group is removed by two 30-minute treatments with a DCM solution containing 50 equivalents of DIC and 20 equivalents of phenylsilane (gently stirred under a stream of argon). The resin is then washed with DMF (5 times) and DCM (5 times). N-hydroxysuccinimide (NHS) ester is introduced to the carboxylic acid side chain of the final polysarcosine compound by 90-minute treatment with a DMF solution containing 50 equivalents of DIC and 60 equivalents of N-hydroxysuccinimide (1.5 mL per 100 mg of resin). The resin is then washed with DMF (4 times) and DCM (4 times). The final polysarcosine compound is then cleaved from the resin (100% TFA 2 times, 30 minutes).

[0253] 4-beta-glucuronide-3 nitro-octopamine intermediate: (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0254] [ka]

[0255] 4-beta-glucuronide-3-nitro-octopamine intermediate 4-beta-glucuronide-3-nitro-octopamine intermediate: (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate can be prepared using Scheme 2.

[0256] [ka]

[0257] The octopamine or octopamine intermediate utilized in the present process can be obtained as a racemic mixture or as an enantiopure compound. The racemic mixture can be subjected to chiral separations known in the art to obtain the enantiopure compound.

[0258] Octopamine (±) hydrochloride (1690 mg / 11 mmol) is suspended in 4 mL of distilled water. The flask is cooled at 0 °C and 4 mL of pre-chilled 65% nitric acid solution is slowly added. The reaction is kept at 0 °C for 20 min and the mononitration is evaluated by HPLC. The mononitrated octopamine precursor is transferred to a pre-chilled 250 mL Erlenmeyer flask and diluted with saturated NaHCO until a pH value of 8-9 is reached. 3 The solution is slowly neutralized at 0° C. Then, 30 mL of dioxane is added, followed by Boc 2 2H2O (7202 mg / 13.2 mmol) is added. The reaction is allowed to reach room temperature and stirred overnight. The reaction is then diluted with EtOAc and washed three times with saturated citric acid solution and once with saturated NaCl solution. The organic phase is washed with MgSO 4The mixture is dried over 100° C., filtered and evaporated under vacuum to give a crude product, which is purified by chromatography on silica gel (petroleum ether / EtOAc, gradient 70:30 to 20:80) to give the mononitrated octopamine precursor: tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate. The resulting mononitrated octopamine precursor is then subjected to chiral separation before further synthetic steps are carried out.

[0259] Chiral separation of racemic tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate is carried out using an MPLC column with a mobile phase of DCM ± 0.2% (v / v) EtOH (isocratic gradient) and a sample solvent of DCM ± 0.2% (v / v) EtOH. To determine the absolute configuration, the phenolic position of both enantiomers is esterified with 1.2 molar equivalents of 4-nitrobenzoyl chloride and 2 molar equivalents of triethylamine in anhydrous THF. The compound is purified by chromatography on silica gel (petroleum ether / EtOAc, gradient 90:10 to 10:90) to give 4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenyl 4-nitrobenzoate. The absolute configuration of the enantiomers (previously dissolved in a 1:1 mixture of heptane / dichloromethane and slowly evaporated for 3 weeks to induce crystal formation) is confirmed by X-ray crystallography.

[0260] Round bottom flash medium, Ag 2 CO 3(1500 mg / 5.4 mmol) and 1,1,4,7,10,10-hexamethyltriethylenetetramine (251 mg / 1.1 mmol) are suspended in 4 mL of anhydrous acetonitrile and stirred at room temperature for 2 h. Enantiopure tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate (292 mg / 0.98 mmol) and 1-bromo-2,3,4-tri-O-acetyl-α-D-glucuronide methyl ester (583 mg / 1.46 mmol) are added at 0 °C and the solution mixture is stirred at room temperature for 4 h. The reaction is then filtered through Celite, diluted with EtOAc, washed three times with saturated citric acid solution and once with saturated NaCl solution. The organic phase is removed with MgSO 4 Drying over, filtration and evaporation in vacuo gives the crude product which is purified by chromatography on silica gel (petroleum ether / EtOAc, gradient 70:30 to 30:70) to give (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate.

[0261] (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (334 mg / 0.54 mmol) and 4-nitrophenyl chloroformate (219 mg / 1.09 mmol) are dissolved in 6 mL of dry DCM at 0° C. Anhydrous pyridine (112 mg / 1.41 mmol) is added and the mixture is stirred at room temperature for 30 min. The reaction is filtered through a 0.45 μm PTFE filter and purified by chromatography on silica gel (petroleum ether / EtOAc, gradient 85:15 to 30:70) to give (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate.

[0262] 4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate: (2S,3R,4S,5S,6S)-2-(4-(2-amino-1-((((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':]6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0263] [ka]

[0264] The 4-beta-glucuronide-3-nitro-octopamine intermediate can be conjugated to exatecan using Scheme 3 to provide the 4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate: (2S,3R,4S,5S,6S)-2-(4-(2-amino-1-((((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid.

[0265] [ka]

[0266] 101 mg (0.13 mmol) of 4-beta-glucuronide-3-nitro-octopamine intermediate (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate, 0.14 mmol of exatecan mesylate, and 18 mg (0.13 mmol) of HOBt are dissolved in 1 mL of an 85:15 (v / v) mixture of anhydrous DMF / pyridine. 16.7 mg (0.13 mmol) of DIPEA are added. The reaction is stirred at 40° C. for 2 h and the volatiles are evaporated under reduced pressure. The crude residue is purified by chromatography on silica gel (DCM / MeOH gradient 99:1 to 95:5) to give the intermediate compound which is then subjected to deprotection.

[0267] 144 mg (0.106 mmol) of this intermediate compound are dissolved in 3 mL of MeOH (75:25) at 0° C. LiOH monohydrate (44.5 mg / 1.06 mmol) is dissolved in water (0.4 mL) and added to the reaction vessel. After stirring, the mixture is neutralized with acetic acid (83 mg / 1.4 mmol) and concentrated under reduced pressure. The resulting crude product is redissolved in a TFA / DCM solution and stirred. The volatiles are evaporated under reduced pressure and the crude residue is collected and purified using HPLC to give (2S,3R,4S,5S,6S)-2-(4-(2-amino-1-((((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':]6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid.

[0268] Conjugation of PSAR intermediate and 4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate to obtain functionalized PSAR-4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate

[0269] [ka]

[0270] The PSAR intermediate and the 4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate can be conjugated using Scheme 4 to provide a functionalized PSAR-4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate.

[0271] [ka]

[0272] Dissolve 100 mg (0.081 mmol) of PSAR intermediate and 51 mg (0.061 mmol) of 4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate in anhydrous DMF. Add 41 mg (0.405 mmol) of triethylamine and stir the reaction at room temperature for 30 min. After complete conversion of the reactants is assessed by HPLC, add piperidine directly to the reaction vial to reach an 8% (v / v) piperidine solution in DMF. The reaction is then stirred at room temperature for 5-10 min until complete Fmoc deprotection is observed by HPLC. Neutralize the reaction slowly with a 10% TFA solution in water / ACN 1:1 (v / v) and purify using HPLC to obtain the functionalized PSAR-4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate.

[0273] Linker-payload of formula (L-P'): (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)acetamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3' ,4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-11,14,17,20,23,26,29,32,35,38-decamethyl-1,6,10,13,16,19,22,25,28,31,34,37,40-tridecaoxo-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecaazatetracontan-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0274] [ka]

[0275] A linker-payload of formula (L-P') can be prepared by conjugating maleimidopropionic acid N-hydroxysuccinimide ester and a functionalized-PSAR-4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate using Scheme 5.

[0276] [ka]

[0277] Maleimidopropionic acid N-hydroxysuccinimide ester and functionalized-PSAR-4-beta-glucuronide-3-nitro-octopamine-exatecan intermediate are dissolved in anhydrous DMF (0.1M concentration of maleimide compound). 1.56 mg (0.015) of triethylamine is added and the reaction is stirred for 2 hours until complete conversion of the reaction is observed by HPLC. The reaction mixture is then diluted with 1% aqueous TFA / ACN 1:1 (v / v) and purified using HPLC preparative method 6 to obtain the linker-payload of formula (L-P').

[0278] Example 2: Humanized anti-Nectin-4 monoclonal antibody (15A7.5mAb) The anti-Nectin-4 mAb utilized in the disclosed ADCs is a humanized derivative of mAb 15A7.5 described in WO 2022 / 207822 and WO 2022 / 207825. As shown, mAb 15A7.5 exhibits selective affinity for tumor cells versus keratinocytes. The mAb utilized to prepare the ETx-22 ADCs disclosed herein is a humanized derivative of 15A7.5 mAb with back mutations introduced into the amino acid sequence of its HC (H1) and the amino acid sequence of its LC (L2), referred to herein as H1L2_15A7.5. H1L2_15A7.5 mAb has a heavy chain amino acid sequence of SEQ ID NO:5 and a light chain amino acid sequence of SEQ ID NO:6. The HC variable region (HCVR) is provided as SEQ ID NO:7 and the LC variable region (LCVR) is provided as SEQ ID NO:8. The HC and LC complementarity determining regions (HCDR and LCDR) are provided as SEQ ID NOs: 9-14 (Kabat) and 15-20 (IMGT).

[0279] Example 3: Preparation of ETx-22 ADC An ADC designated "ETx-22" was prepared by conjugating H1L2-15A7.5 mAb via a cysteine ​​residue to an exatecan payload, referred to herein as a linker-payload of formula (L-P'). A schematic diagram of ETx-22 is provided in FIG.

[0280] To prepare the ADC designated ETx-22, mAb H1L2-15A7.5 (HC: SEQ ID NO: 5, LC: SEQ ID NO: 6) in PBS 1X, 1 mM EDTA was reduced with 14 molar equivalents of TCEP at 37° C. for 2 hours, after which the buffer was changed to 100 mM KPO 4 The buffer was then exchanged into 1 mM EDTA pH 7.4 (Amicon ultra 30 kDa). 12 molar equivalents of linker-payload of formula (L-P') were used for conjugation with the reduced mAb for 35 min at room temperature. The buffer was then changed to 100 mM KPO 4(pH 8.0) and then incubated at 37° C. for 24 h in the absence of oxygen to allow autohydrolysis of the maleimide. The final exchange buffer was performed in 20 mM His (pH 6.0) and then filtered through a 0.22 μM filter to give ETx-22.

[0281] The drug-to-antibody ratio (DAR) of ETx-22 was determined according to LC-MS analysis and found to be 7.77-7.82 toxin per conjugated mAb. Less than 5% of the material was aggregated as determined by SEC-HPLC. Analysis of ETx-22 by hydrophobic interaction chromatography (HIC) revealed a single sharp peak for ETx-22 close to the naked 15A7.5 mAb, indicating high homogenous hydrophilicity. (See Figure 2 and corresponding brief description). The ex vivo stability of ETx-22 was assessed by incubating ETx-22 in mouse, cynomolgus monkey, or human serum and measuring the DAR over time. (See Figure 3 and corresponding brief description). In vitro cytotoxic activity was assessed for the HCT-116-2G10 clone expressing human nectin-4. (See Figure 4 and corresponding brief description).

[0282] As a comparative ADC, HA22 anti-nectin-4 mAb was conjugated to maleimidocaproyl-valine-citrulline-PABC-MMAE to generate enfortumumab vedotin. Briefly, a 15 mg / mL solution of HA22 in 10 mM acetic acid, 1% sorbitol, 3% L-arginine (pH 5.0) was supplemented with 20% volume of 0.1 M Tris, 25 mM EDTA, and 750 mM NaCl (pH 8.4) to adjust the pH of the solution to 7.5, 5 mM EDTA, and 150 mM sodium chloride. HA22 mAb was then partially reduced by adding 2.5 molar equivalents of TCEP and then gently stirred at 37° C. for 2 hours. The partially reduced mAb solution was then cooled to 5° C. and 4.4 molar equivalents of the cysteine ​​reactive linker-exatecan compound were added as a 6% (v / v) solution in DMSO. The mixture was stirred at 5° C. for 60 minutes, followed by the addition of 1 molar equivalent of N-acetylcysteine ​​relative to the cysteine ​​reactive linker-MMAE compound, followed by an additional 15 minutes of stirring. Excess quenched cysteine ​​reactive linker-payload and other reactive components were removed by UF / DF of the antibody drug conjugate (ADC) with 10 volumes of 20 mM histidine, pH 6.0. The drug-antibody ratio (DAR) by LC-MS analysis was 3.79 toxin per conjugated mAb. Less than 1% of the material was aggregated as determined by SEC-HPLC.

[0283] Example 4: In vivo studies in mice Female NOD / SCID / γc null (NSG) and male NMRI mice were obtained from Charles River laboratories. Female NOD / SCID and BALB / c nude mice were obtained from GemPharmatech Co. Mice were housed under sterile conditions, provided with sterile food and water ad libitum, and maintained on a 12-h light and 12-h dark cycle. For NSG mice, cells and PDXs were cultured at 0.5 × 10 cells suspended in 50% phenol red-free Matrigel (Becton-Dickinson Bioscience). 6PDX fragments (2-3 mm diameter) were inoculated subcutaneously into both flanks in the mammary fat pad with 100 μg / mL of 10 ...

[0284] Tumor is 100-200 mm 3 Mice were treated when tumor volumes reached a mean volume of 100 μg / kg. Mice were treated iv with a single or two doses of the ADC at the indicated concentrations. Tumor volumes (length × width) were measured with digital calipers. 2 Tumor growth was monitored by calculating the mean tumor volume in each group (mean tumor volume was 100–200 mm). 3 All animals were randomly assigned to treatment groups such that the mean tumor volume was >1500 mm3. To assess the toxicity of the different treatments, the body weight of the animals was monitored every 3 days. Mice with weight loss >20% and tumor volumes >1500 mm3 were excluded. 3 The following were monitored: ruffled coat and hunched back, weakness, and decreased mobility were considered as end points.

[0285] PK / PD characterization of ETx-22 was evaluated using NSG mice and triple-negative breast cancer (TNBC) PDX 400-engrafted NSG mice (see FIG. 5 and corresponding brief description).

[0286] The mechanism of action of ETx-22 was evaluated in NSG mice implanted with a triple-negative breast cancer PDX model (PDX400) (see Figure 6 and corresponding brief description).

[0287] The in vivo efficacy of ETx-22 in an MMAE-resistant triple-negative breast cancer (TNBC) cell line (SUM190) was evaluated in NSG mice (see FIG. 7 and corresponding brief description).

[0288] The in vivo efficacy of ETx-22 was evaluated in PDX models of esophageal cancer (ES0201) (Figure 8 and corresponding brief description), bladder cancer (BLCU003) (Figure 9 and corresponding brief description), bladder cancer (B521) (Figure 10 and corresponding brief description), head and neck cancer (HN2579) (Figure 11 and corresponding brief description), cervical cancer (CV3035) (Figure 12 and corresponding brief description), cervical cancer (CV3560) (Figure 13 and corresponding brief description), and ovarian cancer (OV2423) (Figure 14 and corresponding brief description).

[0289] The in vivo efficacy of ETx-22 was evaluated in PDX models of triple-negative breast cancer (TNBC): PDX400 (Figure 15 and corresponding brief description), PDX317 (Figure 16 and corresponding brief description), PDX348 (Figure 17 and corresponding brief description), and PDX434 (Figure 18 and corresponding brief description).

[0290] Amino acid sequence SEQ ID NO:1 MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYEC RVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLED QNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVVLMSRYHRRKAQQM TQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV SEQ ID NO:2 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:4 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:5 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVSFISNLAYGINYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGARATGWFAYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:6 DIQMTQSPSSLSASVGDRVTITCKASQNVDTHVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:7 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVSFISNLAYGINYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGARATGWFAYWGQGTLVTVSS SEQ ID NO:8 DIQMTQSPSSLSASVGDRVTITCKASQNVDTHVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK SEQ ID NO:9 NYGMA SEQ ID NO:10 FISNLAYGINYADTVTG SEQ ID NO:11 GARATGWFAY SEQ ID NO:12 KASQNVDTHVA SEQ ID NO:13 SASYRYS SEQ ID NO:14 QQYNSYPLT SEQ ID NO:15 GFTFSNYG SEQ ID NO:16 ISNLAYGI SEQ ID NO:17 ARGARATGWFAY SEQ ID NO:18 QNVDTH SEQ ID NO:19 SAS SEQ ID NO:20 QQYNSYPLT

Claims

1. An antibody-drug conjugate (ADC) of the formula: 【Chemistry 1】 During the ceremony, The Ab is an antibody that binds to human Nectin-4, the Ab comprising a heavy chain (HC) comprising a variable region (HCVR) and a light chain (LC) comprising a variable region (LCVR), the HCVR comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; (i) the HCDR1 comprises SEQ ID NO: 9 (NYGMA), the HCDR2 comprises SEQ ID NO: 10 (FISLAYGINYADTVTG), the HCDR3 comprises SEQ ID NO: 11 (GARATGWFAY), the LCDR1 comprises SEQ ID NO: 12 (KASQNVDTHVA), the LCDR2 comprises SEQ ID NO: 13 (SASYRYS), and the LCDR3 comprises SEQ ID NO: 14 (QQYNSYPLT); or (ii) the HCDR1 comprises SEQ ID NO: 15 (GFTFSNYG), the HCDR2 comprises SEQ ID NO: 16 (ISNLAYGI), the HCDR3 comprises SEQ ID NO: 17 (ARGARATGWFAY), the LCDR1 comprises SEQ ID NO: 18 (QNVDTH), the LCDR2 comprises SEQ ID NO: 19 (SAS), and the LCDR3 comprises SEQ ID NO: 20 (QQYNSYPLT); DAR is 1-8, ADC.

2. 2. The ADC of claim 1 , wherein the HCVR comprises SEQ ID NO:7, the LCVR comprises SEQ ID NO:8, and / or the HC comprises SEQ ID NO:5 and the LC comprises SEQ ID NO:

6.

3. 3. The ADC of claim 1 or 2, wherein the Ab has a human IgG1 or IgG4 isotype, preferably a human IgG1 isotype.

4. having the formula 【Chemistry 2】 4. The ADC of any one of claims 1 to 3, wherein Ab comprises a HC of SEQ ID NO:5 and a LC of SEQ ID NO:6, and said Ab is conjugated via cysteines at positions C222, C228, C231 of said HC and via a cysteine ​​at C214 of said LC.

5. 4. A composition comprising an ADC according to any one of claims 1 to 3, wherein at least about 95% of the ADC in the composition has a DAR of 8.

6. A pharmaceutical composition comprising the ADC of any one of claims 1 to 4 or the composition of claim 5, and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

7. A method for treating cancer, comprising administering to a patient in need thereof an effective amount of the ADC of any one of claims 1 to 4, the composition of claim 5, or the pharmaceutical composition of claim 6.

8. 8. The method of claim 7, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer, preferably urothelial cancer.

9. 9. The method of claim 7 or 8, wherein the patient has previously received enfortumab vedotin as a first-line, second-line, or third-line treatment, and optionally the cancer has recurred after enfortumab vedotin was administered, or the cancer has become refractory to treatment with enfortumab vedotin after enfortumab vedotin was administered.

10. 5. The ADC of any one of claims 1 to 4 for use in therapy.

11. 5. The ADC of any one of claims 1 to 4 for use in the treatment of cancer.

12. 10. A pharmaceutical composition comprising an effective amount of the ADC of any one of claims 1 to 4 or an effective amount of the composition of claim 5 for use in the treatment of cancer.

13. 13. Use of the ADC of any one of claims 1 to 4 for the manufacture of a medicament for the treatment of cancer.

14. 1. A method of generating an ADC, comprising: (i) an antibody (Ab) that binds to human Nectin-4, the Ab comprising a heavy chain (HC) having a variable region (HCVR) and a light chain (LC) having a variable region (LCVR), the HCVR comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; a) the HCDR1 comprises SEQ ID NO:9 (NYGMA), the HCDR2 comprises SEQ ID NO:10 (FISNLAYGINYADTVTG), the HCDR3 comprises SEQ ID NO:11 (GARATGWFAY), the LCDR1 comprises SEQ ID NO:12 (KASQNVDTHVA), the LCDR2 comprises SEQ ID NO:13 (SASYRYS), and the LCDR3 comprises SEQ ID NO:14 (QQYNSYPLT); or b) an Ab, wherein the HCDR1 comprises SEQ ID NO: 15 (GFTFSNYG), the HCDR2 comprises SEQ ID NO: 16 (ISNLAYGI), the HCDR3 comprises SEQ ID NO: 17 (ARGARATGWFAY), the LCDR1 comprises SEQ ID NO: 18 (QNVDTH), the LCDR2 comprises SEQ ID NO: 19 (SAS), and the LCDR3 comprises SEQ ID NO: 20 (QQYNSYPLT); and (ii) a compound of the formula: 【Chemistry 3】 The method of claim 1, further comprising conjugating

15. 16. The method of claim 15, wherein the compound is present in at least about 8 molar excess relative to the Ab during conjugation.

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