Methods of treating cancer with combination therapy comprising Anti-nectin-4 radioimmunoconjugate and PD-1 / PD-l1 inhibitors

Abstract

Described herein is a method of treating cancer, the method comprising administering to a subject in need thereof, one or more anti-nectin-4 immunoconjugate comprising an anti-nectin-4 antibody and a radionuclide, and one or more PD-1 / PD-L1 inhibitor.

Description

TITLE: METHODS OF TREATING CANCER WITH COMBINATION THERAPY COMPRISING ANTI-NECTIN-4 RADIOIMMUNOCONJUGATE AND PD-1 / PD-L1 INHIBITORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 691 ,784, filed September 6, 2024, the contents of which is incorporated herein by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “P76645PC00_96560058_Sequence_Listing” (113,800 bytes) created on September 4, 2025, is herein incorporated by reference.FIELD

[0003] The present disclosure relates to combination treatments for cancer and in particular combinations comprising anti-nectin-4 radioimmunoconjugate and PD-1 inhibitors and PD-L1 inhibitors.BACKGROUND

[0004] Advances in molecular profiling and sequencing of NSCLC (in both adenocarcinoma and squamous histologic subtypes) and triple negative breast cancer (TNBC) have identified Nectin-4 (poliovirus receptor-related protein 4 PVRL4) to be highly upregulated1’2’3. PVRL4 / Nectin-4 is uniquely overexpressed in most cancers of epithelial origin (including gastric, breast, lung, pancreatic, ovarian, bladder and esophageal) but not in non-malignant tissues1. Large clinical studies show 60% of NSCLC overexpress Nectin-44. Immunohistochemical analysis of a panel of 294 healthy tissue specimens from 36 human organs showed very weak homogenous staining mainly in human skin keratinocytes, skin appendages (sweat glands and hair follicles), transitional epithelium of bladder, salivary gland (ducts), esophagus, breast, and stomach, with no staining from the rest5.

[0005] Enfortumab vedotin (EV) is an anti-Nectin-4 antibody drug conjugate (ADC) approved for the treatment of advanced urothelial cancer12. BT8009 is also a peptide drug conjugate targeting Nectin-4 in phase l / ll clinical trials13. In EV, a human anti-Nectin-4 antibody is conjugated to monomethyl auristatin (MMAE) via a cleavable linker (valine citrulline vc). In preclinical studies, EV was effective at inhibiting the growth of patient derived xenograft (PDX) and cell line derived tumors13. Unfortunately, most tumors re-grew after periods of no-treatment.Cabaud et al14showed that the mechanism of resistance to anti-Nectin-4 ADC was due to the expression of ABCBIgene, encoding the multidrug resistance protein MDR1 / P-glycoprotein (P- gp), associated with focal gene amplification and high protein expression. This is one of the common mechanisms of resistance of ADCs, and this mirrors that of chemotherapeutics14’15’16. EV is being evaluated in a phase 2 trial against NSCLC and TNBC (EV-202 trial; NCT04225117)17.

[0006] There are a few other anti-Nectin-4 agents in development EV, 9MW2821 and BT800918’19’20, but there are no preclinical reports or clinical trials of radioimmunotherapy (RIT) against this antigen.

[0007] 225Ac decay characteristics include a ti / 2 of 10.0 days; energy range of as is 6-8MeV (cumulatively 28 MeV / decay), decays with the emission of 4 as, 3 p- per disintegration; a range of 50-80 pm in tissue (linear energy transfer (LET) of 0.16 MeV / μm).

[0008] Beta (β-)161Tb has a ti / 2 of 6.90 days and Eβ-average= 0.154 MeV.

[0009] Improved cancer treatments are desirable.SUMMARY

[0010] Herein, the immune inducing effects of high linear energy transfer (LET) alpha (a) particle-labeled [225Ac]Ac-N ectin-4 have been compared with beta-emitter (P') / Auger electron- labeled antibody [161Tb]Tb-N4MU01 against Nectin-4-positive TNBC and NSCLC. The effectiveness of these radioimmunotherapeutics (RIT) in vitro, and in vivo in patient-derived xenograft (PDX) mouse models has been evaluated. The a and P' / Auger electron emitting RITs have also been evaluated in combination with anti-PD-L1 in immunocompetent syngeneic mouse models.

[0011] Accordingly, an aspect of the disclosure includes a method of treating cancer, the method comprising administering to a subject in need thereof, one or more anti-nectin-4 immunoconjugate comprising an anti-nectin-4 antibody and one or more therapeutic agent, and one or more Programmed Death-1 (PD-1 / Programmed Death Ligand 1 (PD-L1) inhibitor.

[0012] An aspect of the disclosure includes a combination treatment comprising 1) one or more anti-nectin-4 immunoconjugate each immunoconjugate comprising an anti-nectin-4 antibody and a therapeutic agent, and 2) one or more PD-1 / PD-L1 inhibitor.

[0013] Another aspect includes a method of treating cancer in a subject in need thereof according to a method described herein.

[0014] Another aspect of the disclosure includes a kit comprising one or more immunoconjugate described herein and one or more PD-1 / PD-L1 inhibitor.

[0015] The preceding section is provided by way of example only and is not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions and methods of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional advantages objects and embodiments are expressly included within the scope of the present disclosure. The publications and other materials used herein to illuminate the background of the disclosure, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are listed in the appended reference section.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure, in which:

[0017] FIGs.1A-1 D show the production and quality control of [161Tb]Tb-DOTA-N4MU01 in an exemplary embodiment of the disclosure. FIG. 1A shows representative illustration of a SEC-HPLC chromatogram (280 nm) of 25 pg of N4MU01 antibody. FIG. 1 B shows a SEC-HPLC chromatogram (280 nm) of 25 pg of DOTA-N4MU01. FIG. 1C shows the MALDI-TOF spectrum of DOTA-N4MU01 showing a half-mass of 74884 Da. FIG. 1 D shows the structure of [161Tb]Tb- DOTA-N4MU01. FIG. 1 E shows instant thin-layer chromatogram showing > 95% radiochemical yield for [161Tb]Tb-DOTA-N4MU01 (region 1) and < 3% unchelated161Tb (region 2). [161Tb]Tb- DOTA-N4MU01 showed a greater stability in human serum than in PBS at 37 °C (Figure 1) over 6 days. On d1 post-labelling., the percentage of intact [161Tb]Tb-DOTA-N4MU01 in human serum was 95.08 ± 0.66% while that in PBS was similarly 95.50 ± 0.38%. However, by d6 post-labelling, the percentage of intact [161Tb]Tb-DOTA-N4MU01 in human serum had dropped slightly to 88.52 ± 0.13% while that in PBS had dropped remarkably to 60.57 ± 0.67%.

[0018] FIG. 2 shows the in vitro stability of [161Tb]Tb-DOTA-N4MU01 in an exemplary embodiment of the disclosure. Assay was run in duplicates and the percentage of intact radioconjugate determined by iTLC.

[0019] FIGs. 3A-3C show flow cytometry of N4MU01 and DOTA-N4MU01 on nectin-4 expressing; mouse syngeneic TNBC cell lines 4T1.nectin-4and E0771.nectin-4, NSCLC cell line CMT167.nectin-4and human NSCLC cell line NCI-H358 in an exemplary embodiment of the disclosure. FIG. 3A shows a dose response curves for estimation of binding constant (KD) for all 4 cell lines. FIG. 3B shows the log of dose vs normalized response curves for estimation of half maximal effective concentrations (ECso). FIG. 3C shows the low cytometric histograms for DOTA- N4MU01 across 3 mouse cell lines at 3 different concentrations and for N4MU01 (NCI-H358 cell line), compared with control. Values are plotted as mean ± SD of triplicate experiments.

[0020] FIG. 4 shows a Phage Enzyme-Linked Immunosorbent Assay for the binding of N4MU01 to human and mouse nectin-4 proteins in an exemplary embodiment of the disclosure.

[0021] FIG. 5 shows a radioligand assay confirming the great binding affinity of [161Tb]Tb- DOTA-N4MU01 to 4T1.nectin-4cells in an exemplary embodiment of the disclosure. Data is plotted as mean ± SD of a duplicate assay.

[0022] FIG. 6 shows the internalization of N4MU01 and DOTA-N4MU01 on 4T 1.nectin-4and E0771.nectin-4cell lines in an exemplary embodiment of the disclosure. Data was acquired in quadruplicates every 0.5 h for 72 h using live-cell imaging.

[0023] FIG. 7 shows the pharmacokinetics of [161Tb]Tb-DOTA-N4MU01 studied in BALB / c female mice (n = 3) in an exemplary embodiment of the disclosure. Values are plotted as mean with SD.

[0024] FIGs. 8A-8D show the biodistribution of [161Tb]Tb-DOTA-N4MU01 in healthy and tumor-bearing mice in a representative embodiment of the disclosure. FIG. 8A shows the ex vivo biodistribution of [161Tb]Tb-DOTA-N4MU01 in healthy female BALB / c mice injected with 1.74 ± 0.12 M Bq (n = 4 / timepoint). FIG. 8B shows the ex vivo biodistribution of [161Tb]Tb-DOTA-N4MU01 in 4T1.nectin-4tumor-bearing BALB / c mice injected with 1.76 ± 0.30 MBq (n = 4 / timepoint). FIG. 8C shows the microSPECT / CT imaging of a 4T 1.nectin-4tumor-bearing female BALB / c mouse that was injected with two doses of 5 MBq of [161Tb]Tb-N4MU01 , administered 7 days apart and images acquired on d8 p.i. of the first dose (d1 p.i. of second dose) and on d10 p.i. of first dose (d3 p.i. of second dose). FIG. 8D shows the ex vivo biodistribution of [161Tb]Tb-DOTA-N4MU01 in CMT167.nectin-4tumor-bearing C57BL / 6 mice injected with 2.48 ± 0.10 MBq (n = 5 / timepoint). Data is presented as mean ± SD. *p < 0.0332, **p < 0.00021 , ***p < 0.0002, ****p < 0.0001.

[0025] FIGs. 9A-9B show the efficacy of [225Ac]Ac-Macropa-N4MU01 monotherapy in human TNBC cell line MDA-MB-468 in an athymic BALB / c mouse xenograft model in arepresentative embodiment of the disclosure. Mice were either treated with a repeated dose of each treatment, injected intravenously on d0 and on d10 (n = 4-7 mice / group). FIG. 9A shows the average tumor growth curves. FIG. 9B shows Kaplan-Meier survival curves. Study endpoint was considered as tumor volume 1500 ≥ mm3or survival for 90 days.

[0026] FIGs. 10A-10B show efficacy of [225Ac]AC-Macropa-N4MU01 monotherapy in human NSCLC cell line NCI-H358 in an athymic BALB / c mouse xenograft model in an exemplary embodiment of the disclosure. Mice were either treated with a repeated dose of [225Ac]AC- Macropa-N4MU01 (n = 6 mice) injected intravenously on d0 and on d10 or with saline (n = 4 mice). FIG. 10A shows average tumor growth curves. FIG. 10B shows the Kaplan Meier survival curve of treatment groups shows Log-rank (mantel-Cox) test for survival comparison. All data is plotted as mean ± SD. Study endpoint was considered as tumor volume 650 mm3or su ≥rvival for 50 days. [225Ac]ACN4MU01 = [225Ac]AC-Macropa-N4MU01.

[0027] FIGs. 11A-11 B show the efficacy of [225Ac]AC-Macropa-N4MU01 monotherapy in human NSCLC cell line NCI-H2170 in an athymic BALB / c mouse xenograft model in an exemplary embodiment of the disclosure. Mice were either treated with a repeated dose of [225Ac]AC- Macropa-N4MU01 (n = 8 mice) injected intravenously on d0 and on d10 or with saline (n = 3 mice). FIG. 11 A shows the average tumor growth curves. FIG. 11 B shows average body weights. All data is plotted as mean ± SD. Study endpoint was considered as tumor volume 1500 mm3≥ or survival for 37 days. [225Ac]ACN4MU01 = [225Ac]AC-Macropa-N4MU01

[0028] FIGs. 12A-12C shows the efficacy of [161Tb]Tb-DOTA-N4MU01 monotherapy in human TNBC cell line MDA-MB-468 in an athymic BALB / c mouse xenograft model in an exemplary embodiment of the disclosure. Mice were either treated with a repeated dose of [161Tb]Tb-DOTA-N4MU01 injected intravenously on d0 and on d7 or with saline (n = 7 mice / group). FIG. 12A shows average tumor growth curves with a t-test performed on d13 following start of treatment. FIG. 12B shows a Kaplan Meier survival curve of treatment groups that shows Log-rank (mantel-Cox) test for survival comparison. FIG. 12C shows the average body weight of treatment groups over time. All data is plotted as mean ± SD. Study endpoint was considered as tumor volume 1500 ≥ mm3or survival for 97 days. [161Tb]Tb-N4MU01 = [161Tb]Tb- DOTA-N4MU01.

[0029] FIGs. 13A-13K show the effectiveness of [161Tb]Tb-DOTA-N4MU01 with / without anti-PDL-1 antibody in an exemplary embodiment of the disclosure. Single-dose treatment using [161Tb]Tb-DOTA-N4MU01 (A, B, and C), and 2-dose treatment (d0 and d7) regimen of [161Tb]Tb- DOTA-N4MU01 (d, e, f, j, and k) with or without anti-PDL-1 treatment. All groups that receivedanti- PD- L1 had three doses (250 μg / mouse per dose) intraperitoneally on d3, d5, and d7 following start of treatment which was defined as injection of saline or [161Tb]Tb-DOTA-N4MU01, intravenously. FIG. 13A shows a representative illustration of the study scheme for single dose treatment with [161Tb]Tb-DOTA-N4MU01 at either 5 M Bq or at 2.5 MBq, with / without anti-PD-L1 in a representative embodiment of the disclosure. FIG. 13B shows the average tumor growth curve for single dose treatment with [161Tb]Tb-DOTA-N4MU01 at either 5 MBq or at 2.5 MBq, and in combination with anti-PD-L1. FIG. 13C shows a Kaplan Meier survival curve for single dose treatment with [161Tb]Tb-DOTA-N4MU01 at either 5 MBq or at 2.5 MBq, in combination with anti- PD-L1. FIG. 13D shows a representative illustration of the study scheme for 2-dose treatment with [161Tb]Tb-DOTA-N4MU01 at 5 MBq, and in combination with anti-PD-L1 in an exemplary embodiment of the disclosure. FIG. 13E shows individual tumor growth curves for the 2x [161Tb]Tb-DOTA-N4MU01 monotherapy group. FIG.13F shows individual tumor growth curves for the 2x [161Tb]Tb-DOTA-N4MU01 + anti-PD-L1. FIG. 13G shows individual tumor growth curves for the saline + anti-PD-L1 . FIG. 13H shows individual tumor growth curves for 2x treatment with N4MU01 (25 μg / mouse per injected) group. FIG. 131 shows individual tumor growth curves for the saline group. FIG. 13J shows an average tumor volume growth curve that shows growth curve comparison between groups. FIG. 13K shows a Kaplan Meier survival curve of treatment groups that shows Log-rank (mantel-Cox) test for survival comparison. Study endpoint was considered as tumor volume ≥ 1500 mm3or survival for 96 days. CR = complete remission, RC = xenograft rechallenge, d = day, [161Tb]Tb-N4MU01 = [161Tb]Tb-DOTA-N4MU01 , *p < 0.0332, **p < 0.00021, ***p < 0.0002, ****p < 0.0001.

[0030] FIGs. 14A-14H shows [161Tb]Tb-DOTA-N4MU01 with / without anti-PD-L1 in a breast cancer E0771nectin-4model. FIG. 14A shows a representative illustration of the study scheme for 2x dose treatment with [161Tb]Tb-DOTA-N4MU01 at 5 MBq, in combination with anti- PD-L1 in an exemplary embodiment of the disclosure. FIG. 14B shows an average tumor growth curve that shows growth curve comparison between groups. FIG. 14C shows the individual tumor growth curves for the 2x [161Tb]Tb-DOTA-N4MU01 monotherapy group. FIG. 14D shows the individual tumor growth curves for the 2x [161Tb]Tb-DOTA-N4MU01 + anti-PD-L1. FIG. 14E shows the individual tumor growth curves for the saline + anti-PD-L1 group. FIG. 14F shows the individual tumor growth curves for the 2x treatment with N4MU01 (25 μg / mouse per injected). FIG. 14G shows the individual tumor growth curves for saline. FIG. 14H shows a Kaplan Meier survival curve of treatment groups that shows Log-rank (mantel-Cox) test for survival comparison. Study endpoint was considered as tumor volume ≥ 1500 mm3or survival for 81 days. CR =complete remission, RC = xenograft rechallenge, d = day, [161Tb]Tb-N4MU01 = [161Tb]Tb-DOTA- N4MU01.

[0031] FIGs. 15A-15B show the efficacy of [225Ac]Ac-Macropa-N4MU01 monotherapy in 4T1.nectin-4syngeneic BALB / c mouse model (n = 5-6 mice / group) in an exemplary embodiment of the disclosure. Each treatment group received two doses of the treatment on d0 and on d10. FIG. 15A shows average tumor growth curves that show complete remission for all mice in [225Ac]Ac- Macropa-N4MU01 treatment group. FIG. 15B shows a Kaplan-Meier survival curve. Study endpoint was considered as tumor volume ≥ 1500 mm3or survival for 30 days.

[0032] FIGs. 16A-E show that [161Tb]Tb-DOTA-N4MU01 induces differential release of pro- immune checkpoint inhibition therapy-associated cytokines in Nectin-4+ syngeneic TNBC mouse models. All cytokines were analyzed in duplicates for each mouse and the mean values used for computations. Fig. 16A is a schematic of the immune priming study design. Created in BioRender. Fig. 16 B and C shows time dependent blood plasma cancer-associated cytokine release changes after an intravenous injection of 5 MBq of [161Tb]Tb-DOTA-N4MU01 , plotted as normalized means (heatmap) and key differences in some cytokines (mean ± SD) f Fig. 16B) 4T1 Nectin-4 female BALB / c syngeneic model, and Fig. 16C) E0771 Nectin-4 female C57BL / 6 syngeneic model. Differences between groups for each cytokine were computed using Two-tailed student t-test. Pearson correlation matrices of blood cytokine changes on days 1 , 3, and 7 (and saline control on day 3) for d) 4T 1 Nectin-4 female BALB / c syngeneic model, and e) E0771 Nectin- 4 female C57BL / 6 syngeneic model. Cd3: saline control (day 3), d1 : 5 MBq radioligand (day 1), d3: 5 MBq radioligand (day 3), d7: 5 MBq radioligand (day 7), G-CSF: Granulocyte colonystimulating factor, IL: Interleukin, INF: interferon, IP-10: interferon- -induced protein-10, KC: Keratinocyte-derived chemokine, LIF: Leukemia inhibitory factor, LIX: lipopolysaccharide-induced CXC chemokine, TNF: tumor necrotic factor, VEGF: vascular endothelial growth factor.

[0033] FIGs. 17A-C: Combination treatment of 4T1 Nectin-4 BALB / c syngeneic mice with anti-Nectin-4 161Tb-radioligand and anti-PD-L1 induce immune changes that rejects tumor rechallenge. All values are plotted as mean ± SD (n=4-6 mice / group). Fig. 17A Heat map of normalized mean analyte values on d96 for mice that had complete tumor responses in the 2x [161Tb]Tb-DOTA-N4MU01 + anti-PD-L1 (161TbN5.0+aPD-L1 , n = 4 mice) and 2x [161Tb]Tb- DOTA-N4MU01 (161TbN5.0, n = 1 mouse) treatment groups compared with control values (saline_p_d3, Fig 16A-E). Fig. 17B) Two tailed student t-test comparison graphs for key blood cytokines / chemokines. Blood samples from mice with a complete tumor response were collected at the end of the study period (d96). Fig. 17C is an Illustration of treatment and responses in thetumor immune microenvironment (TME) for the four mice with complete tumor response in the 2x [161Tb]Tb-DOTA-N4MU01 + anti-PD-L1 therapy group. Day 3 priming cytokine data is used as a baseline for Fig. 17C) to observe long term changes that resulted in rejection of tumor rechallenge. Illustration created in BioRender.

[0034] FIGs. 18A-F: Safety and toxicity of [161Tb]Tb-DOTA-N4MU01 in healthy female BALB / c mice (n = 3-5 / group). Fig. 18A: Projected human tissue absorbed doses for [161Tb]Tb- DOTA-N4MU01 in mGy / MBq. Values are projected from female BALB / c mice % lA / g data (n = 4 / time point) using OLINDA software. Fig. 18B: Schematic representation of study design. Fig. 18C: Body weight of mice measured over corresponding cohort time points shows general tolerability of radioligand at 2x 5 MBq. Fig. 18D: Changes in blood biochemistry parameters of saline control vs radioligand at acute (d2), chronic (d14) and sub-chronic (d28) time points. Dashed lines indicate lower and upper limits of the 95% confidence interval for 8-10-week-old female BALB / c mice, North American colonies (Charle River data). Groups were compared using One-Way ANOVA. Fig. 18E: Changes in complete blood count of saline control vs radioligand at sub-chronic time point. Dashed lines in graphs indicate lower and upper limits of the 95% Cl and comparison between groups was done using Two-tailed student t-test. Blood biochemistry and CBC parameters were analyzed using the Cobas c311 biochemistry and ADVIA 2120i hematology analyzers, respectively. Fig.18F: Hematoxylin & Eosin-stained tissues of major organs. Images are displayed at a final resolution of 200x. All results are presented as mean ± SD. ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, ALP: Alkaline phosphatase, GDH: Glutamate dehydrogenase, CK: Creatinine kinase, NEU / SEG: Segmented neutrophils, WBC: white blood cells, RBC: red blood cells, 161TbN5.0: 5 MBq of [161Tb]Tb- DOTA-N4MU01.

[0035] FIGs. 19A-D: [161Tb]Tb-DOTA-N4MU01 is highly efficacious against cell line derived xenografts in mice . Fig.19A: Average tumor growth curves for MDA-MB-468 xenograftbearing female athymic nude mice and average body weights of the mice treated with 2x 5 MBq [161Tb]Tb-DOTA-N4MU01 (161TbN5.0) or saline on dayO and d7 intravenously. Average tumor growth curves for Fig. 19B: 4T1Nectin4 and Fig. 19C: E0771Nectin-4 xenograft-bearing female BALB / c and C57BL / 6 mice, respectively, treated with 2x 5 MBq [161Tb]Tb-DOTA-N4MU01 (161TbN5.0) or N4MU01 (25 pg) or saline on d0 and d7 intravenously. Fig. 19D: Median survival (MS) values of various treatment groups across 3 cell line derived xenografts. Survival values were obtained from Kaplan Meier survival curves of treatment groups, while Log-rank (Mantel- Cox) test for survival comparisons was used to compare MS values between two groups. Studyendpoint was considered as tumor volume 1500 m ≥m3 or survival for 97 days (MDA-MB-468), 96 days (4T1 Nectin4), and 81 days (E0771 Nectin-4), post treatment. All data is plotted as mean ± SD. 97#: MS > 97 days.

[0036] FIGs. 20A-H: Radioligand induces anti-PD-L1 response in checkpoint inhibition therapy resistant 4T1 Nectin-4 syngeneic tumors in BALB / c mice and prevents tumor reoccurrence. All groups that received anti-PD-L1 (aPD-L1) were administered three 250 / zg doses intraperitoneally on d3, d5, and d7 following start of intravenous saline or [161Tb]Tb- DOTA-N4MU01 (5 MBq) treatment. Fig. 20A: Therapeutic study scheme (designed using BioRender. Individual tumor growth curves for: Fig. 20B: 2x [161Tb]Tb-DOTA-N4MU01 monotherapy group (161TbN5.0); Fig. 20C: 2x [161Tb]Tb-DOTA-N4MU01 + anti-PD-L1 (161TbN5.0+aPD-L1); Fig. 20D: Saline + anti-PD-L1 (Saline+aPD-L1); Fig. 20E: 2x 25 / zg of N4MU01 (N4MU01); or Fig. 20F: Saline. Fig. 20G: Average tumor growth curve shows tumor volume comparison of each treatment group with saline (d16) using Two-tailed student t-test. Fig. 20H: Kaplan Meier survival curve of treatment groups shows Log-rank (Mantel-Cox) test for survival comparisons and a bar-chart of MS values (days, d) for the different treatment groups. Study endpoint was considered as tumor volume 1500 m ≥m3 or survival for 96 days post treatment. The individual growth curves in c and d in ‘bold’, indicate mice with complete tumor remission. CR = complete remission, RC = xenograft rechallenge, d = day.

[0037] FIGs. 21A-H: Radioligand co-treatment enhances anti-PD-L1 efficacy in checkpoint inhibition therapy responsive E0771 Nectin-4 tumors in a syngeneic C57BL / 6 mouse model. All groups that received anti-PD-L1 (aPD-L1) were given three 250 pg doses intraperitoneally on d3, d5, and d7 following intravenous saline or [161Tb]Tb-DOTA-N4MU01 (5 MBq) treatment. Fig. 21A: Therapeutic study scheme (designed using BioRender). Individual tumor growth curves for: Fig. 21 B: 2x [161Tb]Tb-DOTA-N4MU01 monotherapy group (161TbN5.0); Fig. 21C: 2x [161Tb]Tb-DOTA-N4MU01 + anti-PD-L1 (161TbN5.0+aPD-L1); Fig.21 D: Saline + anti-PD-L1 (Saline+aPD-L1); Fig. 21 E: 2x 25 / zg of N4MU01 (N4MU01); and Fig. 21 F: Saline. Fig. 21G: Average tumor growth curve shows tumor volume comparison of each treatment group with saline (d9) and between 161TbN5.0 and 161TbN5.0+aPD-L1 treatment groups (d14). These were compared using Two-tailed student t-test. Fig. 21 H: Kaplan Meier survival curve of treatment groups shows Log-rank (Mantel-Cox) test for survival comparisons and a bar-chart of MS values (days, d) for the different treatment groups. Study endpoint was considered as tumor volume 1500 ≥ mm3 or survival for 81 days post treatment. The individualgrowth curves in c and d in ‘bold’, indicate mice with complete tumor remission. CR = complete remission, RC = xenograft rechallenge, d = day.

[0038] FIGs. 22A-F: CMT167.n4 161-Tb combination therapy end point 1000mm. Combination therapy in CMT167.nectin-4 syngeneic mouse model with two doses of [161Tb]Tb- N4MU01 and three doses of anti-PD-L1. 5 MBq of [161Tb]Tb-N4MU01 was injected on day 0 and on day 7 through the tail vein, while 250 μg / mouse of anti-PD-L1 antibody was injected on days 3, 5, and 7, intra-peritoneally. Plots of tumor volumes over time after treatment of, Fig. 22A: [161Tb]Tb-N4MU01 group, Fig. 22B: [161Tb]Tb-N4MU01 with anti-PD-L1 group, Fig. 22C: saline with anti-PD-L1 group, Fig. 22D: saline group and Fig. 22E: average tumor volume. Fig. 22F: Kaplan Meier survival curve of treatment groups shows Log-rank (mantel-Cox) test for curve comparison.DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

[0039] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0040] The following non-limiting examples are illustrative of the present application:L Definitions

[0041] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0042] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently beincluded in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the description.

[0043] The term “about” as used herein may be used to take into account experimental error and variations that would be expected by a person having ordinary skill in the art. For example, “about” may mean plus or minus 10%, or plus or minus 5%, of the indicated value to which reference is being made.

[0044] As used herein the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0045] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified.

[0046] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of" or, when used in the claims, "consisting of" will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0047] As used herein, the term "PD-1 / PD-L1 inhibitor" refers to any compound or agent that inhibits the activity of PD-L1 and the term PD-1 inhibitor refers to any compound or agent that inhibits activity of PD-1. Programmed cell death-1 (PD-1), an important inhibitory receptor and PD-1 signaling is induced upon binding of its ligands, PD-1 ligand-1 (PD-L1). Several PD-1 and PD-L1 antibodies are in clinical use for the treatment of various solid cancers and lymphomas, and blocking of the PD-1 pathway. Reference to PD-1 / PD-L1 inhibitor refers to an inhibitor of PD- 1 , PD-L1 or both.

[0048] The term “complementarity determining region” or “CDR” as used herein refers to particular hypervariable regions of antibodies that to contribute to epitope binding. Computational methods for identifying CDR sequences include Kabat, Chothia, Martin (AbM), AHo and IMGT. The CDRs listed in the present disclosure are identified using the Kabat definition or the IMGT definition, as indicated. A person skilled in the art having regard to the sequences comprised herein would also be able to identify CDR sequences based on IMGT, Kabat, and Chothia etc (e.g., see Giudicelli et al., 2022, Swindells et al., 2017). Such antibodies are similarly encompassed.

[0049] The term “framework region” or “FR” as used herein refers to regions interspersed before and between CDRs. For example, the light chain or the heavy chain can comprise FR1 N- terminal to CDR1 , FR2 between CDR1 and CDR2, FR3 between CDR2 and CDR3, and FR4 C- terminal to CDR3.

[0050] The term "antibody" as used herein is intended to encompass for example monoclonal antibodies, polyclonal antibodies, fully human antibodies, humanized and other chimeric antibodies, and binding fragments thereof, including for example a single chain Fab fragment, Fab’2 fragment, or single chain Fv fragment. The antibody may be from recombinant sources and / or produced in transgenic animals. Also included are human antibodies that can be produced in transgenic animals or using biochemical techniques, or can be isolated from a library such as a phage display library. Antibody backbones may comprise any suitable variable heavy chain or variable light chain sequences. Antibodies, including humanized and / or other chimeric antibodies may include sequences from one or more than one isotype, class, or species. Antibodies may be any class of immunoglobulins including: IgG, IgM, IgD, IgA, or IgE; and any isotype thereof, including lgG1, lgG2 (e.g. lgG2a, lgG2b), lgG3 and lgG4. Further, these antibodies can be produced as antigen binding fragments such as Fab, Fab' F(ab')2, Fd, Fv and single domain antibody fragments, or as single chain antibodies in which the heavy and light chains are linked by a spacer.

[0051] The term "binding fragment" as used herein refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain and which binds the antigen or competes with intact antibody. Exemplary binding fragments include without limitations Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, and diabodies, and includes multimers thereof. Fragments can be obtained via chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. For example, F(ab')2 fragments can begenerated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be constructed by recombinant expression techniques.

[0052] The term “cell” as used herein refers to a single cell or a plurality of cells.

[0053] A "conservative amino acid substitution" as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. The phrase “conservative substitution” also includes the use of a chemically derivatized residue or non-natural amino acid in place of a non-derivatized residue provided that such polypeptide displays the requisite activity.

[0054] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to any chain of two or more natural or unnatural amino acid residues, regardless of post-translational modifications (e.g., glycosylation or phosphorylation). Included are proteins that are a single polypeptide chain and multisubunit proteins (e.g., composed of 2 or more polypeptides).

[0055] The term "sequence identity" as used herein refers to the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. , % identity = [number of identical overlapping positions] / [total number of positions] X 100%). Thedetermination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBI_AST programs of Altschul et al., 1990. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g. for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g. to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. of XBLAST and NBLAST) can be used (see, e.g. the NCBI website). Another nonlimiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0056] For antibodies, percentage sequence identities can be determined when antibody sequences are maximally aligned by Kabat, IMGT, or other numbering conventions. The terms “Kabat numbering”, ”IMGT numbering”, etc., which are recognized in the art, refer to systems of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or antigen binding portion thereof. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted,multiplied by 100 to convert to percentage. Accordingly, Kabat, IMGT, and other alignment systems can also be used to identify or annotate CDRs in an antibody sequence.

[0057] The term "nucleic acid” or “nucleic acid molecule", as used herein, are intended to include unmodified DNA or RNA or modified DNA or RNA. The nucleic acid molecules of the disclosure may contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. Unless otherwise indicated, standard IUPAC-IUB nomenclature is used herein. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms. The term "polynucleotide" shall have a corresponding meaning. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid molecule" includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents. The term "isolated nucleic acid molecules" as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.

[0058] The term “pharmaceutically acceptable” means compatible with the treatment of animals, in particular, humans.

[0059] The term “treating” or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, inhibiting spread or progression of disease, delay or slowing of disease progression or onset, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. “Treating” and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0060] As used herein, the term “cancer” includes “Nectin-4-expressing cancers” also referred to as “Nectin-4 positive cancers”. Cancer refers to one of a group of diseases caused by the uncontrolled, abnormal growth of cells that can spread to adjoining tissues or other parts of the body. Cancer can include solid tumors (e.g., cancer cells can form a solid tumor, in which the cancer cells are massed together), or cancer cells which exist as dispersed cells, such as in blood cancers. “Nectin-4-expressing cancers” also referred to as “Nectin-4 positive cancers”, exhibit increased expression of nectin-4 in cancer cells or tissue as compared to corresponding and / orsurrounding normal cells or tissue, such as cancers of epithelial origin (e.g., breast cancer, triple negative breast cancer, ovarian cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, lung cancer, bladder cancer, for example urothelial cancer, gastric cancer among others). For example, a Nectin-4- expressing cancer comprises cancer cells that have a level of nectin-4 expression that is detectable by immunohistochemistry, e.g., as compared to normal cells. For example, a Nectin- 4-expressing cancer may comprise cancer cells that have a level of nectin-4 expression that is for example, detectable (e.g. above background), or is about 20% increased or is 2X-3X higher as compared to corresponding normal cells, using for example an H-score immunohistochemistry (IHC) numerical method. Normal cells typically show undetectable or background levels of nectin- 4 detectable by immunohistochemistry. A person skilled in the art would understand how to assess if a cancer is a nectin-4 positive cancer. Examples of characterization of nectin-4 using IHC staining H-score grading as well as lists of cancers comprising nectin-4 expression are described in for example, Challita-Eid, Pia M et al. “Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models.” Cancer research vol. 76,10 (2016): 3003-13.

[0061] The term “cancer cell” includes a Nectin-4-expressing cancer cell, e.g., a cell characterized by uncontrolled, abnormal growth and the ability to invade another tissue or a cell derived from such a cell, which exhibits increased expression of nectin-4 as compared to corresponding and / or surrounding normal cells or tissue. Cancer cells include, for example, a primary cancer cell obtained from a patient with cancer or cell line derived from such a cell. For example, a Nectin-4-expressing cancer cell has a level of nectin-4 expression that is detectable by immunohistochemistry, e.g., as compared to a normal cell which may not have a level of nectin- 4 detectable by immunohistochemistry.

[0062] The term "administered" as used herein means administration of a therapeutically effective dose of an antibody, immunoconjugate, compound and / or composition of the disclosure to a cell or subject. The term includes administration of combinations which may have various administration profiles, for example a first component may be administered within about 18 hours to about 24 hours, optionally about 18 hours or about 24 hours, or about 2 days or about 3 days or about 4 days of a second component as part of a treatment regimen.

[0063] The term “day”” as used herein means about a day for example, at least 18 hours and up to 30 hours. For example, “a day after” or “a day prior” refers to at least or about 18 hours after or prior and up to about 30 hours after or prior. For example, an administration that takesplace about a day after a first administration may be 18 hours after (e.g. first administration at 2 pm on day 1 and second administration at 8 am on day 2 or 8 pm on day 2). Similarly reference to “on day 3” in reference to “on day 0” as used herein refers to about 3 days, or at least or about 54 hours to at least or about 90 hours.

[0064] As used herein, the phrase "effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, in the context of treating cancer, an effective amount is an amount that for example induces remission, reduces tumor burden, and / or prevents tumor spread or growth of cancer cells compared to the response obtained without administration of the compound. Effective amounts may vary according to factors such as the disease state, age, sex and weight of the subject. The amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given antibody or immunoconjugate, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

[0065] The term "subject" as used herein includes all members of the animal kingdom including mammals and suitably refers to humans. Optionally, the term “subject” includes mammals that have or have been diagnosed with cancer, such as breast cancer, optionally triple negative breast cancer, ovarian cancer , lung cancer, optionally non-small cell lung cancer, optionally adenocarcinoma or squamous cell lung cancer, gastric cancer, urothelial cancer, bladder cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, esophageal cancer or pancreatic cancer, or are in remission. The term “subject” can also refer to a human having, or suspected of having, cancer such as breast cancer optionally triple negative breast cancer, ovarian cancer, lung cancer, optionally non-small cell lung cancer, optionally adenocarcinoma or squamous cell lung cancer, optionally small cell lung cancer, gastric cancer, urothelial cancer, bladder cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, esophageal cancer or pancreatic cancer.

[0066] As used herein, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of” and "consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively

[0067] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0068] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.II. Methods, Uses, Combinations, Compositions and Kits

[0069] An aspect of the disclosure includes a method of treating cancer, the method comprising administering to a subject in need thereof, 1) one or more anti-nectin-4 immunoconjugate each comprising an anti-nectin-4 antibody and one or more therapeutic agent, and 2) one or more Programmed Death-1 (PD-1 / Programmed Death Ligand 1 (PD-L1) inhibitor.

[0070] The anti-nectin-4 immunoconjugate can be administered before the one or more PD-1 / PD-L1 inhibitor. For example, a first dose of the anti-nectin-4 immunoconjugate is administered before a first dose of the one or more PD-1 / PD-L1 inhibitor of the treatment regimen and for example a second dose of the anti-nectin-4 immunoconjugate is administered before a second dose of the one or more PD-1 / PD-L1 inhibitor of the treatment regimen. In some embodiments, the anti-nectin-4 immunoconjugate is administered prior to administering the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered at least or about 18 hours after administration of the one or more anti-nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered between about 18 hours and about 24 hours after administration of the anti-nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered at least about 24 hours after administration of the anti-nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered between about 18 hours and about 7 days after administration of the anti-nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered between about 18 hours and about 3 days after administration of the anti-nectin-4 immunoconjugate. In some embodiments, the one or morePD-1 / PD-L1 inhibitor is administered between about 1 day and about 3 days after administration of the anti-nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered between about 1 day and about 7 days after administration of the anti- nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered between about 3 days and about 7 days after administration of the anti-nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered between about 5 days and about 7 days after administration of the anti-nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered about 2 days after administration of the anti-nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered about 3 days after administration of the anti- nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered about 4 days after administration of the anti-nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered about 5 days after administration of the anti-nectin-4 immunoconjugate. In some embodiments, the one or more PD- 1 / PD-L1 inhibitor is administered about 6 days after administration of the anti-nectin-4 immunoconjugate. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered about 7 days after administration of the anti-nectin-4 immunoconjugate. For example, at least one dose, such as the first dose, or a plurality of doses, such as the first and second doses, or optionally each dose of the one or more PD-1 / PD-L1 inhibitor can be administered at least or about 18 hours after administration of the anti-nectin-4 immunoconjugate or administered between about 18 hours and about 24 hours after administration of the anti-nectin-4 immunoconjugate or between about 18 hours and about 7 days after administration of the anti- nectin-4 immunoconjugate, etc. A person skilled in the art would also understand that the foregoing includes reciting wherein the anti-nectin-4 immunoconjugate is administered prior to the one or more PD-1 / PD-L1 inhibitor

[0071] In some embodiments, the one or more therapeutic agent is one or more radionuclide. In some embodiments, the one or more therapeutic agent is a radionuclide and a cytotoxin.

[0072] In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody.

[0073] In some embodiments, the anti-PD-1 inhibitor is nivolumab. For example, inhibitors of PD-1 biological activity (e.g., ligands of PD-1) disclosed in U.S. Patent Nos. 7,029,674; 6,808,710; or U.S. Patent Application Nos. 20050250106 and 20050159351 canbe used in the methods provided herein. Exemplary antibodies against PD-1 include: anti-mouse PD-1 antibody clone J43 from BioXcell (catalog number BE0033-2); anti-mouse PD-1 antibody clone RMP1-14 from BioXcell (catalog number BE0146); mouse anti-PD-1 antibody clone EH12; Merck's MK-3475 anti-mouse PD-1 antibody (Keytruda, pembrolizumab, lambrolizumab); and AnaptysBio's anti-PD-1 antibody known as ANB011 ; antibody MDX-1 106 (ONO-4538); Bristol- Myers Squibb's human lgG4 monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106); AstraZeneca's AMP-514 and AMP-224; and CureTech Ltd's pidilizumab (CT-011).

[0074] Exemplary PD-L1 inhibitors include antibodies (e.g., anti-PD-L1 antibodies), RNAi molecules (e.g., anti-PD-L1 RNAi), antisense molecules (e.g., anti-PD-L1 antisense RNA), dominant negative proteins (e.g., dominant negative PD-L1 proteins), and small molecule inhibitors. Exemplary anti-PD-L1 antibodies include clone EH12. Exemplary antibodies against PD-L1 include: MPDL3280A (RG7446) from Genentech; anti-mouse PD-L1 antibody clone 10F from BioXcell. 9G2 (catalog no. BE0101); anti-PD-L1 monoclonal antibodies MDX-1105 (BMS- 936559) and BMS-935559 from Bristol-Meyer's Squibb; MSB0010718C; mouse anti-PD-L1 clone 29E.2A3; and MEDI4736 from AstraZeneca, atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi) .

[0075] The PD-1 inhibitor can include inhibitors of PD-L2.

[0076] Exemplary PD-L2 inhibitors include antibodies (e.g., anti-PD-L2 antibodies), RNAi molecules (e.g., anti-PD-L2 RNAi), antisense molecules (e.g., anti-PD-L2 antisense RNA), dominant negative proteins (e.g., dominant negative PD-L2 proteins), and small molecule inhibitors. Antibodies include monoclonal antibodies, humanized antibodies, deimmunized antibodies, and Ig fusion proteins.

[0077] In some embodiments, the cytotoxin is selected from the group comprising or consisting of maytansine and derivatives thereof (maytansinoids), auristatins, calicheamicins, duocarmycins, doxorubicin, a nth racy clines, amanitins and camptothecins. In some embodiments, the antibody drug conjugate comprises a plurality of cytotoxins conjugated to the antibody, preferably 2 - 8 cytotoxins. In some embodiments, the cytotoxin is Monomethyl Auristatin E (MMAE).

[0078] In some embodiments, the radionuclide is an a emitting radiopharmaceutical or a P' / Auger electron emitting radionuclide.

[0079] In some embodiments, the antibody is conjugated with the radionuclide using a bifunctional chelator, optionally desferrioxamine (DFO), NOTA, DOTA, NODAGA, TETA, 3p-C-NETA, HYNIC, DTPA, CB-TE2A, H2dedpa, H4octapa, NODAGA, HBED-CC, 3p-C-DEPA, HOPO. In some embodiments, the chelator is 18-membered macrocyclic bifunctional chelator 6-((16-((6- carboxypyridin-2-yl)methyl)-1 ,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4- isothiocyanatopicolinic acid (macropa-SCN). In some embodiments, the chelator-antibody ratio (CAR) is 4:1 to 1 :1 , preferably 3:1 to1 :1. In some embodiments, the CAR is 1 :1. In some embodiments, the CAR is 3:1. In some embodiments, the CAR is 2:1. In some embodiments, the CAR is 4:1.. In some embodiments, the chelator is DFO. In some embodiments, the chelator is DFO and the CAR is 3:1. In some embodiments, the chelator is macropa-SCN. In some embodiments, the chelator is macropa-SCN and the CAR is 1 :1.

[0080] In some embodiments, the radionuclide is selected from Fluorine-18 (half-life 109.8 min), iodine-131 (half-life 8 days), idodine-123 (half-life 13.2 hours), idodine-125 (half-life 60 days), copper-67 (half-life 61.8 days), indium-111 (half-life 2.8 days), thorium-227 (half-life 18.7 days), astatine-211 (half-life 7.2 hours), bismutt-212 (half-life 45 minutes), lead-212 (half-life 10.6 hours), radium-223 (half-life 11.4 days), rhenium-188 (half-life 16.9 hours), technecium-99m (halflife 6.6 hours), yttrium-90 (half-life 64.6 hours), lutetium-177 (half-life 6.8 days), copper-61 (halflife 3.3 hours), copper-64 (half-life 12.7h). In-111 (half-life 2.8 days), terbium-152 (half-life 17.5 hours), terbium-156 (half-life 5.3 days), terbium-161 (half-life 6.8 days), terbium-149 (half-life 4.1 hours), iodine-124, gallium-67 (half-life 78.3 hours), or actinium-225 (half-life 10 days).

[0081] In some embodiments, the radionuclide is161Tb or225Ac. In some embodiments, the radionuclide is161Tb. In some embodiments, the radionuclide is225Ac. In some embodiments, the radionuclide is177Lu.

[0082] Immunoconjugates may be generated using any suitable technique. Bifunctional chelating agents may be used to make radionuclide-conjugated antibodies. Suitable bifunctional chelating agents are known in the art, including desferrioxamine (DFO). Other suitable chelating agents include DTPA (diethylenetriamine pentaacetic acid), TCMC (1 , 4,7,10-tetraaza-1 , 4, 7,10- tetra(2-carbamoylmethyl)cyclododecane), TETA (1 ,4,8,11-Tetraazacyclotetradecane-1 ,4,8,11- tetraacetic acid), DOTA (1,4,7,10-tetraazacyclododecane tetraacetic acid), macropa (N,N'-bis[(6- carboxy-2-pyridil)methyl]-4, 13-diaza-18-crown-6).

[0083] In some embodiments, the one or more PD-1 / PD-L1 inhibitor is one or more antibody. In some embodiments, the one or PD-1 / PD-L1 inhibitor is an anti-PD-L1 antibody.

[0084] In some embodiments, in which the inhibitor is an antibody, the antibody may be a monoclonal antibody, a synthetic antibody, a polyclonal antibody, a multispecific antibody(including a bispecific antibody), a human antibody, a humanized antibody, a chimeric antibody, a single chain Fv (scFv) (including a bispecific scFv), a single chain antibody, a Fab fragment, an F(ab') fragment, a disulfide-linked Fv (sdFv), or other binding fragment of any of the above antibody types. In some embodiments, the antibody is a single chain antibody. In some embodiments, the antibody is or comprises a Fab fragment.

[0085] In some embodiments, the subject is a subject that has failed treatment with an immune checkpoint inhibitor optionally an immune checkpoint inhibitor monotherapy.

[0086] The treatment regimen may be altered for example to maximize the priming effect of the anti-nectin-4 immunoconjugate. For example, the subject may lack an increase in peripheral levels of one or more cytokine after administration of the anti-nectin-4 immunoconjugate described herein. Where the subject lacks an increase in peripheral levels of one or more cytokine after administration of the anti-nectin-4 immunoconjugate, an increased dose or additional dose of the anti-nectin-4 immunoconjugate may be administered to the subject. Accordingly in some embodiments, the method comprises measuring the level of one or more of IP-10, IL-5, G-CSF, Eotaxin, TNFα, IFN- VEGF, IL-2, IL-10, and / or M-CSF in a blood sample obtained after administration of the one or more anti-nectin-4 immunoconjugate. For example, said level may be measured in a blood sample obtained 1 day, 2 days, 3 days, 4 days, 5 days or more after administration of the one or more anti-nectin-4 immunoconjugate. In some embodiments, the subject lacks an increase in peripheral levels of one or more cytokine after administration of the anti-nectin-4 immunoconjugate after about 18 hours, after about 1 day, after about 2 days, after about 3 days, after about 4 days, after about 5 days, after about 6 days, or after about 7 days. In some embodiments, the subject lacks an increase in peripheral levels of one or more cytokine after administration of the anti-nectin-4 immunoconjugate after about 1 day to about 3 days, after about 1 days to about 7 days, after about 3 days to about 7 days, or after about 5 days to about 7 days. In some embodiments, the one or more cytokine include IP-10, IL-5, G-CSF, Eotaxin, TNFα, IFN- VEGF, IL-2, IL-10, and / or M-CSF. In some embodiments, the one or more cytokine includes IL-2, IL-10, IFN- VEGF, and TNFα. In some embodiments, the one or more cytokine includes one or more cytokines described in the Examples and / or Figures. In some embodiments, the increased level of the one or more cytokine is considered increased when it is higher than a control, e.g., a baseline level of the subject or a reference value. In some embodiments, the increased level of the one or more cytokine is considered increased when it is higher than e.g., a subject’s baseline level of the one or more cytokine. Subjects without an increase in the one or more cytokines may be administered an additional dose of the one or more anti-nectin-4immunoconjugate or an increased dose thereof for example prior to the administration of a dose or the next dose of the one or more PD-1 / PD-L1 inhibitor.

[0087] In some embodiments, the cancer is selected from breast cancer, preferably triple negative breast cancer, ovarian cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, gastric cancer, urothelial cancer, bladder cancer, and lung cancer, optionally non-small cell lung cancer, optionally adenocarcinoma or squamous cell lung cancer, optionally small cell lung cancer.

[0088] In some embodiments, the cancer is a cancer that is resistant or refractory to immune checkpoint monotherapy. In some embodiments, the cancer is a cancer that is responsive to immune checkpoint monotherapy. The resistance can for example be primary resistance or acquired resistance.

[0089] In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple negative breast cancer.

[0090] In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is, colorectal cancer. In some embodiments, the cancer is hepatocellular cancer. In some embodiments, the cancer is gallbladder cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is urothelial cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is lung cancer, optionally non-small cell lung cancer, optionally adenocarcinoma or squamous cell lung cancer. In some embodiments, the lung cancer is small cell lung cancer.

[0091] In some embodiments, the subject is a human.

[0092] In some embodiments, one or more dose of the one or more inhibitor or anti-nectin-4 immunoconjugate is administered prior to one or more dose of the one or more PD-1 / PD-L1 inhibitor. For example, two dose of the one or more inhibitor or anti-nectin-4 immunoconjugate can be administered prior to the first two dose of the one or more PD-1 / PD-L1 inhibitor. The subject may receive additional dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the one or more dose of the one or more inhibitor or anti-nectin-4 immunoconjugate is administered prior to every dose of the one or more PD-1 / PD-L1 inhibitor. For example, if a subject is administered 4 doses of the one or more PD-1 / PD-L1 inhibitor, a dose a of the anti- nectin-4 immunoconjugate is administered to the subject prior to each of the 4 doses of the oneor more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered between at least about 18 hours to about 7 days prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered between at least about 18 hours to about 3 days prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered between at least about 24 hours to about 7 days prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered between at least about 24 hours to about 3 days prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered between at least about 3 days to about 7 days prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered between at least about 5 days to about 7 days prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered at least about 18 hours prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered at least about 24 hours prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered about 2 days prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered about 3 days prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered about 4 days prior to each dose of the one or more PD-1 / PD- L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered about 5 days prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti- nectin-4 immunoconjugate is administered about 6 days prior to each dose of the one or more PD-1 / PD-L1 inhibitor. In some embodiments, the anti-nectin-4 immunoconjugate is administered about 7 days prior to each dose of the one or more PD-1 / PD-L1 inhibitor.

[0093] In some embodiments, the dose of the one or more anti-nectin-4 immunoconjugate comprises between about 2.5 MBq and 5 M Bq or an equivalent dose according to the subject (e.g. mouse or human). In some embodiments, the dose of the one or more anti-nectin-4 immunoconjugate comprises about 2.5 MBq. In some embodiments, the dose of the one or more anti-nectin-4 immunoconjugate comprises about 5 MBq. For example, the human dose may be 2-5 GBq / dose, for example for a 70 kg patient. In some embodiments, the dose is any 0.1 increment between 2 and 5 GBq, for example 2.1 GBq, 2.2 GBq, 2.5 GBq, 3 GBq, 3.5 GBq etc.

[0094] In some embodiments, at least one dose of the one or more anti-nectin-4 immunoconjugate is administered to the subject. In some embodiments, at least two doses of the one or more anti-nectin-4 immunoconjugate is administered to the subject. In some embodiments, at least 3, at least 4, at least 5, at least, 6, at least 7, at least 8, at least 9, at least 10 doses of the one or more anti-nectin-4 immunoconjugate is administered to the subject. In some embodiments, at least 3, at least 4, at least 5, at least, 6, doses of the one or more anti-nectin-4 immunoconjugate is administered to the subject. In some embodiments, at least 4 doses of the one or more anti- nectin-4 immunoconjugate is administered to the subject.

[0095] In some embodiments, each of the at least two doses of the one or more anti- nectin-4 immunoconjugate is administered on different days.

[0096] In some embodiments, a first dose of the at least two doses of the anti-nectin-4 immunoconjugate is administered 7 days prior to a second dose of the at least two doses. In some embodiments, a first dose of the at least two doses of the anti-nectin-4 immunoconjugate is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days prior to a second dose of the at least two doses.

[0097] In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered in at least one dose. In some embodiments, the one or more PD-1 / PD-L1 inhibitor is administered in a plurality of doses.

[0098] In some embodiments, each of the plurality of doses of the one or more PD-1 / PD- L1 inhibitor is administered to the subject on different days.

[0099] In some embodiments, the dose of the PD-1 / PD-L1 inhibitor is for example about 250ug. In some embodiments, the dosage can be decreased by about 10%, about 15%, about 20% or about 25%.

[0100] In some embodiments, each of the plurality of doses is administered at leastl day apart. In some embodiments, each of the plurality of doses is administered 2 days apart. In some embodiments, each of the plurality of doses is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days apart.

[0101] In some embodiments, the plurality of doses of the one or more PD-1 / PD-L1 inhibitor is three or more doses. In some embodiments, the plurality of doses of the one or more PD-1 / PD-L1 inhibitor is two doses. In some embodiments, the plurality of doses of the one ormore PD-1 / PD-L1 is three doses. In some embodiments, the plurality of doses of the one or more PD-1 / PD-L1 is four doses.

[0102] In some embodiments, a first dose of the one or more anti-nectin-4 immunoconjugate is administered to the subject on day 0, a first dose of the one or more PD- 1 / PD-L1 inhibitor is administered to the subject on day 3, a second dose of the one or more PD- 1 / PD-L1 inhibitor is administered to the subject on day 5, a second dose of the one or more anti- nectin-4 immunoconjugate is administered to the subject on day 7, and a third dose of the one or more PD-1 / PD-L1 inhibitor is administered to the subject on day 7.

[0103] In some embodiments, the one or more anti-nectin-4 immunoconjugate and / or one or more PD-1 / PD-L1 inhibitor can be administered for example, by parenteral, intravenous, intramuscular, intracranial, intraventricular, intrathecal, intracisternal, intraperitoneal. In some embodiments, the PD-1 / PD-L1 inhibitor can be administered for example by intranasal, aerosol or oral administration.

[0104] An aspect of the disclosure includes a combination treatment comprising 1 ) one or more anti-nectin-4 immunoconjugate each immunoconjugate comprising an anti-nectin-4 antibody and a therapeutic agent, and 2) one or more PD-1 / PD-L1 inhibitor. In some embodiments, the one or more anti-nectin-4 immunoconjugate is comprised in a composition. In some embodiments, the one or more one or more PD-1 / PD-L1 inhibitor is comprised in a composition.

[0105] An aspect of the disclosure includes a method of priming a tumor for immune checkpoint therapy, the method comprising administering one or more anti-nectin-4 immunoconjugate, each immunoconjugate comprising an anti-nectin-4 antibody and a therapeutic agent to the tumor prior to immune checkpoint therapy, optionally wherein the immune checkpoint therapy comprises administering one or more immune checkpoint inhibitor e.g., PD-1 / PD-L1.

[0106] An aspect of the disclosure includes use of one or more anti-nectin-4 immunoconjugate, each immunoconjugate comprising an anti-nectin-4 antibody and a therapeutic agent for priming a tumor for immune checkpoint therapy, optionally prior to with the immune checkpoint therapy. A kit comprising a first container comprising one or more anti-nectin-4 immunoconjugate comprising an anti-nectin-4 antibody and a therapeutic agent, and a second container comprising one or more PD-1 / PD-L1 inhibitor.

[0107] In some embodiments, the one or more therapeutic agent is a radionuclide.

[0108] In some embodiments, the one or more therapeutic agent is a radionuclide and a cytotoxin.

[0109] In some embodiments, the anti-nectin-4 antibody is a fully human antibody.

[0110] Another aspect includes a method of treating cancer in a subject in need thereof according to a method described herein. In some embodiments, the subject has been previously determined to have a Nectin-4 expressing cancer. The subject can for example have been determined to have a Nectin-4 expressing cancer by imaging, or by testing a biopsy of the cancer.

[0111] In some embodiments, the anti-nectin-4 antibody is an antibody which specifically binds Nectin-4 comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising complementarity determining regions CDR-L1 , CDR-L2, and CDR-L3, and the heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2, and CDR-H3, wherein the amino acid sequences of said CDRs, as defined us g IMGT numbering, are:CDR-L1 QSV X1X2X3Y (SEQ ID NO: 1);CDR-L2 GACDR-L3 QQR X4X5X6PPT (SEQ ID NO: 3);CDR-H1 GF X7F X8SYG (SEQ ID NO: 4);CDR-H2 IWYDG X9NK (SEQ ID NO: 5); andCDR-H3 AR D X10W X11X12G X13Y (SEQ I D NO: 6) wherein, X1is an amino acid selected from S, K, F, and R,X2is an amino acid selected from N, H, A, T, S, and M,X3is an amino acid selected from S, G, and R,X4is an amino acid selected from S, Y, W, and T,X5is an amino acid selected from N, G, D, S, Y, T, H, F, and E,X6is an amino acid selected from W, F, and Y,X7is an amino acid selected from T, A, and N,X8is an amino acid selected from R, M, K, and G,X9is an amino acid selected from S and T,X10is an amino acid selected from D and G;X11is an amino acid selected from T, D, N, A, S, and R;X12is an amino acid selected from N, K, Y, H, R, and D; and / orX13. is an amino acid selected from W, F, and Y; b)CDR-L1 QSVSRY (SEQ ID NO: 68);CDR-L2 DA;CDR-L3 QQRYNWPPD (SEQ ID NO: 70);CDR-H1 GFTFSSYG (SEQ ID NO: 71);CDR-H2 ISYDGSNK (SEQ ID NO: 72); andCDR-H3 AKSTLHSSGWYMDY (SEQ ID NO: 73); or c)CDR-L1 QDISNY (SEQ ID NO: 74);CDR-L2 AA;CDR-L3 QQSYTTRTT (SEQ ID NO: 76);CDR-H1 GFRFSGYP (SEQ ID NO: 77);CDR-H2 IWYDGRNR (SEQ ID NO: 78); andCDR-H3 AKEGKWGEWYFDL (SEQ ID NO: 79).In some embodiments, the CDRs, as defined using IMGT, areCDR-L1 QSV X1X2X3Y (SEQ ID NO: 1);CDR-L2 GACDR-L3 QQR X4X5X6PPT (SEQ ID NO: 3);CDR-H1 GF X7F X8SYG (SEQ ID NO: 4);CDR-H2 IWYDG X9NK (SEQ ID NO: 5); andCDR-H3 AR D X10WX11X12G X13Y (SEQ ID NO: 6) wherein, X1is an amino acid selected from S, K, F, and R,X2is an amino acid selected from N, H, A, T, S, and M,X8is an amino acid selected from S, G, and R,X4is an amino acid selected from S, Y, W, and T,X4is an amino acid selected from N, G, D, S, Y, T, H, F, and E,X6is an amino acid selected from W, F, and Y,X7is an amino acid selected from T, A, and N,X8is an amino acid selected from R, M, K, and G,X9is an amino acid selected from S and T,X10is an amino acid selected from D and G;X11is an amino acid selected from T, D, N, A, S, and R;X12is an amino acid selected from N, K, Y, H, R, and D; and / orX13. is an amino acid selected from W, F, and Y.

[0112] In some embodiments, X1is S. In some embodiments, X1is K. In some embodiments, X1is F. In some embodiments, X1is R. In some embodiments, X2is N. In some embodiments, X2is H. In some embodiments, X2is A. In some embodiments, X2is T. In some embodiments, X2is S. In some embodiments, X2is M.

[0113] In some embodiments, X3is S. In some embodiments, X3is G. In some embodiments, X3is R. In some embodiments, X4is S. In some embodiments, X4is Y. In some embodiments, X4is W. In some embodiments, X4is T.

[0114] In some embodiments, X5is N. In some embodiments, X5is G. In some embodiments, X5is D. In some embodiments, X5is S. In some embodiments, X5is Y. In someembodiments, X5is T. In some embodiments, X5is H. In some embodiments, X5is F. In some embodiments, X5is E.

[0115] In some embodiments, X6is W. In some embodiments, X6is F. In some embodiments, X6is Y. In some embodiments, X7is T. In some embodiments, X7is A. In some embodiments, X7is N. In some embodiments, X8is R. In some embodiments, X8is M. In some embodiments, X8is K. In some embodiments, X8is K. In some embodiments, X8is G. In some embodiments, X9is S. In some embodiments, X9is T.

[0116] In some embodiments, X10is D. In some embodiments, Xw is G. In some embodiments, X11is T. In some embodiments, X11is D. In some embodiments, X11is N. In some embodiments, X11is A. In some embodiments, X11is S. In some embodiments, X11is R. In some embodiments, X12is N. In some embodiments, X12is K. In some embodiments, X12is Y. In some embodiments, X12is H. In some embodiments, X12is R. In some embodiments, X12is D. In some embodiments, X13is W. In some embodiments, X13is F. In some embodiments, X13is Y.

[0117] In some embodiments, X1is an amino acid selected from S, K, F, and R. In some embodiments, X2is an amino acid selected from N, H, A, T, S, and M. In some embodiments, X3is an amino acid selected from S, G, and R. In some embodiments, X4is an amino acid selected from S, Y, W, and T. In some embodiments, X5is an amino acid selected from N, G, D, S, Y, T, H, F, and E. In some embodiments, X6is an amino acid selected from W, F, and Y. In some embodiments, X7is an amino acid selected from T, A, and N. In some embodiments, X5is an amino acid selected from R, M, K, and G. In some embodiments, X9is an amino acid selected from S and T. In some embodiments, X10is selected from D and G. In some embodiments, X11is an amino acid selected from T, D, N, A, S, and R. In some embodiments, X12is an amino acid selected from N, K, Y, H, R, and D. In some embodiments, X13is an amino acid selected from W, F, and Y. In some embodiments, each are individually selected.

[0118] In some embodiments, the CDRs are those identified in Table 1. In some embodiments, position L53 as defined by Kabat is R, V, K, G, S, T or M (CDR-L2). Position L53 as defined by Kabat is position L67 as defined by IMGT. In some embodiments, position H58 as defined by Kabat is Y, L or F (CDR-H2).

[0119] In some embodiments, the CDRs, as defined using IMGT, are:CDR-L1 QSVSNSY (SEQ ID NO: 80);CDR-L2 GA;CDR-L3 QQRSNWPPT (SEQ ID NO: 81);CDR-H1 GFTFRSYG (SEQ ID NO: 82);CDR-H2 IWYDGSNK (SEQ ID NO: 83); andCDR-H3 ARDDWTNGWY (SEQ ID NO: 84).

[0120] In some embodiments, the light chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 7-35 or 62; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 7-35 or 62, wherein the CDR-L1 is or comprises SEQ ID NO: 1, CDR-L2 is or comprises amino acid sequence GA, and CDR-L3 is or comprises SEQ ID NO: 3; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR-L1 is or comprises SEQ ID NO: 1 , CDR-L2 is or comprises amino acid sequence GA, and CDR-L3 is or comprises SEQ ID NO: 3.

[0121] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 36-61 or 63; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 36-61 or 63, wherein the CDR sequences are as set forth in SEQ ID NOs: 4-6; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 4-6.

[0122] In some embodiments, the CDRs, as defined using IMGT, are:

[0123] In some embodiments, the light chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 64; ii) a polypeptide having an amino acidsequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 64, wherein the CDR-L1 is or comprises SEQ ID NO: 68, CDR-L2 is or comprises amino acid sequence DA, and CDR-L3 is or comprises SEQ ID NO: 70; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR-L1 is or comprises SEQ ID NO: 68, CDR-L2 is or comprises amino acid sequence DA, and CDR-L3 is or comprises SEQ ID NO: 70.

[0124] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 65; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 65, wherein the CDR sequences are as set forth in SEQ ID NOs: 71-73; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 71-73.

[0125] In some embodiments, the CDRs, as defined using IMGT, are:

[0126] In some embodiments, the light chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 66; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 66, wherein the CDR-L1 is or comprises SEQ ID NO: 74, CDR-L2 is or comprises amino acid sequence AA, and CDR-L3 is or comprises SEQ ID NO: 76; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR-L1 is or comprises SEQ ID NO: 74, CDR-L2 is or comprises amino acid sequence AA, and CDR-L3 is or comprises SEQ ID NO: 76.

[0127] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 67; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID Ns:67, wherein the CDR sequences are as set forth in SEQ ID NOs: 77-79; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 77-79. Uses of the methods described herein are also contemplated.

[0128] An aspect of the disclosures includes one or more anti-nectin-4 immunoconjugate comprising an anti-nectin-4 antibody and one or more therapeutic agent for use in a combination treatment with one or more Programmed Death-1 (PD-1 / Programmed Death Ligand 1 (PD-L1) inhibitor for treating cancer

[0129] An aspect of the disclosures includes one or more Programmed Death-1 (PD-1 / Programmed Death Ligand 1 (PD-L1) for use in a combination treatment with one or more anti- nectin-4 immunoconjugate comprising an anti-nectin-4 antibody and one or more therapeutic agent for treating cancer. In some embodiments, the method or use is for decreased recurrence, increased survival, or increased likelihood of complete response in a subject with cancer.

[0130] Another aspect of the disclosure includes a kit comprising one or more anti-nectin- 4 immunoconjugate described herein and / or one or more PD-1 / PD-L1 inhibitor. In some embodiments, the kit further comprises instructions for use. In some embodiments, the kit further comprises one or more vials or containers.

[0131] In some embodiments, the one or more anti-nectin-4 immunoconjugate described herein is formulated in a composition. In some embodiments, the one or more PD-1 / PD-L1 inhibitor described herein is formulated in a composition . In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or diluent. In some embodiments, the one or more anti-nectin-4 immunoconjugate described herein is formulated in a liposome or micelle. In some embodiments, the one or more PD-1 / PD-L1 inhibitor described herein is formulated in a liposome or micelle. In some embodiments, composition is a lyophilized composition. The lyophilized composition can be reconstituted e.g., with sterile water or other diluent. In some embodiments, the diluent may include sterile water, dextrose, and / or saline.

[0132] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.ExamplesExample 1Materials and methodsCell lines and xenografts

[0133] Human breast cancer MDA-MB-468, MCF-7 and NSCLC NCI-H358, and NCI- H2170 cell lines which express Nectin-4, and TNBC MDA-MB-231 cell line, known for Nectin-4 negative expression, were purchased from ATCC (Rockville, MD). For syngeneic mouse model, mouse breast cancer 4T1 , E0771 and NSCLC mouse cell lines CMT167 and LLC cell lines was purchased from ATCC (Rockville, MD) and transfected with full-length Nectin-4 protein to yield 4T1.Nectin-4, E0771.Nectin-4, CMT167.Nectin-4, and LLC.Nectin-4, Nectin-4-positive cell lines. Animals used in this study were maintained following the guidelines of the University of Saskatchewan Animal Care Committee (protocol # 20170084 and 20220021). Cell lines were authenticated using short tandem repeat (STR) profiling (Centre for Applied Genomics, Hospital for SickKids, Toronto, ON) and had no detectable mycoplasma prior to their use.Conjugation with bifunctional chelators and radiolabeling with161Tb and225Ac

[0134] N4MU01 was conjugated with S-2-(4-lsothiocyanatobenzyl)-1 ,4,7,10- tetraazacyclododecane tetraacetic acid (p-SCN-Bz-DOTA) for labeling with161Tb as described previously44. Quality control of the immunoconjugate was done using size exclusion chromatography (SEC) high-performance liquid chromatography (HPLC) (SEC-HPLC), flow cytometry, and automated electrophoresis (2100 Bioanalyzer, Agilent, Santa Clara, CA, USA). Radiolabeling of DOTA-N4MU01 with161Tb and purification were done using lab SOP44.

[0135] 18-membered macrocyclic bifunctional chelator 6-((16-((6-carboxypyridin-2- y I) methyl) - 1 ,4,10,13-tetraoxa-7, 16-diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinic acid (p-SCN-Macropa) was synthesized as reported earlier45. The conjugation of N4MU01 with Macropa for the labeling with225Ac was performed following the lab SOP (supplementary methods). Quality control of the immunoconjugate was done using SEC-HPLC, matrix-assisted laser desorption (MALDI) time of flight (TOF) (MALDI-TOF) and flow cytometry. Radiolabeling with225Ac was performed as previously reported44.

[0136] The stability of [161Tb]Tb-DOTA-labeled and [225Ac]Ac-Macropa-labeled N4MU01 conjugates at 37 °C was investigated using instant thin-layer chromatography (iTLC) by analyzing aliquots of the radiolabeled over time. [161Tb]Tb-DOTA-labeled or [225Ac]Ac-Macropa-labeledconjugates were mixed with human serum or 1x PBS solution at a final concentration of 15 MBq / mL and 2.5 MBq / mL, respectively and incubated at 37 °C. Aliquots of the solution were taken every 24 h for five days and analyzed for radiochemical purity using iTLC.

[0137] In addition,161Tb also emits characteristic gamma (y) photons [Ey = 49 keV, I = 17.0%; Ey = 75 keV, I = 10.2%] suitable for SPECT imaging. Furthermore, other diagnostic [152Tb (β+emitter, ti / 2 = 17.5 h, and155Tb (EC, y emitter, t1 / 2= 5.32 days, Ey = 0.105 MeV] and therapeutic [149Tb (alpha a emitter, ti / 2 = 4.12 h, Ea = 3.97 MeV] isotopes of terbium are available.Characterization of N4MU01 and DOTA-N4MU01 binding using flow cytometry

[0138] The binding affinity and specificity of N4MU01 and DOTA-N4MU01 were evaluated by flow cytometry using Nectin-4 expressing TNBC cell lines 4T1.nectin-4, E0771.nectin-4, and MDA- MB-468, and NSCLC cell lines CMT167.nectin-4and NCI-H358 as described previously46.Internalization of N4MU01 and DOTA-N4MU01

[0139] The internalization of N4MU01 and DOTA-N4MU01 was studied for 72 h on4T1.nectin-4and E0771.nectin-4cell lines using the Incucyte live-cell imaging system as described previously46.Radioligand binding assay of [161Tb]Tb-DOTA-N4MU01 and [225Ac]Ac-Macropa-N4MU01

[0140] The binding of [161Tb]Tb-DOTA-N4MU01 to nectin-4 positive 4T1.nectin-4and [225Ac]Ac-Macropa-N4MU01 to nectin-4 positive MDA-MB-468 was determined using a saturation radioligand binding assay as described previously47.MicroSPECT / CT imaging and biodistribution of [161Tb]Tb-DOTA-N4MU01

[0141] MicroSPECT / CT imaging and ex vivo biodistribution of [161Tb]Tb-DOTA-N4MU01 was carried in female BALB / c and C57BL / 6 mice. Biodistribution was initially performed in 7- week-old healthy female BALB / c mice (n = 4 mice / timepoint). These mice were injected via tail vein with 1.74 ± 0.12 MBq of [161Tb]Tb-DOTA-N4MU01. Similarly, 8-week-old female BALB / c (n = 4 mice / timepoint) and C57BL / 6 (n = 5 mice / timepoint) mice bearing 4T 1.nectin-4and CMT167.nectin-4xenografts were injected with 1.76 ± 0.30 MBq and 2.48 ± 0.10 MBq of [161Tb]Tb-DOTA- N4MU01 , respectively, via tail vein. Biodistribution mice were then sacrificed at different timepoints post injection (p.i.) and the carcass disserted to cleanly recover major organs and blood and these were then analyzed as previously described by Njotu et al.48to obtain percentage injected activity (%IA) and %IA / g values. MicroSPECT / CT imaging was performed in female BALB / C mice bearing 4T 1.nectin-4xenograft (n = 2). Mice were injected via tail vein with 5 MBq (25pg) of [161Tb]Tb-DOTA-N4MU01 on d0 and on d7 and images were acquired on d8 and d10 with reconstruction done at a161Tb energy window ranges of 84.8 ± 2.3 keV and 12.7 ± 2.0 keV.Biodistribution and dosimetry of [225Ac]Ac-Macropa-N4MU01

[0142] To estimate radiation dose to healthy tissues, normal BALB / c nude mice (n ≥ 4 / group) were administered 13 kBq of [225Ac]Ac-Macropa-N4MU01 via tail vein and sacrificed at 1 , 24, 48, 120, and 264 h p.i. followed by biodistribution studies. The mouse biodistribution % injected activity per gram (%IA / g) data was extrapolated to human data (%IA) using the formula % IA (human) = % I A / g (mouse) total b ×ody weight of mouse (in kg) mass of human × organ (in g) I total body weight of human (in kg). For each organ, this was plotted against sampling time and used to obtain an estimate of the residence time of the agent in the organ in MBq-h / MBq, represented by the area under the time-activity function integrated to infinity (complete decay) of the225Ac. The residence time was fitted into the OLINDA kinetics model (OLINDA / EXM V2.2, Hermes Medical Solutions Montreal QC) to generate absorbed doses in units of cGy (centi-gray) per millicurie (cGy / mCi) of225Ac administered.Pharmacokinetic of [161Tb]Tb-DOTA-N4MU01The pharmacokinetics of [161Tb]Tb-DOTA-N4MU01 was studied in 7-week-old healthy female BALB / c mice (n = 3 / group) as described previously49.Toxicity study of [225Ac]Ac-Macropa-N4MU01 and [161Tb]Tb-DOTA-N4MU01 in mice

[0143] Toxicity studies were conducted on 8-week-old female Balb / C mice (n = 4-5 per group) from Charles Rivers. The toxicity of [225Ac]Ac-Macropa-N4MU01 was investigated at a dose of 15 kBq. Acute (Day 2) and delayed (Day 10) toxicity was studied after a single injection of the radiopharmaceutical while chronic (Day 20) toxicity was studied after repeated dosage (two injections, 10 days apart). Similarly, the toxicity of [161Tb]Tb-Macropa-N4MU01was investigated at a dose of 5 MBq. Acute (Day 2) toxicity was studied after a single injection of the radiopharmaceutical while chronic (Day 14) and sub-chronic (Day 28) toxicity was studied after repeated dosage (two injections, 7 days apart). Mice were monitored for body weight and behavioral changes, with was saline used as the vehicle for tail vein injection. The control group consisted of mice treated with saline and sacrificed on Day 2. On the day of sacrifice, cardiac puncture was performed to collect blood for hematological and clinical chemistry analysis, followed by mouse sacrifice and harvesting of organs. Harvested organs (kidneys, spleen, liver, heart, lungs) were stored in 10% formaldehyde, and subsequently processed for histopathological examination. Throughout the study, all animals were regularly observed for signs of mortality,morbidity, injury, and availability of food and water. Individual body weights were recorded during the quarantine period and every other day during the experimental period.Radioimmunotherapy of [225Ac]Ac-Macropa-N4MU01 and [161Tb]Tb-DOTA-N4MU01 against Nectin-4 positive human TNBC and NSCLC xenografts

[0144] In vivo efficacy studies in for human derived xenografts were done using athymic nude BALB / c nude mice aged 6-8 weeks bearing TNBC MDA-MB-468, and NSCLC NCI-H358 and NCI-H2170 xenografts. All the experiments and euthanasia were performed in accordance with LIACC guidelines. Mice bearing MDA-MB-468, NCI-H358 or NCI-H2170 xenograft were divided into one of 4 groups (n 4 / grou ≥p), namely, [225Ac]Ac-Macropa-N4MU01 (two doses of 13 kBq / dose), saline treatment, and unlabeled N4MU01 at either 100 mg / dose or 1.3 mg / dose. All treated mice received two treatment doses via a tail vein on days 0 and 10.

[0145] Mice bearing MDA-MB-468, NCI-H358 or NCI-H2170 tumors also treated with two doses of 5 MBq / dose of [161Tb]Tb-DOTA-N4MU01. Controls were saline and / or N4MU01 at 25 mg / dose. All treated mice received two treatment doses via a tail vein on days 0 and 7.

[0146] Tumor growth was monitored by measuring the length and width using a digital caliper (tumor volume = (length width2×) / 2). The study was terminated when tumors reached a volume ≥ 1500 mm3. These volumes were used to determine survival in the different groups using Kaplan-Meier curves. The body weight of each mouse was recorded during the experimental period.Priming the tumor microenvironment of Nectin-4 positive tumors using [161Tb]Tb-DOTA- N4MU01 radioimmunoconjugates

[0147] To investigate the priming of the tumor immune microenvironment of TNBC by [161Tb]Tb-DOTA-N4MU01 or [225Ac]Ac-N4MU01 , twenty-four each of 6-week-old BALB / c and C57BL / 6 mice were injected subcutaneously with 2.5 106× cells of 4T1. nectin-4 and E0771. nectin-4 and the xenografts allowed to grow for 5 days to about 50 mm3. For each xenograft model, mice were randomized into 4 groups (n = 5 mice / group) and injected via tail vein with either saline (control, n = 6) or 5 MBq of [161Tb]Tb-DOTA-N4MU01( n = 18) formulated in saline as vehicle. Six mice each of the 18 mice injected with [161Tb]Tb-DOTA-N4MU01 were sacrificed on d1 , d3, and d7, prior to collection of blood by cardiac puncture in heparinized vacutainers. Tumor was then excised from the mice, transferred into labeled 5 mL Eppendorf tubes filled with RPMI1640, and kept on ice for isolation of single cells. Blood plasma was prepared from the collected blood, diluted 2x and allowed to decay at -80 °C in 75 pL aliquots.Decayed blood plasma was then shipped for cytokine / chemokine panel analysis at Eve Technologies Corporation (Calgary, Alberta). Tumor microenvironment cells were isolated using a mouse tumor dissociation kit (Cat # 130-096-730, Miltenyi Biotec) with the gentleMACS™ dissociator (Cat # 130-093-235, Miltenyi Biotec). Isolated tumor microenvironment cells were counted, and the percentage of live cells determined using trypan blue staining on an automated cell counter. Cells were then allowed to decay by freezing at -80 °C in freezing medium which consisted of 50% fetal bovine serum, 40 % RPMI and 10% DMSO. Cells will be stained for changes in immune biomarkers using innate and adaptive immune cell panels (CD45, CD3, CD10.3, NK1.1 , ybTCR, FoxP3, PD1 , CD11b, Gr-1 , CD11c, CD80, CD86, CD206, CD4, and CD8) using flow cytometry / FACS using relevant antibodies.

[0148] In addition, using the rest of the tumor samples, single-cell RNAseq (scRNAseq) of pooled tumor samples from different treatment groups will be conducted using 10x Genomics Chromium iX and NovaSeq 6000 system located at the CHU de Quebec Genomics Facility - Universite Laval. Single-cell suspensions will be processed for 10x Genomics as per the manufacturer’s guidelines.Radioimmunotherapy of [225Ac]Ac-Macropa-N4MU01 and [161Tb]Tb-DOTA-N4MU01 with / without immune checkpoint blockage against Nectin-4 positive mouse syngeneic TNBC allografts

[0149] Effectiveness of [225Ac]Ac-Macropa-N4MU01 and [161Tb]Tb-DOTA-N4MU01 with / without immune checkpoint blockage was studied in immune competent mice bearing Nectin- 4 positive syngeneic allografts. Mice were inoculated using Nectin-4 positive transduced / transfected TNBC 4T1Nectin-4, E0771Nectin-4and NSCLC CMT167Nectin-4and LLCNectin-4. Immune competent mice BALB / c (for 4T1Nectin-4) or C57BL / 6 (for CMT167Nectin-4, E0771Nectin-4and LLCNectin-4) mice aged 5-6-week-old were inoculated subcutaneously with 1.5 - 2.5 × 106cells and the xenografts were allowed to grow to 50-100 mm3prior to initiation of therapy. All the experiments and euthanasia were performed in accordance with UACC guidelines. Tumor bearing mice were treated with [225Ac]Ac-Macropa-N4MU01 (two doses of 13 kBq / dose), saline treatment, unlabeled N4MU01 , or using anti-mouse PD-L-1 antibody (Cat # BE0101, Bio X Cell). Combination effect with anti-PD-L1 was studied in additional group of mice treated using [225Ac]Ac-Macropa-N4MU01 (two doses of 13 kBq / dose on days 0 and 10), followed by injection of anti-mouse PD-L-1 antibody on days 3, 5 and 7 after the initiation of [225Ac]Ac-Macropa- N4MU01 treatment. Mice in any treatment group with complete tumor remissions wererechallenge with same number of cells and allograft regrowth / growth was monitored and compared with naive mice inoculated with the xenografts.

[0150] Similarly, mice bearing 4T1Nectin-4, E0771Nectin-4allografts were treated with [161Tb]Tb-DOTA-N4MU01 (two doses at 5 MBq / dose), saline treatment, unlabeled N4MU01 (25 pg), or anti-mouse PD-L-1 antibody. Combination treatment with anti-PD-L1 was studied in additional group of mice treated with [161Tb]Tb-DOTA-N4MU01 (two doses of 5 MBq / dose on days 0 and 7), followed by injection of anti-mouse PD-L-1 antibody on days 3, 5 and 7 after the initiation of [161Tb]Tb-DOTA-N4MU01 treatment. Mice in any treatment group with complete tumor remissions were rechallenged with same number of cells and allograft regrowth / growth was monitored and compared with naive mice inoculated with the same number of cells xenografts. All treatments except anti-mouse PD-L1 were injected via tail vein on d0 and on d7.Statistical Tests

[0151] Prior to data analysis, data processing was done on Microsoft Excel 2016. Statistical analyses were performed on Prism 10 (GraphPad). Differential tissue uptake and clearance of [161Tb]Tb-DOTA-N4MU01 was tested using Two-way ANOVA with Bonferroni Post- hoc test, while comparison of blood chemistry and CBC parameters between experimented groups for toxicological studies were performed using One-way ANOVA with Bonferroni Post-hoc test. For efficacy studies, unpaired t-test was performed to compare tumor growth inhibition between groups. Kaplan-Meier curves for survival were compared using Log-rank (Mantel-Cox) test. All values are reported as mean ± SD, except otherwise stated.ResultsProduction of [161Tb]Tb-DOTA-N4MU01

[0152] The purity of protein-A affinity-purified anti-human nectin-4 antibody, N4MU01 was confirmed by SEC-HPLC (280 nm) to be > 95% (FIG. 1A). Conjugation of N4MU01 with161Tb- compatible bifunctional chelator, p-SCN-Bn-DOTA produced DOTA-N4MU01 with a SEC-HPLC (280 nm) purity of 97.57 ± 3.43 % (FIG. 1 B), with some small amounts of aggregated conjugates (< 3%). Further investigation to determine the number of p-SCN-Bn-DOTA residues per antibody was carried out by analysing the MALDI-TOF spectrum of DOTA-N4MU01 (FIG. 1 C) in comparison to that of N4MU01 and the molecular mass of p-SCN-Bn-DOTA, with a resulting 2.94 molecules of p-SCN-Bn-DOTA per molecule of N4MU01 .161Tb was chelated with DOTA-N4MU01 to produce [161Tb]Tb-DOTA-N4MU01 (FIG. 1 D) at an Asof 0.2 MBq / ug and with a radiochemical yield (RCY) of 99.01 ± 0.75 % by iTLC (FIG. 1 E).Binding affinity of N4MU01 to human nectin-4 is retained after p-SCN-Bn-DOTA conjugation and 161Tb radiolabeling

[0153] A retention in the binding affinity of Macropa-N4MU01 , DFO-N4MU01 , [225Ac]Ac- Macropa-N4MU01, and [189Zr]Zr-DFO-N4MU01 in comparison with N4MU01 on nectin-4 expressing TNBC cells by either flow cytometry or RLBA was reported50. Similarly, the binding affinities of DOTA-N4MU01 , and [161Tb]Tb-DOTA-N4MU01 were estimated by flow cytometry and RLBA, respectively (FIG. 3, FIG. 4). No significant difference in EC50 values were observed between N4MU01 and DOTA-N4MU01 (p = 0.9953), although the values for DOTA-N4MU01 looked slightly higher (FIG. 3B, Table 1). The least KD and ECso values of DOTA-N4MU01 were observed for E0771.nectin-4cell line, followed by 4T1.nectin-4and then CMT167.nectin-4cell lines, albeit they were similar (Table 1).

[0154] Table 1 : Estimated KD and ECso values of DOTA-N4MU01 on nectin-4 transduced / transfected syngeneic murine TNBC and NSCLC cell lines

[0155] The cross-binding of N4MU01 to mouse nectin-4 protein, in comparison to human nectin-4 protein was investigated by phage ELISA. N4MU01 has strong binding signals to both proteins when compared with background signals. However, the binding signal to human nectin- 4 protein was stronger than that for mouse nectin-4 protein (p = 0.0007) (FIG. 4).

[0156] Specific binding obtained through RLBA confirmed high binding affinity of [161Tb]Tb-DOTA-N4MU01 to nectin-4 -expressing 4T1.nectin-4. This low estimated KD of 10.92 ± 3.54 nM showed that the chelation of 161Tb to DOTA-N4MU01 does not alter its binding to nectin- 4 (FIG. 5).Internalization of N4MU01 and DOTA-N4MU01 on nectin-4 expressing cells

[0157] The anti-tumor activity of161Tb can be greatly attributed to its auger electrons51. Although auger electrons have a high LET compared with beta-particles, their range in tissue isbarely one nucleus diameter. This therefore necessitate the internalization of161Tb-based radiopharmaceuticals to achieve anti-tumor activity. The161Tb vehicle, DOTA-N4MU01 , showed great internalization on both 4T1.nectin-4and E0771.nectin-4cell lines, which was not altered from that of N4MU01 for the same models (p > 0.999 for both models) (FIG. 6).Pharmacokinetics of [161Tb]Tb-DOTA-N4MU01

[0158] Blood pharmacokinetics of [161Tb]Tb-DOTA-N4MU01 in healthy female BALB / c mice showed a biphasic distribution with distribution and elimination half-lives of 2.73 ± 0.97 h and 119.57 ± 20.7 h, respectively. The area under the curve for the first 120 h p.i. was 5475.17 ± 903.28 %IAh / mL (FIG. 7).Biodistribution of [161Tb]Tb-DOTA-N4MU01

[0159] Biodistribution of [161Tb]Tb-DOTA-N4MU01 was studied in both healthy mice and in tumor bearing mice. In healthy female BALB / c mice, tissue distribution was generally observed to decrease or remain stable from 1 h p.i. to 120 h p.i. (FIG. 8A). The highest uptakes were observed in the lungs, spleen, and kidneys, in addition to blood, with %l A / g at 1 h p.i. being 35.86 ± 7.24, 21.01 ± 3.54, 23.48 ± 1.82, and 55.33 ± 2.73, respectively. These initial uptakes cleared off from the tissues overtime except for the spleen where they remained high over 120 h p.i. (FIG. 8A, Table 2). In 4T1.nectin-4tumor-bearing female BALB / c mice, uptake was high for organs such as the liver and spleen. But unlike in healthy mice where spleen uptake was sustained, this was not the case in 4T 1.nectin-4tumor-bearing female BALB / c mice as clearance was observed for the spleen (p < 0.0001), liver (p < 0.0001), and lungs (p = 0.0005) from 24 h p.i. to 120 h p.i. (FIG. 8B). 4T1.nectin-4tumor uptake was equally high though. This uptake dropped from 24 h p.i. (15.26 ± 2.68 %IA / g) to 120 h p.i. (10.62 ± 1.06 %IA / g) (p = 0.0141) (FIG. 8B). A microSPECT / CT image of a female BALB / c mouse bearing 4T1.nectin-4tumor which was injected with two doses of [161Tb]Tb-DOTA-N4MU01at 5 MBq (7 days apart) and imaged on d1 and on d3 p.i. of the second dose shows very high and sustained tumor uptake, with minimal background activity (FIG. 80). To further evaluate nectin-4-expressing tumor uptake of [161Tb]Tb-DOTA-N4MU01 in a different mouse specie, biodistribution was carried out in CMT167.nectin-4tumor-bearing C57BL / 6 mice (FIG. 8D). In this model, tumor uptake was highest of all tissues and was sustained from 24 h p.i. (20.65 ± 2.75 %IA / g) to 168 h p.i. (19.77 ± 1.28 %IA / g) (p > 0.9999), agreeing with microSPECT / CT imaging data. Also observed for this model was a general clearance from tissues over time (FIG. 8D).

[0160] Table 2. Ex vivo biodistribution of [161Tb]Tb-DOTA-N4MU01 in healthy female BALB / c mice (n = 4 / timepoint). Values are presented as mean ± SD.Efficacy of [225Ac]Ac-Macropa-N4MU01 and [161Tb]Tb-DOTA-N4MU01 monotherapies in human cancer cell lines

[0161] To investigate the anti-tumor effect of a combination of [225Ac]Ac-Macropa- N4MU01 or [161Tb]Tb-DOTA-N4MU01 with anti-PD-L1, the mono-therapeutic effects of [225Ac]Ac- Macropa-N4MU01 and [161Tb]Tb-DOTA-N4MU01 in nectin-4 expressing human derived TNBC and NSCLC xenograft were investigated.

[0162] In a TNBC MDA-MB-468 athymic BALB / c nude mouse xenograft model, 2 doses of [225Ac]Ac-Macropa-N4MU01 at either 13 kBq / dose or 18 kBq / dose, administered on d0 and on d10, led to complete tumor remission of 100% of mice by d20 (Fig. 9A). N4MU01 at 1.3 mg / dose and saline control injected on d0 and d10 produced similar effects on tumor growth where no antitumor activity was observed. In both cases, 100% of mice reached tumor volume endpoint by d25 (Fig. 9A). Similarly, Kaplan Meier curve for survival shows that all mice that received [225Ac]Ac- Macropa-N4MU01 survived until the end of study date (d90) in comparison to mice that received saline or N4MU01(Fig. 9B).

[0163] In a NSCLC NCI-H358 athymic BALB / c nude mouse xenograft model, 2 doses of [225Ac]Ac-Macropa-N4MU01 at 13 kBq / dose (n = 6), administered on d0 and on d10, led to great anti-tumor activity compared with saline treated group (n = 4). The difference in average tumor volume on d31 between [225Ac]Ac-Macropa-N4MU01 and saline treated mice was 552 ± 46 mm3( p < 0.0001). At the end of study defined by d50, 100% of mice that received [225Ac]Ac-Macropa- N4MU01 were still alive and with an average tumor volume of 21.4 ± 14.2 mm3. This resulted in prolonged survival compared with saline treated mice (p = 0.0018) (Fig 10A and B).

[0164] In a NSCLC NCI-H2170 athymic BALB / c nude mouse xenograft model, 2 doses of [225Ac]Ac-Macropa-N4MU01 at 13 kBq / dose (n = 8), administered on d0 and on d10, led to partial anti-tumor activity compared with saline treated group (n = 3) (Fig. 11A). Low nectin-4 expression was observed in NCI-H2170 through flow cytometry. The difference in average tumor volume on d34 between [225Ac]Ac-Macropa-N4MU01 and saline treated mice was 898.2 ± 133.5 mm3( p < 0.0001). By d34, 33% (1 / 3) of mice in saline group reached tumor volume endpoint ( ≥ 1500 mm3) while 100% (8 / 8) of mice in the [225Ac]Ac-Macropa-N4MU01 treated group had not reached study endpoint and had a tumor volume < 855 mm3(Fig. 11A). Average body weights over time showed no differences between saline and [225Ac]Ac-Macropa-N4MU01 treated mice, indicating the tolerability of the treatment at 13 kBq / dose at 2 doses (Fig. 11 B).Anti-PD-L1 therapeutic enhancement with [161Tb]Tb-DOTA-N4MU01

[0165] Combination therapeutic studies were investigated in syngeneic mouse models of human nectin-4 transfected 4T1 cells (4T1.nectin-4) and E0771 cells (E0771.nectin-4). These models were chosen because of their varying degree of response to anti-PD-L1 checkpoint inhibition therapy, where reported data concludes that 4T 1 does not respond or responds minimally, while E0771 respond well52’53’54’55.

[0166] In the 4T1.nectin-4female BALB / c syngeneic model, an initial study with a single intravenous dose of [161Tb]Tb-DOTA-N4MU01 or saline on d0 and / or 3 doses of anti-PD-L1 injected intraperitoneally on d3, d5, and d7 was investigated for therapeutic efficacy (FIG. 13A, B, and C). While [161Tb]Tb-DOTA-N4MU01 monotherapy at both 5 MBq and 2.5 MBq showed antitumor efficacy when compared with saline control (p < 0.0001 and p = 0.0021, respectively), anti-PD-L1 monotherapy showed no tumor growth inhibition compared with saline control (p = 0.1166) (FIG. 13A and B) by d16 following start of treatment. Interestingly, 5 MBq of [161Tb]Tb- DOTA-N4MU01 showed superior tumor growth inhibition compared with 2.5 MBq (p = 0.0.0216) and there was no difference in the tumor growth inhibition between monotherapy of 5 MBq of [161Tb]Tb-DOTA-N4MU01 and combination therapy group of 5MBq of [161Tb]Tb-DOTA- N4MU01 together with anti-PD-L1 (p = 0. 1032) (FIG. 13A and B). The median survivals for 5 MBq [161Tb]Tb-DOTA-N4MU01 , 2.5 MBq [161Tb]Tb-DOTA-N4MU01 , 5 MBq [161Tb]Tb-DOTA-N4MU01 + anti-PD-L1, 2.5 MBq [161Tb]Tb-DOTA-N4MU01 with anti-PD-L1, saline with anti-PD-L1, and saline groups were 35, 21 , 26, 24, 24, and 18 days, respectively. Notably, survival was prolongedfor all treatment groups when compared with saline control. While survival was prolonged for the 5 MBq [161Tb]Tb-DOTA-N4MU01 group when compared with 2.5 MBq [161Tb]Tb-DOTA-N4MU01 group (p = 0.0002), this was not the case when compared with 5 MBq [161Tb]Tb-DOTA-N4MU01 + anti-PD-L1 combination group (p = 0.2345). These survival results correlate greatly with tumor growth inhibition (FIG. 13C).

[0167] With the observation that tumor growth was well inhibited for treatment groups until about day 10 before tumor regrowth became rapid, a 2-dose treatment regimen for the [161Tb]Tb- DOTA-N4MU01 at 5 MBq, injected on d0 and on d7 p.i. alone or in combination with anti-PD-L1 (FIG. 13D, E, F, G, H, I, J, and K) was further investigated. Two doses (2x 5MBq) administered on d0 and d7 resulted in significant growth inhibition. There was strong synergistic effect in groups were mice received combination treatment of the radiopharmaceutical with anti-PD-L1 ICI treatment.

[0168] FIGs. 14A-14H show efficacy of [161Tb]Tb-DOTA-N4MU01 with / without anti-PD-L1 in a breast cancer E0771nectin-4model. Combination therapy in E0771.nectin-4syngeneic C57BL / 6 mouse model. All groups that received anti-PD-L1 had three doses (250 pg / mouse per dose) intraperitoneally on d3, d5, and d7 following start of treatment which was defined as injection of saline or 5 MBq of [161Tb]Tb-DOTA-N4MU01 , intravenously. FIG. 14C shows the individual tumor growth curves for the 2x [161Tb]Tb-DOTA-N4MU01 monotherapy group. FIG. 14D shows the individual tumor growth curves for the 2x [161Tb]Tb-DOTA-N4MU01 + anti-PD-L1. FIG. 14E shows the individual tumor growth curves for the saline + anti-PD-L1 group. FIG. 14F shows the individual tumor growth curves for the 2x treatment with N4MU01 (25 pg / mouse per injected). 2x 5 MBq [161Tb]Tb-DOTA-N4MU01 administered on d0 and d7 resulted in significant tumor growth inhibition (p < 0.05) of E0771nectin-4tumors in mice compared with non-treated mice. This effect was more pronounced when 2x 5 MBq [161Tb]Tb-DOTA-N4MU01 treatment was combined with anti-PD-L1 antibody treatment indicative of a synergistic enhancement in effect. It should be noted that E0771nectin-4are sensitive to anti-PD-L1 treatment and there was no significant difference between 2x 5 MBq [161Tb]Tb-DOTA-N4MU01 and anti-PD-L1 treatment monotherapy groups. However, the combination resulted in a synergistic effect of better tumor control. FIG. 14G shows the individual tumor growth curves for saline. FIG. 14H shows a Kaplan Meier survival curve of treatment groups that shows Log-rank (mantel-Cox) test for survival comparison.Therapeutic efficacy of [225Ac]Ac-Macropa-N4MU01 and anti-PD-L1 in syngeneic modelsThe efficacy of [225Ac]Ac-Macropa-N4MU01 monotherapy was investigated in a 4T1.nectin-4syngeneic BALB / c mouse model (n = 5-6 mice / group). Each treatment group received two dosesof the treatment on d0 and on d10. Average tumor growth curves showed complete remission for all mice in [225Ac]Ac-Macropa-N4MU01 treatment group (FIG. 15A). Kaplan-Meier survival curve (FIG. 15B). The study endpoint was considered as tumor volume ≥ 1500 mm3or survival for 30 days.Example 2

[0169] Fully human monoclonal antibodies against nectin-4 (anti-N4) were developed using phage display. The anti-N4 antibody N4-Fab1 (SEQ ID NOs: 62 and 63) was conjugated with p-SCN-Bn-DFO or macropa and radiolabeled with89Zr or225Ac, respectively, for imaging or radiotherapy using TNBC mouse xenograft models. Characterization of binding affinity of chelator-conjugated and radiolabeled antibodies was performed using flow cytometry and radioligand binding assay in Nectin4 expressing cell lines. Pharmacokinetic profile, biodistribution and microPET / CT imaging of89Zr-anti-Nectin4 radioimmunoconjugate (RIC) was studied in mice bearing nectin-4 positive xenografts. Cytotoxicity of the225Ac-anti-N4 antibody was evaluated in MDA-MB-468 cells. Biodistribution of225Ac-anti-N4 RIC was studied in healthy mice, and the therapeutic efficacy was evaluated in mice bearing MDA- MB-468 xenograft. Mice were treated with two doses of 350 nCi or 500 nCi administered at 10 days apart. Tumor growth was monitored using a digital caliper.

[0170] Macropa and DFO conjugated anti-Nectin4 antibodies retained high affinity binding (3nM) to cell lines and appeared to have better binding affinity compared to the radiolabeled antibody (10 nM) which was not expected. The pharmacokinetic profile of89Zr- anti-N4 RIC showed fast distribution half-life t1 / 2a of 3.4 h and a moderate elimination tl / 213. of 63 h. MicroPET / CT imaging and biodistribution of89Zr-anti-N4 in mice bearing MDA-MB-468 xenograft showed high tumor uptake of (14.14 ± 5.1 %IA / g) and (13.2 ± 1.12 %IA / g) injected dose per gram (%l A / g) at 24 h and 120 h post injection (p.i.), respectively. Tumor uptake of89Zr-anti-N4 RIC in BT-474 and MCF-7 xenografts was (13.2 ± 8.2 % lA / g; 16.4 ± 7.0 % lA / g), respectively at 120 h p.i.225Ac-anti-N4 RIC was effectively internalized in MDA-MB-468, and was cytotoxic to the cells with an IC50 of (1.2 kBq / mL). The therapeutic efficacy of225Ac-anti- N4 RIC in TNBC tumor bearing mice for 90 days was evaluated. Treatment with225Ac-anti-N4 (350 nCi or 500 nCi) led to complete tumor remission in all mice in these groups.

[0171] In addition to the MDA-MB-468 cell line that has endogenous expression of nectin- 4, murine cancer cell line 4T1 was transduced to express human nectin-4 (4T1 -nectin-4, syngeneic model). Radiotherapy was studied in mice bearing 4T1 -nectin-4 xenograft afterinjection of 350 nCi (two doses at 10 days apart). All mice treated with vehicle alone reached end point (1500 mm3) after 22 days while 7 / 8 mice treated with 225Ac-anti-N-4 had complete tumor remission, and the remaining 1 / 8 had a tumor volume of 36 mm3.Materials and methodsCell lines and xenografts

[0172] Human breast cancer cell lines MDA-MB-468, MCF-7 and BT-474 that express nectin-4 and MDA-MB-231 known for nectin-4 negative expression were purchased from ATCC (Rockville, MD). MDA-MB-468 and MDA- MB-231 cells were propagated in Leibovitz's L-15 medium (HyClone Laboratories, Logan, UT, USA) supplemented with 10% FBS. MCF-7 cells were cultured in DMEM medium (HyClone Laboratories, Logan, UT, USA), supplemented with 10 % fetal bovine serum (FBS) (Biochrom). BT-474 cells were cultured in ATCC Hybri-Care medium supplemented with 1.5 g / L sodium bicarbonate and 10% FBS. MCF-7 and BT-474 cells were incubated at 37 °C in a humidified atmosphere of 5 % CO2, while MDA-MB-468 and MDA-MB- 231 cells did not require CO2. Female CD-1 nude and athymic nude mice of 4 weeks of age were obtained from Charles River Canada (St-Constant, QC, Canada). Animals used in this study were maintained following the guidelines of the University of Saskatchewan Animal Care Committee (protocol # 20170084 and 20220021). At five weeks of age, CD-1 nude mice were subcutaneously injected at the right flank with 10 × 106MDA-MB-468 cells in 100 pL suspension of a 1:1 mixture of medium and matrigel matrix basement membrane (Discovery Laboware, Inc. Bedford, MA). Athymic nude mice were subcutaneous implanted with 17 beta- Estradiol 1.7 mg / 90 days release pellets, as per manufacturer recommendations (Innovative Research of America, Sarasota, FL), using a 10-gauge precision trochar. Seven days later, the mice were injected with 1.4 × 107MCF- 7 and BT-474 cells in the left and right flanks, respectively. Tumor growth was measured using a digital calliper.Mammalian expression and purification of recombinant nectin-4 domain I

[0173] The DNA sequence encoding IgV-type domain of nectin-4 (amino acids 32-144, Uniprot reference Q96NY8) was amplified from cDNA (GenBank Accession #BC010423) using the primers; AS53-5’-CTTGTCACGAATTCGATAGGTGAGCTGGAGACCTCAG-3’ (SEQ ID NO: 102), AS60-5’-CAGATCTAACCATGGC AGGCACCAGCACTCGGAG-3’ (SEQ ID NO: 103).

[0174] The sequence was cloned into pFUSE-hlgG1-Fc2 (InvivoGen, San Diego, CA, USA) using restriction enzymes Ncol and EcoRI. The recombinant protein for nectin-4 IgV-Fcfusion was expressed and purified using transient transfection of sequence verified plasmids in Expi293F suspension cells (Thermo Fisher Scientific Waltham, MA, USA). Expi293F cells were cultured using Expi293F complete media (Thermo Fisher Scientific Waltham, MA, USA). 75 x 106Expi293F cells were transfected with 30 ug of nectin-4 IgV-Fc plasmid DNA at 37 °C. Cells were harvested and pelleted after five days, and recombinant protein was purified from the supernatant using affinity chromatography (MabSelectSure resin, GE Healthcare Chicago, IL, USA) following manufacturer recommended protocol. The integrity and the purity of the protein was confirmed using SDS-PAGE.Screening naive antibody libraries using phage display

[0175] A naive antibody library previously developed was used

[0021] , This library was panned against the recombinant nectin-4 IgV-Fc fusion protein using previously described protocols

[0022] . Three rounds of selection were performed, and the selection pool was tested for specific binding to nectin-4 IgV-Fc fusion protein. Clonal ELISA was performed using 24 clones isolated from round 3 pool to test for specific binding to the target protein following standard ELISA protocols

[0023] , Clones specific to target recombinant nectin-4 IgV-Fc fusion protein were identified by DNA sequencing.

[0176] Expression of anti-N4 lgG1 antibody N4-Fab1 To express the full-length lgG1 version of nectin-4 antibody, the VL and VH coding sequences were cloned into pFUSE2ss-CLIg- hK and pFUSE2ss-CHIg-hG1 vectors (InvivoGen, San Diego, CA, USA), respectively. The verified clones were transiently transfected into Expi293F cells for secretory expression of full length anti-N4 lgG1 following previously described protocols

[0024] , The purification of the anti-N4 antibody N4-Fab1 (Seq IDs 62 and 63) was performed using MabSelectSure (GE Healthcare, Chicago, IL, USA) affinity resin using manufacturer’s recommended protocols. Bioanalyzer (Agilent 2100 Bioanalyzer using Agilent High Sensitivity Protein 230 Kit, Santa Clara, CA, USA) was used to confirm the purity of the protein following manufacturer's protocol.Characterization of anti-N4 binding using flow cytometry

[0177] In a 96 well non-binding plate, different concentrations of the anti-N4 antibody N4- Fab1 in 1 × PBS of (500 nM, 250 nM, 125 nM, 62 nM, 30 nM, 10 nM and 3 nM) were added to nectin-4 expressing breast cancer cells MDA-MB-468, MCF-7 and nectin-4 negative MDA-MB- 231 cells at 3 x 105cells / well. After 30 min of binding at 4 °C, cells were washed two times and re-suspended in ice-cold 1x PBS. FITC labeled goat F(ab')2 fragment anti-human IgG (H + L) antibody (Beckman Coulter, Brea, CA, USA) was added to cells in (1 in 50) dilution and allowedto bind for 30 min at 4 °C. Cells were washed with ice-cold 1 PBS three × times before reading the plate using CytoFLEX (Beckman Coulter) on the FL1 channel. Flow cytometry data were acquired using CytoFLEX and analyzed using FlowJo (version 10.7.2; FlowJo LLC, Ashland, OR, USA). GraphPad Prism v9 (GraphPad software, San Diego, CA, USA) was used to determine the binding constant (Kd) and half maximal effective concentration (EC50)

[0025] ,Internalization of anti-N4

[0178] In a flat-bottom 96-well plate, nectin-4 expressing MDA-MB-468, MCF-7 and nectin-4 negative MDA-MB-231 cells were seeded and incubated at 37 °C overnight. Twenty-four hours later, anti-N4 antibody N4-Fab1 (Seq IDs 62 and 63) was added with a final concentration of 4 ug / mL to a 3x molar-excess of FabFluor pH red antibody internalization reagent (Essen Bioscience Ann Arbor, Ml, USA) and incubated for 15 minutes at 37 °C. The labeled antibody was then added to the cells, and images were taken at 10x magnification every 2 hours with phase contrast and red fluorescence filters using the Incucyte live-cell imaging system (Essen BioScience Ann Arbor, Ml, USA). The mean red object area (pm2 / well) was calculated using the Incucyte software and was used to quantify antibody internalization.Conjugation with bifunctional chelators, and radiolabeling with 89Zr and 225Ac

[0179] The anti-N4 antibody N4-Fab1 was conjugated with p-SCN-Bn-deferoxamine (DFO) for labeling with89Zr as described previously

[0026] , Quality control of the immunoconjugate was done using SEC HPLC, flow cytometry, and automated electrophoresis (2100 Bioanalyzer, Agilent, Santa Clara, CA, USA). Radiolabeling and purification of DFO-N4 with89Zr was done as reported previously

[0026] , 18-membered macrocyclic bifunctional chelator 6-((16-((6- carboxypyridin-2-yl)methyl)-1 ,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4- isothiocyanatopicolinic acid (macropa-SCN) (p-SCN- macropa) was synthesized as reported by Thiele et al

[0027] , To conjugate the antibody with the chelator, a stock of 20 mg / mL of macropa- SCN was prepared for conjugation reactions. Briefly, 1 mg each of the above antibodies were buffer exchanged with 0.1 M sodium bicarbonate buffer containing 0.15 M sodium chloride using a spin cap column. After buffer exchange, the desired volume of macropa-SCN (15-fold excess) was added to the antibody solution and incubated at 4°C for 18 h. Following incubation, excess unreacted macropa was removed using Amicon Ultra-4 10K centrifugal filters (EMD Millipore, Burlington, MA, USA). Radiolabeling with225Ac was performed as previously reported

[0026] ,

[0180] The stability of89Zr-labeled and225Ac-labeled anti-N4 antibody conjugates at 37 °C was investigated using iTLC by analyzing aliquots of the radiolabeled over time.89Zr-labeledor225Ac-labeled conjugates was mixed with human plasma or 1x PBS solution at a final concentration of 15 MBq / mL and incubated at 37 °C. Aliquots of the solution were taken every 24 h for five days and analyzed for radiochemical purity using iTLC.Radioligand binding assay of 89Zr-anti-N4

[0181] The binding of89Zr-labeled anti-N4 antibody to nectin-4 positive MCF-7 cells was determined using a saturation radioligand binding assay. Briefly, 106cells were incubated with increasing concentrations of89Zr-nectin-4 antibody (0.17 to 375 nmol / L in 100 pL of PBS) for 4 h at 4°C. Non-specific binding was determined in a similar assay using a 10-fold molar excess (3750 nmol / L) of unlabeled anti-nectin-4 antibody. A non-linear regression analysis with one-site - total and non-specific binding equation was used to determine the Kd and Bmax using GraphPad Prism version 9 (La Jolla, CA).MicroPET / CT imaging and biodistribution of 89Zr-anti-N4

[0182] Female CD-1 nude mice (n = 4 / group) bearing nectin-4 positive MDA-MB-468 xenograft and female Balb-C athymic nude mice (n = 4) bearing nectin-4 positive BT-474 and MCF-7 xenografts were injected intravenously using 12 ± 1 MBq (21 - 26 pg)89Zr-anti-N4 antibody. Additionally, mice bearing MDA-MB-468 xenograft (n = 4) were injected intravenously with 900 ug of unlabeled anti-N4 antibody four hours prior to the injection of89Zr- anti-N4 antibody to pre-block nectin-4 receptors. Additionally, to understand uptake of89Zr- anti-N4 in immunocompetent mice murine 4T1 breast cancer cell line were transduced to stably express nectin-4 (4T1 -nectin-4). 4T1-nectin-4 was injected subcutaneously in healthy Balb-C mice to grow xenografts for microPET and radioimmunotherapy studies. MicroPET / CT imaging was performed at 24, 48, 72, 96, and 120 h post-injection using the Vector4CT scanner (Ml Labs B.V., Utrecht). PET scans were acquired in a list- mode data format using a high-energy ultra-high resolution (HE-UHR-1.0 mm) mouse / rat pinhole collimator. Images were reconstructed using PMOD 3.8 software (PMOD, Switzerland). Immediately after imaging, mice were sacrificed at 120 h post injection for biodistribution studies. To complete the biodistribution studies, additional group (n=4) of tumor-bearing mice were sacrificed 24 h post injection. All major organs and blood were harvested and collected in fared tubes and weighted. The radioactivity in all organs and blood were measured using an automated gamma counter (Wallac Wizard 1480, PerkinElmer, Waltham, MA), and expressed as percent injected activity per gram (% I A / g).Pharmacokinetic profile of 89Zr-anti-N4

[0183] Female CD-1 nude mice (n = 4) were injected intravenously with 5 - 6 MBq of89Zr- anti-N4 antibody (10 - 12 pg of antibody). Blood was collected from a saphenous vein in heparinized capillary tubes at different time points (0.5 - 120 h). The height of the capillary tube occupied by blood was determined using a digital caliper. Blood volume in the capillary tube (mL) was measured using V= ππ r2h. The radioactivity in the samples was measured using a gamma counter and expressed as % injected activity / mL (%IA / mL). Pharmacokinetic parameters, including the distribution and elimination half-lives (t1 / 2a and t1 / 2p), volume of distribution at steady-state (Vss), clearance (CL) and volume of the central compartment (V1), were calculated by fitting the blood radioactivity versus time curve to a two-compartment model with i.v. bolus input.In vitro cytotoxicity

[0184] The cytotoxicity (IC50 values) of225Ac-anti-N4 and control immunoconjugates (225Ac-rituximab, non-labeled anti-N4) in nectin-4 positive MDA-MB-468, MCF-7, and nectin-4 negative MDA-MB-231 was determined using IncuCyte Cytotox Red reagent in an IncuCyte S3 live-cell imager (Essen BioScience, AnnArbor, Ml) as previously reported

[0028] , Live-cell images were captured every 2 h using a 10A~ objective lens using phase contrast and fluorescence channel. Images were processed and analyzed using IncuCyte S3 software. The red fluorescent values were generated, and IC50 values for individual compounds were calculated using GraphPad prism v9.Biodistribution and dosimetry of 225Ac-anti-N4

[0185] To estimate radiation dose to healthy tissues, normal BALB / c nude mice (n ≥ 4 / group) were administered 350 nCi of225Ac-anti-N4 antibody via a tail vein and sacrificed at 1, 24, 48, 120, and 264 post injection followed by biodistribution studies. Carcasses were collected and analyzed using a gamma counter and activity was expressed as injected activity per gram organ weight (% lA / g) and % IA. For each organ or tissue, the effective data (non- decay-corrected % lA / g) were plotted against sampling time. This data was used to obtain an estimate of the microcurie-hours per microcurie administered, represented by the area under the time-activity function integrated to infinity (complete decay) of the225Ac. Absorbed doses in units of cGy (centi- gray) per millicurie (cGy / mCi) of225Ac administered were calculated. Mouse organ doses were extrapolated hypothetically to the human and recalculated by recalculating the residence times for the human model from the mouse model using OLINDA1.1 (Hermes Medical Solutions, Montreal QC) [29, 30],225Ac-anti-nectin-4 radioimmunotherapy

[0186] In vivo efficacy studies were done using athymic BALB / c mice (Charles River Laboratories, Hartford, CT) aged 4 - 6 weeks bearing MDA-MB-468 xenograft. Mice were obtained and housed in accordance with University Animal Care Committee (UACC) guidelines (protocol # 20220021). All the experiments and euthanasia were performed in accordance with UACC guidelines. Mice bearing MDA-MB-468 xenograft were divided into 5 groups (n ≥ 4 / group) namely;225Ac-anti-N4 antibody (two doses of 350 nCi / dose),225Ac-anti-N4 antibody (two doses of 500 nCi / dose), PBS treatment, unlabeled anti-N4 antibody, and225Ac-rituximab (an anti-CD20 non- specific control IgG, two doses of 350 nCi / dose). Additionally, mice (n=8 / group) bearing 4T1- nectin-4 xenografts were treated using225Ac-anti-N4 antibody (two doses of 350 nCi / dose) or saline. Treated mice received two doses of treatment via a tail vein on days 0 and 10. Tumor growth was monitored by measuring the greatest length and width using a digital caliper (tumor volume = length wid ×th2 / 2). The MDA-MB-468 xenograft groups were terminated when xenograft reached a volume ≥ 400 mm3(compared with the 1500 mm3endpoint we routinely employ for therapy studies, the ≥ 400 mm3was chosen due to the slow-growing nature of this xenograft), and this was used to determine survival in the different groups using Kaplan Meier curves. For 4T1-nectin-4 xenograft, the study was terminated when tumors reached >= 1500 mm3. The body weight of each mouse was recorded during the quarantine and experimental period.Statistical analysis

[0187] All data were expressed as the mean ± standard deviation of at least three independent experiments. A two- tailed Student's t-test or analysis of variance (ANOVA) with Bonferoni post hoc test were used to assess the statistical significance between the groups. All graphs were prepared and analyzed using GraphPad Prism (version 9; GraphPad, La Jolla, CA).ResultsGeneration and in vitro characterization of anti-nectin-4 antibody

[0188] Panning of naive antibody Fab-phage library on recombinant nectin-4 IgV-Fc protein resulted in three clones that bind specifically to the recombinant protein. N4-Fab1 was selected as the lead clone after ranking by binding affinity in a competitive ELISA. Phage ELISA showed specific binding of N4-Fab1 phage to nectin-4 IgV-Fc and not to negative control Fc fusion proteins. Full-length human lgG1 version of N4- Fab1 was expressed in human origin Expi293F cells by transient transfection and purified using affinity chromatography. The purity and integrityof the purified full length anti-N4 antibody was confirmed using the bioanalyzer and HPLC. In vitro characterization using flow cytometry was performed to determine the binding of anti-N4 antibody to nectin-4 positive MDA-MB-468, MCF-7, and nectin-4 negative MDA-MB-231 cell lines. Dose dependent specific binding was observed with both MDA-MB-468 and MCF-7 and not with MDA- MB-231 cells.

[0189] Table 3: CDRs and Heavy Chain and Light Chains Variable Domains of Fabs 1-3Anti-nectin-4 antibody internalization

[0190] A time-dependent increase in red fluorescence was observed in nectin-4 expressing MDA-MB-468 (high expression) and MCF-7(moderate expression) cell lines, respectively, but not in the negative control MDA-MB- 231 cells or media control at 4 - 48 h post incubation. The anti-N4 antibody showed 72- and 1100- fold higher internalization in nectin-4 positive MDA-MB-468 cells compared with the negative control MDA- MB-231 cells at 24 and 48 h, respectively. In MCF-7 cells, the anti-N4 antibody showed 71- and 365-fold higher internalization compared to the negative control MDA-MB-231 cells.Conjugation and quality control of anti-nectin-4 radioimmunoconjugates

[0191] HPLC analysis showed that the conjugation of p-SCN-DFO to anti-N4 antibody resulted in -100% pure DFO- anti-N4 antibody with no aggregates. Similarly, the conjugation ofmacropa to anti-N4 antibody resulted in 100% pure immunoconjugate with no aggregates. The size and purity of DFO-anti-N4 and macropa-anti-N4 antibodies were evaluated using bioanalyzer. Automated electrophoresis using Bioanalyzer showed that DFO-anti-N4 and macropa-anti-N4 immunoconjugates was 90% and 92.4% pure with molecular weight of 153.2 kDa and 150.3 kDa, respectively (vs 150.2 kDa for unconjugated anti-N4 antibody). This indicates that there were 4.0 DFO and one macropa chelator molecule(s) / antibody molecule.

[0192] In vitro saturation binding assay of unconjugated anti-N4 and DFO-anti-N4 antibody was done using nectin-4 positive MCF-7 cells using flow cytometry. The estimated Kd values for unconjugated anti-N4 antibody and DFO-anti-N4 were 2.9 and 3 nM, respectively. The estimated EC50 values for unconjugated anti-N4 antibody and DFO-anti-N4 were 7.3 and 20.0 nM, respectively. Additionally, a saturation binding assay of unconjugated anti-N4 was done using nectin-4 positive TNBC MDA-MB-468 cells using flow cytometry. This indicated that the conjugation did not affect the binding affinity of the antibody. Similarly, the Kd and EC50 of macropa-anti-N4 antibody were 0.3 and 1.6 nM, respectively.

[0193] The anti-N4 antibodies conjugated with DFO or macropa were labeled with89Zr or 225Ac respectively. The radiochemical yield (RCY) of89Zr-anti-N4 antibody was -100% at a specific activity of 0.5 MBq / pg. Similarly, the RCY of225Ac-anti-N4 was > 95% at a specific activity of 10 kBq / pg. The stability of89Zr-anti-N4 antibody was determined at different time points at 37 °C in PBS and human plasma using iTLC. More than 95% of89Zr- anti-N4 antibody remained intact for 96 h in human serum and for 72 h in PBS, respectively. The saturation radioligand binding assay using89Zr-anti-N4 antibody on MCF7 cells showed dose- dependent increase in specific binding. The estimated Kd of89Zr-anti-N4 antibody was 10 nM which is 4-fold lower affinity (p < 0.05) than naked anti-nectin-4 antibody and 2.9-fold lower affinity (p < 0.05) than the DFO- anti-N4 antibody.Pharmacokinetics, biodistribution and microPET / CT of 89Zr-anti-N 4

[0194] The pharmacokinetic profile of89Zr-anti-N4 injected in CD-1 nude mice are shown in Table 4.

[0196] MicroPET / CT imaging experiments were conducted with89Zr-anti-N4 in mice carrying xenografts of MDA-MB-468, MCF7, BT-474 and 4T1-nectin-4 cells. High tumor uptake was delineated in all nectin-4 positive xenografts as shown by microPET / CT imaging at 24 - 120 h p.i.. There was visibly very low tumor uptake of89Zr-anti-N4 antibody when pre-blocked with unlabeled antibody. Uptake of89Zr-anti-N4 in nectin-4 positive 4T1-nectin-4 xenograft (immunocompetent mice) was similar to those observed in MDA-MB-468, MCF7, BT-474 as delineated by microPET imaging and ex vivo biodistribution studies.

[0197] Biodistribution of89Zr-anti-N4 antibody was performed at 24 and 120 h p.i. in female athymic CD-1 nude mice bearing nectin-4 positive MDA-MB-468 (high expression) xenograft, and at 120 h in BALB / c athymic nude mice bearing nectin-4 positive BT-474 and MCF- 7 (medium expression) xenografts, and in a syngeneic mouse model bearing 4T1-nectin-4 xenograft. To investigate specificity, mice bearing MDA-MB-468 xenografts were pre-blocked using 900 pg of unlabeled anti-N4 antibody 4 h prior to injection of89Zr-anti-N4 antibody. In athymic CD-1 nude mice, tumor uptake of MDA-MB-468 was highest at 24 h p.i. (14.1 ± 5.1 % lA / g) and this decreased slightly (insignificantly, p > 0.05) to 13.2 ± 1.1 %IA / g at 120 h p.i.. When pre-blocked using unlabeled antibody, tumor uptake of MDA-MB-468 decreased significantly (p < 0.0005) to 2.8 ± 1.3 %IA / g at 120 h p.i. There was a slightly high uptake of89Zr-anti-N4 in the lungs and heart (6.0 ± 1.5 % lA / g and 4.7 ± 2.3 % lA / g, respectively) at 24 h p.i., likely reflecting blood pool activity. However, this decreased to 3.4 ± 1.5 %IA / g and 2.7 ± 0.5 %IA / g) at 120 h p.i., respectively. Tumor to blood ratio for MDA-MB-468 was 1.0 and 3.1 at 24 and 120 h p.i., respectively. Tumor to muscle ratio for MDA-MB-468 was 13.2 compared with 1.7 for pre-blocked at 120 h p.i. Tumor uptake in BT-474, MCF-7 and 4T1-nectin-4 xenografts was 13.2 ± 8.2 % lA / g, 16.4 ± 7.0 % I A / g and 11% ± 2.46 % I A / g, respectively, at 120 h p.i. Tumor to blood ratio was 6.9, 8.6 and 5.5 for BT- 474, MCF-7 and 4T1-nectin-4, respectively at 120 h p.i.In vitro cytotoxicity of 225Ac-anti-N4

[0198] Live cell imaging was used to study the in vitro cytotoxicity of unlabeled anti-N4 antibody and225Ac- anti-N4 antibody on MDA-MB-468, MCF7 and MDA-MB-231 cells (Table 5). Phase contrast images showed potent cytotoxicity with225Ac- anti-N4 antibody when compared with unlabeled anti-N4 antibody. Increased cytotoxicity was observed in MDA-MB-468 at 24 h using225Ac- anti-N4 antibody with IC50 of 1.2 kBq / ml whereas unlabeled N4 antibody had no effect on MDA-MB-468 cells. The IC50 values for MCF7 and MDA-MB- 231 cells were consistent with moderate and no expression of nectin-4 respectively.

[0199] Table 5: IC50 values of immunoconjugates in different in nectin-4 expressing TNBC cell lines.Biodistribution of 225Ac-anti-N4

[0200] Biodistribution of225Ac- anti-N4 antibody was studied in healthy BALB / c mice. The uptake of225Ac- anti-N4 antibody was high in the kidney, liver, spleen, lungs, and blood at early time points but this uptake decreased overtime. Ten days post injection, the highest uptake was observed in the spleen (4 ± 1.3 %IA / g), lungs (6 ± 1 ,6%IA / g) and the blood (5.8 ± 0.9 %IA / g) (Table 6).

[0201] Table 6: Biodistribution of 225Ac-anti-N4 antibody in healthy BALB / c mice at different time points post injection expressed as % injected activity per gram (%l A / g) ± SD.Efficacy of 225Ac-anti-N4

[0202] The efficacy of225Ac-anti-N4 RIT, and control225Ac-rituximab in TNBC cell line MDA-MB-468 mouse xenograft model was studied. Tumor growth inhibition was evaluated using a digital caliper. All mice treated with two doses of 350 nCi225Ac-anti-N4 or 500 nCi225Ac-anti-N4 antibody had complete tumor remission by day 22 or 24, respectively. There was no tumor regrowth in the two groups at the end of the study period of 90 days. Two out of four mice treated with two doses of 350 nCi225Ac-rituximab control IgG reached study endpoint (3400 mm3) by day 66 and 78, respectively, while the other two mice reached 200 mm3by day 90. All the mice in PBS group reached the study endpoint of3400 mm3by day 73. Two out of four mice treated with unlabeled N4 antibody reached the study endpoint by day 72 and the rest of the mice reached the study endpoint by day 90. The Kaplan Meier survival curve showed that mice treated with 225Ac-anti-N4 antibody survived for the whole period of the therapy study of 90 days. However, the median survival of the PBS and unlabeled N4 antibody treated mice was 72.5 and 77.5 days, respectively. The median survival for mice treated with225Ac- rituximab was 84 days.

[0203] Two doses of225Ac-anti-N4 administered 10 days apart led to complete tumor remission in 7 / 8 immunocompetent mice bearing 4T1-nectin-4 tumor by day 26, with the remaining 1 / 8 xenograft having a tumor volume of 36 mm3at day 26. In contrast, all 6 saline-treated mice bearing 4T1-nectin-4 tumor reached end point of >= 1500 mm3by day 23 following start of treatment. No regrowth of 4T1-nectin-4 has been observed. Median survival was 21 days for saline-treated 4T1-nectin-4 tumor bearing mice, and had not reached for the225Ac-anti-N4 treated group.

[0204] For clinical application, fully human antibodies are advantageous for therapy for which the concern of immunogenicity is eliminated

[0032] , This study describes the development of a fully human anti-nectin-4 antibody and its evaluation as a PET imaging probe for the early detection of TNBCs and its development as a radioimmunotherapy (RIT) agent. Using phage display, an anti-N4 antibody was developed, which was selected for further evaluation as a PET imaging probe based on its low Kd of 2.9 nM in nectin-4 positive TNBC cell line (MDA-MB-468).89Zr is considered an ideal PET isotope because of its long half-life of 78.4 h and high resolution due to the emission of positrons. To evaluate anti-N4 IgG as immunoPET imaging probe, the antibody was conjugated with p-SCN- Bn-DFO, and the immunoconjugate had similar low nanomolar affinity (3 nM) compared with the unconjugated antibody. As expected, radiolabeling with89Zr resulted in a stable probe. The imaging89Zr-anti-N4 probe was evaluated (microPET and biodistribution studies) using nectin-4 positive TNBC cell lines with high and medium receptor densities. Tumor uptake of99mTc-HYNIC-mAbNectin-4 in nectin-4 positive MDA-MB-468 xenograft has been studied. Maximum tumor uptake in MDA-MB-468 xenograft was 15.32 ± 1.04% ID / g and when pre-blocked was 4.33 ± 0.48% ID / g which is similar to the present results. Additionally, the tumor-to-blood and tumor-to- muscle ratios for the present data are comparable with those for99mTc-HYNIC--mAbNectin-4 tracer. Evaluation of89Zr-AGS-22M6 in tumor bearing mice and cynomolgus monkeys has been done. Tumor uptake of89Zr-AGS- 22M6 in nectin-4 transduced MDA-MB-231 cells (MDA-MB-231-Nectine-4) transduced to express the receptor ranged from an average of 38.8 ± 2.8 % ID / g on day 1 to an average of 39.9 ± 5.9 %l D / g on day 6 with an average high of 45.3 ± 2.4 %l D / g on day 3 compared with non-transduced receptor negative MDA-MB-231-Neo) that ranged from ranged from an average of 16.3 ± 1.8 %ID / g on day 1 to an average of 17.2 ± 1.3 %ID / g on day 6 with an average high of 18.2 ± 2.8 % ID / g on day 2. Similar tumor uptake ratios were found for89Zr-AGS-22M6 in nectin-4 positive patient- derived xenografts (PDX) compared with nectin-4 negative PDX5, In the current study, tumor uptake of89Zr-anti-N4 in MDA-MB-468 decreased from 13.2 %l A / g to 2.8 %IA / g when pre-blocked with cold antibody which is indicative for better specificity compared with89Zr-AGS-22M6 in spite of its apparent lower Kd of 0.01 nM [8], In addition,89Zr-anti-N4 displayed fast distribution half-life t1 / 2a of 3.37 h and a relatively moderate clearance t1 / 2p of 63 h compared with other very slow- clearing IgGs such as trastuzumab which is advantageous for imaging.

[0205] Eighteen-membered ring macrocylic chelator macropa forms a highly stable and inert complex with225Ac. To investigate the antitumor effects of the radioimmunoconjugate, the in vitro cytotoxicity was studied using IncuCyte S3 live-cell imaging with Cytotox Red reagent, that allows for real-time quantification of dead cells.225Ac-anti-N4 displayed enhanced cytotoxicity to nectin-4 expressing cells while the unlabeled N4 antibody was not cytotoxic to nectin-4 expressing TNBC MDA-MB-468. The IC50 of the present radioimmunoconjugate (1.2 kBq / mL) was similar to other highly cytotoxic225Ac-labeled radioimmunoconjugates.

[0206] The therapeutic efficacy of the unlabeled N4 antibody and225Ac-anti-N4 antibody at controlling the growth of the TNBC MDA-MB-468 xenograft or 4T1-nectin-4 syngeneic mouse model were then investigated. For MDA-MB-468 xenograft mice were administered two doses of 350 nCi (low dose) or 500 nCi (high dose) on days 0, and 10, while for 4T1 -nectin-4 syngeneic model mice were only administered two doses of 350 nCi . Interestingly, all mice bearing MDA- MB-468 xenograft treated with both the low and the high dose had complete tumor remission following the administration of only two doses of225Ac-anti-N4. Tumors in mice treated with PBS, unlabeled N4 antibody and225Ac-rituximab controls continued to grow until they reached the study end point of3400 mm3, which was chosen as an end point for the study as MDA-MB-468 xenograft tumor growth is relatively slow

[0034] , compared to other cell lines.

[0207] All mice treated with225Ac-anti-N4 RIT that had complete tumor remission on day 20 with no regrowth until the end of the study (90 days). There was also no apparent toxicity evident from weight gain throughout in the RIT treated group. M-Rabet et al [7] evaluated an anti- nectin-4 ADC in which the antibody is conjugated to monomethyl auristatin E (MMAE) via a valine- citruline cleavable linker in cell line-derived TNBC xenograft SUM190 and several PDX models, where, in contrast to the present results, regrowth of tumors was observed following period of complete regression. Continued regrowth was observed after re-treatment ended.Example 3: Affinity Maturation of Anti-N4 (N4-Fab1)Materials and Methods:Affinity maturation library construction

[0208] The phagemid encoding the parent clone N4-Fab1, was used as a template for site-directed mutagenesis and stop codons were introduced in CDRL3 and CDRH3 using the following oligonucleotides (Integrated DNA Technologies):

[0209] 5’- GTTTATTACTGTCAGCAGTAATAAACTTTTGGCCAGGGG-3’ (CDRL3) (SEQID NO: 104)

[0210] 5’- GTATTACTGTGCAAGATAATAAGGCCAGGGAACCCTG-3’ (CDRH3) (SEQID NO: 105)

[0211] This step eliminates the high prevalence of parent clone in the library, as site- directed mutagenesis usually results in >10% of the library being the unmutated template. Using the CDRH3-stop codon plasmid as a template, two libraries were constructed using the following mutagenic random oligonucleotides (Integrated DNA technologies):

[0212] For library 1 , CDRL1 , CDRL2 and CDRL3 were mutated. For Library 2, CDRH1 , H2 and H3 were mutated. Both libraries were constructed using established protocols as described previously (Fellouse et.al.) and resulted in ~ 3 × 109transformants for each library.Panning of libraries and characterization of individual clones

[0213] Three rounds of library panning were performed using standard protocols. The recombinant protein Nectin-ECD-Fc protein was immobilized at 2pg / ml concentration of NUNCmaxisorp plates and each library was panned separately. After three rounds, 48 clones from each library were picked and characterized by clonal phage ELISA followed by sequencing.Results and DiscussionLibrary Design

[0214] Upon sequence alignment with germline genes in the IMGT repertoire, N4-Fab1 is closely aligned with genes IGKV3-20*2 and IGHV3-33 for VL and VH respectively. The crystal structure PDB 5I1C (representing an antibody derived from IGHV3-33) and 5GGU (representing an antibody derived from IGKV3-20) were used as a guide to ascertain solvent exposed residues in CDRH1 , H2, L1 and L2. Based on these structures, positions 29-32 (IMGT numbering) in CDRL1, positions 50,53 in CDRL2, positions 91-96 in CDRL3, positions 28, 30, 31 in CDRH1, positions 52, 52A, 53, 55, 56, 58 in CDRH2, loop positions (95-101) and flanking residue 103 (mutation from lgHJ4 gene) in CDRH3 were selected for mutagenesis. A soft-randomization scheme was used where nucleotide sequences for the corresponding amino acids were biased with hand-mixed nucleotides where:A was replaced with a mix of (70%A, 10%C, 10%G, 10%T)-Named (N 1), C was replaced with a mix of (10%A, 70%C, 10%G, 10%T)-Named (N2), G was replaced with a mix of (10%A, 10%C, 70%G, 10%T)-Named (N3), T was replaced with a mix of (10%A. 10%C, 10%G, 70%T)-Named (N4).

[0215] This allowed for a bias of -40% for native amino acid found in the parent clone while still allowing for other 19 amino acids to occur in this position in the library of mutants.Library panning and characterization of individual clones

[0216] Both libraries showed enhanced binding to Nectin4-ECD-Fc when compared to control proteins in each round of selection and 48 clones from Round 3 selection were picked and analyzed for specific binding to Nectin-4 in a phage ELISA assay. Positive clones were sequenced and shown below.Table 7: Sequences of specific unique clones from light chain library. Differences from parent clone N4-Fab1-LC are bolded and underlined.

[0217] Table 8. Summary of mutations in light-chain positions (IMGT numbering) compatible with binding to Nectin-4 (first row are parent clone amino acids).

[0218] Positions 38, 56, 107, 115 and 116 are conserved. Position 114 highly favors F, Y over W found in N4-Fab1-LC. Position L67 is a mutation in the framework close to CDRL2 as defined by IMGT (and in CDR-L2 as defined by Kabat) and was randomized.

[0219] Table 9: Sequences of specific unique clones from heavy chain library. Differences from parent clone N4-Fab1-HC are underlined and bolded.

[0220] Table 10. Summary of mutations in heavy-chain positions (IMGT numbering) compatible with binding to Nectin-4 (first row are parent clone amino acids).

[0221] Positions 36, 57, 58, 59, 64, 107, 109, 115 and 118 are conserved. Positions 29, 35, 63, 66, 116 seem to favor amino acids found in the parent clone N4-Fab1-HC. Positions 113 and 114 seem to be highly variable.

[0222] Position H66 (Kabat position 58) is part of CDRH2 in Kabat scheme. Position H 118 is a mutation in the framework close to CDRH3 in IMGT and was randomized.

[0223] Table 11A: Light Chain IMGT Numbering and CDR Annotation for N4-Fab1(SEQ ID NO: 62).

[0224] Table 11B Heavy Chain IMGT Numbering and CDR Annotation for N4-Fab1 (SEQ ID NO: 63).

[0225] The alignment of selected antibodies with respective germline V genes and J genes. Mutations are highlighted and CDR regions are underlined.Pharmacokinetics of fully human anti-Nectin-4 antibody (N4MU01)

[0226] Pharmacokinetic (PK) profile of [89Zr]Zr-DFO-Nectin-4, comprising the fully human anti-nectin-4 antibody described herein, injected in CD-1 nude mice was biphasic with fast distribution half-life t1 / 2a of 3.4 h and a moderate elimination tl / 213. of 63 h. Over 85% of the antibody (% injected activity %IA) was eliminated in the first 10 hours, followed by a slow elimination half-life. The area under the curve AUC (%IA.h / mL), clearance Cl (mL / h) and volume of distribution at steady state Vss (mL / h*102) was 729.4, 0.14 and 9.57, respectively.

[0227] In contrast the PK profile of enfortumab antibody AGS-22ME in humans showed a very slow monophasic half-life. The t1 / 2 of AGS-22ME in humans was 3.6 days

[0040] . 9MW2821 is another anti-Nectin-4 antibody that is currently in clinical development. The preclinical characteristics including pharmacokinetics of enfortumab vedotin and 9MW2821 has been evaluated and it has been shown that the pharmacokinetics of enfortumab vedotin and 9MW2821 are similar and that their monophasic PK is significantly slower than that exhibited by the fully human anti-nectin-4 antibody described herein, N4MU01. As a result of the slower PK and differences in the binding characteristics of enfortumab vedotin and 9MW2821 , non-target tissues uptake are significantly higher particularly in highly vascularized non-target tissues such as the lungs, heart, spleen and liver compared with those of N4MU01. The faster PK of N4MU01 allows for significantly reduced radiation doses to healthy tissues at an optimized dosing to nectin-4 expressing tumors.Example 4

[0228] Tissue radiation absorbed doses serve as a gauge for potential radiopharmaceutical safety. The human radiation effective dose for clinically approved therapeutic [177Lu]Lu-DOTATATE has been reported to range from 0.069 to 0.115 milliGray per megaBecquerel (mGy / MBq) in women (59, 60). Human absorbed doses of [161Tb]Tb-DOTA- N4MU01 for women were projected from healthy female BALB / c mice biodistribution %IA / g data (Fig 18a). The highest radiation absorbed doses were recorded in the spleen (1.54 mGy / MBq),lungs (1.16 mGy / MBq) and kidneys (1.03 mGy / MBq) while effective dose and total-body dose were 0.27 mGy / MBq and 0.06 mGy / MBq, respectively (Fig. 18a).

[0229] To further evaluate the safety of [161Tb]Tb-DOTA-N4MU01 , acute (d2), chronic (d14), and sub-chronic (d28) toxicity studies were performed in healthy female BALB / c mice after a tail vein administration of a single or repeated dose of 5 MBq [161Tb]Tb-DOTA-N4MU01 (study design schematic presented in Fig 18b). Recorded body weights of mice showed general tolerance in all the groups and there were no recorded deaths or loss in body weight greater than 15% of initial body weight (Fig. 18c). An analysis of blood chemistry revealed elevated (compared with saline control (SD2)) creatinine (p = 0.0001) and total protein (p = 0.0430) in the sub-chronic cohort (received 2 injections of 5 MBq, 7 days apart), although the elevated creatinine was within the reference range as published by Charles River (Fig. 18d). No changes of toxicological concern were recorded for blood chemistry. A complete blood count analysis of the sub-chronic group (2D28) compared with saline control revealed a reduced RBC count (p = 0.0031) which was within the reference range and a reduced WBC count (p = 0.0329), which together with that of the saline group were below the lower reference value (Fig 18e). Other recorded changes in blood counts were reduced values for lymphocytes (p = 0.0144), hemoglobin concentration (p = 0.0172) and hematocrit (p = 0.0033), with increased mean corpuscular hemoglobin concentrations (p = 0.0042).

[0230] Major organs (liver, lungs, spleen, heart, and kidneys) from the toxicity study mice were stained with hematoxylin and eosin (H&E) and then scanned at a final resolution of 200x (Fig 18f). Notably and in coherence with no observed decreases in body weights, no pathological findings of severe grade (>2+) were observed for any organ and study group.[161Tb]Tb-DOTA-N4MU01 is highly efficacious against Nectin-4-positive xenografts

[0231] Upon validating the safety profile of 2x 5 MBq dose (administered intravenously on d0 and d7) of [161Tb]Tb-DOTA-N4MU01 , the anti-tumor efficacy of [161Tb]Tb-DOTA-N4MU01 was investigated (Figs. 19A-D). In MDA-MB-468 subcutaneous xenografts in athymic nude mice (n = 7 / group), [161Tb]Tb-DOTA-N4MU01 greatly controlled tumor volume resulting in an average tumor volume of 231.1 ± 97.4 mm3on d97 (end of study) compared with saline control tumor volumes (p = 0.0002 for untreated vs treated on d13) being > 1500 mm3by d20 (Fig. 4a). The average body weight of [161Tb]Tb-DOTA-N4MU01 treated mice decrease slightly from d0 to d25and then increased steadily until d97 (Fig. 4a). 100% of mice in the radioligand treated group survive till study endpoint on 97 days while survival in the saline treated group was 0% on d20. Median survival for mice treated with radioligand was more than 7-fold greater than that for those treated with saline (p = 0.0004).

[0232] The anti-tumor efficacy of 2x 5 MBq [161Tb]Tb-DOTA-N4MU01 against Nectin-4 positive subcutaneous syngeneic allografts 4T1Nectin-4and E0771Nectin-4was investigated (Fig. 19b, c). For the lCI irresponsive 4T1Nectin-4model, administration of 2x 5 MBq [161Tb]Tb-DOTA-N4MU01 on d0 and d7 resulted in 20% (n = 1 / 5 mice) complete response (CR) by day 14. On d16, tumor growth was significantly controlled in radioligand treated mice compared with 25 pg dose (~ 1.25 mg / kg) of N4MU01 (p = 0.0085) and saline groups (p < 0.0001) (Fig. 19b). Median survival was prolonged for [161Tb]Tb-DOTA-N4MU01 treated mice compared with 25 pg N4MU01 dose (p = 0.0227) and saline groups (p = 0.0008) (Fig. 19d). Similarly, in the ICI-responsive E077lNectin-4model, administration of 2x 5 MBq [161Tb]Tb-DOTA-N4MU01 on d0 and d7 resulted in significant tumor growth control by d9 compared with 25 pg of N4MU01 (p = 0.0143) and saline groups (p < 0.0316) (Fig. 19c). Median survival was prolonged for [161Tb]Tb-DOTA-N4MU01 treated mice compared with 25 pg of N4MU01 (p = 0.0004) and saline groups (p = 0.0004) (Fig. 19d) and 25 pg of N4MU01 treatment had no effect on tumor growth (p = 0.3855, d9) and mouse survival (p = 0.2542) (Fig. 19c, d).

[0233] Single administrations of 2.5 MBq and 5 MBq of radioligand showed significantly increased anti-tumor and survival effects compared with saline in the 4T1Nectin-4tumor model. [161Tb]Tb-DOTA-N4MU01 therapy was well tolerated in all models as evidenced by body weights (Fig. 19a).

[0234] The tissue-specific absorbed radiation doses of [161Tb]Tb-DOTA-N4MU01 were estimated using % IA and %IA / g values obtained from naive BALB / c mice following tail vein injection of the radioligand as previously reported (70). %l A / g data were extrapolated to human %IA data for each organ and plotted against sampling time. Residence time of the radioligand for that organ in MBq.h / MBq, represented by the area under the time-activity function curve integrated to infinity (complete decay) of161Tb was determined. The residence times were fitted into OLINDA kinetics model (OLINDA / EXM V2.2, Hermes Medical Solutions, Montreal QC) togenerate absorbed doses in units of milliGray per megaBecquerel (mGy / MBq) of injected [161Tb]Tb.Multiplex analysis of cytokines

[0235] This study used Luminex xMAP technology for multiplexed quantification of 32 Mouse cytokines, chemokines, and growth factors. The multiplexing analysis was performed using the Luminex™ 200 system (Luminex, Austin, TX, USA) by Eve Technologies Corp. (Calgary, Alberta). Thirty-two markers were simultaneously measured in the samples using Eve Technologies' Mouse Cytokine 32-Plex Discovery Assay® (MilliporeSigma, Burlington, Massachusetts, USA) according to the manufacturer's protocol. The 32-plex consisted of Eotaxin, G-CSF, GM-CSF, IFNy, IL-1a, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL- 12(p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1 , M-CSF, MIG, MIP-1a, MIP-ip, MIP-2, RANTES, TNFα, and VEGF. Assay sensitivities of these markers range from 0.3 - 30.6 pg / mL for the 32-plex. Individual analyte sensitivity values are available in the MilliporeSigma MILLIPLEX® MAP protocol. All analytes were assayed in duplicates per sample and the average value used for analysis. In cases where the analyte was below instrument threshold of detection, half the value of the lowest assayed value for that analyte was assigned to the sample.

[0236] Although immune checkpoint inhibitions (ICI) have been successful for other cancers, response rates against TNBC are low. Described herein is a fully human radioimmunotherapeutic [161Tb]Tb-DOTA-N4MU01 against Nectin-4-positive TNBC, which is well tolerated and provides synergistic effects when used with ICI in mouse models. Using syngeneic mouse models changes in peripheral blood cancer-associated cytokines and chemokines at 3-7 days post [161Tb]Tb-DOTA-N4MU01 treatment associated with immune responses were shown. Consequently, [161Tb]Tb-DOTA-N4MU01 synergized with anti-PD-L1 therapy in both ICI-resistant (4T1Nectin-4) and ICI-responsive (E077lNectin-4) syngeneic mouse models and invoked long-term immune changes which resulted in rejection of tumors after mice were rechallenged following complete responses. These findings reveal the potential clinical benefits of [161Tb]Tb-DOTA- N4MU01 in combination with ICI against TNBC.161Tb]Tb-DOTA-N4MU01 induces pro-ICI therapy response immune changes in peripheral blood of Nectin-4+ TNBC mice

[0237] Response rates to ICI therapies remain low in TNBC patients Demonstrated herein is cancer-associated immune changes in peripheral blood (cytokines and chemokines) over time in ICI-irresponsive (4T1 N ectin-4) and ICI-responsive (E0771 Nectin-4) Nectin-4- expressing syngeneic tumor models after the administration of 5.0 MBq of the beta-particle, Auger and conversion electron Nectin-4 binding [161Tb]Tb-DOTA-N4MU01 (Fig. 16 for experimental scheme). In the 4T1 Nectin-4 model, blood levels of interferon gamma (IFN- / , interleukin (IL)-2, IL-10, tumor necrotic factor alpha (TNFα), and vascular endothelial growth factor (VEGF) were highly elevated on day 3 (d3) compared with d1 , d7, and saline control following intravenous injection of 5.0 MBq of [161Tb]Tb-DOTA-N4MU01 (Fig. 16b).

[0238] For the E0771 Nectin-4 model, peripheral blood cytokine pattern of change was slightly different (Fig. 16a, c). Markedly TNFα levels were highest on d7, while VEGF levels were highest and similar for d7 and control group (Cd3) and significantly lowered on d3 (Fig. 16c). Likewise, IFN- / and IL- 10 were most elevated (although not significant) on d1 and just moderately elevated on d3. Notably, granulocyte colony stimulating factor (G-CSF) was moderately to highly elevated in blood in both TNBC tumor models between d1 and d3 but dropped significantly by d7 (Fig. 16b-c). A correlation analysis of the different cytokines and chemokines measured in peripheral blood in the two TNBC models is presented in Figs. 16d, e. IFN- is T-cell activatorand can thus be associated positively with cancer response to immunotherapy, although its’ indiscriminate action on T-cell proliferation means that even regulatory T-cells (Tregs) are equally activated by it. It was observed that IFN- rrelated strongly with IL-2 (r = 0.990, p = 0.01), IL-10(r = 0.862, p = 0.138), MCP-1 (r = 0.981 , p = 0.019), MIP-1α (r = 0.935, p = 0.065), TNFα (r = 0.994, p = 0.006 ), and VEGF (r = 0.833, p = 0.163) in the 4T1 Nectin-4 while a different correlation pattern could be observed in the E0771 Nectin-4 model (Fig. 16d and e). Notably in the E0771 Nectin-4 model, there was a correlation of IFN- w h the leukemia inhibitory factor (LIF: r= -0.955, p = 0.045) and IFN- duced protein-10 (IP-10) (r = -0.804) which is a chemokine thatis secreted by immune cells in response to IFN- (F g. 16e). CXCL10 (IP10) through its bindingto either CXCR3-A or CXCR3-B will either promote tumor migration and growth or exert immunogenic cell death, respectively. Driven by IFN-gamma, CXCL10 through its actions on theCXCR3-CXCL9 / 10 / 11 axis, is a chemoattractant of cytotoxic CXCR3+T cells to the TME where it induces cell lyses. In addition, depending on the presence or absence of Glu-Leu-Arg (ELR) motif, chemokines exert anti / pro angiogenic effects. CXCL10 is an ELR-negative chemokine and exerts antitumor effects through attenuation of angiogenesis. Peripheral blood levels of I P-10 were highest on d7 in the E0771 Nectin-4 model and on d1 in the 4T1 Nectin-4 model (Fig. 16b, c).

[0239] The present TNBC tumor priming data herein suggest a multitude of possible intervention immunotherapy approaches after [161Tb]Tb-DOTA-N4MU01 administration. A suitable day for intervention immunotherapy appears to be d3 especially in the 4T 1 Nectin-4 model as levels of IL-2, IL-10, IFN- VEGF, and TNFα were highest.

[0240] All mice that had complete tumor response were reinoculated with same number of cells at the same site of initial xenograft inoculation at least a month after complete tumor response was recorded. Rechallenged mice (on d44) in the combination group (n = 4) had no tumor regrowth (Fig. 20a, c). A second rechallenge of the same mice a month later (d76) resulted in small regrowth of tumors to a maximum average size of 10.2 mm3 by d91 , which was shortlived and was followed by complete responses in all animals by d95 (Fig. 20c). Similarly, the mouse in the [161Tb]Tb-DOTA-N4MU01 treatment group with complete response was rechallenged once on d76. A maximum regrowth of 13.5 mm3 was recorded on d91 but again, complete response was recoded on d95 (Fig. 20b). Noticeably, all treatment groups had superior anti-tumor activity and improved survivals compared with saline control mice (Fig. 20b, c, d, e, f, g and h).

[0241] The tumor rechallenge study findings prompted investigation of if [161Tb]Tb- DOTA-N4MU01 administration either induced or enhanced long-term immune memory against the recurrence of 4T1 Nectin-4 TNBC in these female BALB / c mice. To investigate this, blood was collected from all 5 mice (4 from combination group and 1 from TRT group) with complete responses at the end of the study period (d97) and analyzed the level of tumor-associated cytokines and chemokines. The results are represented together with those of saline_d3 as control data (Fig. 17a-c). In general, the levels of peripheral blood cytokines and chemokines in the rechallenged mouse from [161Tb]Tb-DOTA-N4MU01 alone treated group were just slightly elevated except for G-CSF and IL-5 which were greay reduced, and IP10 and Eotaxin that werehighly elevated, from control values (Fig. 17a). G-CSF promotes metastasis, tumor migration and an anti-immunogenic microenvironment(61), while IL-5 has been shown to be anti-(62) and pro- tumorigenic(63). Hence, decrease levels of G-CSF and IL-5 compared with baseline control values favor anti-tumor immunity as observed in this cohort. Strikingly, except for Eotaxin and IL- 5, all assayed cytokines and chemokine levels in mice with complete tumor response from the combination treatment group were highly elevated (Fig. 17a, b). These findings (n=5 mice) on Eotaxin and IL-5 in BALB / c are in line with clinical data for TNBC. Also, immune checkpoint blockade has been strongly associated with elevated levels of pro-inflammatory cytokines, including INF- IL-1 , IL-6 and GM-CSF(64, 65). The presence of tumor infiltrating lymphocytes (TILs), and consequently, high levels of peripheral blood cancer-associated cytokines and chemokines correlates with immune memory to cancer(66). A schematic overview of these changes in mice with complete remission in the combination group is presented (Fig. 17c). TNBCs are known to have higher levels of immune cells in the tumor microenvironment than other breast cancers(67). Herein, in mice (n = 4) with complete tumor response (even after tumor rechallenge) after combination treatment with [161 Tb]Tb-DOTA-N4MU01 and anti-PD-L1 (Fig. 17a-c) indicates the potential for clinical benefits for patients who will have complete response.

[0242] To further validate the superior anti-tumor efficacy and improved survival of the combination of 5.0 MBq [161Tb]Tb-DOTA-N4MU01 with anti-PD-L1 observed in the PD1 / PD-L1- insensitive 4T1Nectin-4syngeneic TNBC BALB / c model, similar therapeutic studies were performed in the ICI-responsive E077lNectin-4syngeneic TNBC female C57BL / 6 mouse model (Figs. 21A-H). The anti-tumor efficacy of a combination of [161Tb]Tb-DOTA-N4MU01 with anti-PD-L1 was superior to [161Tb]Tb-DOTA-N4MU01 monotherapy (d19, p = 0.0211) (Fig. 21b, c, g). The combination treatment had a greater prolonged survival compared with saline + anti-PD-L1 treatment (Fig. 21 b, c, d, e, f, g and h). [161Tb]Tb-DOTA-N4MU01 + anti-PD-L1 treatment had better tumor growth inhibition than [161Tb]Tb-DOTA-N4MU01 and a longer median survival than both saline + anti-PD-L1 and [161Tb]Tb-DOTA-N4MU01 (Fig. 32g, h). All mice that had complete tumor response were reinoculated with same number of cells at the same site of initial xenograft inoculation and at least a month after complete remission. Upon rechallenge of mice in both groups (n = 1 / group) on d56, no regrowth was observed until the end of the study on d81 (Fig. 21a, c and d).

[0243] Although immune checkpoint inhibition therapy has proven to be very effective in selected cancer types, benefits remain confined to a small fraction of patients(57, 58). More still, benefits remain dismal for most solid cancers including TNBC and this is mostly attributed to immunologically “cold” TMEs which are deficient of tumor-associated immune cells and or to the presence of cytokines / chemokines and expression of checkpoint proteins(68). Described herein, is 1) the development of an anti-Nectin-4 radioimmunoconjugate ([161Tb]Tb-DOTA-N4MU01), 2) the efficacy of the anti-nectin-4 radioimmunoconjugate, and 3) the TME priming and synergism achieved with anti-PD-L1 , using ICI irresponsive 4T1Nectin-4and ICI responsive E0771Nectin-4syngeneic allograft models.

[0244] A detailed analysis of the safety of [161Tb]Tb-DOTA-N4MU01 revealed a good pharmacological profile with the potential for clinical translation. Notably, the human effective dose for females (Fig 18a). [161Tb]Tb-DOTA-N4MU01 was effective as monotherapy against both human cell line derived xenograft (MDA-MB-468) and ICI-irresponsive and responsive syngeneic mouse models (Fig. 19A-D).

[0245] The priming of 4T1Nectin-4and E0771Nectin-4tumors with 5 MBq [161Tb]Tb-DOTA- N4MU01 revealed changes in peripheral blood tumor-associated cytokines / chemokines from d1 to d7 (Fig. 16A-E). Highest levels of prognostic markers for ICI therapy benefits in TNBC (IL-2, IL-10, and TNF-a)(69) were recorded on d3 in the 4T1Nectin-4model, resulting in synergized therapeutic and survival benefits of saline + anti-PD-L1 and [161Tb]Tb-DOTA-N4MU01 (median survivals: 40 days + 22 days < 96 days; CRs: 0 + 1 < 4) (Fig. 20A-H). Local rechallenge of xenografts for mice with CRs in the 4T1Nectin-4model resulted in tumor rejection in all mice which was associated with highly elevated levels of peripheral blood anti-tumor cytokines recorded on d96 following and compared with cytokine levels on d3 measured after [161Tb]Tb-DOTA-N4MU01 injection. In E0771Nectin-4model, tumor growth was more delayed, and survival was prolonged when compared with [161Tb]Tb-DOTA-N4MU01 and saline + anti-PD-L1 treatments alone. Provided herein is a beta-particle, auger electron, and electron capture emitting radioimmunoconjugate that specifically targets Nectin-4 overexpressing TNBC and it was shown that it is well tolerated and it converts the TME of nectin-4 expressing TNBC from immunologically “cold” to “hot” (in the case of ICI-resistant 4T1 model) or further improves immunity (in the caseof ICI-responsive E0771 model), resulting in enhanced therapeutic benefit and tumor rejection when combined with anti-PD-L1 therapy.Example 5To further validate the superior anti-tumor efficacy and improved survival of the combination of 5.0 MBq [161Tb]Tb-DOTA-N4MU01 with anti-PD-L1 observed in the TNBC BALB / c models, similar therapeutic studies were performed using a Nectin-4 positive non-small cell lung cancer (NSCLC) CMTNectin-4syngeneic model xenografted on C57BL / 6 mice (Figs. 22A-G). The anti-tumor efficacy of a combination of [161Tb]Tb-DOTA-N4MU01 with anti-PD-L1 was superior to [161Tb]Tb-DOTA- N4MU01 monotherapy. The combination treatment had a greater prolonged survival compared with saline + anti-PD-L1 treatment (Figs. 22A-G).). [161Tb]Tb-DOTA-N4MU01 + anti-PD-L1 treatment had better tumor growth inhibition than [161Tb]Tb-DOTA-N4MU01 and a longer median survival than both saline + anti-PD-L1 and [161Tb]Tb-DOTA-N4MU01. Median survival in the saline, anti-PD-L1 and [161Tb]Tb-N4MU01 monotherapy groups were 23, 21 and 30 days, respectively while in the [161Tb]Tb-N4MU01 + ICI group, the study end point was not reached after 37 days of treatment. Study end-point was considered as tumor volume 1000 mm3or ≥ survival for 30 days.Example 6

[0246] A patient with an anti-nectin-4 positive cancer e.g., based on immunohistochemical analysis) is treated with one or more anti-nectin-4 immunoconjugate described herein and one or more PD-1 / PD-L1 inhibitor described herein. The patient is administered an anti-nectin-4 immunoconjugate comprising a radionuclide and subsequently for example 2 days, 3 days or 4 days later, administered one or more PD-1 / PD-L1 inhibitor for example atezolizumab (Tecentriq), avelumab (Bavencio), or ordurvalumab (Imfinzi). The one or more anti-nectin-4 immunoconjugate and the one or more PD-1 / PD-L1 inhibitor are administered as part of a treatment regimen.Example 7

[0247] The mouse models described in Examples 1-5 are tested with N4MU01 labelled with a cytotoxin with and without combination with a PD-1 / PD-L1 inhibitor.

[0248] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0249] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and / or biomarker sequences (e.g. protein and / or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.

[0250] The scope of the claims should not be limited by the preferred embodiments and examples but should be given the broadest interpretation consistent with the description as a whole.REFERENCES1 . Bouleftour W, Guillot A, Magne N. The Anti-Nectin 4: A Promising Tumor Cells Target. A Systematic Review. Mol Cancer Ther 21 , 493-501 (2022).2. Takano A, et al. Identification of nectin-4 oncoprotein as a diagnostic and therapeutic target for lung cancer. Cancer Res 69, 6694-6703 (2009).3. Chatterjee S, Sinha S, Kundu CN. Nectin cell adhesion molecule-4 (NECTIN-4): A potential target for cancer therapy. Eur J Pharmacol 911 , 174516 (2021).4. Challita-Eid PM, et al. Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models. Cancer Res 76, 3003- 3013 (2016).5. M MR, et al. Nectin-4: a new prognostic biomarker for efficient therapeutic targeting of primary and metastatic triple-negative breast cancer. 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Claims

CLAIMS1. A method of treating cancer, the method comprising administering to a subject in need thereof, 1) one or more anti-nectin-4 immunoconjugate comprising an anti-nectin-4 antibody and one or more therapeutic agent, and 2) one or more Programmed Death-1 (PD-1) / Programmed Death Ligand 1 (PD-L1) inhibitor.

2. The method of claim 1 , wherein the one or more therapeutic agent comprises or is a radionuclide.

3. The method of claim 1 or 2, wherein the one or more therapeutic agent comprises or is a radionuclide and a cytotoxin.

4. The method of any one of claims 1 to 3, wherein the radionuclide comprise or is an a emitting radiopharmaceutical or a β- / Auger electron emitting radionuclide.

5. The method of any one of claims 1 to 4, wherein the radionuclide comprise or is161Tb or 225Ac.

6. The method of any one of claims 1 to 5, wherein the radionuclide comprise or is161Tb.

7. The method of any one of claims 1 to 6, wherein the one or more PD-1 / PD-L1 inhibitor is one or more antibody, optionally a PD-L1 antibody.

8. The method of any one of claims 1 to 7, wherein the cancer is breast cancer.

9. The method of any one of claims 1 to 8, wherein the cancer is triple negative breast cancer.

10. The method of any one of claims 1 to 9, wherein the subject is a human.

11. The method of any one of claims 1 to 10, wherein one or more dose of the one or more anti-nectin-4 immunoconjugate is administered prior to one or more dose of the one or more PD-1 / PD-L1 inhibitor.

12. The method of any one of claims 1 to 11 , wherein at least one, a plurality or each of the one or more dose of the one or more anti-nectin-4 immunoconjugates is administered at least about a day, about 2 days, about 3 days or about 4 days prior to at least one, a plurality or each of the one or more PD-1 / PD-L1 inhibitor.

13. The method of any one of claims 1 to 11 , wherein a plurality of the one or more dose of the one or more anti-nectin-4 immunoconjugates is administered at least about a day, about 2 days, about 3 days or about 4 days prior to a plurality of the one or more PD- 1 / PD-L1 inhibitor.

14. The method of any one of claims 1 to 11 , wherein each of the one or more dose of the one or more anti-nectin-4 immunoconjugates is administered at least about a day, about 2 days, about 3 days or about 4 days prior to each of the one or more PD-1 / PD-L1 inhibitor.

15. The method of any one of claims 1 to 11 , wherein at least one of the one or more dose of the one or more anti-nectin-4 immunoconjugates is administered at least about a day, about 2 days, about 3 days or about 4 days prior to at least one of the one or more PD- 1 / PD-L1 inhibitor.

16. The method of any one of claims 1 to 11, wherein at least two doses of the one or more anti-nectin-4 immunoconjugate is administered to the subject, optionally wherein each of the at least two doses of the one or more anti-nectin-4 immunoconjugate is administered on different days and / or wherein a first dose of the at least two doses is administered 7 days prior to a second dose of the at least two doses.

17. The method of any one of claims 1 to 14, wherein the one or more PD-1 / PD-L1 is administered in a plurality of doses, optionally at least 3 doses, optionally wherein each of the plurality of doses of the one or more PD-1 / PD-L1 is administered to the subject on different days and / or wherein each of the plurality of doses is administered 2 days apart.

18. The method of any one of claims 11 to 17, wherein the one or more dose of the one or more PD-1 / PD-L1 inhibitor and / or the one or more anti-nectin-4 immunoconjugate is 4 doses.

19. The method of any one of claims 1 to 18, wherein a first dose of the one or more anti- nectin-4 immunoconjugate is administered to the subject on day 0, and a first dose of the one or more PD-1 / PD-L1 inhibitor is administered to the subject on day 3, optionally wherein a second dose of the one or more PD-1 / PD-L1 inhibitor is administered to the subject on day 5, and a second dose of the one or more anti-nectin-4 immunoconjugate is administered to the subject on day 7, and further optionally wherein a third dose of the one or more PD-1 / PD-L1 inhibitor is administered to the subject on day 7.

20. The method of any one of claims 1 to 19, wherein the anti-nectin-4 antibody is a fully human antibody.

21. A combination treatment comprising 1) one or more anti-nectin-4 immunoconjugate, each immunoconjugate comprising an anti-nectin-4 antibody and a radionuclide and / or a cytotoxin, and 2) one or more PD-1 / PD-L1 inhibitor, optionally wherein the one or moreanti-nectin-4 immunoconjugate is comprised in a composition and / or the one or more PD- 1 / PD-L1 inhibitor is comprised in a composition.

22. A kit comprising a first container comprising one or more anti-nectin-4 immunoconjugate comprising an anti-nectin-4 antibody and one or more therapeutic agent, and a second container comprising one or more PD-1 / PD-L1 inhibitor.

23. The method any one of claims 1 to 20, the combination treatment of claim 21 , or the kit of claim 22, wherein the anti-nectin-4 antibody is an antibody which specifically binds Nectin- 4 comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising complementarity determining regions CDR-L1 , CDR-L2, and CDR-L3, and the heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2, and CDR-H3, wherein the amino acid sequences of said CDRs, as defined using IMGT numbering, are:CDR-L1 QSV X1X2X3Y (SEQ ID NO: 1);CDR-L2 GACDR-L3 QQR X4X5X6PPT (SEQ ID NO: 3);CDR-H1 GF X7F X8SYG (SEQ ID NO: 4);CDR-H2 IWYDG X9NK (SEQ ID NO: 5); andCDR-H3 AR D X10WX11X12G X13Y (SEQ ID NO: 6) wherein, X1is an amino acid selected from S, K, F, and R,X2is an amino acid selected from N, H, A, T, S, and M,X3is an amino acid selected from S, G, and R,X4is an amino acid selected from S, Y, W, and T, X5is an amino acid selected from N, G, D, S, Y, T, H, F, and E, X6is an amino acid selected from W, F, and Y,X7is an amino acid selected from T, A, and N, X8is an amino acid selected from R, M, K, and G,X9is an amino acid selected from S and T,X10is an amino acid selected from D and G;X11is an amino acid selected from T, D, N, A, S, and R; X12is an amino acid selected from N, K, Y, H, R, and D; and / or X13.is an amino acid selected from W, F, and Y; b)CDR-L1 QSVSRY (SEQ ID NO: 68);CDR-L2 DA;CDR-L3 QQRYNWPPD (SEQ ID NO: 70);CDR-H1 GFTFSSYG (SEQ ID NO: 71);CDR-H2 ISYDGSNK (SEQ ID NO: 72); andCDR-H3 AKSTLHSSGWYMDY (SEQ ID NO: 73); or c)CDR-L1 QDISNY (SEQ ID NO: 74);CDR-L2 AA;CDR-L3 QQSYTTRTT (SEQ ID NO: 76);CDR-H1 GFRFSGYP (SEQ ID NO: 77);CDR-H2 IWYDGRNR (SEQ ID NO: 78); andCDR-H3 AKEGKWGEWYFDL (SEQ ID NO: 79).

24. The combination of claim 21 for use in treating a cancer.

25. Use of one or more anti-nectin-4 immunoconjugate comprising an anti-nectin-4 antibody and one or more therapeutic agent as defined in any one of claims 1 to 23, and 2) one or more Programmed Death-1 / Programmed Death Ligand 1 (PD-L1) inhibitor as defined in any one of claims 1 to 23 for treating a cancer.

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