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

Abstract

The present invention provides an antibody-drug conjugate (ADC) containing exatecan and a pharmaceutical composition thereof, and a method of using such ADC to treat cancer.

Description

Prior Technology

[0001] This invention relates to the medical field. More specifically, this invention relates to Nectin-4 antibody-drug conjugates and pharmaceutical compositions thereof, and their use in the treatment of cancer.

[0002] Cell adhesion protein-4 (CAP-4) is a member of the CAP family of Ca2+-independent immunoglobulin-like cell adhesion molecules. Unlike other members of this family, CAP-4 is primarily expressed in the placenta or embryo in healthy tissues. However, CAP-4 is overexpressed in several tumor types, including urothelial carcinoma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, and ovarian cancer. Studies have associated high CAP-4 expression with tumor incidence in several cancer types.

[0003] Antibody-drug conjugates (ADCs) containing antibodies against cell adhesion protein-4 have been developed. These include an ADC containing auristatin E (MMAE) (WO201247724), ADCs containing camptothecin analogs (WO2022112356 and WO2021151984), and an ADC containing exatecan (WO2022207825).

[0004] Designing antibody-drug conjugates (ADCs) for oncology is extremely challenging because the molecule must balance multiple aspects, including sufficient specificity against tumor targets exceeding that of healthy cells, acceptable toxicity while maintaining desired activity against bystander tumor cells, and controlled instability of the drug delivery to allow intracellular delivery while maintaining good physical and chemical stability. Specifically, one of the challenges in ADC development is selecting a suitable linker to bind the drug delivery to the antibody in the ADC. The chemical structure of the linker affects various properties of the ADC, including toxicity, specificity, stability, and potency.

[0005] Currently, there is an FDA-approved anti-cell adhesion protein-4 ADC, enfortumab vedotin (ejfv), marketed under the brand name PADCEV®. Enfortumab vedotin is an ADC containing a monoclonal anti-cell adhesion protein-4 antibody and monomethylorisstatin-E (MMAE) as a cytotoxic drug delivery system. Unfortunately, enfortumab vedotin exhibits some undesirable skin toxicities, which may be at least partially associated with the non-discriminatory binding of enfortumab to skin cells rather than targeting tumor cells. In addition, long-term administration of enfortumab vedotin can lead to drug-resistant cancers in human patients. Cabaud et al. (Mol Cancer Ther 21 (2022):1227-1235) found that in preclinical mouse models, the expression of ABCB1, which encodes the multidrug resistance protein MDR1 / P-glycoprotein (P-gp), was upregulated in resistant tumors. The sensitivity of the resistant mouse model to the anti-cell adhesion protein-4 antibody-MMAE conjugate can be restored in vitro and in vivo by the P-gp inhibitor tariquidar.

[0006] Specifically, there remains a need for anti-cell adhesion protein-4 (ADC) drugs containing topoisomerase I. Specifically, there remains a need for anti-cell adhesion protein-4 ADCs that avoid or allow for better management of dermatological events observed in some anti-cell adhesion protein-4 ADCs. Specifically, there remains a need for anti-cell adhesion protein-4 ADCs that avoid ocular and / or peripheral neuropathy signals observed in some anti-cell adhesion protein-4 ADCs. Specifically, there remains a need for anti-cell adhesion protein-4 ADCs with low immunogenicity, stable in vivo pharmacokinetics, and adequate chemical and physical stability. Additionally, there remains a need for anti-cell adhesion protein-4 ADCs possessing one or more of the following characteristics: improved antitumor activity measured in certain tumor models, enhanced bystander activity against low-tumor cells of cell adhesion protein-4, lower immunogenicity, undetectable antibody effector function, and / or better physical and chemical stability.

[0007] Here, the inventors disclose a novel anti-cell adhesion protein-4 ADC that addresses one or more of these needs. The disclosed ADC comprises an anti-cell adhesion protein-4 antibody exhibiting selectivity for cell adhesion protein-4, a tumor-specific protein, and a novel linker drug delivery containing essanotecan. The inventors demonstrate that the disclosed anti-cell adhesion protein-4 ADC exhibits one or more of these improved properties related to aggregation, toxicity, specificity, stability, and potency. The inventors also demonstrate that this novel anti-cell adhesion protein-4 ADC can be used to treat auristatin-resistant cancers, including cancers resistant to enrofloxacin (vitolstatin). Summary of the Invention

[0008] This article provides anti-cell adhesion protein-4 ADCs and compositions comprising anti-cell adhesion protein-4 ADCs. This article also provides a method for treating a patient's cancer (such as cancers exhibiting cell adhesion protein-4) using the anti-cell adhesion protein-4 ADCs or compositions comprising the anti-cell adhesion protein-4 ADCs.

[0009] In one embodiment, this document provides an ADC comprising an antibody binding to human cell adhesion protein-4 and a drug delivery vehicle. In another embodiment, this document provides an ADC prepared by conjugating an antibody binding to human cell adhesion protein-4 to a drug delivery vehicle. In this ADC, the antibody and the drug delivery vehicle are directly or covalently linked or bound via a linker. In one embodiment of the disclosed ADC, the antibody and the drug delivery vehicle are covalently linked or bound via a linker. In one embodiment, the linker provides a suitable and stable bond or binding between the antibody and the drug delivery vehicle when the ADC is systemically administered, and provides suitable instability when the drug delivery vehicle is released from the ADC by internalization of the ADC into a target cell expressing cell adhesion protein-4.

[0010] In one embodiment, this article provides an antibody or an ADC prepared therefrom that binds to human cell adhesion protein-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 (HCDR) HCDR1, HCDR2, and HCDR3, and the LCVR comprises light chain complementarity-determining regions (LCDR) LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 contains SEQ ID NO: 9 (NYGMA), HCDR2 contains SEQ ID NO: 10 (FISNLAYGINYADTVTG), HCDR3 contains SEQ ID NO: 11 (GARATGWFAY), LCDR1 contains SEQ ID NO: 12 (KASQNVDTHVA), LCDR2 contains SEQ ID NO: 13 (SASYRYS), and LCDR3 contains SEQ ID NO: 14 (QQYNSYPLT) (all Kabat numbers); or (b) HCDR1 contains SEQ ID NO: 15 (GFTFSNYG), HCDR2 contains SEQ ID NO: 16 (ISNLAYGI), HCDR3 contains SEQ ID NO: 17 (ARGARATGWFAY), LCDR1 contains SEQ ID NO: 18 (QNVDTH), LCDR2 contains SEQ ID NO: 19 (SAS), and LCDR3 contains SEQ ID NO: 20 (QQYNSYPLT) (all IMGT numbers).

[0011] In another embodiment, this document provides an antibody or ADC comprising binding to human cell adhesion protein-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.

[0012] In another embodiment, this document provides an antibody or ADC comprising binding to human cell adhesion protein-4, wherein the antibody comprises one or more mutations in its constant region that alter Fc effector function as disclosed herein, such as mutations in the constant region that reduce the binding of the antibody to the Fc receptor. In some embodiments, the antibody contains an IgG1 heavy chain (HC) comprising one or more mutations selected from L234F, L235E, and P331S (EU designations) or one or more mutations selected from L247F, L248E, and P350S (Kabat designations). In some embodiments, the antibody contains an IgG1 heavy chain (HC) comprising each of the mutations L234F, L235E, and P331S (EU designations) or each of the mutations L247F, L248E, and P350S (Kabat designations). In some embodiments, the antibody contains a heavy chain (HC) comprising one or more mutant chains selected from L234F, L235E, and P331S relative to the wild-type sequence shown in SEQ ID NO: 2. In some embodiments, the antibody contains a heavy chain comprising each of the mutants L234F, L235E, and P331S relative to the wild-type sequence shown in SEQ ID NO: 2. In some embodiments, the antibody contains a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 3.

[0013] In another embodiment, this document provides an antibody or ADC comprising binding to human cell adhesion protein-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.

[0014] In another embodiment, this document provides an ADC comprising a cell adhesion protein-4 antibody directly or via a linker bound to a drug delivery system, the drug delivery system potentially comprising a cytotoxic agent, such as ethanotecan or an analogue thereof. In some embodiments, the cell adhesion protein-4 antibody is bound to the drug delivery system via a thiol group of one or more cysteine ​​residues.

[0015] In some embodiments, the ADC comprises or is prepared from an anti-cell adhesion protein-4 antibody bound via a thiol group of one or more cysteine ​​residues present in the HC of the anti-cell adhesion protein-4 antibody, which, if desired, is present in a 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 numbers) and combinations thereof, or at positions selected from C233, C239, C242 and combinations thereof (Kabat numbers). In some embodiments, the anti-cell adhesion protein-4 antibody is bound at positions C220, C226, C229 (EU index numbers), or at positions C233, C239, C242 (Kabat numbers) via a thiol group of each of the cysteine ​​residues present in the HC. In some embodiments, the anti-cell adhesion protein-4 antibody binds via a thiol group of one or more cysteine ​​residues present in the HC of the antibody, wherein the HC has the amino acid sequence shown in SEQ ID NO: 5 and the cysteine ​​residues are present in the HC at positions selected from C222, C228, C231 and combinations thereof, and if desired, wherein the anti-cell adhesion protein-4 antibody binds via a thiol group of each of the cysteine ​​residues present at positions C222, C228 and C231. In some embodiments, the anti-cell adhesion protein-4 antibody binds via a thiol group of cysteine ​​residues present in the LC. In some embodiments, the anti-cell adhesion protein-4 antibody binds via a thiol group of cysteine ​​residues present at the C-terminus of the LC, and if desired, wherein the LC is a κ light chain and the cysteine ​​residue is present at position C214. In some embodiments, the anti-cell adhesion protein-4 antibody binds via a thiol group of one or more cysteine ​​residues present in the LC of the antibody, wherein the LC has the amino acid sequence shown in SEQ ID NO: 6 and the cysteine ​​residue is present in the LC at position C214. In some embodiments, the anti-cell adhesion protein-4 antibody binds via the thiol group of the cysteine ​​residue present in the HC at positions C220, C226, C229 (EU index number), or at positions C233, C239, C242 (Kabat number) and via the thiol group of the cysteine ​​residue present in the LC at position C214.In some embodiments, the anti-cell adhesion protein-4 antibody binds via a thiol group of a cysteine ​​residue present in the HC, wherein the HC has the amino acid sequence shown in SEQ ID NO: 5 and the cysteines are present at positions C222, C228, and C231, and the cell adhesion protein-4 antibody binds via a thiol group of a cysteine ​​residue present in the LC, wherein the LC has the amino acid sequence shown in SEQ ID NO: 6 and the cysteine ​​is present at position C214.

[0016] In another embodiment, this document provides an ADC comprising or prepared from the following: (i) an antibody binding to human cell adhesion protein-4, (ii) a linker as desired, and (iii) a drug load. In another embodiment, this document provides an ADC comprising an antibody binding to human cell adhesion protein-4, the antibody being directly or indirectly bound to a drug load via a linker. In yet another embodiment, this document provides an ADC prepared by directly or indirectly binding an antibody binding to human cell adhesion protein-4 to a drug load via a linker.

[0017] In another embodiment, this document provides an ADC comprising or prepared from the following: (i) an antibody binding to human cell adhesion protein-4, (ii) a linker as desired, and (iii) ethatecan or an analogue thereof. In another embodiment, this document provides an ADC comprising an antibody binding to human cell adhesion protein-4, the antibody being directly or via a linker to ethatecan or an analogue thereof. In yet another embodiment, this document provides an ADC prepared by directly or indirectly via a linker binding an antibody binding to human cell adhesion protein-4 and ethatecan or an analogue thereof.

[0018] In another embodiment, this paper provides an ADC comprising an antibody that binds to human cell adhesion protein-4, a linker as needed, and a compound of formula (P), which in some embodiments may be referred to as a "drug carrier": Formula (P)

[0019] The compound of formula (P) may refer to ethathecan.

[0020] In another embodiment, this document provides an ADC comprising an antibody that binds to human cell adhesion protein-4 directly or via a linker to a drug delivery of formula (P). In yet another embodiment, this document provides an ADC prepared by directly or indirectly binding an antibody that binds to human cell adhesion protein-4 to a drug delivery of formula (P).

[0021] In another state, compounds are provided herein that can be used as linkers-drug loaders for ADCs in some states. In one state, a compound of formula (LP) is disclosed that can be used as a linker-drug loader for the ADC disclosed herein: Formula (LP) Where: m is selected from 1 to 6; n is selected from 1 to 6; and p is selected from 1 to 20.

[0022] In some embodiments of the linker-drug carrier of formula (LP), m is 2.

[0023] In some embodiments of the linker-drug carrier of formula (LP), n is 2.

[0024] In some embodiments of the linker-drug carrier of formula (LP), p is 10.

[0025] In another embodiment, this article provides an ADC comprising an antibody and a compound of formula (LP) that binds to human cell adhesion protein-4, or an ADC prepared by binding an antibody and a compound of formula (LP) that binds to human cell adhesion protein-4.

[0026] In another embodiment, this paper provides a compound of formula (L-P') that can be used as a linker-drug carrier for the ADC disclosed herein: Formula (L-P')

[0027] In another state, the compound is provided herein as: (2 S,3 S,4 S,5 R,6 S)-6-(4-((3 S,9 S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxy-2,5-dihydro-1H-pyrrolo-1-yl)propionicamin)ethoxy)ethoxy)ethoxy)acetamino)-1-(((1 S,9 S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12 H-Benzo[de]piperano[3',4':6,7]inzizo[1,2-b]quinoline-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-tetrazoxy-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecazatoracocarbamate-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-piperano-2-carboxylic acid; This compound can be used as a linker-drug carrier for the ADC disclosed herein in some forms.

[0028] In another embodiment, this invention provides an ADC comprising an antibody that binds to human cell adhesion protein-4 and is drug-loaded by a linker of formula (LP). In yet another embodiment, this invention provides an ADC prepared by binding an antibody that binds to human cell adhesion protein-4 and a linker of formula (LP) to a drug-loaded substance.

[0029] In one state, this paper provides an antibody-drug conjugate (ADC) of formula (ADC): Formula (ADC) Wherein: Ab is an antibody that binds to cell adhesion protein-4 as 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.

[0030] In some embodiments of the ADC of formula (ADC), m is 2.

[0031] In some embodiments of the ADC of formula (ADC), n is 2.

[0032] In some embodiments of the ADC of formula (ADC), p is 10.

[0033] In some embodiments of the ADC of formula (ADC), DAR is 8.

[0034] In some embodiments of the ADC of formula (ADC), at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC has a DAR of 8. In some embodiments of the ADC of formula (ADC), the ADC is present in a composition, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC in the composition has a DAR of 8.

[0035] In some embodiments of the ADC of formula (ADC), the Ab is bound to the linker-drug carrier via a thiol group of one or more cysteine ​​residues. In some embodiments, the Ab is bound via a thiol group of one or more cysteine ​​residues in the HC of the Ab, wherein the HC has the amino acid sequence shown in SEQ ID NO: 5 and the cysteines are selected from positions C222, C228, C231 and combinations thereof. In some embodiments, the Ab is bound via a thiol group of one or more cysteine ​​residues in the LC of the Ab, wherein the LC has the amino acid sequence shown in SEQ ID NO: 6 and the cysteine ​​is present at position C214. In some embodiments, the Ab is bound via all of C222, C228 and C231 of the HC shown in SEQ ID NO: 5; and C214 of the LC shown in SEQ ID NO: 6.

[0036] In another embodiment, this invention provides an ADC comprising an antibody binding to human cell adhesion protein-4 via a linker of formula (L-P'). In yet another embodiment, this invention provides an ADC prepared by binding an antibody to human cell adhesion protein-4 and a linker of formula (L-P') to a drug delivery system.

[0037] In one state, this paper provides an antibody-drug conjugate (ADC) of formula (ADC'): Formula (ADC') The DAR value ranges from 1 to 8 (inclusive) and represents the drug-antibody ratio (DAR) of the ADC.

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

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

[0040] In some embodiments of the ADC of formula (ADC'), the Ab is bound to the linker-drug carrier via a thiol group of one or more cysteine ​​residues. In some embodiments, the Ab is bound via a thiol group of one or more cysteine ​​residues in the HC of the Ab, wherein the HC has the amino acid sequence shown in SEQ ID NO: 5 and the cysteines are selected from positions C222, C228, C231 and combinations thereof. In some embodiments, the Ab is bound via a thiol group of one or more cysteine ​​residues in the LC of the Ab, wherein the LC has the amino acid sequence shown in SEQ ID NO: 6 and the cysteine ​​is present at position C214. In some embodiments, the Ab is bound via all of C222, C228 and C231 of the HC shown in SEQ ID NO: 5; and C214 of the LC shown in SEQ ID NO: 6.

[0041] In another embodiment, this paper provides an antibody-drug conjugate (ADC) of formula (ETx-22): Equation (ETx-22) Ab is an antibody that binds to cell adhesion protein-4 as disclosed herein. Ab 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. Ab is bound to the linker-drug carrier via thiol groups of cysteine ​​residues (including C222, C228, and C231 of the HC shown in SEQ ID NO: 5; and C214 of the LC shown in SEQ ID NO: 6).

[0042] In another embodiment, this document provides a composition comprising the anti-cell adhesion protein-4 ADC disclosed herein. In one embodiment of the disclosed composition comprising the anti-cell adhesion protein-4 ADC, at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC in the composition has a DAR of 8.

[0043] In another embodiment, this document provides a pharmaceutical composition comprising the anti-cell adhesion protein-4 antibody disclosed herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients. In another embodiment, this document provides a pharmaceutical composition comprising the anti-cell adhesion protein-4 ADC disclosed herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients. In another embodiment, this document provides a pharmaceutical composition comprising the anti-cell adhesion protein-4 ADC disclosed herein, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC in the composition has a DAR of 8.

[0044] In another embodiment, this article provides a method of treating cancer comprising administering to a patient in need an effective dose of an anti-cell adhesion protein-4 ADC as disclosed herein, such as cancers expressing cell adhesion protein-4. In another embodiment, this article provides a method of treating cancer comprising administering to a patient in need an effective dose of an anti-cell adhesion protein-4 ADC as disclosed herein, wherein the cancer is urothelial carcinoma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer. In another embodiment, this article provides a method of treating cancer comprising administering to a patient in need an effective dose of an anti-cell adhesion protein-4 ADC as disclosed herein, as needed, wherein the cancer is resistant to treatment with an ADC comprising a cell adhesion protein-4 binder and orlistatine (such as monomethyl orlistatine E (MMAE)). In another embodiment, this article provides a method of treating cancer comprising administering to a patient in need an effective dose of an anti-cell adhesion protein-4 ADC as disclosed herein, wherein the patient has previously been administered enrofloxacin, which, if necessary, is enrofloxacin-ejfv marketed under the brand name Padcev®.

[0045] In another embodiment, this document provides an ADC comprising a cell adhesion protein-4 binder and ezetine, used in a method of administering the ADC to a patient suffering from a cancer as disclosed herein (such as cancers expressing cell adhesion protein-4). If desired, the cancer is resistant to treatment with an ADC comprising a cell adhesion protein-4 binder and orlistatine (such as monomethyl orlistatine E (MMAE)). In another embodiment, this document provides an ADC comprising a cell adhesion protein-4 binder and ezetine, used in a method of administering the ADC to a patient suffering from a cancer expressing cell adhesion protein-4, wherein the patient has previously received enrofloxacin, which, if desired, is enrofloxacin-ejfv marketed under the brand name Padcev®.

[0046] In another embodiment, this document provides a pharmaceutical composition comprising a cell adhesion protein-4 binder and ezetine for the treatment of cancers, such as cancers expressing cell adhesion protein-4. If desired, the cancer is resistant to treatment with an ADC comprising a cell adhesion protein-4 binder and an orlistatine (such as monomethyl orlistatine E (MMAE)). In yet another embodiment, this document provides a pharmaceutical composition comprising a cell adhesion protein-4 binder and ezetine for the treatment of cancer in a patient previously treated with enrofloxacin, which, if desired, is enrofloxacin-ejfv marketed under the brand name Padcev®.

[0047] In another embodiment, this document provides the use of an ADC comprising a cell adhesion protein-4 binder and ezetine in the manufacture of a medicament for treating patients with cancers exhibiting cell adhesion protein-4. If desired, the cancer is resistant to treatment with an ADC comprising a cell adhesion protein-4 binder and orlistatine (such as monomethyl orlistatine E (MMAE)). In another embodiment, this document provides the use of an ADC comprising a cell adhesion protein-4 binder and ezetine in the manufacture of a medicament for treating patients with cancers exhibiting cell adhesion protein-4, wherein the patient has previously been treated with enrofloxacin, which, if desired, is enrofloxacin-ejfv marketed under the brand name Padcev®. Simple Explanation of the Diagram

[0048] Figure 1. Schematic diagram of ETx-22.

[0049] Figure 2. Comparison between naked humanization 15A7.5 and ETx-22 HIC.

[0050] Figure 3. In vitro stability assessment. ETx-22 was cultured in mouse, cynomolgus monkey, or human serum. At specified time points, ETx-22 was affinity-captured via anti-human LC-κ (mouse) or Fc-cell adhesion protein-4 fragment (cynomolgus monkey and human), and the drug-antibody ratio was measured by LC / MS.

[0051] Figure 4. In vitro cytotoxic activity of ETx-22. ETx-22 (solid triangles) at a specific dose range or an IgG1 isotype control conjugated to the same linker (open rhombuses) was co-cultured with a pure HCT-116-2G10 line expressing human cell adhesion protein-4 for 8 days. Viability was monitored using a BMG Labtech fluorometer via mitochondrial oxidation (cell titer blue reagent).

[0052] Figure 5. PK / PD characterization of ETx-22 ADC. NSG mice and TNBC PDX400-transplanted NSG mice (n=3 / time point) were intravenously injected with 10 mg / kg Etx-22 when the mean tumor size was 150 mm3 (T0). At specified time points, limb blood samples were collected, and plasma was prepared from naïve and PDX 400-transplanted NSG mice. A, Pharmacokinetic analysis of ETx-22 (ADC + total antibody) using mesoscale detection technology. B, In vivo stability measurement of ETx-22 after capture with anti-human LC-κ affinity by LC / MS. C, Circulating free ethathecan concentration in plasma was determined by LC / MS.

[0053] Figure 6. Mechanism of action of ETx-22. TNBC PDX400 transplanted NSG mice (n=3 / time point) were intravenously injected with 10 mg / kg Etx-22 at a mean tumor size of 150 mm³ (T0). Animals were euthanized at designated time points, and their tumors were collected, measured, and weighed. A, IHC analysis of ETx-22 infiltration and pharmacokinetics in PDX400 tumors. Tumors were fixed and embedded in paraffin. ETx-22 was detected using rabbit anti-human IgG (top inset), which was visualized using secondary anti-rabbit IgG conjugated to horseradish peroxidase and a ChromoMap DAB kit. Phosphorylated H2A.X was detected using mouse antiphosphohistone H2A.X (bottom inset), a marker of topoisomerase I inhibitor activity. This antiphosphohistone H2A.X was visualized using secondary rabbit anti-mouse IgG and tertiary anti-rabbit IgG conjugated to horseradish peroxidase, and a ChromoMap DAB panel. B, Western ink dot analysis of tumor lysis products. ETx-22 was detected using goat anti-human IgG conjugated to horseradish peroxidase. C, LC / MS determination of ethathecan per gram of tumor. D, Quantification of phosphorylated H2A.X-positive cells. The slides in A were quantified using a Hamamatsu scanner, and the percentage of phosphorylated H2A.X-positive cells was quantified using Tribun Calopix software. E, Tumor volume measured using calipers (V = (L x W x H) x π / 6)).

[0054] Figure 7. In vivo efficacy of ETx-22 in the MMAE-resistant TNBC cell line (SUM190). MMAE-resistant SUM190 cells were bilaterally orthotopically transplanted into NSG mice (n=5 / group). At specified time points (black arrows), three different ADCs were administered intravenously: isotype control (8 mg / kg), ETx-22 (2, 4, and 8 mg / kg), and enrofloxacin (4 and 8 mg / kg). The control group received ADC diluents. Tumor growth was monitored twice weekly (V (mm³) = L x W 2 x π / 6).

[0055] Figure 8. In vivo efficacy of ETx-22 in the PDX model (ES0201, esophageal cancer). Tumor fragments were subcutaneously implanted in BALB / c Nu mice. The designated ADC was intravenously injected twice at specified times (black arrows). Cell adhesion protein-4 performance was rapidly scored by immunohistochemistry. Tumor growth was monitored twice weekly (V (mm 3) = L x W 2x π / 6).

[0056] Figure 9. In vivo efficacy of ETx-22 in a PDX model (BLCU003, bladder cancer). Tumor fragments were subcutaneously implanted in nude mice via NMRI. The designated ADC was administered intravenously twice at specified time points (black arrows). Rapid scoring of cell adhesion protein-4 expression was determined by immunohistochemical staining and is shown here. Tumor growth was monitored twice weekly (V (mm³) = L x W 2 x π / 6).

[0057] Figure 10. In vivo efficacy of ETx-22 in a PDX model (B521, bladder cancer). This bladder cancer PDX model, B521, exhibits relatively high expression of cell adhesion protein-4 and possesses a homozygous mutation in FGFR3, thus confers resistance to erdafitinib. Administration: cisplatin / gemcitabine: 4 mg / kg Q3W ×2 / 60 mg / kg QW ×4.

[0058] Figure 11. In vivo efficacy of ETx-22 in a PDX model (HN2579, head and neck cancer). The head and neck PDX model HN2579 exhibited moderate to high expression of cell adhesion protein-4. Tumor fragments were subcutaneously implanted into NOD / SCID nude mice. The designated ADC was administered intravenously twice at specified time points (black arrows). A rapid score of cell adhesion protein-4 expression was determined by immunohistochemical staining and is shown here. Tumor growth was monitored twice weekly (V (mm³) = L x W 2 x π / 6).

[0059] Figure 12. In vivo efficacy of ETx-22 in a PDX model (CV3035, cervical cancer). Tumor fragments were subcutaneously implanted in BALB / c Nu mice. The designated ADC was intravenously injected twice at specified time points (black arrows). Rapid scoring of cell adhesion protein-4 expression was determined by immunohistochemical staining and is shown here. Tumor growth was monitored twice weekly (V (mm 3) = L x W 2x π / 6).

[0060] Figure 13. In vivo efficacy of ETx-22 in a PDX model (CV3560, cervical cancer). This cervical cancer PDX model CV3560 exhibits moderate to high expression of cell adhesion protein-4.

[0061] Figure 14. In vivo efficacy of ETx-22 in a PDX model (OV2423, ovarian cancer). The tumor fragment was subcutaneously implanted in NOD / SCID mice. The designated ADC was administered intravenously twice at specified time points (black arrows). Rapid scores of cell adhesion protein-4 expression were determined by immunohistochemical staining in each PDX model and are shown here. Tumor growth was monitored twice weekly (V (mm³) = L x W 2 x π / 6).

[0062] Figure 15. In vivo efficacy of ETx-22 in the TNBC PDX400 model. Tumor fragments were bilaterally implanted into NSG mice. The designated ADC was intravenously injected at specified time points (black arrows). Rapid score of cell adhesion protein-4 performance as determined by immunohistochemical staining is shown. Rod = 500 µm.

[0063] Figure 16. In vivo efficacy of ETx-22 in the TNBC PDX317 model. Tumor fragments were bilaterally implanted in NSG mice. The designated ADC was intravenously injected at specified time points (black arrows). Rapid scores of cell adhesion protein-4 performance, as determined by immunohistochemical staining, are shown. Rod = 500 µm.

[0064] Figure 17. In vivo efficacy of ETx-22 in the TNBC PDX348 model. Tumor fragments were bilaterally implanted in NSG mice. The designated ADC was intravenously injected at specified time points (black arrows). Rapid scores of cell adhesion protein-4 performance, as determined by immunohistochemical staining, are shown. Rod = 500 µm.

[0065] Figure 18. In vivo efficacy of ETx-22 in the TNBC PDX434 model. This TNBC PDX434 model exhibits low cell adhesion protein-4 expression. Tumor fragments were bilaterally implanted into NSG mice. The designated ADC was intravenously injected at specified time points (black arrows). Rapid scores of cell adhesion protein-4 expression as determined by immunohistochemical staining are shown. Rod = 500 µm. Implementation

[0066] Reference to the sequence listing: This application claims the benefits of EP 23201214.6, filed on October 2, 2023; the disclosure of that application is incorporated herein by reference.

[0067] This application is filed together with a sequence list in ST.26 XML format. This sequence list is provided as a file titled "txt_P30825_FR" created on September 27, 2023, and is 7,935 bytes in size. The full text of the ST.26 XML format sequence list information is incorporated herein by reference. [Cell adhesion proteins] [-4]

[0068] As used herein, "human cell adhesion protein-4" refers to the human cell adhesion protein-4 protein or polypeptide, also known as poliovirus receptor-associated 4, Ig superfamily receptor LNIR, or poliovirus receptor-associated protein 4 (PVRL4). The amino acid sequence of human cell adhesion protein-4 can be found in NP_112178.2 and is provided as in SEQ ID NO: 1. [Antibody] []

[0069] The disclosed ADCs contain antibodies. The term "antibody," as used herein, refers to an immunoglobulin molecule that binds to an antigen. These antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0070] An exemplary antibody of the present invention is an immunoglobulin G (IgG) antibody comprising four polypeptide chains cross-linked via interchain disulfide bonds: two heavy chains (HC) and two light chains (LC). The amino-terminal portion of each of the four polypeptide chains includes a variable region of approximately 100 to 125 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function. Each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region. Each light chain includes a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further subdivided into subtypes (e.g., IgG1, IgG2, IgG3, and IgG4).

[0071] 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). These CDRs are exposed on the protein surface and are important regions for antibody-antigen binding specificity. Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this document, 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." These CDRs contain most of the residues that form specific interactions with the antigen. Assigning amino acid residues to these CDRs can be done according to well-known protocols, including those described by them in the following: 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 is available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999;27:209-212). The CDR of this invention was determined by North.

[0072] Some antibodies of the ADCs described herein contain an IgG1 Fc region or Fc region derived from human IgG1, such as a modified IgG1 Fc region with altered Fc effector function. IgG1 is known to induce antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Some antibodies of the disclosed ADCs contain amino acid substitutions introduced into the IgG1 Fc region, which alter the Fc effector function. In some morphologies of the antibodies of the disclosed ADCs, mutations are introduced in the Fc region at positions 234 and 235 (EU index numbers) or at positions 247 and 248 (Kabat numbers). In some morphologies 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 numbers) or 247, 248, and 350 (Kabat numbers). In some embodiments, the anti-cell adhesion protein-4 antibody of the present invention contains a modified human IgG1 Fc region comprising one or more mutations selected from L234E, L235E, P331S and combinations thereof (EU index number) or selected from L247F, L248E, P350S and combinations thereof (Kabat number). In another embodiment, the amino acid substitutions introduced into the IgG1 Fc region reduce or eliminate measurable antibody effector function, including (but not limited to) reduced binding to the Fc receptor.

[0073] Mammal expression of antibodies from IgG subclasses can result in the cleavage of C-terminal amino acids from one or both heavy chains; for example, one or both C-terminal amino acids of an IgG1 antibody can be removed. For IgG1 antibodies, if a C-terminal lysine is present, it can be truncated or cleaved from the heavy chain during expression. Additionally, the penultimate glycine can also be truncated or cleaved from the heavy chain. The anti-cell adhesion protein-4 antibodies described herein may accordingly include one or more truncations and cleavages.

[0074] The mammalian expression of antibodies can also lead to modifications of the N-terminal amino acids. For example, if the N-terminal amino acid of the heavy or light chain is glutamic acid or glutamine, it can be modified to pyroglutamic acid. Similarly, if the C-terminal amino acid of the heavy or light chain is lysine or glycine, it can be removed. The anti-cell adhesion protein-4 antibody disclosed herein may accordingly include one or more modifications or removals.

[0075] The terms "nucleic acid" or "polynucleotide," as used interchangeably herein, refer to polymers of nucleotides, including molecules containing single-stranded and / or double-stranded nucleotides, such as DNA, cDNA, and RNA molecules, which incorporate natural nucleotides, modified nucleotides, and / or analogs of nucleotides. The polynucleotides of this invention may also include, for example, acceptors incorporated therein by DNA or RNA polymerases or synthetic reactions.

[0076] The polynucleotides of this invention can be expressed in host cells, for example, after such polynucleotides have been operatively linked to an expression control sequence. Expression control sequences capable of expressing the operatively linked polynucleotides are well known in the art. For example, the expression vector may include sequences encoding one or more signal peptides that promote the secretion of the polypeptide(s) from the host cell. For example, the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide. Expression vectors containing the polynucleotide of interest (e.g., polynucleotides encoding polypeptides of antibodies) can be transferred into host cells using well-known methods. Additionally, the expression vector may contain one or more selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to assist in the detection of host cells transformed with the desired polynucleotide sequence.

[0077] The host cell includes cells stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing all or part of the antibodies of the present invention. According to some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with an expression vector expressing an HC polypeptide of the antibody of the present invention and an expression vector expressing an LC polypeptide. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with both HC and LC polypeptide expression vectors expressing the antibodies of the present invention. The antibodies of the present invention can be produced in mammalian cells (such as CHO, NSO, HEK293, or COS cells) according to techniques well known in the art.

[0078] The medium in which the antibody of this invention has been secreted can be purified using conventional techniques, such as a mixed-mode approach of ion exchange and hydrophobic interaction chromatography. For example, the medium can be applied to and dissociated from a protein A or G column using conventional methods; a mixed-mode approach of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and polymers can be effectively removed using common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, for example, refrigerated at -70°C, or lyophilized. Various methods of protein purification can be used, and such methods are known in this art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd ed., Springer, NY (1994). [anti] [-] [Cell adhesion proteins] [-4] [Antibody] []

[0079] The antibodies used in the disclosed ADCs exhibit specific binding to cell adhesion protein-4 relative to other proteins in the human cell adhesion protein family, such as cell adhesion protein-1. As used herein, the terms "binding" and "specific binding" refer to the binding of the antibody to the antigenic determinant of the cell adhesion protein-4 antigen. The measure of the strength of antibody binding is called affinity. Methods for determining this binding and / or affinity using in vitro assays are known to those skilled in the art. According to the invention, detection is described herein and particularly preferred using flow cytometry, immunohistochemistry, and / or fluorescence. The affinity of an antibody for an antigen can be defined by the terms Ka (rate constant of antibody dissociation from the antibody / antigen complex), KD (dissociation constant), and Kdis (KD / Ka).

[0080] In some embodiments, the revealed ADC may comprise an anti-cell adhesion protein-4 antibody, either a tumor-selective anti-cell adhesion protein-4 antibody or an antibody prepared therefrom. Compared to cell adhesion protein-4 expressed by normal cells, a tumor-selective anti-cell adhesion protein-4 antibody can be characterized as an anti-cell adhesion protein-4 antibody that binds to tumor-expressed cell adhesion protein-4 with higher affinity. For comparison, in some embodiments, binding affinity can be detected by flow cytometry, immunohistochemistry, and / or fluorescence detection. Normal cells may include normal human endothelial keratinocytes (NHEK). Normal cells may include human endothelial keratinocytes expressing in vitro differentiated cell adhesion protein-4. In one embodiment, the tumor-selective anti-cell adhesion protein-4 antibody may have a dissociation constant KD for binding to cell adhesion protein-4 expressed on keratinocytes, which is greater than the dissociation constant for binding to cell adhesion protein-4 expressed on tumor cells. In one state, compared to tumor cells, tumor-selective anti-cell adhesion protein-4 antibodies exhibited lower internalization and / or cytotoxic activity against keratinocytes. In one state, compared to tumor cells, tumor-selective anti-cell adhesion protein-4 antibodies exhibited lower binding affinity, lower internalization, and lower cytotoxic activity against keratinocytes. In one state, compared to the reference antibody HA22 mAb (enfortumab), the revealed ADCs exhibited lower binding affinity, lower internalization, or lower cytotoxic activity against keratinocytes compared to tumor cells.

[0081] The tumor-selective anti-cell adhesion protein-4 antibody used in the disclosed ADC may comprise the antibody disclosed in WO2022 / 207822 and WO2022 / 207825. In one state, the ADC disclosed herein comprises a monoclonal antibody called 15A7.5 or a humanized variant thereof called H1L2_15A7.5, which comprises the recurrent mutation as disclosed in WO2022 / 207822 and WO2022 / 207825 and has an HC comprising the amino acid sequence shown in SEQ ID NO: 5 and an LC comprising the amino acid sequence shown in SEQ ID NO: 6.

[0082] In one embodiment, this document provides an antibody or an ADC prepared therefrom that binds to human cell adhesion protein-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 (HCDR) HCDR1, HCDR2, and HCDR3, and the LCVR comprises light chain complementarity-determining regions (LCDR) 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 numbers). In another embodiment, this paper provides an antibody that binds to human cell adhesion protein-4, wherein the HCDR1 contains SEQ ID NO: 15 (GFTFSNYG), the HCDR2 contains SEQ ID NO: 16 (ISNLAYGI), the HCDR3 contains SEQ ID NO: 17 (ARGARATGWFAY), the LCDR1 contains SEQ ID NO: 18 (QNVDTH), the LCDR2 contains SEQ ID NO: 19 (SAS), and the LCDR3 contains SEQ ID NO: 20 (QQYNSYPLT) (all IMGT numbers).

[0083] In one embodiment, the ADC disclosed herein comprises or is prepared from an anti-cell adhesion protein-4 antibody, wherein the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the antibody contains an HCVR comprising SEQ ID NO: 7 and an LCVR comprising SEQ ID NO: 8.

[0084] In another embodiment, the ADC disclosed herein comprises or is prepared from an anti-cell adhesion protein-4 antibody, wherein the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), and wherein the antibody has a human IgG1 or IgG4 isotype. In another embodiment, the anti-cell adhesion protein-4 antibody has a human IgG1 isotype. In another embodiment, the anti-cell adhesion protein-4 antibody has a human IgG1 isotype, and the HC of the anti-cell adhesion protein-4 antibody contains one or more mutations at positions L234, L235, and P331 (EU index number) or at positions L247, L248, and P350 (Kabat number). In another embodiment, the anti-cell adhesion protein-4 antibody contains one or more mutations selected from L234F, L235E, and P331S (EU index number) or L247F, L248E, and P350S (Kabat number).

[0085] In one embodiment, this document provides an antibody or ADC comprising binding to human cell adhesion protein-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.

[0086] In another embodiment, this article provides an antibody or ADC comprising binding to human cell adhesion protein-4, wherein the HC is composed of SEQ ID NO: 5 and the LC is composed of SEQ ID NO: 6.

[0087] In another embodiment, this document provides mammalian cells comprising DNA molecules containing a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequence shown in SEQ ID NO: 6, or both the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, these cells are capable of expressing cell adhesion protein-4 antibodies as disclosed herein.

[0088] In another embodiment, this document provides 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 shown in SEQ ID NO: 5, and wherein the second DNA molecule comprises a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6.

[0089] In another embodiment, this paper provides a method for generating a cell adhesion protein-4 antibody, which includes culturing one of the mammalian cells disclosed herein under conditions that allow the antibody to be expressed, and recovering the expressed antibody.

[0090] In another embodiment, this document provides an antibody produced by culturing mammalian cells containing DNA molecules under conditions that allow the antibody to be expressed, the DNA molecules containing a polynucleotide sequence encoding a polypeptide encoding an amino acid sequence comprising both SEQ ID NO: 5, SEQ ID NO: 6, or both SEQ ID NO: 5 and SEQ ID NO: 6, and recovering the expressed antibody.

[0091] In another embodiment, this document provides an antibody produced by culturing mammalian cells containing a first DNA molecule and a second DNA molecule under conditions that allow the antibody to be expressed, wherein the first DNA molecule contains a polynucleotide sequence encoding a polypeptide containing the amino acid sequence shown in SEQ ID NO: 5, and wherein the second DNA molecule contains a polynucleotide sequence encoding a polypeptide containing the amino acid sequence shown in SEQ ID NO: 6, and recovering the expressed antibody.

[0092] The term "enrofloxacin" as used herein refers to a fully human anti-cell adhesion protein 4 IgG1κ monoclonal antibody having the sequence disclosed in WO2012047724 (Figures 3A and 3B), expressed and purified using standard conditions. Enrofloxacin may also be referred to herein as HA22 mAb. The term enrofloxacin-ejfv refers to an antibody-drug conjugate marketed under the brand name PADCEV®, comprising enrofloxacin bound to a monomethyl orrisstatin E (MMAE) drug delivery via a peptide linker, wherein the linker-drug delivery has the formula maleiminohexanoyl (MC)-valine-citrulline-PABC-MMAE. [Drug Carrier] []

[0093] The cell adhesion protein-4 antibody of the present invention can be bound to various drug carriers (including pharmaceutically acceptable salts thereof) to form antibody-drug conjugates (ADCs). In one embodiment, the disclosed ADC comprises or is prepared from: (i) an antibody binding to human cell adhesion protein-4, (ii) a linker as desired, and (iii) a drug carrier. In another embodiment, the disclosed ADC comprises an antibody binding to human cell adhesion protein-4 directly or indirectly via a linker to a drug carrier. In yet another embodiment, the disclosed ADC is prepared by directly or indirectly binding an antibody binding to human cell adhesion protein-4 to a drug carrier via a linker. In yet another embodiment, the disclosed ADC releases the drug carrier after the ADC binds to target cells expressing cell adhesion protein-4 and the ADC is internalized by the target cells.

[0094] Suitable portions for binding to the cell adhesion protein-4 antibody disclosed herein include cytotoxic agents (e.g., chemotherapeutic agents), prodrug-converting enzymes, radioisotopes or compounds, toxins, and other known drug loadings in this art.

[0095] The exemplary ADC described in this article utilizes an ethathecan-based drug delivery system (e.g., ethathecan or ethathecan analogues). Ethatecan is a topoisomerase I (TOPO 1) inhibitor that has been shown to have anticancer activity. Ethatecan and its analogues bind to this TOPO 1 / DNA complex, preventing re-annealing due to the accumulation of partially cleaved DNA, which leads to cell death.

[0096] In another embodiment, the disclosed ADC comprises an antibody binding to human cell adhesion protein-4, a linker as needed, and ethatecan or an analogue thereof. In another embodiment, the disclosed ADC comprises an antibody binding to human cell adhesion protein-4 that is directly or via a linker bound to ethatecan or an analogue thereof. In another embodiment, the disclosed ADC is prepared by directly or indirectly binding an antibody binding to human cell adhesion protein-4 and ethatecan or an analogue thereof via a linker. In the disclosed ADC, ethatecan can be directly or indirectly bound to the anti-cell adhesion protein-4 antibody via its free NH2 group (e.g., by forming a amide bond between the NH2 group of ethatecan and the linker).

[0097] In another embodiment, the disclosed ADC comprises or is prepared from the following: (i) an antibody binding to human cell adhesion protein-4, (ii) a linker as desired, and a compound of formula (P), which may be referred to as a "drug carrier": Formula (P)

[0098] Compound of formula (P) may refer to ethathecan.

[0099] In another embodiment, the disclosed ADC comprises an antibody binding to human cell adhesion protein-4 directly or via a linker to a drug delivery of formula (P). In yet another embodiment, the disclosed ADC is prepared by directly or via a linker binding an antibody binding to human cell adhesion protein-4 to a drug delivery of formula (P). [Connector] []

[0100] As disclosed herein, a drug delivery vehicle can be bound to a cell adhesion protein-4 antibody to form the cell adhesion protein-4 ADC described herein, using methods understood by those skilled in the art. One example of such binding would involve linking the drug delivery vehicle described herein to the cell adhesion protein-4 antibody described herein via a linker.

[0101] The linker used in ADCs is designed to achieve stability in plasma, allowing the ADC time to localize to target cells. Premature drug delivery reduces the therapeutic index of the ADC by disrupting various non-target tissues. Once the ADC is internalized into target cells, the linker should provide a release mechanism for the drug delivery so that it can function as designed.

[0102] Those skilled in the art know that linkers contain, for example, cleavable and non-cleavable portions. Therefore, this article provides an ADC in which the drug delivery (e.g., ethatec or an ethatec analog) binds to an anti-cell adhesion protein-4 antibody via a linker having a cleavable portion or to an antibody via a linker having a non-cleavable portion.

[0103] Any suitable linker known in this art can be used to prepare the ADC of the present invention. In some embodiments, such linkers contain reactive groups capable of binding to both the antibody and the drug delivery system of the present invention. Examples include (but are not limited to) linkers containing a maleimine group of a thiol that can bind to a cysteine ​​residue of the antibody and a succinimine ester group (such as N-hydroxysuccinimine 3-(maleimino)propionic acid) that can bind to the drug delivery system.

[0104] In some embodiments of the present invention, the ADC includes a linker having a cleavable portion. The cleavable portion may include a β-glucuronide that can be cleaved by intracellular β-glucuronidase.

[0105] In some embodiments of the present invention, the ADC includes a linker comprising a spacer unit, referred to herein as spacer unit A, which links the cysteine ​​of the antibody disclosed herein to the drug delivery unit described herein. Some chemicals used in this art for binding the drug delivery unit to the thiol group of the cysteine ​​include maleimide or succinimidide chemicals, and can be used in the ADC of the present invention. In other embodiments of the present invention, maleimide spacers (such as maleiminopropyl) are used in the linker disclosed herein.

[0106] In some embodiments of the present invention, an ADC having a spacer subunit A with the following formula is provided: Where z ranges from 1 to 5. The spacer unit A is bound to the anti-cell adhesion protein-4 antibody via a reduced maleic imine group. The spacer unit A can be bound to the drug delivery unit directly or indirectly via a linker through the methylene portion (i.e., via the (CH2)z portion). In some states of spacer unit A, z is 2. In some states, the spacer unit A "spaces" or "locates" the remaining portion of the linker and the drug delivery unit away from the antibody in the ADC. [Contains polyinosine] [(PSAR)] [and polyethylene glycol] [(PEG)] [Connector to the next part.] []

[0107] In some samples, the disclosed ADC contains a linker comprising a polysarcosine moiety. ADCs comprising a polysarcosine moiety are known in this art. (See WO2019 / 081455 and WO2022 / 207699; and Conilh et al., "Exatecan antibody drug conjugates based on a hydrophilic polysarcosine drug-linker platform" (Pharmaceuticals 14 (2021), 247).)

[0108] In some embodiments, the disclosed linker comprises at least one ethylene glycol unit, such as 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 acetyl, aminomethyl, and / or sulfonamide groups) added to at least one ethylene glycol unit, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more ethylene glycol units.

[0109] In some embodiments, the linker is a hydrophilic polysarcosine linker comprising, for example, 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), wherein the linker can be cleaved by glycosidase, and in particular by glucuronidase.

[0110] In some embodiments, the linker is a hydrophilic polysarcosine linker comprising, for example, about 8 to 12 sarcosine units and at least one ethylene glycol unit (e.g., up to 10 ethylene glycol units), and wherein the linker can be cleaved by glycosidase, and in particular by glucuronidase.

[0111] In some embodiments, the linker is a hydrophilic polysarcosine linker comprising 10 sarcosine units and 2 ethylene glycol units, and wherein the linker can be cleaved by glucuronidase.

[0112] Another aspect of the present invention relates to a linker-drug conjugate comprising: (i) a hydrophilic polysarcosine linker comprising, for example, about 8 to 12 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), wherein the linker is cleavable by a glycosidase, and particularly by a glucuronidase, and wherein the linker comprises a thiol reactive group, such as a maleimine group, capable of reacting with cysteine ​​residues on an antibody or its antigen-binding fragment; and (ii) ethatecan covalently linked to the linker.

[0113] In a particular embodiment, the linker-drug conjugate comprises: (i) a hydrophilic polysarcosine group (e.g., a polysarcosine group consisting of 10 sarcosine units) and a polyethyl group (e.g., a polyethylene glycol group consisting of 2 ethylene glycol units), wherein the linker is cleavable by glycosidase, and particularly by glucuronidase, and wherein the linker comprises a maleimine group capable of reacting with cysteine ​​residues on an antibody or its antigen-binding fragment; and (ii) ethatecan covalently linked to the linker. In some embodiments, the polysarcosine group is located in an orthogonal position relative to the polyethylene glycol group and ethatecan in the linker. [Self-sacrifice] [type] [unit] []

[0114] A self-sacrificing or self-removing portion of the ADC can be designed into its structure, for example, within the ADC's linker. Self-sacrifice typically involves a self-sacrificing unit that is activated upon removal of a trigger group bound to it. In some forms of the self-sacrificing unit, after the ADC is internalized by a target cell, the trigger group is enzymatically cleaved from the self-sacrificing unit by cellular enzymes. Suitable trigger groups may include β-glucuronide, which is cleaved from the self-sacrificing unit by intracellular β-glucuronidase. Then, further biological or chemical reactions cause the self-sacrificing unit to spontaneously eliminate itself from the linker. This self-sacrificing unit can provide positive properties to the ADC, such as providing space to reduce steric hindrance for cellular proteases to reach peptide cleavage sites within the ADC.

[0115] In some embodiments of the present invention, the ADC described herein contains a self-sacrificing unit. When present, the self-sacrificing unit may be located within the linker and may be activated by the trigger group following enzymatic cleavage of the self-sacrificing unit. In some embodiments, the self-sacrificing unit comprises a 3-nitro-octopamine group bound to a β-glucuronide via its 4-hydroxyl group, the β-glucuronide being cleaved by intracellular β-glucuronidase expressed by tumors. [Combination] []

[0116] In the disclosed ADCs, the drug delivery (particularly ethatecan or ethatecan analogs) is covalently bound to the anti-cell adhesion protein-4 antibody, either directly or indirectly via a linker. Methods for binding the antibody disclosed herein to the disclosed drug delivery are known in the art. In some methods, the antibody is bound to the linker in a first reaction, and then the antibody and linker are bound to the drug delivery in a second reaction. In some methods, the antibody is bound to the drug delivery or drug delivery / linker in a single reaction.

[0117] The drug delivery system can bind to any suitable site on the anti-cell adhesion protein-4 antibody without disabling the antibody's binding to cell adhesion protein-4. For example, the drug delivery system can bind to reactive amino acid residues on the antibody, such as cysteine ​​residues with thiol groups.

[0118] In some samples, the ADC has a drug-antibody / antibody fragment ratio (DAR) greater than 1, meaning that more than one drug molecule is linked to an antibody / antibody fragment. Typically, the conjugate has a DAR of about 2:1 to about 16:1, particularly about 4:1 to about 10:1, and more particularly about 6:1 to about 8:1. This DAR can be calculated from a statistical distribution using known methods. In some samples, the ADC has a DAR of 8:1. In some samples, the ADC has a DAR of 8, or the ADC has a substantially uniform DAR of 8, for example, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the ADC have a DAR of 8.

[0119] In some samples, it may be necessary to obtain a composition comprising an ADC (or a group of ADCs) wherein the DAR of the ADC (or the group of ADCs) is substantially homogeneous, wherein at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADCs have a selected DAR of 1 to 8. In some samples of the disclosed ADC and compositions comprising the disclosed ADC, the compositions comprising the ADC have substantially homogeneous DAR, wherein 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 samples of the disclosed ADC and compositions comprising the disclosed ADC, the compositions comprising the ADC have substantially homogeneous DAR, wherein at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADCs have a DAR of 8.

[0120] Compositions comprising the disclosed ADC (or ADC group) may comprise an ADC having an average DAR. In some embodiments, compositions comprising the disclosed ADC (or ADC group) comprise an ADC having an average DAR in 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. [Contains ethatheca-loaded antibodies] [-] [Cell adhesion proteins] [-4 ADC]

[0121] In another embodiment, this paper provides an ADC comprising an antibody binding to human cell adhesion protein-4 and an ethanotecan drug delivery system. In some embodiments, the ADC is prepared from a compound of formula (LP) or a compound of formula (LP) that can be used as a linker-drug delivery system. Formula (LP) Where: m is selected from 1 to 6; n is selected from 1 to 6; and p is selected from 1 to 20.

[0122] In some embodiments of the linker-drug carrier of formula (LP), m is 2.

[0123] In some embodiments of the linker-drug carrier of formula (LP), n is 2.

[0124] In some embodiments of the linker-drug carrier of formula (LP), p is 10.

[0125] In some samples, the ADC contains a compound of formula (L-P') or is prepared therefrom, which can be used as a linker-drug carrier: Formula (L-P')

[0126] In some samples, the disclosed ADC contains or is prepared using the following compound: (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxy-2,5-dihydro-1H-pyrrolo-1-yl)propionic amino)ethoxy)ethoxy)ethoxy)acetylamino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12 H-Benzo[de]piperano[3',4':6,7]inzizo[1,2-b]quinoline-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-tetrazoxy-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecazatoratezotecan-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-piperano-2-carboxylic acid; This compound can be used as a linker-drug carrier.

[0127] In one state, the ADC revealed in this paper is expressed as equation (ADC): Formula (ADC) Wherein: Ab is an antibody that binds to cell adhesion protein-4 as 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 and represents the drug-antibody ratio (DAR) of the ADC.

[0128] In some embodiments of the ADC of formula (ADC), m is a sufficiently large value such that the linker-drug carrier 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 cell adhesion protein-4. In some embodiments of the ADC of formula (ADC), m is greater than 1, and preferably, m is 2.

[0129] In some embodiments of the ADC of formula (ADC), m is a sufficiently small value to avoid compromising the stability of the linker-drug delivery. In some embodiments of the ADC of formula (ADC), m is less than 6, 5, 4, or 3, and preferably, m is 2.

[0130] In some embodiments of the ADC of formula (ADC), n is a sufficiently large value to provide sufficient hydrophilicity and reduce aggregation of the ADC. In some embodiments of the ADC of formula (ADC), n is greater than 1, and preferably, n is 2.

[0131] In some embodiments of the ADC of formula (ADC), n is a sufficiently small value to avoid compromising the stability of the linker-drug delivery. In some embodiments of the ADC of formula (ADC), n is less than 6, 5, 4, or 3, and preferably, n is 2.

[0132] In some embodiments of the ADC of formula (ADC), p is a sufficiently large value 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, and preferably, p is 10.

[0133] In some embodiments of the ADC of formula (ADC), p is a sufficiently small value to avoid impairing the stability of the linker-drug delivery. 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.

[0134] In some embodiments of the ADC of formula (ADC), the DAR is a sufficiently large value to provide sufficient activity (such as sufficient toxicity) for the ADC. In some embodiments of the ADC of formula (ADC), the DAR is greater than 1, 2, 3, 4, 5, 6, or 7, and preferably, the DAR is 8.

[0135] In some embodiments of the ADC of formula (ADC), at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC in the composition has a DAR of 8. In some embodiments, the ADC of formula (ADC) is present in a composition, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC in the composition has a DAR of 8. In some embodiments, the ADC of formula (ADC) is present in a composition, wherein the ADC present in the composition has an average DAR greater than about 1, 2, 3, 4, 5, 6, or 7.

[0136] In some embodiments of the ADC of formula (ADC), the Ab is bound to the linker-drug carrier via a thiol group of one or more cysteine ​​residues. In some embodiments, the Ab is bound via a thiol group of one or more cysteine ​​residues in the HC of the Ab, wherein the HC has the amino acid sequence shown in SEQ ID NO: 5 and the cysteines are selected from C222, C228, C231 and combinations thereof. In some embodiments, the Ab is bound via a thiol group of one or more cysteine ​​residues in the LC of the Ab, wherein the LC has the amino acid sequence shown in SEQ ID NO: 6 and the cysteine ​​is C214. In some embodiments of the ADC of formula (ADC), the Ab contains an HC comprising the amino acid sequence shown in SEQ ID NO: 5 and an LC comprising the amino acid sequence shown in SEQ ID NO: 6, and the Ab is bound to the linker-drug carrier via a thiol group of cysteine ​​residues (including all of C222, C228, and C231 of the HC; and C214 of the LC).

[0137] In some embodiments, the ADC disclosed herein is of formula (ADC'). The DAR ranges from 1 to 8 and represents the drug-antibody ratio (DAR) of the ADC.

[0138] In some embodiments of the ADC of formula (ADC'), the DAR is a sufficiently large value 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, or 7, and preferably, the DAR is 8.

[0139] In some embodiments of the ADC of formula (ADC'), at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC in the composition has a DAR of 8. In some embodiments of the ADC of formula (ADC'), the ADC is present in a composition, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the ADC in the composition has a DAR of 8. In some embodiments, the ADC of formula (ADC') is present in a composition, wherein the ADC present in the composition has an average DAR greater than about 1, 2, 3, 4, 5, 6, or 7.

[0140] In some embodiments of the ADC of formula (ADC'), the Ab is bound to the linker-drug carrier via a thiol group of one or more cysteine ​​residues. In some embodiments, the Ab is bound via a thiol group of one or more cysteine ​​residues in the HC of the Ab, wherein the HC has the amino acid sequence shown in SEQ ID NO: 5 and the cysteines are selected from C222, C228, C231 and combinations thereof. In some embodiments, the Ab is bound via a thiol group of one or more cysteine ​​residues in the LC of the Ab, wherein the LC has the amino acid sequence shown in SEQ ID NO: 6 and the cysteine ​​is C214. In some embodiments of the ADC of formula (ADC), the Ab contains an HC comprising the amino acid sequence shown in SEQ ID NO: 5 and an LC comprising the amino acid sequence shown in SEQ ID NO: 6, and the Ab is bound to the linker-drug carrier via a thiol group of cysteine ​​residues (including all of C222, C228, and C231 of the HC; and C214 of the LC).

[0141] In another sample, this paper provides the ADC of equation (Etx-22): Equation (ETx-22) Ab is an antibody that binds to cell adhesion protein-4 as disclosed herein. The Ab of formula (ETx-22) comprises two HCs containing the amino acid sequence shown in SEQ ID NO: 5 and two LCs containing the amino acid sequence shown in SEQ ID NO: 6. The Ab is bound to the linker drug carrier in formula (Etx-22) via the thiol groups in C222, C228 and C231 of the two HCs of the Ab and via the thiol group in C214 of the two chains of the LC, thereby providing 8 DARs (i.e., 4 cysteine-maleimine bonds in each arm of the Ab).

[0142] As shown, the ADC of formula (ETx-22) contains a 3-maleimino-propionic spacer, a PEG2 spacer (i.e., -CH2-CH2-O-CH2-CH2-O-), a PSAR10 group (i.e., -(N(CH3)-CH2-C(O))10-) located in the linker in a position orthogonal to the drug load, and a linker containing a self-sacrificing unit of 4-β-glucuronide-3-nitro-octopamine.

[0143] In some forms, Ab of formula (ETx-22) includes a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR includes heavy chain complementarity determination regions (HCDR) HCDR1, HCDR2, and HCDR3, and the LCVR includes light chain complementarity determination regions (LCDR) LCDR1, LCDR2, and LCDR3, wherein: (a) HCDR1 includes SEQ ID NO: 9, HCDR2 includes SEQ ID NO: 10, HCDR3 includes SEQ ID NO: 11, LCDR1 includes SEQ ID NO: 12, LCDR2 includes SEQ ID NO: 13, and LCDR3 includes SEQ ID NO: 14 (all Kabat numbers); or (b) HCDR1 includes SEQ ID NO: 15, HCDR2 includes SEQ ID NO: 16, HCDR3 includes SEQ ID NO: 17, LCDR1 includes SEQ ID NO: 18, and LCDR2 includes SEQ ID NO: 19. 19, and the LCDR3 contains SEQ ID NO: 20 (both are IMGT numbers).

[0144] In some states, the Ab of the ADC of formula (ETx-22) includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the HCVR includes SEQ ID NO: 7 and the LCVR includes SEQ ID NO: 8.

[0145] In some states, the Ab of the ADC of formula (ETx-22) contains a heavy chain (HC) and a light chain (LC), wherein the HC contains SEQ ID NO: 5 and the LC contains SEQ ID NO: 6.

[0146] In some formulations, the ADC of formula (ETx-22) contains the following compound or is prepared using the following compound as a linker-drug carrier: (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxy-2,5-dihydro-1H-pyrrolo-1-yl)propionic)ethoxy)ethoxy)ethoxy)acetylamino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12 H-Benzo[de]piperano[3',4':6,7]inzizo[1,2-b]quinoline-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-tetrazoxy-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecazatetratetradecano-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-piperano-2-carboxylic acid.

[0147] In some samples, the ADC of formula (ETx-22) is prepared by combining the following: (i) mAb; and (ii) a linker-drug delivery: (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxy-2,5-dihydro-1H-pyrrolo-1-yl)propionic)ethoxy)ethoxy)ethoxy)acetylamino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12 H-Benzo[de]piperano[3',4':6,7]inzizo[1,2-b]quinoline-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-tetrazoxy-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecazatoratriacande-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-piperano-2-carboxylic acid. In some samples, the ADCs prepared by combining (i) and (ii) exhibit a DAR of 8.

[0148] In some samples, the ADC of formula (ETx-22) is prepared by: (a) reducing the mAb; and (b) binding the reduced mAb to the following linker-drug carrier: (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxy-2,5-dihydro-1H-pyrrolo-1-yl)propionic)ethoxy)ethoxy)ethoxy)acetylamin)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12 H-Benzo[de]piperano[3',4':6,7]inzizo[1,2-b]quinoline-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-tetrazoxy-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecazatoratriacande-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-piperano-2-carboxylic acid. In some samples, the ADCs prepared by steps (a) and (b) exhibit a DAR of 8.

[0149] In some samples, the disclosed ADC is prepared by combining the following: (i) mAb containing HC comprising the amino sequence shown in SEQ ID NO: 5 and LC comprising LC comprising the LC shown in SEQ ID NO: 6; and (ii) (2S,3S,4S,5R,6S)-6-(4-((3S,9S)-40-amino-9-(2-(2-(2-(3-(2,5-dioxy-2,5-dihydro-1H-pyrrole-1-yl)propionic acid)ethoxy)ethoxy)ethoxy)acetylamin)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12 H-Benzo[de]piperano[3',4':6,7]inzizo[1,2-b]quinoline-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-tetrazoxy-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecazatoracocarbamate-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-piperano-2-carboxylic acid. In some samples, the revealed ADC is prepared by first reducing the mAb, then binding it, and then binding the reduced mAb to the linker drug carrier. In some samples, the ADC prepared by combining the reduced mAb with the linker-drug delivery unit exhibits a DAR of 8. [Mode] [(ETx-22)] [Of] [ADC] [Structural characteristics and activity] []

[0150] The ADC called ETx-22 has the following formula: [] [] The Ab is an antibody that binds to cell adhesion protein-4 as disclosed herein, comprising two HCs containing the amino acid sequence shown in SEQ ID NO: 5 and two LCs containing the amino acid sequence shown in SEQ ID NO: 6. The Ab binds to the linker drug delivery in formula (ETx-22) via thiol groups at C222, C228, and C231 of the two HCs of the Ab and via thiol groups at C214 of the two chains of the LC, thereby providing 8 DARs (i.e., where ETx-22 comprises 4 cysteine-maleimine bonds in each arm of the Ab).

[0151] As shown, the ADC of formula (ETx-22) possesses unique structural features associated with the anti-cell adhesion protein-4 antibody and the linker-drug delivery of the ADC of formula (ETx-22). In some embodiments, these unique structural features, or combinations thereof, of the ADC of formula (ETx-22) impart the desired activity to the ADC of formula (ETx-22). []

[0152] In one state, the ADC of formula (ETx-22) comprises H1L2_15A7.5 anti-cell adhesion protein-4 mAb, which comprises HC as shown in SEQ ID NO: 5 and LC as shown in SEQ ID NO: 6, exhibiting desired selectivity. In one state, the H1L2_15A7.5 anti-cell adhesion protein-4 mAb exhibits desired selectivity for human cell adhesion protein-4 relative to other cell adhesion proteins (such as human cell adhesion protein-1, cell adhesion protein-2, or cell adhesion protein-3). In one state, the H1L2_15A7.5 anti-cell adhesion protein-4 mAb exhibits desired selectivity for cell adhesion protein-4 expressed by tumors, compared to cell adhesion protein-4 expressed by normal cells, such as normal human endothelial keratinocytes. []

[0153] In one state, the ADC of formula (ETx-22) has a DAR of 8. In one state, the ADC of formula (ETx-22) has a DAR of 8, providing the desired toxicity and potency. In one state, the ADC of formula (ETx-22) has a DAR of 8, and the ADC exhibits minimal aggregation. In one state, the ADC of formula (ETx-22) has a DAR of 8, and the linker of ETx-22 minimizes aggregation. In one state, the linker has sufficient length to minimize aggregation. In one state, the linker has sufficient hydrophilicity to minimize aggregation. []

[0154] As shown, the ADC of formula (ETx-22) includes a linker comprising a 3-maleimino-propionic spacer, a PEG2 spacer (i.e., -CH2-CH2-O-CH2-CH2-O-), a PSAR10 group (i.e., -(N(CH3)-CH2-C(O))10-) at a position characterized as orthogonal to the drug delivery, and a self-sacrificing unit comprising a 4-β-glucuronide-3-nitro-octopamine group; or prepared therefrom. In one state, one or more of these features of the linker minimize the aggregation of the ADC of formula (ETx-22). In one state, one or more of these structural features provide sufficient length to minimize the aggregation of the ADC of formula (ETx-22). In a state, one or more of these structural features of the connector provide sufficient hydrophilicity to the ADC of Equation (ETx-22) to minimize the aggregation of the ADC. []

[0155] In one embodiment, the ADC of formula (ETx-22) includes a linker containing a spacer of sufficient length to position the linker-drug carrier at a distance from the anti-cell adhesion protein-4 antibody such that the bound linker-drug carrier does not substantially interfere with the activity of the anti-cell adhesion protein-4 antibody (such as cell adhesion protein-4 binding activity and affinity). In another embodiment, the ADC of formula (ETx-22) includes a linker containing a spacer of sufficiently small length to avoid impairing the stability of the linker-drug carrier. []

[0156] As shown, the ADC of formula (ETx-22) includes a linker comprising a 3-maleimino-propionic spacer, a PEG2 spacer (i.e., -CH2-CH2-O-CH2-CH2-O-), a PSAR10 group (i.e., -(N(CH3)-CH2-C(O))10-) that may be positioned orthogonally relative to the drug delivery, and a self-sacrificing unit comprising a 4-β-glucuronide-3-nitro-octopamine group; or prepared therefrom. In one state, one or more of these structural features may provide the desired spacer between the linker-drug delivery and the anti-cell adhesion protein-4 antibody. []

[0157] In one state, the ADC of formula (ETx-22) includes a linker exhibiting the desired stability. In one state, the ADC of formula (ETx-22) includes a linker exhibiting the desired stability in plasma. In one state, the stability in plasma can be measured by determining the DAR of the ADC relative to the time after the ADC has been placed in plasma. []

[0158] As shown, the ADC of formula (ETx-22) includes a linker comprising a 3-maleimino-propionic spacer, a PEG2 spacer (i.e., -CH2-CH2-O-CH2-CH2-O-), a PSAR10 group (i.e., -(N(CH3)-CH2-C(O))10-) that may be positioned orthogonally relative to the drug loading, and a self-sacrificing unit comprising a 4-β-glucuronide-3-nitro-octopamine group; or prepared therefrom. In one state, one or more of these structural features may provide the desired stability of the linker-drug loading in the bloodstream. []

[0159] In one state, the ADC of formula (ETx-22) includes a linker exhibiting selective cleavability. In another state, the ADC of formula (ETx-22) includes a linker that remains intact while the ADC is in the bloodstream and cleaves only after the ADC has been delivered to the tumor site. In some states, the tumor targeted by the ADC of formula (ETx-22) exhibits an enzyme that cleaves the components of the linker. In some states, the tumor exhibits a β-glucuronidase that cleaves the β-glucuronide of the linker. []

[0160] As shown, the ADC of formula (ETx-22) comprises a self-sacrificing unit containing a β-glucuronide group or prepared from a β-glucuronide group that can be used as a triggering group; and a 3-nitro-octopamine group undergoing rearrangement and self-sacrificing cleavage. In one state, the self-sacrificing unit provides selective cleavability, wherein cleavage is not triggered until the β-glucuronide is cleaved by β-glucuronidase in targeted tumor cells. In one state, the ADC of ETx-22 is delivered to a tumor exhibiting intracellular β-glucuronidase, preferably at a relatively high level. In another state, the β-glucuronide group is cleaved from the self-sacrificing unit, and then the self-sacrificing group undergoes rearrangement and self-sacrificing cleavage, thereby releasing the ethatecan drug load. [] [Therapeutic Applications] []

[0161] In another embodiment, this document provides a method of treating cancer comprising administering to a patient in need an effective amount of the cell adhesion protein-4 ADC or pharmaceutical composition described herein. In yet another embodiment, this document provides a method of treating cancer comprising administering to a patient in need an effective amount of the ADC or pharmaceutical composition described herein, wherein the cancer is bladder cancer, breast cancer, lung cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0162] In another embodiment, this document provides an ADC comprising a cell adhesion protein-4 binder and ethatecan, used in a method comprising administering the ADC to a patient with cancer exhibiting cell adhesion protein-4, wherein, if desired, the cancer is resistant to treatment with an ADC comprising a cell adhesion protein-4 binder and orlistatine (such as monomethylorlistatine E (MMAE)). In yet another embodiment, this document provides the use of an ADC comprising a cell adhesion protein-4 binder and ethatecan in the manufacture of a medicament for treating a patient with cancer exhibiting cell adhesion protein-4, wherein, if desired, the cancer is resistant to treatment with an ADC comprising a cell adhesion protein-4 binder and orlistatine (such as monomethylorlistatine E (MMAE)). In another embodiment, this article provides a method for treating cancers exhibiting cell adhesion protein-4, comprising administering to a patient in need a therapeutically effective amount of a conjugate comprising a cell adhesion protein-4 binder and ethatecan, as needed, wherein the condition is a cancer resistant to treatment with an ADC comprising a cell adhesion protein-4 binder and orlistatine (such as monomethyl orlistatine E (MMAE)).

[0163] In another embodiment, a method for treating cancer is provided, wherein the cancer is bladder cancer. In another embodiment, a method for treating cancer is provided, wherein the cancer is urothelial carcinoma. In another embodiment, a method for treating cancer is provided, wherein the cancer is breast cancer. In another embodiment, a method for treating cancer is provided, wherein the cancer is lung cancer. In another embodiment, a method for treating cancer is provided, wherein the cancer is stomach cancer. In another embodiment, a method for treating cancer is provided, wherein the cancer is esophageal cancer. In another embodiment, a method for treating cancer is provided, wherein the cancer is colorectal cancer. In another embodiment, a method for treating cancer is provided, wherein the cancer is pancreatic cancer. In another embodiment, a method for treating cancer is provided, wherein the cancer is head and neck cancer. In another embodiment, a method for treating cancer is provided, wherein the cancer is ovarian cancer. In another embodiment, a method for treating cancer is provided, wherein the cancer is prostate cancer.

[0164] In another scenario, the patient has relapsed after administration of enrofloxacin or vedoltin, or the patient has become refractory to enrofloxacin or standard care. In another scenario, the patient treated with the ADCs or pharmaceutical compositions described herein is not suitable for treatment with enrofloxacin or vedoltin. In yet another scenario, the patient has cancer resistant to orlistatine (such as monomethyl orlistatine E (MMAE)).

[0165] In another scenario, patients treated with the ADC or pharmaceutical composition described herein were previously treated with programmed death receptor-1 (PD-1) or programmed death ligand 1 (PD-L1) inhibitors and platinum-containing chemotherapy in neoadjuvant / adjuvant, locally advanced, or metastatic settings. In yet another scenario, patients treated with a combination of the ADC or pharmaceutical composition described herein and a PD-1 inhibitor or PD-L1 inhibitor were not suitable for treatment with cisplatin-containing chemotherapy.

[0166] In another embodiment, a method is provided comprising administering an effective amount of the ADC or pharmaceutical composition described herein in combination with one or more antitumor agents, either simultaneously, separately, or sequentially. In yet another embodiment, a method is provided comprising administering an effective amount of the ADC or pharmaceutical composition described herein in combination with a PD-1 inhibitor or a PD-L1 inhibitor, either simultaneously, separately, or sequentially. In one embodiment, the patient undergoes a diagnostic test measuring the patient's cancer's response to PD-1 or PD-L1 prior to simultaneous, separate, or sequential administration of the PD-1 inhibitor or PD-L1 inhibitor.

[0167] In another embodiment, this document provides a method of administering an effective amount of the cell adhesion protein-4 ADC or pharmaceutical composition described herein, either simultaneously, alone, or in combination with an FGFR compound. In another embodiment, the cancer is urothelial carcinoma. In another embodiment, the FGFR compound is erdatinib, LOXO-435, futibatinib, vofatamab, bemarituzumab, derazantinib, infigratinib, pemigatinib, rogaratinib, FGF401, or pemigatinib. In some embodiments, the cell adhesion protein-4 ADC is administered to a patient with cancer resistant to erdatinib treatment.

[0168] In another embodiment, this document provides a cell adhesion protein-4 ADC or pharmaceutical composition described herein for use in a therapy. In another embodiment, this document provides an ADC or pharmaceutical composition described herein for the treatment of cancer. In yet another embodiment, the cancer is urothelial carcinoma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0169] In another embodiment, this document provides the cell adhesion protein-4 ADC or pharmaceutical composition described herein for the treatment of bladder cancer. In another embodiment, this document provides the cell adhesion protein-4 ADC or pharmaceutical composition described herein for the treatment of urothelial carcinoma. In another embodiment, this document provides the ADC or pharmaceutical composition described herein for the treatment of breast cancer. In another embodiment, this document provides the ADC or pharmaceutical composition described herein for the treatment of lung cancer. In another embodiment, this document provides the ADC or pharmaceutical composition described herein for the treatment of gastric cancer. In another embodiment, this document provides the ADC or pharmaceutical composition described herein for the treatment of esophageal cancer. In another embodiment, this document provides the ADC or pharmaceutical composition described herein for the treatment of colorectal cancer. In another embodiment, this document provides the ADC or pharmaceutical composition described herein for the treatment of pancreatic cancer. In another embodiment, this document provides the ADC or pharmaceutical composition described herein for the treatment of head and neck cancer. In another embodiment, this document provides the ADC or pharmaceutical composition described herein for the treatment of ovarian cancer. In another instance, this document provides the ADC or pharmaceutical composition described herein for the treatment of prostate cancer.

[0170] In another embodiment, this document provides a cell adhesion protein-4 ADC or pharmaceutical composition described herein for the treatment of cancer in which the cancer has relapsed after treatment with enrofloxacin or has become refractory to enrofloxacin. In another embodiment, this document provides a cell adhesion protein-4 ADC of a pharmaceutical composition described herein for the treatment of cancer in which the cancer is resistant to orlistatine (such as monomethyl orlistatine E (MMAE)). In another embodiment, this document provides a cell adhesion protein-4 ADC or pharmaceutical composition described herein for the treatment of cancer in which the cancer has relapsed after treatment with enrofloxacin or has become refractory to standard care. In another embodiment, this document provides an ADC or pharmaceutical composition described herein for the treatment of cancer in which prior use of enrofloxacin is contraindicated.

[0171] In another embodiment, this document provides the ADC or pharmaceutical composition described herein for the treatment of cancer in which prior treatment with a PD-1 or PD-L1 inhibitor and platinum-containing chemotherapy has been administered in neoadjuvant / adjuvant, locally advanced, or metastatic settings. In yet another embodiment, this document provides the ADC or pharmaceutical composition described herein in combination with a PD-1 inhibitor or PD-L1 inhibitor, either simultaneously, alone, or sequentially, for the treatment of cancer in which the cancer is not treatable with cisplatin-containing chemotherapy.

[0172] In another embodiment, this document provides a combination of the ADC or pharmaceutical composition described herein with one or more antitumor agents for the treatment of cancer. In yet another embodiment, the antitumor agent is a PD-1 inhibitor or a PD-L1 inhibitor.

[0173] In another embodiment, this document provides the cell adhesion protein-4 ADC or pharmaceutical composition described herein for use in conjunction with, alone or sequentially in combination with an FGFR compound for the treatment of cancer. In another embodiment, the cancer is urothelial carcinoma. In another embodiment, the FGFR compound is erdatinib, LOXO-435, fobatinib, vorfamab, bematumab, dezatinib, inflavinib, pemitinib, rogatinib, FGF401 or pemitinib. In yet another embodiment, this document provides a cell adhesion protein-4 ADC pharmaceutical composition for the treatment of cancers resistant to erdatinib.

[0174] In another embodiment, this document provides the use of the cell adhesion protein-4 ADC or pharmaceutical composition described herein in the manufacture of a medicament for treating cancer. In yet another embodiment, this document provides the use of the ADC or pharmaceutical composition disclosed herein in the manufacture of a medicament for treating cancer, wherein the cancer is bladder cancer, breast cancer, lung cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0175] In another embodiment, this document provides the use of the cell adhesion protein-4 ADC or pharmaceutical composition described herein in the manufacture of a medicament for treating cancer, wherein the cancer has relapsed after treatment with enrofloxacin or vitolamine, or the cancer has become refractory to enrofloxacin or standard care. In another embodiment, this document provides the use of the ADC or pharmaceutical composition described herein in the manufacture of a medicament for treating cancer, wherein prior use of enrofloxacin or vitolamine is contraindicated. In another embodiment, this document provides the use of the cell adhesion protein-4 ADC or pharmaceutical composition described herein in the manufacture of a medicament for treating cancer, wherein the cancer is resistant to treatment with orlistatine (such as monomethyl orlistatine E (MMAE)).

[0176] In another embodiment, this document provides the use of the ADC or pharmaceutical composition described herein in the manufacture of a medicament for treating cancer, wherein prior treatment with a PD-1 or PD-L1 inhibitor and platinum-containing chemotherapy is given in neoadjuvant / adjuvant, locally advanced, or metastatic settings. In yet another embodiment, this document provides the use of the ADC or pharmaceutical composition described herein in the manufacture of a medicament for treating cancer, wherein the cancer is untreatable with cisplatin-containing chemotherapy and wherein the medicament is intended to be administered simultaneously, separately, or sequentially with a PD-1 inhibitor or PD-L1 inhibitor.

[0177] In another embodiment, this document provides the use of the ADC or pharmaceutical composition described herein in the manufacture of a medicament for treating cancer, wherein the medicament is intended to be administered simultaneously, separately, or sequentially with one or more antitumor agents. In yet another embodiment, this document provides the use of the ADC or pharmaceutical composition described herein in the manufacture of a medicament for treating cancer, wherein the medicament is intended to be administered simultaneously, separately, or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor.

[0178] In another embodiment, this document provides the use of the cell adhesion protein-4 ADC or pharmaceutical composition described herein in the manufacture of an agent for treating cancer, wherein the agent is intended to be administered simultaneously, separately, or sequentially with an FGFR compound. In another embodiment, the cancer is urothelial carcinoma. In another embodiment, the FGFR compound is erdatinib, LOXO-435, fobatinib, vorfamab, bematumab, dezatinib, inflaviginib, pemitinib, rogatinib FGF401, or pemitinib. In yet another embodiment, this document provides the use of the cell adhesion protein-4 ADC or pharmaceutical composition described herein in the manufacture of an agent for treating cancers resistant to erdatinib treatment.

[0179] In another scenario, the bladder cancer is urothelial carcinoma, squamous cell carcinoma, or adenocarcinoma. In another scenario, the bladder cancer is non-invasive, non-muscle-invasive, or muscle-invasive. In another scenario, the bladder cancer is located in the bladder, renal pelvis, ureter, or urethra. In another scenario, the breast cancer is HR-positive, HER2-negative, or triple-negative breast cancer (TNBC). In another scenario, the breast cancer is located in the mammary ducts or lobules. In another scenario, the lung cancer is squamous non-small cell lung cancer (NSCLC) or non-squamous NSCLC. In another scenario, the lung cancer is squamous carcinoma, adenocarcinoma, or small cell carcinoma. In another scenario, the prostate cancer is metastatic castration-resistant prostate cancer. In another scenario, the gastric cancer is stomach cancer. In another scenario, the stomach or esophageal cancer is adenocarcinoma of the gastroesophageal junction. In another scenario, the ovarian cancer is serous or mucous. In another scenario, the ovarian cancer is in the fallopian tube or peritoneum.

[0180] In another embodiment, these antitumor agents may be chemotherapy agents, including platinum-containing chemotherapy, and / or may include cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel, paclitaxel protein-bound particles for injectable suspensions, ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed, and cetuximab. In another category, these antitumor agents may be immunotumor agents, including those selected from the group consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat, and durvalumab. [Pharmaceutical Compositions and Administration Methods] []

[0181] The ADC described herein can be formulated to administer a pharmaceutical composition via any route that makes the antibody or ADC biologically available, including, for example, oral, topical, or subcutaneous administration.

[0182] This article also provides a pharmaceutical composition comprising the ADC provided herein and one or more agents selected from the group consisting of physiologically acceptable carriers, diluents, excipients and adjuvants.

[0183] The ADC of the present invention, or pharmaceutical compositions comprising it, can be administered via non-enteral routes (e.g., subcutaneous and intravenous). The ADC of the present invention can be administered to a patient alone in single or multiple doses with a pharmaceutically 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 as disclosed herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0184] In one embodiment, this document discloses a pharmaceutical composition comprising the antibody disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. In another embodiment, this document discloses a pharmaceutical composition comprising the ADC disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. [Dosage, regimen, and] [cycle] []

[0185] The disclosed cell adhesion protein-4 ADC can be administered in a method of cancer treatment that delivers an effective amount of the cell adhesion protein-4 ADC or pharmaceutical composition described herein to a patient in need. The effective amount can be delivered by adjusting the dosage, dosing regimen, or dosing cycle of the cell adhesion protein-4 ADC.

[0186] In one embodiment, the ADC of the present invention or a pharmaceutical composition comprising it may be administered via non-enteric routes (e.g., subcutaneous and intravenous). [definition] []

[0187] As used herein, unless otherwise indicated herein or the context clearly contradicts it, the terms “a,” “an,” “the,” and similar terms used in the context of this invention (especially in the context of the claims) shall be construed as covering both singular and plural forms.

[0188] Unless otherwise indicated, the terms "bind" and "binds" as used herein are intended to mean the ability of a protein or molecule to form a chemical bond or to attract another protein or molecule, resulting in proximity of the two proteins or molecules, as determined by commonly known methods in this art.

[0189] As used herein, the term "effective amount" refers to the amount (in terms of time period and means of administration) required to achieve the desired therapeutic effect. The effective amount of a protein or conjugate can vary depending on factors such as an individual's disease state, age, sex, weight, and the ability of the protein or conjugate to elicit the desired response in the individual. The effective amount is also the amount in which any toxic or adverse effects of the protein or conjugate outweigh the beneficial therapeutic effects.

[0190] As used herein, the terms “treating,” “treatment,” or “to treat” refer to all processes that may involve the reduction, control, delay, or cessation of the progression of the condition or disease disclosed herein, or the improvement of the symptoms of the condition or disease, but do not necessarily indicate the complete elimination of all symptoms of the condition or disease.

[0191] The terms "patient" or "individual" as used herein refer to a human patient or a human individual, respectively. The terms "patient" and "individual" may be used interchangeably herein.

[0192] Some abbreviations are defined as follows: "ACN" refers to acetonitrile; "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]undecyl-7-ene; "DMTMM" refers to (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium 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 methylformamide; "HMPA" refers to hexamethylphosphatamine; "h" refers to hour; "HEPE" refers to... "S" refers to (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid); "HMTTA" refers to hexamethyltriethylenetetramine; "HATU" refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxyhexafluorophosphate; "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 p-toluenesulfonic acid pyridinium; "THF" refers to tetrahydrofuran; "TsOH" refers to p-toluenesulfonic acid; and "TCEP" refers to (2-carboxyethyl)phosphine; "TFA" refers to trifluoroacetic acid; "TEA" refers to triethylamine. Example

[0193] The following examples are illustrative and should not be construed as limiting the scope of the patent application.

[0194] Example 1. An antibody-drug conjugate (ADC) of the following formula: Wherein: Ab is an antibody that binds to human cell adhesion protein-4, wherein Ab contains a heavy chain (HC) containing a variable region (HCVR) and a light chain (LC) containing a variable region (LCVR), wherein the HCVR contains heavy chain complementarity-determining regions (HCDR) HCDR1, HCDR2 and HCDR3, and the LCVR contains light chain complementarity-determining regions (LCDR) LCDR1, LCDR2 and LCDR3, wherein: (i) the HCDR1 contains SEQ ID NO: 9 (NYGMA), the HCDR2 contains SEQ ID NO: 10 (FISNLAYGINYADTVTG), the HCDR3 contains SEQ ID NO: 11 (GARATGWFAY), the LCDR1 contains SEQ ID NO: 12 (KASQNVDTHVA), the LCDR2 contains SEQ ID NO: 13 (SASYRYS), and the LCDR3 contains SEQ ID NO: 14 (QQYNSYPLT) (all Kabat numbers); or (ii) the HCDR1 contains SEQ ID NO: 15 (GFTFSNYG), HCDR2 contains SEQ ID NO: 16 (ISNLAYGI), HCDR3 contains SEQ ID NO: 17 (ARGARATGWFAY), LCDR1 contains SEQ ID NO: 18 (QNVDTH), LCDR2 contains SEQ ID NO: 19 (SAS), and LCDR3 contains SEQ ID NO: 20 (QQYNSYPLT) (all IMGT numbers); and DAR is 1 to 8.

[0195] Example 2. The ADC of Example 1, wherein the HCVR contains SEQ ID NO: 7 and the LCVR contains SEQ ID NO: 8.

[0196] Example 3. An ADC as in Example 1 or 2, wherein the HC contains SEQ ID NO: 5 and the LC contains SEQ ID NO: 6.

[0197] Example 4. An ADC as described in any of Examples 1 to 3, wherein the Ab has a human IgG1 or IgG4 isotype.

[0198] Example 5. An ADC as described in any of Examples 1 to 4, wherein the Ab has a human IgG1 isotype.

[0199] Example 6. An ADC as in any of Examples 1 to 5, wherein the Ab is bound via one or more thiol groups of cysteine ​​residues present in the HC or LC.

[0200] Example 7. An ADC as described in any of Examples 1 to 6, wherein the Ab comprises an HC having the amino acid sequence shown in SEQ ID NO: 5 and an LC having the amino acid sequence shown in SEQ ID NO: 6, and the Ab is connected via a thiol group of C222, C228 or C231 of the HC; a thiol group of C214 of the LC, or a combination thereof.

[0201] Example 8. An ADC as in Example 7, wherein the ADC has an 8-fold DAR and the Ab is bound by thiol groups of C222, C228 and C231 of the HC; and thiol group of C214 of the LC.

[0202] Example 9. An ADC as described in any of Examples 1 to 8, wherein at least 95% of the ADC has a DAR of 8.

[0203] Example 10. An ADC as described in any of Examples 1 to 8 has the following formula: .

[0204] Example 11. The ADC of Example 10, wherein the Ab contains the HC shown in SEQ ID NO: 5 and the LC shown in SEQ ID NO: 6.

[0205] Example 12. An ADC as in Example 11, wherein the Ab is bound via cysteine ​​at positions C222, C228, and C231 of the HC and via cysteine ​​at C214 of the LC.

[0206] Example 13. A composition comprising an ADC as described in any of Examples 1 to 8, wherein at least about 95% of the ADC in the composition has a DAR of 8.

[0207] Example 14. A pharmaceutical composition comprising an ADC as described in any of Examples 1 to 12 or a composition as described in Example 13; and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0208] Example 15. A method for treating cancer, the method comprising administering to a patient in need an effective amount of an ADC as described in any of Examples 1 to 11, a composition as described in Example 13, or a pharmaceutical composition as described in Example 14.

[0209] Example 16. The method of Example 15, wherein the cancer is urothelial carcinoma, breast cancer, lung cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0210] Example 17. The method as in Example 15 or 16, wherein the cancer is urothelial carcinoma.

[0211] Example 18. The method of any of Examples 15 to 17, wherein the cancer has recurred after administration of enrofloxacin or the cancer has become refractory to enrofloxacin treatment.

[0212] Example 19. The method of any of Examples 15 to 18, wherein enrofloxacin is administered to the patient as a first-line, second-line, or third-line treatment before administering an effective amount of the ADC of any of Examples 1 to 11, the composition of Example 13, or the pharmaceutical composition of Example 14.

[0213] Example 20. The method of any of Examples 15 to 17, wherein the patient is not suitable for treatment with enrofloxacin and vedocin.

[0214] Example 21. The method of any of Examples 15 to 17, wherein the cancer is resistant to treatment with monomethylorisstatin E (MMAE).

[0215] Example 22. The method of any of Examples 15 to 21, wherein the cancer is characterized by the growth of the primary tumor, the occurrence and / or recurrence of tumor metastasis, and / or an increase in at least one tumor marker, and / or an upregulation of ABCB1 expression.

[0216] Example 23. The method of any of Examples 15 to 22, further comprising administering a PD-1 inhibitor or a PD-L1 inhibitor to the patient simultaneously, separately, or sequentially.

[0217] Example 24. The method of Example 23, wherein the patient is given a diagnostic test to measure the cancer’s response to PD-1 or PD-L1 before administration of the PD-1 inhibitor or the PD-L1 inhibitor.

[0218] Example 25. An ADC as described in any of Examples 1 to 12, used in a therapy.

[0219] Example 26. An ADC of any of Examples 1 to 12, used for the treatment of cancer.

[0220] Example 27. The ADC used in Example 26, wherein the cancer is urothelial carcinoma, breast cancer, lung cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0221] Example 28. The ADC used in Example 26, wherein the cancer is urothelial carcinoma.

[0222] Example 29. An ADC used in any of Examples 26 to 28, wherein the cancer has relapsed after treatment with enrofloxacin or the cancer has become refractory to enrofloxacin.

[0223] Example 30. An ADC used in any of Examples 26 to 29, wherein enrofloxacin is administered to the patient as a first-line, second-line, or third-line treatment before administering an effective amount of an ADC as in any of Examples 1 to 11, a composition as in Example 13, or a pharmaceutical composition as in Example 14.

[0224] Example 31. An ADC used in any of Examples 26 to 28, wherein the previous use of enrofloxacin vedotin is contraindicated.

[0225] Example 32. An ADC used in any of Examples 26 to 28, wherein the cancer is resistant to treatment with monomethylorisstatin E (MMAE).

[0226] Example 33. An ADC used in any of Examples 26 to 32, wherein the cancer is characterized by the growth of the primary tumor, the occurrence and / or recurrence of tumor metastasis, and / or an increase in at least one tumor marker, and / or an upregulation of ABCB1 expression.

[0227] Example 34. The ADC used in any of Examples 24 to 33 is combined with a PD-1 inhibitor or a PD-L1 inhibitor simultaneously, separately or sequentially.

[0228] Example 35. The ADC used in Example 34 was combined with a PD-1 inhibitor or a PD-L1 inhibitor simultaneously, separately, or sequentially after the patient had been diagnosed with cancer exhibiting PD-1 or PD-L1.

[0229] Example 36. A pharmaceutical composition comprising an effective amount of an ADC as in any of Examples 1 to 12 or an effective amount of a composition as in Example 13, for the treatment of cancer.

[0230] Example 37. The composition used in Example 36, wherein the cancer is urothelial carcinoma, breast cancer, lung cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0231] Example 38. The composition of Example 36 or 37, administered simultaneously, separately or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor.

[0232] Example 39. The composition of Example 36, after the cancer has been determined to be a PD-1 or PD-L1 cancer, is administered simultaneously, separately or sequentially in combination with a PD-1 inhibitor or a PD-L1 inhibitor.

[0233] Example 40. Use of an ADC as described in any of Examples 1 to 12 in the manufacture of a drug for treating cancer.

[0234] Example 41. As used in Example 40, wherein the cancer is urothelial carcinoma, breast cancer, lung cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

[0235] Example 42. Used as in Example 40 or 41, wherein the agent further comprises a PD-1 inhibitor or a PD-L1 inhibitor.

[0236] Example 43. A method for generating an ADC, the method comprising binding: (i) an antibody (Ab) to human cell adhesion protein-4, wherein the Ab comprises a heavy chain (HC) having a variable region (HCVR) and a light chain (LC) having a variable region (LCVR), wherein the HCVR comprises heavy chain complementarity-determining regions (HCDR) HCDR1, HCDR2, and HCDR3, and the LCVR comprises light chain complementarity-determining regions (LCDR) LCDR1, LCDR2, and LCDR3, wherein: (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). (all are Kabat numbers); or (b) the HCDR1 contains SEQ ID NO: 15 (GFTFSNYG), the HCDR2 contains SEQ ID NO: 16 (ISNLAYGI), the HCDR3 contains SEQ ID NO: 17 (ARGARATGWFAY), the LCDR1 contains SEQ ID NO: 18 (QNVDTH), the LCDR2 contains SEQ ID NO: 19 (SAS), and the LCDR3 contains SEQ ID NO: 20 (QQYNSYPLT) (all are IMGT numbers); and (ii) compounds of the following formula: .

[0237] Example 44. The method of Example 43, wherein the Ab and the compound are bound under conditions sufficient to bind the Ab and the compound and provide an ADC with a DAR of 8.

[0238] Example 45. The method of Example 43 or 44, further comprising reducing the Ab with a reducing agent prior to binding to produce a reduced Ab.

[0239] Example 46. The method of Example 45, wherein the reducing agent is DTT or TCEP.

[0240] Example 47. The method of any of Examples 43 to 46, wherein the compound is present in excess of at least about 8 moles relative to the Ab during binding.

[0241] Example 48. The method of any of Examples 43 to 46, wherein the compound is present in excess of at least about 12 moles relative to the Ab during binding. Example [Example] [1] [:composition] [(L-P')] [Connector] [-] [Drug Carrier] [:] [(2, S , ,3 , S , ,4 , S , ,5 , R , ,6 , S , )-6-(4-((3 , S , ,9 , S , )-40- ] [Amine] [-9-(2-(2-(2-(3-(2,5-] [Dilateral oxygen group] [-2,5-] [Dihydrogen] [-1H-] [Pyrrole] [-1-] [base] [)] [Acetoamino] [)] [Ethoxy] [)] [Ethoxy] [)] [Acetamino] [)-1-(((1 , S , ,9 , S , )-9- ] [Ethyl] [-5-] [fluorine] [-9-] [Hydroxy] [-4-] [methyl] [-10,13-] [Dihydroxy] [-2,3,9,10,13,15-] [Hexane] [-1, H , ,12 , H , - ] [Benzoan] [[de]] [piperanone] [[3',4':6,7]] [Indazine] [[1,2-b]] [Quinoline] [-1-] [base] [)] [Amine] [)-11,14,17,20,23,26,29,32,35,38-] [Decamethyl] [-1,6,10,13,16,19,22,25,28,31,34,37,40-] [Decathoxyl] [-2-] [Oxygen-containing compounds] [-5,11,14,17,20,23,26,29,32,35,38-] [Undecylate] [-3-] [base] [)-2-] [Nitrophenoxy] [)-3,4,5-] [Three-metallic base four-hydrogen] [-2, H , - ] [piran] [-2-] [Formic acid] [] [Mode] [(L-P')] [:connector] [-] [Drug Carrier] []

[0242] Linker-drug carriers derived from precursors or intermediates of formula (L-P'), the precursors or intermediates comprising: polyinosinic acid (PSAR) compounds; 4-β-glucuronide-3-nitro-octopamine compounds; ethatecan compounds; and maleimino-propionic compounds. The linker-drug carriers are synthesized using the methods disclosed in WO2019081455 and WO2022 / 207699. []

[0243] [PSAR] [Intermediate item:] [FmocNH-PEG2-Glu(Su)-PSAR10-NH , 2, ] [PSAR] [Intermediate substance] []

[0244] The PSAR intermediate can be prepared using Scheme 1.

[0245] Polyinosine was synthesized on resin using an iterative process for the synthesis of Rink amides with commercially available Fmoc-Sar-Sar-OH dipeptide constructs. All reactions were carried out at room temperature unless otherwise indicated.

[0246] Rinkamide preloaded with the first Fmoc-sarcosine residue was used as the starting material. The Fmoc-sarcosine preloaded Rinkamide resin was treated with 20% piperidine in DMF (1 mL per 100 mg resin) for 2×, 15 min at room temperature. The resin was then washed with DMF (4×) and DCM (4×). A solution of Fmoc-Sar-Sar-OH (3 equivalents), HATU (2.9 equivalents), and DIPEA (6 equivalents) in DMF was added to the resin (1 mL per 100 mg resin). The reaction vessel was stirred for 2 hours and the resin was washed with DMF (4×) and DCM (4×). The resin was then treated twice for 15 min at room temperature with 20% piperidine in DMF (1 mL per 100 mg resin). The resin was then washed with DMF (4×) and DCM (4×) to provide a Rink resin having n = 3 polyinosine oligomers. []

[0247] The n = 3 polystyrene oligomers were elongated using a submonomer synthesis iterative procedure until the desired length was achieved through alternating bromoacetylation and amine substitution steps. Bromoacetylation was performed by adding 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide contained in DMF (2 mL per 100 mg resin). The mixture was agitated for 30 min, drained, and washed with DMF (4×). For the amine substitution step, 40% (by weight) methylamine contained in aqueous solution (1.5 mL per 100 mg resin) was added, and the container was agitated for 30 min, drained, and washed with DMF (4×) and DCM (4×). []

[0248] When the desired polyinosine oligomer length (e.g., PSAR 10-polymer) is reached, orthogonal chemical functionalization is performed. Following orthogonal chemical functionalization, final end-capping with Fmoc-protected amino acid groups or other groups can be carried out. The Fmoc protecting group can be removed before or after resin degradation. []

[0249] The polyinosine can be functionalized with glutamic acid and amino-3,6-dioxanoic acid as follows: Fmoc-Glu(OAll)-OH (3 equivalents), HATU (2.9 equivalents), and DIPEA (6 equivalents) contained in DMF were added to the Rink resin (1 mL per 100 mg resin). The reaction vessel was stirred for 90 min, and the resin was washed with DMF (4×) and DCM (4×). Then, the resin was treated with 20% piperidine contained in DMF (1 mL per 100 mg resin) for 2×, 15 min at room temperature. The resin was washed with DMF (4×) and DCM (4×), followed by coupling with Fmoc-amino-3,6-dioxanoic acid (3 equivalents), HATU (2.9 equivalents), and DIPEA (6 equivalents) contained in DMF (1 mL per 100 mg resin) for 1 hour. The resin was washed with DMF (4×) and DCM (4×). The alloprotective group was removed by treatment with a DCM solution containing 0.25 equivalents of Pd(PPh3)4 and 20 equivalents of phenylsilane (with gentle agitation under an argon flow) for 2×, 30 min. The resin was then washed with DMF (5×) and DCM (5×). N-hydroxysuccinimide (NHS) ester was introduced into the carboxylic acid side chain of the final polyinosine compound by treatment with a DMF solution containing 50 equivalents of DIC and 60 equivalents of N-hydroxysuccinimide for 90 min (1.5 mL per 100 mg resin). The resin was then washed with DMF (4×) and DCM (4×). The final polyinosine compound was then cleaved from the resin (100% TFA 2×, 30 min). []

[0250] [4-β-] [Glucuronide] [-3-] [Nitro] [-] [Octopine intermediate:] Triacetic acid [(2, S , ,3 , R , ,4 , S , ,5 , S , ,6 , S , )-2-(4-(2-(( ] [Third butoxycarbonyl] [)] [Amine] [)-1-(((4-] [Nitrophenoxy] [)] [Carbonyl] [)] [Oxygen group] [)] [Ethyl] [)-2-] [Nitrophenoxy] [)-6-(] [Methoxycarbonyl] [)] [Tetrahydrogen] [-2, H , - ] [piran] [-3,4,5-] [Triester] [] [4-β-] [Glucuronide] [-3-] [Nitro] [-] [Octopamine intermediate] []

[0251] The 4-β-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-piperan-3,4,5-trimethyltriacetate can be prepared using Scheme 2.

[0252] Octopamine or octopamine intermediates used in this method can be obtained as racemic mixtures or as enantiomerically pure compounds. Racemic mixtures can be subjected to anticoagulant separation as known in this technique to obtain enantiomerically pure compounds.

[0253] Octopamine hydrochloride (±) (1690 mg / 11 mmol) was suspended in 4 mL of distilled water. The flask was cooled at 0 °C and 4 mL of pre-cooled 65% nitric acid solution was slowly added. The reaction was maintained at 0 °C for 20 min, and mononitration was evaluated by HPLC. The mononitrated octopamine precursor was transferred to a 250 mL pre-cooled Erlenmeyer flask and slowly neutralized with saturated NaHCO3 solution at 0 °C until a pH of 8 to 9 was reached. Then, 30 mL of dioxane was added, followed by Boc2O (7202 mg / 13.2 mmol). The reaction was allowed to reach room temperature and stirred overnight. The reaction was then diluted with EtOAc and washed 3 × 10⁻⁶ with saturated citric acid solution, followed by one wash with saturated NaCl solution. The organic phase was dried over MgSO4, filtered, and then evaporated under vacuum to provide a crude product, which was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 70:30 to 20:80) to provide the mononitrated octopamine precursor: (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)aminocarbamate tributyl ester. The resulting mononitrated octopamine precursor was then subjected to piezoresistive separation before proceeding to further synthetic steps.

[0254] Racemic separation of tert-butyl 2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)aminocarbamate was performed 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 positions of the two enantiomers were esterified with 1.2 molar equivalents of 4-nitrobenzoyl chloride and 2 molar equivalents of triethylamine in anhydrous THF. The compound was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 90:10 to 10:90) to provide 4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenol 4-nitrobenzoate. The absolute configuration of the enantiomers was confirmed by X-ray crystallography (previously dissolved in a 1:1 mixture of heptane and dichloromethane and allowed to slowly evaporate for 3 weeks to induce crystal formation).

[0255] In a round-bottom flask, Ag₂CO₃ (1500 mg / 5.4 mmol) and 1,1,4,7,10,10-hexamethyltriethylenetetramine (251 mg / 1.1 mmol) were suspended in 4 mL of anhydrous acetonitrile and stirred at room temperature for 2 hours. Enantiomerically pure (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)aminocarbamate tributyl ester (292 mg / 0.98 mmol) and 1-bromo-2,3,4-tri-O-acetylglucuronide methyl ester (583 mg / 1.46 mmol) were added at 0 °C, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was then filtered through diatomaceous earth, diluted with EtOAc, and washed three times with saturated citric acid solution followed by one wash with saturated NaCl solution. The organic phase was dried over MgSO4, filtered, and evaporated under vacuum to give a crude product, which was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 70:30 to 30:70) to provide triacetic acid (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-piperan-3,4,5-triester).

[0256] At 0 °C, (2 S,3 R,4 S,5 S,6 S)-2-(4-(2-((tert-oxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-piperan-3,4,5-trimethyltriacetate (334 mg / 0.54 mmol) and 4-nitrobenzene chloroformate (219 mg / 1.09 mmol) were dissolved in 6 mL of anhydrous DCM. Anhydrous pyridine (112 mg / 1.41 mmol) was added and the mixture was stirred at room temperature for 30 min. The reaction was filtered through a 0.45 µm PTFE filter and purified by chromatography on silicone (gradients of petroleum ether / EtOAc, 85:15 to 30:70) to provide triacetic acid (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-piperan-3,4,5-triester).

[0257] [4-β-] [Glucuronide] [-3-] [Nitro] [-] [Octopamine] [-] [Exatecan intermediate:] [(2, S , ,3 , R , ,4 , S , ,5 , S , ,6 , S , )-2-(4-(2- ] [Amine] [-1-((((1 , R , ,9 , R , )-9- ] [Ethyl] [-5-] [fluorine] [-9-] [Hydroxy] [-4-] [methyl] [-10,13-] [Dilateral oxygen group] [-2,3,9,10,13,15-] [Hexahydrogen] [-1, H , ,12 , H , - ] [Benzoan] [[de]] [piperanone] [[3',4':6,7]] [Indazine] [[1,2- , b , ] ] [Quinoline] [-1-] [base] [)] [Aminomethoxy] [)] [Oxygen group] [)] [Ethyl] [)-2-] [Nitrophenoxy] [)-3,4,5-] [Trihydroxytetrahydro] [-2, H , - ] [piran] [-2-] [Formic acid] [] [4-β-] [Glucuronide] [-3-] [Nitro] [-] [Octopamine] [-] [Exanotecan intermediate] []

[0258] The 4-β-glucuronide-3-nitro-octopamine intermediate can be used in combination with ethatecan using scheme 3 to provide the 4-β-glucuronide-3-nitro-octopamine-ethatecan intermediate: (2S,3R,4S,5S,6S)-2-(4-(2-amino-1-((((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]piperano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)aminomethoxy)ethyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-piperano-2-carboxylic acid.

[0259] 101 mg (0.13 mmol) of 4-β-glucuronide-3-nitro-octopamine intermediate triacetic acid (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-piperan-3,4,5-triester), 0.14 mmol of ethatecan mesylate, and 18 mg (0.13 mmol) of HOBt were dissolved in 1 mL of anhydrous DMF / pyridine 85:15 (v / v) mixture. 16.7 mg (0.13 mmol) of DIPEA was added. The reaction was stirred at 40 °C for 2 hours, and the evaporation was evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM / MeOH gradient from 99:1 to 95:5) to provide an intermediate compound, which was then subjected to deprotection.

[0260] At 0 °C, 144 mg (0.106 mmol) of the intermediate compound was dissolved in 3 mL of MeOH (75:25). LiOH monohydrate (44.5 mg / 1.06 mmol) was dissolved in water (0.4 mL) and added to the reaction vessel. After stirring, the mixture was neutralized with acetic acid (83 mg / 1.4 mmol) and concentrated under reduced pressure. The resulting crude product was redissolved in TFA / DCM solution and stirred. The evaporator was evaporated under reduced pressure, and the crude residue was dissolved and purified by HPLC to provide (2S,3R,4S,5S,6S)-2-(4-(2-amino-1-((((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]piperano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)aminomethoxy)ethyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-piperano-2-carboxylic acid.

[0261] [PSAR] [Intermediate substance] [and] [4-β-] [Glucuronide] [-3-] [Nitro] [-] [Octopamine] [-] [Exatecan intermediates are bound to provide functionalization] [PSAR-] [4-β-] [Glucuronide] [-3-] [Nitro] [-] [Octopamine] [-] [Exanotecan intermediate] [] [Functionalization] [PSAR-] [4-β-] [Glucuronide] [-3-] [Nitro] [-] [Octopamine] [-] [Exanotecan intermediate] []

[0262] The PSAR intermediate and the 4-β-glucuronide-3-nitro-octopamine-exatecan intermediate can be combined using scheme 4 to provide a functionalized PSAR-4-β-glucuronide-3-nitro-octopamine-exatecan intermediate.

[0263] 100 mg (0.081 mmol) of the PSAR intermediate and 51 mg (0.061 mmol) of the 4-β-glucuronide-3-nitro-octopamine-ethanotecan intermediate were dissolved in anhydrous DMF. 41 mg (0.405 mmol) of triethylamine was added, and the reaction was stirred at room temperature for 30 min. After complete conversion was assessed by HPLC, piperidine was added directly to the reaction vial to achieve an 8% (v / v) piperidine solution in DMF. The reaction was then stirred at room temperature for 5 to 10 min until the entire Fmoc- group was observed to have deprotected by HPLC. The reaction was slowly neutralized with a 10% TFA solution in water / ACN 1:1 (v / v) and purified by HPLC to provide the functionalized -PSAR-4-β-glucuronide-3-nitro-octopamine-ethanotecan intermediate.

[0264] [Mode] [(L-P')] [Connector] [-] [Drug Carrier] [:] [(2, S , ,3 , S , ,4 , S , ,5 , R , ,6 , S , )-6-(4-((3 , S , ,9 , S , )-40- ] [Amine] [-9-(2-(2-(2-(3-(2,5-] [Dilateral oxygen group] [-2,5-] [Dihydrogen] [-1, H , - ] [Pyrrole] [-1-] [base] [)] [Acetoamino] [)] [Ethoxy] [)] [Ethoxy] [)] [Acetamino] [)-1-(((1 , S , ,9 , S , )-9- ] [Ethyl] [-5-] [fluorine] [-9-] [Hydroxy] [-4-] [methyl] [-10,13-] [Dilateral oxygen group] [-2,3,9,10,13,15-] [Hexahydrogen] [-1H, 12H-] [Benzoan] [[de]] [piperanone] [[3',4':6,7]] [Indazine] [[1,2-b]] [Quinoline] [-1-] [base] [)] [Amine] [)-11,14,17,20,23,26,29,32,35,38-] [Decamethyl] [-1,6,10,13,16,19,22,25,28,31,34,37,40-] [Decathoxyl] [-2-] [Oxygen-based] [-5,11,14,17,20,23,26,29,32,35,38-] [Undecazazetane] [-3-] [base] [)-2-] [Nitrophenoxy] [)-3,4,5-] [Trihydroxytetrahydro] [-2, H , - ] [piran] [-2-] [Formic acid] [] [Mode] [(L-P')] [Connector] [-] [Drug Carrier] []

[0265] The linker-drug carrier of formula (L-P') can be prepared by combining maleiminopropionic acid N-hydroxysuccinimino ester and the functionalized -PSAR-4-β-glucuronide-3-nitro-octopamine-ethanotecan intermediate using scheme 5. []

[0266] The intermediates of maleimide N-hydroxysuccinimide ester and functionalized -PSAR-4-β-glucuronide-3-nitro-octopamine-ethanotecan were dissolved in anhydrous DMF (0.1 M maleimide compound). 1.56 mg (0.015) of triethylamine was added, and the reaction was stirred for 2 hours until complete conversion was observed by HPLC. The reaction mixture was then diluted with 1% TFA solution in water / ACN 1:1 (v / v) and purified using HPLC preparation method 6 to provide the linker-drug carrier of formula (L-P'). [Example] [2] [Humanized resistance] [-] [Cell adhesion proteins] [4] [Single-strain antibody] [(15A 7.5 mAb)]

[0267] The anti-cell adhesion protein-4 mAb used in the disclosed ADC is a humanized derivative of mAb 15A7.5, described in WO2022 / 207822 and WO2022 / 207825. As shown, mAb 15A7.5 exhibits selective affinity for tumor cells relative to keratinocytes. The mAb used to prepare the ETx-22 ADC disclosed herein is a humanized derivative of 15A7.5 mAb having a recurve mutation introduced into its HC amino acid sequence (H1) and its LC amino acid sequence (L2), and is referred to herein as H1L2_15A7.5. The H1L2_15A7.5 mAb has the heavy chain amino acid sequence shown in SEQ ID NO: 5 and the light chain amino acid sequence shown in SEQ ID NO: 6. The HC variable region (HCVR) is shown in SEQ ID NO: 7 and the LC variable region (LCVR) is shown in SEQ ID NO: 8. These HC and LC complementary determinant regions (HCDR and LCDR) are shown in SEQ ID No: 9 to 14 (Kabat) and SEQ ID No: 15 to 20 (IMGT). [Example] [3] [:] [ETx-22 ADC] [Preparation] []

[0268] The ADC, referred to as "ETx-22", was prepared by binding the H1L2-15A7.5 mAb to an ethatecan drug delivery vehicle via a linker of formula (L-P') referred to herein. A schematic diagram of ETx-22 is provided in Figure 1.

[0269] To prepare the ADC termed ETx-22, mAb H1L2-15A7.5 (HC:SEQ ID NO: 5; LC:SEQ ID NO: 6) in PBS 1X was reduced with 1 mM EDTA at 37°C for 2 hours using 14 molar equivalents of TCEP. The buffer was then exchanged (Amicon ultra 30 kDa) to 100 mM KPO4, 1 mM EDTA, pH 7.4. A 12 molar equivalent of the linker (L-P') was used to bind the reduced mAb to the ADC at room temperature for 35 min. The buffer was then exchanged to 100 mM KPO4, pH 8.0, followed by incubation at 37°C in the absence of oxygen for 24 hours to allow the maleimine to self-hydrolyze. The final buffer exchange was performed at 20 mM His at pH 6.0, followed by filtration through a 0.22 µM filter to provide ETx-22.

[0270] The drug-antibody ratio (DAR) of ETx-22 was determined by LC-MS analysis and found to be between 7.77 and 7.82 toxins per bound mAb. Less than 5% material aggregation was observed by SEC-HPLC. Hydrophobic interaction chromatography (HIC) analysis of ETx-22 showed a single, sharp peak close to that of naked 15A at 7.5 mAb, indicating high homogeneous hydrophilicity (see Figure 2 and corresponding brief description). The in vitro stability of ETx-22 was assessed by culturing it in mouse, cynomolgus monkey, or human serum and measuring the DAR over time (see Figure 3 and corresponding brief description). The in vitro cytotoxic activity of the HCT-116-2G10 conjugate expressing human cell adhesion protein-4 was evaluated (see Figure 4 and corresponding brief description).

[0271] As a comparative ADC, the HA22 anti-cell adhesion protein-4 mAb was bound to maleiminohexylvaline-citrulline-PABC-MMAE to produce enrofloxacin. In short, a 15 mg / mL solution of HA22 in 10 mM acetate, 1% sorbitol, and 3% L-arginine (pH 5.0) was adjusted to pH 7.5 by adding 20% ​​volume of 0.1 M Tris, 25 mM EDTA, and 750 mM NaCl (pH 8.4), 5 mM EDTA, and 150 mM sodium chloride. Then, the HA22 mAb was partially reduced by adding 2.5 molar equivalents of TCEP, followed by gentle stirring 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-ethatecan compound were added in 6% (v / v) DMSO solution. The mixture was stirred at 5°C for 60 minutes, followed by the addition of 1 molar equivalent of N-acetycysteine ​​relative to the cysteine-reactive linker-MMAE compound and stirring for another 15 minutes. The antibody-drug conjugate (ADC) was then subjected to ultrafiltration / dialysis with 10 volumes of 20 mM histamine at pH 6.0 to remove the mass-quenched cysteine-reactive linker-drug carrier and other reaction components. The drug-antibody ratio (DAR) analyzed by LC-MS was 3.79 toxins per bound mAb. Less than 1% material aggregation was determined by SEC-HPLC. [Example] [4] [In vivo study in mice] []

[0272] Female NOD / SCID / γc knockout (NSG) and male NMRI nude mice were obtained from the Charles River Laboratory. Female NOD / SCID and BALB / c nude mice were obtained from GemPharmatech Co. Mice were housed under sterile conditions with sterilized food and freely available water, maintaining a 12-hour light-12-hour dark cycle. For NSG mice, cells and PDX were seeded into the mammary fat pads on both sides at a concentration of 0.5 × 10⁶ cells in 50% phenol red-free matrix gel (Becton-Dickinson Bioscience). Otherwise, PDX fragments (2 to 3 mm in diameter) were subcutaneously seeded.

[0273] Mice were treated when the tumor reached an average volume of 100 to 200 mm³. Mice were treated with a single or two-dose ADC at a specified concentration intravenously. Tumor growth was monitored by measuring the tumor volume (length × width 2 × π / 6) using digital calipers. All animals were randomly assigned to treatment groups such that the average tumor volume in each group was 100 to 200 mm³. Animal weight was monitored every 3 days to assess the toxicity of different treatments. Endpoints were defined as weight loss >20%, tumor volume >1500 mm³, ruffled coat and hunched back, weakness, and reduced movement.

[0274] The PK / PD characterization of ETx-22 was evaluated using NSG mice and NSG mice with triple-negative breast cancer (TNBC) PDX400 transplantation. (See Figure 5 and corresponding brief explanation).

[0275] The mechanism of action of ETx-22 was evaluated in NSG mice transplanted with a PDX triple-negative breast cancer model (PDX400). (See Figure 6 and corresponding brief explanation).

[0276] Evaluation of the in vivo efficacy of ETx-22 in the MMAE-resistant triple-negative breast cancer (TNBC) cell line (SUM190) in NSG mice. (See Figure 7 and corresponding brief description).

[0277] The in vivo efficacy of ETx-22 was evaluated in the following models: PDX esophageal cancer (ES0201) (Figure 8 and brief description); bladder cancer (BLCU003) (Figure 9 and brief description); bladder cancer (B521) (Figure 10 and brief description); head and neck cancer (HN2579) (Figure 11 and brief description); cervical cancer (CV3035) (Figure 12 and brief description); cervical cancer (CV3560) (Figure 13 and brief description); and ovarian cancer (OV2423) (Figure 14 and brief description).

[0278] The in vivo efficacy of ETx-22 was evaluated in PDX triple-negative breast cancer (TNBC) models: 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). [Amino acid sequence] []

[0279] TW202530217A_113137434_SEQL.xml

Claims

1. An antibody-drug conjugate (ADC) of the following formula: Wherein: Ab is an antibody that binds to human cell adhesion protein (Nectin)-4, and Ab contains a heavy chain (HC) containing a variable region (HCVR) and a light chain (LC) containing a variable region (LCVR), wherein the HCVR contains heavy chain complementarity-determining regions (HCDR) HCDR1, HCDR2, and HCDR3, and the LCVR contains light chain complementarity-determining regions (LCDR) LCDR1, LCDR2, and LCDR3, wherein: (i) HCDR1 contains SEQ ID NO: 9 (NYGMA), HCDR2 contains SEQ ID NO: 10 (FISNLAYGINYADTVTG), HCDR3 contains SEQ ID NO: 11 (GARATGWFAY), LCDR1 contains SEQ ID NO: 12 (KASQNVDTHVA), LCDR2 contains SEQ ID NO: 13 (SASYRYS), and LCDR3 contains SEQ ID NO: 14 (QQYNSYPLT); or (ii) HCDR1 contains SEQ ID NO: 15 (GFTFSNYG), HCDR2 contains SEQ ID NO: 16 (ISNLAYGI), HCDR3 contains SEQ ID NO: 17 (ARGARATGWFAY), LCDR1 contains SEQ ID NO: 18 (QNVDTH), LCDR2 contains SEQ ID NO: 19 (SAS), and LCDR3 contains SEQ ID NO: 20 (QQYNSYPLT); and DAR is 1 to 8.

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

6.

3. An ADC such as that in either request 1 or 2, wherein the Ab has a human IgG1 or IgG4 isotype.

4. An ADC as requested in either 1 or 2, wherein the antibody is of human IgG1 isotype.

5. The ADC of claim 1, having the following formula: wherein Ab comprises the HC shown in SEQ ID NO: 5 and the LC shown in SEQ ID NO: 6, and wherein the Ab is bound via cysteine ​​at positions C222, C228, and C231 of the HC and via cysteine ​​at C214 of the LC.

6. A composition comprising an ADC as claimed in any one of claims 1 to 5, wherein at least 95% of the ADC in the composition has a DAR of 8.

7. A pharmaceutical composition comprising an ADC as claimed in any one of claims 1 to 5 or a composition as claimed in claim 6, and one or more pharmaceutically acceptable carriers, diluents or excipients.

8. The use of an ADC of any one of claims 1 to 5, a composition of claim 6, or a pharmaceutical composition of claim 7 in the manufacture of a pharmaceutical product for treating cancer in patients in need.

9. As claimed in claim 8, wherein the cancer is urothelial carcinoma, breast cancer, lung cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

10. As requested in claim 8, wherein the cancer is urothelial carcinoma.

11. For any of the uses in requests 8 to 10, where the patient has previously received enfortumab vedotin as a first-, second-, or third-line treatment and, if necessary, the cancer has recurred after receiving enfortumab vedotin, or the cancer has become difficult to treat with enfortumab vedotin after receiving it.

12. A method for generating an ADC, the method comprising binding: (i) an antibody (Ab) that binds to human cell adhesion protein-4, wherein the Ab comprises a heavy chain (HC) having a variable region (HCVR) and a light chain (LC) having a variable region (LCVR), wherein the HCVR comprises heavy chain complementarity-determining regions (HCDR) HCDR1, HCDR2, and HCDR3, and the LCVR comprises light chain complementarity-determining regions (LCDR) LCDR1, LCDR2, and LCDR3, wherein: a) HCDR1 contains SEQ ID NO: 9 (NYGMA), HCDR2 contains SEQ ID NO: 10 (FISNLAYGINYADTVTG), HCDR3 contains SEQ ID NO: 11 (GARATGWFAY), LCDR1 contains SEQ ID NO: 12 (KASQNVDTHVA), LCDR2 contains SEQ ID NO: 13 (SASYRYS), and LCDR3 contains SEQ ID NO: 14 (QQYNSYPLT); or b) HCDR1 contains SEQ ID NO: 15 (GFTFSNYG), HCDR2 contains SEQ ID NO: 16 (ISNLAYGI), HCDR3 contains SEQ ID NO: 17 (ARGARATGWFAY), LCDR1 contains SEQ ID NO: 18 (QNVDTH), LCDR2 contains SEQ ID NO: 19 (SAS), and LCDR3 contains SEQ ID NO:

20. (QQYNSYPLT); and (ii) compounds of the following formula: .

13. The method of claim 12, wherein during binding, the compound is present in excess of at least 8 moles relative to the Ab.

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