CN116916954A - Methods and compositions for neoadjuvant and adjuvant therapy of urothelial cancer - Google Patents

Abstract

The present application provides methods and compositions for treating urothelial cancer in a patient, for example, by administering a treatment regimen comprising a PD-1 axis binding antagonist (e.g., alemtuzumab) to the patient as a neoadjuvant therapy or adjuvant therapy based on the presence or level of ctDNA in a biological sample obtained from the patient. Also provided are compositions (e.g., PD-1 axis binding antagonists (e.g., alemtuzumab), pharmaceutical compositions thereof, kits thereof, and articles of manufacture thereof) for treating urothelial cancer in a patient.

Description

Methods and compositions for neoadjuvant and adjuvant therapy of urothelial cancer

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application serial No. 63/120,643 filed on 12/2/2020 and U.S. provisional patent application serial No. 63/210,950 filed on 15/6/2021, the entire contents of which are incorporated herein by reference.

Sequence listing

The present application contains a sequence listing that has been electronically submitted in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy was created at 2021, 11/29, and was named 50474_242wo3_sequence_listing_11_29_21_st25, of 9,574 bytes in size.

Technical Field

The present invention relates to methods and compositions for treating Urothelial Cancer (UC) in a patient, for example, by administering to the patient a treatment regimen comprising a PD-1 axis binding antagonist (e.g., atezolizumab).

Background

UC is the most common cancer of the urinary system worldwide. Most cases originate in the bladder. UC can be diagnosed as a non-myogenic, myogenic or metastatic disease, with one third of new cases being diagnosed as myogenic disease (classified according to tumor, lymph node and metastasis (TNM), cT2-T4a Nx M0). Myogenic Invasive UC (MIUC) refers to Myogenic Invasive Bladder Cancer (MIBC) and myogenic invasive Urinary Tract Urothelial Cancer (UTUC). In 2018, there were estimated 549,393 new cases of bladder cancer and 199,922 deaths worldwide. In europe, there are estimated 197,110 new cases of bladder cancer and 64,970 deaths, including 164,450 new cases and 52,930 deaths in 28 member countries of the european union. In the united states, there are estimated 81,400 new cases of bladder cancer and 17,980 deaths in 2020. The median age of patients diagnosed with UC in the united states is 73 years, the highest age at diagnosis among all tumor types.

For MIBC, radical cystectomy and bilateral pelvic lymphadenopathy are the mainstay of treatment. Surgery involves the excision of the bladder, adjacent organs and regional lymph nodes. Surgical methods also have sex differences: for men, the procedure involves removal of the prostate and seminal vesicles; whereas for women, surgery involves removal of the uterus, cervix, ovary and anterior vagina. Urinary diversion is required after bladder removal. The perioperative mortality rate was approximately 2% to 3% when cystectomy was performed in an excellent center.

Despite this surgery, many patients relapse MIBC and they are associated with pain or systemic symptoms such as fatigue, weight loss, anorexia and developmental arrest. About half of MIBC patients will develop localized and/or metastatic recurrence of their disease within 2 years after cystectomy and will eventually die from the disease. For those patients with high risk profile (pT 3-T4a or pn+) who did not receive neoadjuvant chemotherapy (NAC), the overall 5-year survival ranged from 10% to 40%. Despite numerous clinical trial attempts, no adjuvant therapy has so far shown improved MIBC survival.

Thus, there remains a need in the art for improved new adjunctive and adjunctive therapies for UC.

Disclosure of Invention

The invention relates, inter alia, to methods, compositions (e.g., pharmaceutical compositions), uses, kits and articles of manufacture for the adjunctive treatment of UC.

In one aspect, the invention features a method of treating Myometrial Invasive Urothelial Cancer (MIUC) in a patient in need thereof, the method comprising administering to the patient an effective amount of a treatment regimen comprising an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody comprises (a) hypervariable region (HVR) -H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively, wherein the treatment regimen is adjuvant therapy, and wherein the patient has been identified as likely to benefit from the treatment regimen based on the presence of circulating tumor DNA (ctDNA) in a biological sample obtained from the patient.

In another aspect, the invention features a method of treating MIUC in a patient in need thereof, the method comprising: (a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising a PD-L1 antibody; and (b) administering to the patient an effective amount of a treatment regimen comprising a PD-L1 antibody according to the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy, and wherein the anti-PD-L1 antibody comprises (a) HVR-H1, HVR-H2 and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4) and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2 and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7) and QQYLYHPAT (SEQ ID NO: 8), respectively.

In another aspect, the invention features a method of identifying a patient having MIUC who is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody, the method comprising determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample identifies the patient as likely to benefit from treatment with a treatment regimen comprising an anti-PD-L1 antibody, wherein the treatment regimen is adjuvant therapy, and wherein the anti-PD-L1 antibody comprises (a) HVR-H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively.

In another aspect, the invention features a method of selecting a therapy for a patient having MIUC, the method including (a) determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody; and (b) selecting a treatment regimen comprising an anti-PD-L1 antibody based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy, and wherein the anti-PD-L1 antibody comprises (a) HVR-H1, HVR-H2 and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4) and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2 and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7) and QQYLYHPAT (SEQ ID NO: 8), respectively.

In another aspect, the invention features a method of monitoring a response in a patient having MIUC, the patient having been administered at least a first dose of a treatment regimen comprising an anti-PD-L1 antibody, wherein the treatment regimen is neoadjuvant therapy or adjuvant therapy, and wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen, the method comprising determining whether ctDNA is present in a biological sample obtained from the patient at a time point after administration of the first dose of the treatment regimen, thereby monitoring the response in the patient, wherein the anti-PD-L1 antibody comprises (a) HVR-H1, HVR-L2 and HVR-L3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4) and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2 and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7) and QQYLYHPAT (SEQ ID NO: 8), respectively. In some embodiments, the treatment regimen is neoadjuvant therapy. In other embodiments, the treatment regimen is adjuvant therapy.

In another aspect, the invention features a method of identifying a patient having MIUC who is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody, wherein the treatment regimen is neoadjuvant or adjuvant therapy, and the patient has been administered at least a first dose of the treatment regimen, and wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen, the method comprising: determining whether ctDNA is present in a biological sample obtained from the patient at a time point after administration of the first dose of the treatment regimen, wherein the absence of ctDNA in the biological sample at the time point after administration of the treatment regimen identifies the patient as likely to benefit from treatment with a treatment regimen comprising an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody comprises (a) HVR-H1, HVR-H2 and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4) and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2 and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7) and QQYLYHPAT (SEQ ID NO: 8), respectively. In some embodiments, the treatment regimen is neoadjuvant therapy. In other embodiments, the treatment regimen is adjuvant therapy.

In another aspect, the invention features an anti-PD-L1 antibody or a pharmaceutical composition comprising an anti-PD-L1 antibody for use in treating MIUC in a patient in need thereof, wherein the treatment includes administering an effective amount of a treatment regimen comprising an anti-PD-L1 antibody, wherein the treatment regimen is adjuvant therapy, and wherein the patient has been identified as likely to benefit from the treatment regimen based on the presence of ctDNA in a biological sample obtained from the patient, and wherein the anti-PD-L1 antibody comprises (a) HVR-H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively.

In another aspect, the invention features an anti-PD-L1 antibody or a pharmaceutical composition comprising an anti-PD-L1 antibody for use in treating MIUC in a patient in need thereof, the treatment comprising: (a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody; and (b) administering to the patient an effective amount of a treatment regimen comprising an anti-PD-L1 antibody according to the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy, and wherein the anti-PD-L1 antibody comprises (a) HVR-H1, HVR-H2 and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4) and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2 and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7) and QQYLYHPAT (SEQ ID NO: 8), respectively.

In another aspect, the invention features an anti-PD-L1 antibody or a pharmaceutical composition comprising an anti-PD-L1 antibody for use in treating a patient having MIUC, the patient having been administered at least a first dose of a treatment regimen comprising an anti-PD-L1 antibody, wherein the treatment regimen is neoadjuvant therapy or adjuvant therapy, and wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen, wherein the patient's response has been monitored by a method comprising the steps of: whether ctDNA is present in a biological sample obtained from the patient at a time point after administration of the first dose of the treatment regimen, wherein the anti-PD-L1 antibody comprises (a) HVR-H1, HVR-H2 and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4) and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2 and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7) and QQYLYHPAT (SEQ ID NO: 8), respectively. In some embodiments, the treatment regimen is neoadjuvant therapy. In other embodiments, the treatment regimen is adjuvant therapy.

Drawings

Fig. 1A is a schematic diagram showing inclusion criteria for ctDNA Biomarker Evaluable Populations (BEPs) in an IMvigor010 study.

FIGS. 1B and 1C are a series of graphs showing Kaplan-Meier plots comparing patients treated with alemtuzumab (dark gray) with observations (light gray), stratified according to lymph node status, PD-L1 status and tumor stage for probability of disease-free survival (DFS) in ctDNA BEP population (FIG. 1B); and stratification according to lymph node status, PD-L1 status and tumor stage for mid-term probability of total survival (OS) in ctDNA BEP population (fig. 1C). HR, hazard ratio.

Fig. 2A to 2D are a series of graphs showing Kaplan-Meier graphs comparing ctDNA (+) (dark gray) and ctDNA (-) (light gray) states at C1D1 for DFS in the arm of atrazumab (fig. 2A), DFS in the arm of observation (fig. 2B), OS in the arm of atrazumab (fig. 2C), and OS in the arm of observation (fig. 2D). The probability of DFS and the probability of OS are shown on the y-axis. C1D1, cycle 1, day 1.

Fig. 3 is a histogram showing the distribution of duration between C1D1ctDNA (+) test and radiological recurrence for patients within the C1D1ctDNA (+) subgroup.

Fig. 4A and 4B are a series of graphs showing Kaplan-Meier plots of DFS comparing ctDNA (+) patients treated with alemtuzumab with ctDNA (+) patients on the observation arm and comparing ctDNA (-) patients treated with alemtuzumab with ctDNA (-) patients on the observation arm (fig. 4A), and Kaplan-Meier plots of ctDNA (+) patients treated with alemtuzumab with ctDNA (+) patients on the observation arm and comparing ctDNA (-) patients treated with alemtuzumab with ctDNA (-) patients on the observation arm for ctDNA status assessment (fig. 4B). The probability of DFS and the probability of OS are shown on the y-axis.

Fig. 5A and 5B are a series of forest graphs showing DFS (fig. 5A) and OS (fig. 5B) in BEP comparing alemtuzumab with observations in subgroups defined by established prognostic factors. A subset defined by baseline clinical features and tissue immune biomarkers is shown, including lymph node status; staging of the tumor; number of resected lymph nodes; previous neoadjuvant chemotherapy; PD-L1 status by histoimmunohistochemistry (IHC); TMB status by tissue Whole Exome Sequencing (WES); and transcriptome characteristics including tGE3, TBRS, angiogenesis and TCGA subtypes. Forest plots show HR of recurrence or death estimated using univariate Cox proportional hazards model, and 95% confidence intervals for HR are represented by horizontal bars.

Fig. 5C is a bar graph showing the association of baseline prognostic factors with ctDNA (-) status (light gray) and ctDNA (+) status (dark gray), wherein lymph node positive patients were enriched for ctDNA positive status (lymph node positive patients were 47.5% ctDNA positive and lymph node negative patients were 25.2% ctDNA positive).

Fig. 6A and 6B are a series of forest charts showing alemtuzumab versus DFS under observation for ctDNA (+) patients (fig. 6A) and ctDNA (-) patients (fig. 6B). A subset defined by baseline clinical features and tissue immune biomarkers is shown, including lymph node status; staging of the tumor; number of resected lymph nodes; new adjuvant chemotherapy in the past; organizing the PD-L1 status of IHC; organizing TMB states of WES; and transcriptome characteristics including tGE3, TBRS, angiogenesis and TCGA subtypes. Forest plots HR of death estimated using univariate Cox proportional hazards models, and 95% confidence intervals for HR are represented by horizontal bars.

Fig. 7A and 7B are a series of forest charts showing alemtuzumab versus OS under observation for ctDNA (+) patients (fig. 7A) and ctDNA (-) patients (fig. 7B). A subset defined by baseline clinical features and tissue immune biomarkers is shown, including lymph node status; staging of the tumor; number of resected lymph nodes; new adjuvant chemotherapy in the past; organizing the PD-L1 status of IHC; organizing TMB states of WES; and transcriptome characteristics including tGE3, TBRS, angiogenesis and TCGA subtypes. Forest plots HR of death estimated using univariate Cox proportional hazards models, and 95% confidence intervals for HR are represented by horizontal bars.

Fig. 8A to 8H are a series of charts showing Kaplan-Meier diagrams for TMB or PD-L1 subgroups. Fig. 8A and 8C are a series of graphs showing Kaplan-Meier plots for patients with TMB (+) and on the atrazumab arm, TMB (+) and on the observation arm, TMB (-) and on the atrazumab arm, and TMB (-) and on the observation arm for DFS in all ctDNA BEP patients (fig. 8A) and OS in all ctDNA BEP patients (fig. 8C). FIGS. 8B and 8D are a series of graphs showing Kaplan-Meier graphs for patients with TMB (+)/high and on the Ab arm, TMB (+)/high and on the view arm, TMB (-)/low and on the Ab arm, and TMB (-)/low and on the view arm for DFS in ctDNA (+) patients (FIG. 8B) and OS in ctDNA (+) patients (FIG. 8D). TMB was measured by WES. FIGS. 8E and 8G are a series of graphs showing the Kaplan-Meier graphs for patients with PD-L1 (+) and on the Ab arm, PD-L1 (+) and on the observation arm, PD-L1 (-) and on the Ab arm, and PD-L1 (-) and on the observation arm for DFS in all ctDNA BEP patients (FIG. 8E) and OS in all ctDNA BEP patients (FIG. 8G). FIGS. 8F and 8H are a series of graphs showing Kaplan-Meier plots for patients on PD-L1 (+)/high and on the Ab arm, PD-L1 (+)/high and on the observation arm, PD-L1 (-)/low and on the Ab arm, and PD-L1 (-)/low and on the observation arm for DFS in ctDNA (+) patients (FIG. 8F) and OS in ctDNA (+) patients (FIG. 8H). TMB, tumor mutational burden. PD-L1 IC expressed as PD-L1 on tumor infiltrating Immune Cells (ICs) by IHC.

FIGS. 9A and 9B are a series of graphs showing Kaplan-Meier graphs for DFS in patients with ctDNA (-) and TMB (+) in the arm and the observation arm of alemtuzumab and DFS in patients with ctDNA (-) and TMB (-) in the arm and the observation arm of alemtuzumab (FIG. 9A); and Kaplan-Meier plots for OS in patients with ctDNA (-) and TMB (+) in the arm and OS in patients with ctDNA (-) and TMB (-) in the arm and the arm (fig. 9B).

FIGS. 10A and 10B are a series of graphs showing Kaplan-Meier graphs for DFS in patients with ctDNA (-) and PD-L1 (+) in the arm and observation arm of alemtuzumab and DFS in patients with ctDNA (-) and PD-L1 (-) in the arm and observation of alemtuzumab (FIG. 10A); and Kaplan-Meier plots for OS in patients with ctDNA (-) and PD-L1 (+) in the arm and OS in patients with ctDNA (-) and PD-L1 (-) in the arm and arm (fig. 10B).

Fig. 11A to 11D are a series of graphs showing Kaplan-Meier graphs comparing ctDNA (+) (dark gray) with ctDNA (-) (light gray) states at C3D1 for DFS in the arm of alemtuzumab (fig. 11A), OS in the arm of alemtuzumab (fig. 11B), DFS in the arm of observation (fig. 11C), and OS in the arm of observation (fig. 11D).

FIG. 12A is a graph showing the ratio of patients (Pos > Neg; cleared) who converted ctDNA (-) at C1D1 to ctDNA (+) at C3D1 versus those who maintained ctDNA (+) at C3D1 for the alemtuzumab arm and the observation arm. C3D1, 3 rd cycle, 1 st day; pos, ctDNA (+); neg, ctDNA (-).

FIGS. 12B through 12E are a series of graphs showing Kaplan-Meier graphs showing different ctDNA kinetics from C1D1 to C3D1, including patients with ctDNA clearance at C1D1 (Pos > Neg; deep solid line), patients with ctDNA clearance at C1D1 (Pos > Pos; deep dashed line), patients with ctDNA clearance at C1D1 (C3D 1) (Neg > Neg; light solid line), patients with ctDNA clearance at C1D1 (C3D 1) and ctDNA clearance at C1D1 (C3D 1) (Neg > Pos; light solid line), and patients with ctDNA clearance at C1D1 (C3D 1) for DFS in the arm of Arterzumab (blue) (FIG. 12B), DFS in the arm of Artermtuzumab (FIG. 12D), and OS in the arm of the observation arm (FIG. 12E).

Fig. 12F is a bar graph showing the proportion of ctDNA (+) (dark gray) or ctDNA (-) (light gray) in ABACUS study participants, comparing patients who responded to the alemtuzumab neoadjuvant therapy (complete pathology remission (pCR)/Major Pathology Remission (MPR), left) to non-responding patients (non-responders, right). Pre-treatment and post-treatment time points (x-axis) are shown.

FIG. 12G is a box plot showing ctDNA concentrations (sample MTM/mL) in the participants of the ABACUS study with ctDNA (+) and ctDNA (-) comparing patients (pCR/MPR, left) who responded to the alemtuzumab neoadjuvant therapy with those who did not (non-responders, right). Pre-treatment and post-treatment time points (x-axis) are shown. The sample sizes of the box plot are n=17, 15, 23, and 15, respectively, from left to right. The box plot depicts the median at the midline, the lower and upper edges at the first and third quartiles, respectively, whisker lines showing a minimum to maximum of not more than 1.5 times the quartile spacing, and the remaining outlier data points plotted separately.

Fig. 12H is a bar graph showing the fraction of ctDNA (+) patients with ctDNA clearance (dark gray) or no clearance (light gray) by the time point after treatment, comparing patients who responded to the alemtuzumab neoadjuvant therapy (pCR/MPR, left) with those who did not (non-responders, right).

Fig. 13A is a scatter plot showing ctDNA concentration measured by average tumor molecule number per mL of sample of plasma (sample MTM/mL) versus DFS in months. Solid dots indicate events and open dots indicate deletions. The observation arm ctDNA (+) patient is shown.

FIG. 13B is a Kaplan-Meier graph showing DFS in patients with high ctDNA concentrations (dark gray; greater than or equal to median sample MTM/mL (i.e., sample MTM/mL. Gtoreq. Median)) and low ctDNA concentrations (light gray; less than median sample MTM/mL (i.e., sample MTM/mL < median)). The observation arm ctDNA (+) patient is shown.

Fig. 13C is a forest graph showing the segmentation of DFS in patients with high and low ctDNA levels for sample MTM/mL using different quantile thresholds, including 10%, 25%, 50% (median), 75% and 90%. The observation arm ctDNA (+) patient is shown. Forest plots show HR of recurrence or death estimated using univariate Cox proportional hazards model, and 95% confidence intervals for HR are represented by horizontal bars.

FIG. 13D is a scatter plot showing OS (x-axis) in months versus ctDNA concentration measured by sample MTM/mL. Solid dots indicate events and open dots indicate deletions. The observation arm ctDNA (+) patient is shown.

FIG. 13E is a Kaplan-Meier graph showing OS in patients with high ctDNA concentrations (dark gray; greater than or equal to median sample MTM/mL (i.e., sample MTM/mL. Gtoreq. Median)) and low ctDNA concentrations (light gray; less than median sample MTM/mL (i.e., sample MTM/mL < median)). The observation arm ctDNA (+) patient is shown.

FIG. 13F is a forest graph showing OS in patients with high and low ctDNA concentrations using different quantile thresholds (including 10%, 25%, 50% (median), 75% and 90%) to split ctDNA samples MTM/mL. The observation arm ctDNA (+) patient is shown. Forest plots show HR of recurrence or death estimated using univariate Cox proportional hazards model, and 95% confidence intervals for HR are represented by horizontal bars.

Fig. 14A is a bar graph showing the percentage of C1D1ctDNA (+) patients with reduced ctDNA in the alemtuzumab arms (dark gray) and the observation arms (light gray) by C3D 1. The decrease in C1D1ctDNA (+) patients in C1/C3 BEP was assessed and defined as the decrease in sample MTM/mL from C1 to C3.

Fig. 14B-14E are a series of Kaplan-Meier graphs showing that patients with reduced ctDNA ("reduced" (reduced; dark gray) are compared to patients with increased ctDNA levels ("non-reduced" (increased; light gray)) for DFS in the atrazumab arm (fig. 14B), DFS in the observation arm (fig. 14C), OS in the atrazumab arm (fig. 14D), and OS in the observation arm (fig. 14E). The decrease in C1D1ctDNA (+) patients in C1/C3 BEP was assessed and defined as the decrease in sample MTM/mL from C1D1 to C3D 1.

FIG. 15A is a Kaplan-Meier graph showing DFS, where ctDNA reduction is divided into patients with ctDNA clearance ("reduced and cleared"; dark gray, solid line) and patients with reduced but no ctDNA clearance ("reduced but no clear"; dark gray, dashed line). Patients with an increase in ctDNA ("increase"; light grey, solid line) are also shown.

Fig. 15B is a forest graph showing DFS, patients with ctDNA reduction (from clearance (-100% change) to minimal change in ctDNA (< 0% change)) were compared using different thresholds for percent change in sample MTM/mL, including-100% change (reduced and cleared versus reduced but not cleared), -50% change, -25% change, and-10% change). Note that the scale of the percentage change ranges from-100% (clear) to infinity, with negative values indicating a decrease and positive values indicating an increase.

FIG. 15C is a Kaplan-Meier diagram showing OS, where ctDNA reduction is divided into patients with ctDNA clearance ("reduced and cleared"; dark gray, solid line) and patients with reduced but no ctDNA clearance ("reduced but no clear"; dark gray, dashed line). Patients with an increase in ctDNA ("increase"; light grey, solid line) are also shown.

Fig. 15D is a forest graph showing OS, patients with ctDNA reduction (from clearance (-100% change) to minimal change in ctDNA (< 0% change)) were compared using different thresholds for percent change in sample MTM/mL, including-100% change (reduced and cleared versus reduced but not cleared), -50% change, -25% change, and-10% change). Note that the scale of the percentage change ranges from-100% (clear) to infinity, with negative values indicating a decrease and positive values indicating an increase.

Fig. 16A is a scatter plot showing ctDNA concentration (C1D 1 sample MTM/mL) versus C1D1 acquisition time (days post-surgery) in Myometrial Invasive Bladder Cancer (MIBC) patients.

Fig. 16B is a box plot showing C1D1 acquisition times (y-axis, days post-surgery) for ctDNA negative (x-axis, left box plot, n=339) and ctDNA positive (x-axis, right box plot, n=199) MIBC patients. There was no difference between the time of collection for ctDNA negative patients and ctDNA positive patients (Wilcoxon p=0.18, double sided). The middle line of the box plot is the median, the lower edge and the upper edge correspond to the first and third quartiles, the upper whisker extends from the edge to a maximum no more than 1.5 xIQR from the edge, the lower whisker extends from the edge to a minimum no more than 1.5 xIQR from the edge, and data beyond the ends of the whisker is a separately drawn outlier.

Fig. 16C is a bar graph showing fraction of patients who were ctDNA positive (dark gray filled) for patients with C1D1 collection times less than median collection time (x-axis, left bar graph) and greater than median collection time (x-axis, right bar graph). MIBC patients are shown.

Fig. 16D is a histogram showing the time (days) between surgery and C1D1 for MIBC patients.

Fig. 17A is a confort flow chart showing how patients in the ctDNA biomarker evaluable population (BEP, n=40) are identified from the overall ABACUS study population (n=95).

FIG. 17B is a Kaplan-Meier plot comparing relapse free survival of ctDNA positive patients (light gray) as assessed at baseline (C1D 1) time points prior to neoadjuvant treatment with ctDNA negative patients (dark gray).

FIG. 17C is a Kaplan-Meier plot comparing relapse free survival of ctDNA positive patients (light grey) as assessed at time points after neo-assist with ctDNA negative patients (dark grey).

FIG. 18A is a volcanic plot showing differential gene expression analysis in ctDNA BEP, indicating genes associated with ctDNA positive (ctDNA+) and ctDNA negative (ctDNA-).

FIG. 18B is a graph showing the results of marker gene set enrichment analysis in ctDNA BEP, indicating pathways associated with ctDNA positive (ctDNA+; dark gray) and ctDNA negative (ctDNA-; light gray).

Fig. 18C is a graph showing the results of marker gene set enrichment analysis of ctDNA (+) patients in the arm of atuzumab, demonstrating pathways associated with relapse and non-relapse. DN, down-regulation; EMT, epithelial to mesenchymal transition.

Fig. 18D to 18F are a series of Kaplan-Meier plots showing OS for ctDNA (+) patients in the arm and observation arm of atrazumab in the subgroup defined by immune biomarkers that are responsive to immunotherapy (fig. 18D) and resistant (fig. 18E and 18F). The immunotherapy response biomarker tGE gene expression profile is shown (fig. 18D). Shows the immune biomarker pan-TBRS gene expression profile (fig. 18E), and angiogenic gene expression profile (fig. 18F) that are resistant to immunotherapy. High biomarker expression is indicated by darker shading. Low biomarker expression is indicated by lighter shading.

Figures 19A to 19C are a series of Kaplan-Meier plots showing DFS of ctDNA (+) patients in the arm and observation arm of atrazumab in the subgroup defined by immune biomarkers that are responsive to immunotherapy (figure 19A) and resistant (figures 19B and 19C). The immunotherapy response biomarker tGE gene expression profile is shown (fig. 19A). Shows the immune biomarker pan-TBRS gene expression profile (fig. 19B), and angiogenic gene expression profile (fig. 19C) that are resistant to immunotherapy. High biomarker expression is indicated by darker shading. Low biomarker expression is indicated by lighter shading.

Fig. 19D is a graph showing the results of a marker gene set enrichment analysis of ctdna+ patients in the observation arm, comparing non-relapsers (light grey) with relapsers (dark grey).

FIGS. 20A to 20C are a series of Kaplan-Meier diagrams showing DFS (left) OS (right) for ctDNA (-) patients in the arm of Ab and the observation arm. Transcriptomic features are shown, including tGE (FIG. 20A), pan F-TBRS (FIG. 20B) and angiogenesis (FIG. 20C). High biomarker expression is indicated by darker shading. Low biomarker expression is indicated by lighter shading.

Fig. 21A is a heat map showing that hierarchical clustering in ctDNA biomarker-evaluable populations summarises TCGA subtypes of urothelial cancer. APM, antigen presenting mechanism; ECM, extracellular matrix; IC, tumor infiltrating immune cells; TC, tumor cells.

Fig. 21B to 21E are a series of Kaplan-Meier diagrams showing the OS of the patient in the observation arm and alemtuzumab. The prognostic and/or predictive value of ctDNA status and TCGA subtype in ctDNA BEP was demonstrated for lumen papilla (fig. 21B), lumen infiltration (fig. 21C), lumen (fig. 21D) and basal/squamous (fig. 21E). The ctDNA (-) state and ctDNA (+) state are indicated.

FIG. 21F is a volcanic plot showing differential gene expression analysis in observed (Obs) arm ctDNA (-) patients, demonstrating genes associated with recurrence (left) and non-recurrence (right). ECM, extracellular matrix. IFN, interferon.

FIG. 21G is a graph showing the results of marker gene set enrichment analysis of observation arm (Obs) ctDNA (-) patients, demonstrating pathways associated with relapse and non-relapse.

FIGS. 21H and 21I are a series of bar graphs for ctDNA (-) patients (arm pooled) showing the TCGA subtype distribution (FIG. 21H) sorted by recurrence (left) or non-recurrence (right), and for recurrent patients (arm pooled) showing the fraction of ctDNA (+) (dark gray) and ctDNA (-) (light gray) patients sorted by distant recurrence (left) or local recurrence (right) (FIG. 21I).

FIGS. 22A and 22B are a series of bar graphs showing patient distribution in the TCGA subgroup, comparing between ctDNA (-) and ctDNA (+) populations (FIG. 22A) and between PD-L1 status populations (IC 01 and IC 23) (FIG. 22B).

Fig. 22C to 22H are a series of Kaplan-Meier plots showing the actigizumab of the TCGA subgroup and DFS (fig. 22C to 22F) of ctDNA (+) (dark shading) and ctDNA (-) (light shading) patients in the observation arm, and DFS (fig. 22G) and OS (fig. 22H) in the neuronal TCGA subgroup.

Fig. 23 shows a study protocol of an IMvigor011 phase III, double blind, randomized study of alemtuzumab with placebo as adjuvant therapy in patients with high risk of myometrial invasive bladder cancer who were ctDNA positive after cystectomy. Min, minimum; NAC, neoadjuvant chemotherapy; SOC, standard care; cx, cystectomy; WES, whole exome sequencing.

Detailed Description

The present disclosure provides methods and compositions for the treatment of urothelial cancer. The present invention is based, at least in part, on the following findings: in the prognostic analysis of the IMvigor010 study stage III, baseline ctDNA positivity of urothelial cancer patients receiving adjuvant therapy comprising a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody such as alemtuzumab) was correlated with significantly improved p DFS and OS (see, e.g., example 1). The present invention is also based, at least in part, on the following findings: in the phase III IMvigor010 study and phase II ABACUS study of neoadjuvant alemtuzumab therapy, ctDNA clearance was higher in patients receiving neoadjuvant or adjuvant therapy comprising PD-1 axis binding antagonists than in observations, and clearance was associated with improved DFS and OS (see, e.g., example 1). Thus, the methods and compositions provided herein allow for the identification and treatment of patients who may benefit from neoadjuvant or adjuvant therapy comprising a PD-1 axis binding antagonist (e.g., atrazumab), including patients with MIBC (e.g., high risk MIBC) who are ctDNA positive after surgical excision (e.g., cystectomy). The methods and compositions provided herein also allow monitoring of a patient's response to a neoadjuvant or adjuvant therapy comprising a PD-1 axis binding antagonist.

I. Definition of the definition

As used herein, "circulating tumor DNA" and "ctDNA" refer to DNA of tumor origin that is not associated with cells in the circulatory system. ctDNA is a type of cell-free DNA (cfDNA) that may originate from tumor cells or from Circulating Tumor Cells (CTCs). ctDNA can be found, for example, in the blood stream of a patient or in a biological sample (e.g., blood, serum, plasma, or urine) obtained from a patient. In some embodiments, ctDNA may include abnormal mutations (e.g., patient-specific variants) and/or methylation patterns.

The term "PD-1 axis binding antagonist" refers to a molecule that inhibits interaction of a PD-1 axis binding partner with one or more of its binding partners to eliminate T cell dysfunction resulting from signaling on the PD-1 signaling axis, with the result that T cell function (e.g., proliferation, cytokine production, and/or target cell killing) is restored or enhanced. As used herein, PD-1 axis binding antagonists include PD-L1 binding antagonists, PD-1 binding antagonists, and PD-L2 binding antagonists. In some cases, the PD-1 axis binding antagonist comprises a PD-L1 binding antagonist or a PD-1 binding antagonist. In a preferred aspect, the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1 and/or B7-1). In some cases, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some cases, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from interaction of PD-L1 with one or more of its binding partners (such as PD-1 and/or B7-1). In one case, the PD-L1 binding antagonist reduces a negative co-stimulatory signal mediated by or through signaling by PD-L1 mediated by a cell surface protein expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing the response of an effector to antigen recognition). In some cases, the PD-L1 binding antagonist binds to PD-L1. In some cases, the PD-L1 binding antagonist is an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody). Exemplary anti-PD-L1 antagonist antibodies include Ab, MDX-1105, MEDI4736 (Devalumab), MSB0010718C (aviumab), SHR-1316, CS1001, en Wo Lishan antibody (envafolimab), TQB2450, ZKAB001, LP-002, CX-072, IMC-001, KL-A167, APL-502, ke Xili monoclonal antibody (cosibelimab), lodaplizumab (lodaplimab), FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, RC98, PDL-GEX, KD036, KY1003, YBL-007, and HS-636. In some aspects, the anti-PD-L1 antibody is alemtuzumab, MDX-1105, MEDI4736 (Devaluzumab), or MSB0010718C (avermectin). In a specific aspect, the PD-L1 binding antagonist is MDX-1105. In another specific aspect, the PD-L1 binding antagonist is MEDI4736 (devaluzumab). In another specific aspect, the PD-L1 binding antagonist is MSB0010718C (avilamab). In other aspects, the PD-L1 binding antagonist may be a small molecule, e.g., GS-4224, INCB086550, MAX-10181, INCB090244, CA-170, or ABSK041, which in some cases may be administered orally. Other exemplary PD-L1 binding antagonists include AVA-004, MT-6035, VXM10, LYN192, GB7003 and JS-003. In a preferred aspect, the PD-L1 binding antagonist is alemtuzumab.

The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1 and/or PD-L2). PD-1 (programmed death 1) is also known in the art as "programmed cell death 1", "PDCD1", "CD279" and "SLEB2". An exemplary human PD-1 is shown in UniProtKB/Swiss-Prot accession number Q15116. In some cases, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one case, the PD-1 binding antagonist reduces a negative co-stimulatory signal mediated by or through signaling by PD-1 mediated by a cell surface protein expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some cases, the PD-1 binding antagonist binds to PD-1. In some cases, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist antibody). Exemplary anti-PD-1 antagonist antibodies include nivolumab, pamprouzumab (pembrolizumab), MEDI-0680, PDR001 (Sbarbituzumab), REGN2810 (Simiplimab), BGB-108, palo Li Shan (prolgolimab), carilizumab (camrelizumab), sindi Li Shan (sintilimab), tirilizumab (tisielizumab), terrapu Li Shan (toripalimab), dorlimab (dorlimab), raffinlimab, RETIFAnlimab) and RETIFANLMAB sarface Li Shan antibody (samanlimab), pie An Puli mab (penpulimab), CS1003, HLX10, SCT-I10A, sirolimab (zimbellimab), baterimab (balstilimab), jernomoab (genolimumab), BI 754091, cerilimab (cetrelimimab), YBL-006, BAT1306, HX008, bragg Li Shan antibody (budigalimab), AMG 404, CX-188, JTX-4014, 609A, sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM 388D4, STI-1110, AK-103 and hAb21. In a specific aspect, the PD-1 binding antagonist is MDX-1106 (Nawuzumab). In another specific aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In another specific aspect, the PD-1 binding antagonist is a PD-L2 fusion protein, e.g., AMP-224. In another specific aspect, the PD-1 binding antagonist is MED1-0680. In another specific aspect, the PD-1 binding antagonist is PDR001 (swabber). In another specific aspect, the PD-1 binding antagonist is REGN2810 (cimipn Li Shan antibody). In another specific aspect, the PD-1 binding antagonist is BGB-108. In another specific aspect, the PD-1 binding antagonist is a palo Li Shan antagonist. In another specific aspect, the PD-1 binding antagonist is a karite Li Zhushan antagonist. In another specific aspect, the PD-1 binding antagonist is a syndesmosidic Li Shan antagonist. In another specific aspect, the PD-1 binding antagonist is tirelizumab. In another specific aspect, the PD-1 binding antagonist is terlipressin Li Shan. Other additional exemplary PD-1 binding antagonists include BION-004, CB201, AUNP-012, ADG104, and LBL-006.

The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). PD-L2 (programmed death ligand 2) is also known in the art as "programmed cell death 1 ligand 2", "PDCD1LG2", "CD273", "B7-DC", "Btdc" and "PDL2". An exemplary human PD-L2 is shown in UniProtKB/Swiss-Prot accession number Q9BQ 51. In some cases, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. Exemplary PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signaling resulting from interaction of PD-L2 with one or more of its binding partners (such as PD-1). In one aspect, the PD-L2 binding antagonist reduces a negative co-stimulatory signal mediated by or through signaling by PD-L2 mediated by a cell surface protein expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing the response of an effector to antigen recognition). In some aspects, the PD-L2 binding antagonist binds to PD-L2. In some aspects, the PD-L2 binding antagonist is an immunoadhesin. In other aspects, the PD-L2 binding antagonist is an anti-PD-L2 antagonist antibody.

The terms "programmed death ligand 1" and "PD-L1" refer herein to the native sequence human PD-L1 polypeptide. The native sequence PD-L1 polypeptide is provided under Uniprot accession number Q9NZQ 7. For example, the native sequence PD-L1 may have an amino acid sequence as described in Uniprot accession number Q9NZQ7-1 (isomer 1). In another example, the native sequence PD-L1 may have an amino acid sequence as described in Uniprot accession No. Q9NZQ7-2 (isomer 2). In yet another example, the native sequence PD-L1 may have an amino acid sequence as described in Uniprot accession number Q9NZQ7-3 (isomer 3). PD-L1 is also known in the art as "programmed cell death 1 ligand 1", "PDCD1LG1", "CD274", "B7-H" and "PDL1".

When referring to residues in the variable domain (approximately residues 1-107 of the light chain and 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., kabat et al, sequences of Immunological Interest. 5 th edition, U.S. department of health and public service, national institutes of health, besseda (1991)). When referring to residues in the immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported by Kabat et al, supra). The "EU index as set forth in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

For purposes herein, "alemtuzumab" is an Fc-engineered, humanized, non-glycosylated IgG1 kappa immunoglobulin that binds PD-L1 and comprises the amino acid sequence of SEQ ID NO:1 and SEQ ID NO:2, and a light chain sequence of seq id no. Alemtuzumab comprises a single amino acid substitution (asparagine to alanine) at position 297 on the heavy chain (N297A), using EU numbering of the Fc region amino acid residues, which results in a non-glycosylated antibody that binds minimally to the Fc receptor. Alemtuzumab is also described in the following documents: WHO pharmaceutical information (international pharmaceutical substance non-patent name), proposed INN: listing 112, volume 28, phase 4, 16 days 1 month 2015 (see page 485).

The term "cancer" refers to a disease caused by uncontrolled division of abnormal cells in a part of the body. In one instance, the cancer is urothelial cancer. Cancers may be locally advanced or metastatic. In some cases, the cancer is locally advanced. In other cases, the cancer is metastatic. In some cases, the cancer may be unresectable (e.g., locally advanced or metastatic cancer that is unresectable).

As used herein, "urothelial cancer" and "UC" refer to one type of cancer that commonly occurs in the urinary system, including Myometrial Invasive Bladder Cancer (MIBC) and myometrial invasive Urinary Tract Urothelial Cancer (UTUC). UC is also known in the art as Transitional Cell Carcinoma (TCC).

As used herein, "classification of tumors, lymph nodes, and metastasis" and "TNM classification" refer to the classification of cancer stages described in the united states joint committee for cancer (AJCC) cancer stage manual 7 th edition.

The term "not meeting the conditions for treatment with platinum-based chemotherapy" or "not suitable for treatment with platinum-based chemotherapy" refers to a subject not meeting the conditions for treatment with platinum-based chemotherapy or unsuitable for treatment with a platinum-based chemotherapeutic agent, whether at the discretion of the attending clinician or according to established criteria for platinum-based chemotherapy known in the art. For example, non-compliance with cisplatin treatment conditions may be defined by any of the following criteria: (i) Impaired renal function (glomerular filtration rate (GFR) <60 mL/min); GFR can be assessed by direct measurement (i.e., creatinine clearance or ethylenediamine tetraacetate), and if not available, by serum/plasma creatinine calculation (Cockcroft Gault formula); (ii) Hearing loss of 25dB at two consecutive frequencies (measured by audiometry); (iii) Grade 2 or higher peripheral neuropathy (i.e., sensory changes or paresthesias, including stinging); (iv) ECOG physical ability status is 2.

As used herein, "treatment" includes effective cancer treatment with an effective amount of a therapeutic agent (e.g., a PD-1 axis binding antagonist (e.g., alemtuzumab)) or a combination of therapeutic agents (e.g., a PD-1 axis antagonist and one or more additional therapeutic agents). Treatment herein includes, inter alia, adjuvant therapy, neoadjuvant therapy, non-metastatic cancer therapy (e.g., locally advanced cancer therapy), and metastatic cancer therapy. The treatment may be a first line treatment (e.g., the patient may have not been previously treated or has not received past systemic therapy), or a second line or subsequent treatment. In a preferred example, the treatment is adjuvant therapy. In other preferred examples, the treatment is neoadjuvant therapy.

Herein, "effective amount" refers to the amount of a therapeutic agent (e.g., a PD-1 axis binding antagonist (e.g., alemtuzumab)) or a combination of therapeutic agents (e.g., a PD-1 axis antagonist and one or more additional therapeutic agents) that achieves a therapeutic effect. In some examples, the effective amount of the therapeutic agent or combination of therapeutic agents is to achieve improved total remission rate (ORR), complete Remission (CR), pathological complete remission (pCR), partial Remission (PR), improved survival (e.g., disease-free survival (DFS), disease-specific survival (DSS), distant metastasis-free survival, progression-free survival (PFS), and/or total survival (OS)), improved response Duration (DOR), improved time to functional and quality of life deterioration (QoL), and/or ctDNA clearance. Improvements (e.g., in terms of remission rate (e.g., ORR, CR, and/or PR), survival (e.g., DFS, DSS, no distant metastasis survival, PFS, and/or OS), DOR, time to improved function and QoL deterioration, and/or ctDNA clearance) may be relative to a suitable reference, such as observation or reference treatment (e.g., treatment that does not include a PD-1 axis binding antagonist (e.g., treatment with a placebo)). In some cases, improvement (e.g., in terms of remission rate (e.g., ORR, CR, and/or PR), survival (e.g., DFS, DSS, no distant metastasis survival, PFS, and/or OS), DOR, time to improved performance and QoL deterioration, and/or ctDNA clearance) may be relative to observations.

As used herein, "complete remission" and "CR" refer to the disappearance of all target lesions.

As used herein, "partial remission" and "PR" refer to a reduction in SLD of a target lesion of at least 30% with reference to the sum of baseline longest diameters (SLD) prior to treatment.

As used herein, "total remission rate," "objective remission rate," and "ORR" are interchangeable, referring to the sum of the complete CR rate and PR rate.

As used herein, "disease-free survival" and "DFS" refer to the length of time that a patient survives without cancer recurrence after primary treatment (e.g., surgical excision). In some cases, DFS is defined as the time from randomization to the first occurrence of a DFS event defined as any one of: local (pelvic) recurrence of UC (including soft tissue and regional lymph nodes); urinary tract recurrence of UC (including all pathological stages and malignancy); distal metastasis of UC; or die for any reason.

As used herein, "disease-specific survival" and "DSS" refer to the length of time a patient is not dying from a particular disease (e.g., UC). In some cases, DSS may be defined as the time from randomization to death from UC (e.g., based on a researcher's assessment of cause of death).

As used herein, "no distant metastasis survival" refers to the length of time from the date of diagnosis or the beginning of treatment until the patient is still alive and the cancer does not spread to other parts of the body. In some cases, no distant metastasis survival is defined as the time from randomization to diagnosis of distant (i.e., non-local area) metastasis or death for any reason.

As used herein, "progression free survival" and "PFS" refer to the length of time during and after treatment during which the cancer does not worsen. PFS may include the amount of time a patient has experienced CR or PR, as well as the amount of time a patient has experienced disease stabilization.

As used herein, "total survival" and "OS" refer to the length of time that a patient remains alive from the date of diagnosis or beginning treatment of a disease (e.g., cancer). For example, the OS may be defined as the time from randomization to death for any reason.

As used herein, the terms "response duration" and "DOR" refer to the length of time from recording to tumor response until disease progression or death (whichever occurs first).

As used herein, "time to deterioration of function and QoL" refers to the length of time from the date of diagnosis or the beginning of treatment to deterioration of function or reduction of quality of life. In some cases, the time to function and QoL deterioration is defined as the time from randomization to the date when the patient's score first drops by ≡10 minutes from baseline in the european cancer research and therapy organisation (EORTC) quality of life questionnaire-core 30 (QLQ-C30) body function scale, role function scale and Global Health (GHS)/QoL scale (alone).

As used herein, the term "ctDNA clearance" refers to ctDNA clearance in a patient or patient population that is determined to be ctDNA positive at baseline. In some cases, ctDNA clearance may be defined as the proportion of patients who are ctDNA positive at baseline and ctDNA negative on cycle 3, day 1 or cycle 5, day 1.

As used herein, the term "chemotherapeutic agent" refers to a compound useful in the treatment of cancer (such as urothelial cancer). Examples of chemotherapeutic agents include EGFR inhibitors (including small molecule inhibitors (e.g., erlotinib)Gene tec company/OSI pharmaceutical company); PD 183805 (CI 1033,2-acrylamide, N- [4- [ (3-chloro-4-fluorophenyl) amino group]-7- [3- (4-morpholinyl) propoxy]-6-quinazolinyl]-, dihydrochloride, pfizer inc.); ZD1839, gefitinib (gefitinib)>4- (3 '-chloro-4' -fluoroanilino) -7-methoxy-6- (3- (N-morpholinyl) propoxy) quinazoline, assrileKang (AstraZeneca)); ZM 105180 ((6-amino-4- (3-methylphenyl-amino) -quinazoline, jielikang Co., ltd. (Zeneca)); BIBX-1382 (N8- (3-chloro-4-fluoro-phenyl) -N2- (1-methyl-piperidin-4-yl) -pyrimido [5, 4-d)]Pyrimidine-2, 8-diamine, boilinginvahn (Boehringer Ingelheim)); PKI-166 ((R) -4- [4- [ (1-phenylethyl) amino group ]-1H-pyrrolo [2,3-d]Pyrimidin-6-yl]-phenol); (R) -6- (4-hydroxyphenyl) -4- [ (1-phenylethyl) amino group]-7H-pyrrolo [2,3-d]Pyrimidine); CL-387785 (N- [4- [ (3-bromophenyl) amino)]-6-quinazolinyl]-2-butynamide); EKB-569 (N- [4- [ (3-chloro-4-fluorophenyl) amino group]-3-cyano-7-ethoxy-6-quinolinyl]-4- (dimethylamino) -2-butenamide) (Wyeth), a company; AG1478 (gabbro); AG1571 (SU 5271; part of the company; and dual EGFR/HER2 tyrosine kinase inhibitors such as lapatinib (lapatinib) (-in>GSK572016 or N- [ 3-chloro-4- [ (3-fluorophenyl) methoxy group]Phenyl group]-6- [5- [ [ [ 2-methylsulfonyl) ethyl ]]Amino group]Methyl group]-2-furyl group]-4-quinazolinamine); tyrosine kinase inhibitors (e.g., EGFR inhibitors; small molecule HER2 tyrosine kinase inhibitors such as TAK165 (Takeda), an oral ErbB2 receptor tyrosine kinase selective inhibitor (both of the Jupiter and OSI pharmaceutical), dual HER inhibitors such as EKB-569 (available from Wheater) which preferentially bind EGFR but inhibit cells that overexpress both HER2 and EGFR, PKI-166 (Novartis), pan HER inhibitors such as Kanetinib (canertinib) (CI-1033; french (Pharmacia)), raf-1 inhibitors such as the antisense agent ISIS-5132 (ISIS pharmaceutical), which inhibits Raf-1 signaling, non-HER targeted tyrosine kinase inhibitors such as imatinib mesylate (imatinib mesylate) ((Ci) >Gram smith corporation (Glaxo SmithKline)); multi-targeted tyrosine kinase inhibitors, such as sunitinib (Sunitinib)>The part of the scion company); VEGF receptor tyrosine kinase inhibitors such as Vatalanib (PTK 787/ZK222584, north China/first-come pharmaceutical Co., schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (French Co.); quinazolines, such as PD 153035,4- (3-chloroanilino) oxazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2,3-d]Pyrimidine; curcumin (diferuloylmethane, 4, 5-bis (4-fluoroanilino) phthalimide); tyrosine phosphorylation inhibitors (tyrphostin) containing a nitrothiophene moiety; PD-0183805 (Warner-lambert Co.); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxalines (U.S. patent No. 5,804,396); tyrosine phosphorylation inhibitors (U.S. patent No. 5,804,396); ZD6474 (aslicon corporation); PTK-787 (North China/first come pharmaceutical); pan HER inhibitors such as CI-1033 (gabbro); affinitac (ISIS 3521; isis/Gift corporation (Lilly)); PKI 166 (nowa); GW2016 (glaring smith); CI-1033 (a part of the company; EKB-569 (Hui Corp.); semaxinib (Semaxinib); ZD6474 (aslicon corporation); PTK-787 (North China/first come pharmaceutical); INC-1C11 (Imclone); and rapamycin (sirolimus), ) A) is provided; proteasome inhibitors, such as bortezomib (bortezomib) (-je)>Millennium pharmaceutical company (Millennium pharm); disulfiram; epigallocatechin gallate; salinosporamide A; carfilzomib (carfilzomib); 17-AAG (geldanamycin); radicicol (radicicol); lactate dehydrogenase A (LDH-A); fulvestrant (fulvestrant)>Als (America)Likang Corp.); letrozole (l-letrozole)>North Corp.), finasinate (+.>North Corp); oxaliplatin (+)>Cenofim (Sanofi)); 5-FU (5-fluorouracil); folinic acid; ronafanib (lonafamib) (SCH 66336); sorafenib (sorafenib)>Bayer Labs); AG1478, alkylating agents such as thiotepa and +.>Cyclophosphamide; alkyl sulfonates such as busulfan, imperosulfan (endoprostufan) and piposulfan (piposulfan); aziridines such as benzodopa (benzodopa), carboquinone, meturedepa (meturedopa) and uredopa (uredopa); ethyleneimine and methylmelamine (methyl melamine) including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide and trimethylol melamine; annonaceous acetogenins (especially bullatacin) and bullatacin (bullatacin); camptothecins (including topotecan and irinotecan); bryostatin (bryostatin); calysistatin; CC-1065 (including adozelesin, carbozelesin, and bizelesin synthetic analogs thereof); nostoc (cryptophycin) (in particular, nostoc 1 and nostoc 8); corticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α -reductase, including finasteride (finasteride) and dutasteride (dutasteride); vorinostat (vorinostat), romidepsin (romidepsin), panobinostat (panobinostat), valproic acid, mocetinostat dolastatin; aldi interleukin, talc multicard Mycin (tall duocarmycin) (including synthetic analogs KW-2189 and CB1-TM 1); eleutherobin; a podocarpine (pancratistatin); sarcandbin (sarcandylin), spongostatin (sponagistatin); nitrogen mustards such as chlorambucil, napthalamide, estramustine (estramustine), ifosfamide, dichloromethyldiethylamine, mechlorethamine hydrochloride, melphalan (melphalan), novobiocin (novembichin), benserene cholesterol (phenesterine), prednimustine (prednimustine), qu Luolin amine (trofosfamide), uratemustine (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouremycin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ranimustine (ranimustine); antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1 and calicheamicin ω1); dynemicin, including dynemicin a; bisphosphonates, such as chlorophosphonate; esperamicin (esperamicin); and neocarcinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin (aclacinomycin), actinomycin, amastatin (authamycin), diazoserine, actinomycin C (cactinomycin), carabincin (carminomycin), carminomycin (caminomycin), carcinophilin (carzinophilin), chromomycins (chromomycins), actinomycin D (dactinomycin), ditobacin (detorubicin), 6-diazo-5-oxo-L-norleucine, N-morpholino-doxorubicin (doxorubicin), cyano-N-morpholino-doxorubicin 2- (N-pyrrolinyl) -doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), escorubicin (idarubicin), marcelomycin (marcelomycin), mitomycins such as mitomycin C, mycophenolic acid, norgamycin, olivomycins, pelomycin (peplomycin), methylmitomycin (porfiromycin), puromycin, quelamycin, robobicin (rodobicin), streptocin (strenigrin), streptozocin (streptozocin), tubercidin (tubercidin), ubenimex (zistatin), zorubicin (zorubicin); antimetabolites, such as methotrexate and 5-fluorouracil (5-FU) ) The method comprises the steps of carrying out a first treatment on the surface of the Folic acid analogs such as, for example, dimethyl folic acid (denopterin), methotrexate, ptertrexate (pteroprerin), trimellite (trimellitate); purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thiopurine (thiamiprine), thioguanine; pyrimidine analogs such as ambriseine (ancitabine), azacytidine, 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enoxadine), fluorouridine; androgens such as carbo Lu Gaotong (calasterone), drotasone propionate (dromostanolone propionate), epithioandrol (epiostanol), melandrane (mepistane), testosterone; anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone; aldehyde phosphoramide glycosides; aminolevulinic acid; enuracil (eniluracil); amsacrine (amacrine); bei Labu shake (bestrebicil); bisantrene (bisantrene); edatraxate (edatraxate); difofamin (defofamine); colchicine (demecolcine); deaquinone (diaziquone); efroniornithine (elfornithine); ammonium elide (elliptinium acetate); epothilone (epothilone); ethyleneoxy pyridine (etodolcid); gallium nitrate; hydroxyurea; lentinan; lanidainine; maytansine alkaloids (maytansine), such as maytansine (maytansine) and ansamitocin (ansamitocin); mitoguazone (mitoguazone); mitoxantrone (mitoxantrone); mopidamol; niterine; penstatin (penstatin); egg ammonia nitrogen mustard (phenol); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophylloic acid (podophyllinic acid); 2-ethyl hydrazide; procarbazine; / >Polysaccharide complex (JHS Natural Products); carrying out a process of preparing the raw materials; risperidin (rhizoxin); schizophyllan (sizofuran); germanium spiroamine; tenuazonic acid (tenuazonic acid); triiminoquinone (triaziquone); 2,2',2 "-trichlorotriethylamine; trichothecenes (especially T-2 toxin, wart-bullatacin A (verraCUrin A), verrucella A (roridin A) andserpentine (anguidine)); uratam (urethan); vindesine; dacarbazine; mannitol nitrogen mustard; dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromine (pipobroman); a gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; chlorambucil (chloranil);(gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; etoposide (VP-16); ifosfamide; mitoxantrone; mitoxantrone (Novantrone); teniposide (teniposide), edatraxate (edatrexate); daunomycin; aminopterin; capecitabine (capecitabine)>Ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, prodrugs and derivatives of any of the foregoing.

The chemotherapeutic agent further comprises: (i) Anti-hormonal agents, such as antiestrogens and Selective Estrogen Receptor Modulators (SERMs), including, for example, tamoxifen (includingTamoxifen citrate), raloxifene, droloxifene (droloxifene), iodoxyfene, 4-hydroxy tamoxifen, qu Aoxi-fene (trioxifene), raloxifene hydrochloride (keoxifene), LY117018, onapristone (onapristone) and(tomiphene citrate (toremifine citrate)); (ii) Aromatase inhibitors that inhibit aromatase, which regulates estrogen production of the adrenal glands, such as, for example, 4 (5) -imidazole, aminoglutethimide,/-for example>(megestrol acetate),>(exemestane; pyroxene), formestane (formestanie), method Qu (fadrozole), and +.>(Fu Luo (vorozole)), -, etc.>(letrozole; north Hua Co.) and(anastrozole; assailant Corp.); (iii) Antiandrogens such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin; buserelin (buserelin), triptorelin (tripterlin), medroxyprogesterone acetate, diethylstilbestrol, beclomethasone, fluoxytestosterone, all trans retinoic acid, valatide (fenretinide), and troxacitabine (1, 3-dioxolane nucleoside cytosine analog); (iv) a protein kinase inhibitor; (v) a lipid kinase inhibitor; (vi) Antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways associated with abnormal cell proliferation, such as, for example, PKC- α, ralf, and H-Ras; (vii) Ribozymes, such as inhibitors of VEGF expression (e.g., +. >) And an inhibitor of HER2 expression; (viii) Vaccines, such as gene therapy vaccines, e.g. +.> And(ix) Growth inhibitors, including vinca (e.g., vincristine and vinblastine),/>(vinorelbine), taxanes (e.g., paclitaxel, albumin-bound paclitaxel, and docetaxel), topoisomerase II inhibitors (e.g., doxorubicin, epirubicin, daunomycin, etoposide, and bleomycin), and DNA alkylating agents (e.g., tamoxifen (tamoxigen), prednisone, dacarbazine, dichloromethyl diethylamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C); and (x) pharmaceutically acceptable salts, acids, prodrugs and derivatives of any of the foregoing.

As used herein, the term "cytotoxic agent" refers to any agent that is detrimental to a cell (e.g., causes cell death, inhibits proliferation, or otherwise impedes cell function). Cytotoxic agents include, but are not limited to, radioisotopes (e.g., at ²¹¹ 、I ¹³¹ 、I ¹²⁵ 、Y ⁹⁰ 、Re ¹⁸⁶ 、Re ¹⁸⁸ 、Sm ¹⁵³ 、Bi ²¹² 、P ³² 、Pb ²¹² And a radioisotope of Lu); a chemotherapeutic agent; enzymes and fragments thereof, such as nucleolytic enzymes; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Exemplary cytotoxic agents may be selected from the group consisting of anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormone and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, LDH-a inhibitors, fatty acid biosynthesis inhibitors, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and cancer metabolism inhibitors. In one instance, the cytotoxic agent is a platinum-based chemotherapeutic agent (e.g., carboplatin or cisplatin). In one instance, the cytotoxic agent is an antagonist of EGFR, e.g., N- (3-ethynylphenyl) -6, 7-bis (2-methoxyethoxy) quinazolin-4-amine (e.g., erlotinib). In one instance, the cytotoxic agent is a RAF inhibitor, e.g., a BRAF and/or CRAF inhibitor. In one instance, the RAF inhibitor is Vemurafenib (vemurafenib). In one instance, the cytotoxic agent is a PI3K inhibitor.

Chemotherapeutic agents also include "platinum-based" chemotherapeutic agents that comprise an organic compound containing platinum as part of the molecule. Typically, the platinum-based chemotherapeutic agent is a coordination complex of platinum. Platinum-based chemotherapeutic agents are sometimes referred to in the art as "platinum-based agents". Examples of platinum-based chemotherapeutic agents include, but are not limited to, cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatinum tetranitrate, phenanthriplatin, picoplatin, lipoplatin, and satraplatin. In some cases, the platinum-based chemotherapy may include the administration of a platinum-based chemotherapeutic agent (e.g., cisplatin or carboplatin) in combination with one or more additional chemotherapeutic agents (e.g., nucleoside analogs (e.g., gemcitabine)).

As used herein, "platinum-based chemotherapy" refers to a chemotherapy regimen that includes a platinum-based chemotherapeutic agent. For example, platinum-based chemotherapeutics may include a platinum-based chemotherapeutic agent (e.g., cisplatin or carboplatin) and optionally one or more additional chemotherapeutic agents (e.g., nucleoside analogs (e.g., gemcitabine)) used.

The term "patient" refers to a human patient. For example, the patient may be adult.

The term "antibody" herein specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. In one instance, the antibody is a full length monoclonal antibody.

As used herein, the term IgG "isotype" or "subclass" refers to any subclass of immunoglobulin defined by the chemistry and antigenic characteristics of the immunoglobulin constant region.

Antibodies (immunoglobulins) may be assigned to different classes depending on the amino acid sequence of their heavy chain constant domains. Immunoglobulins are largely divided into five classes: igA, igD, igE, igG and IgM, and some of them can be further divided into subclasses (isotypes), for example, igG1, igG2, igG3, igG4, igA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, γ, ε, γ and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known and are generally described, for example, in the following documents: abbas et al Cellular and mol.immunology, 4 th edition (W.B.Saundrs, co., 2000). The antibody may be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

The terms "full length antibody", "whole antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form rather than an antibody fragment as defined below. The term refers to antibodies comprising an Fc region.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, antibodies produced by the host cell may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise the full-length heavy chain, or the antibody may comprise a cleaved variant of the full-length heavy chain. This may be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447). Thus, the C-terminal lysine (Lys 447) or C-terminal glycine (Gly 446) and lysine (Lys 447) of the Fc region may or may not be present. If not otherwise indicated, the amino acid sequence of the heavy chain comprising the Fc region is herein denoted without the C-terminal lysine (Lys 447). In one aspect, a heavy chain comprising an Fc region as specified herein, comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447), is included in an antibody according to the disclosure herein. In one aspect, a heavy chain comprising an Fc region as specified herein, comprising an additional C-terminal glycine residue (G446), is included in an antibody according to the disclosure herein. In one aspect, a heavy chain comprising an Fc region as specified herein, comprising an additional C-terminal lysine residue (K447), is included in an antibody according to the disclosure herein. In one embodiment, the Fc region contains the single amino acid substitution N297A of the heavy chain. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD, 1991.

"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabeled. The naked antibody may be present in a pharmaceutical composition.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising an antigen-binding region thereof. In some cases, the antibody fragments described herein are antigen binding fragments. Examples of antibody fragments include Fab, fab ', F (ab') ₂ And Fv fragments; a diabody antibody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope except for possible variant antibodies (e.g., containing naturally occurring mutations or occurring during production of monoclonal antibodies, such variants typically being present in minor forms). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the invention can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals comprising all or part of the human immunoglobulin loci.

The term "hypervariable region" or "HVR" as used herein refers to the individual regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, e.g., the "complementarity determining regions" ("CDRs").

Typically, an antibody comprises six CDRs; three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

(a) Highly variable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) (Chothia and Lesk, J.mol. Biol.196:901-917 (1987));

(b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) (Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991)); and

(c) Antigen contact occurs at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al J.mol.biol.262:732-745 (1996)).

The CDRs are determined according to the method described by Kabat et al (supra), unless otherwise indicated. Those skilled in the art will appreciate that CDR names may also be determined according to the methods described by Chothia (supra), mccallium (supra), or any other scientifically accepted naming system.

"framework" or "FR" refers to the variable domain residues other than the Complementarity Determining Regions (CDRs). The FR of the variable domain typically consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, CDR and FR sequences typically occur in VH (or VL) with the following sequences: FR1-CDR-H1 (CDR-L1) -FR2-CDR-H2 (CDR-L2) -FR3-CDR-H3 (CDR-L3) -FR4.

The term "Kabat-described variable domain residue number" or "Kabat-described amino acid position number" and variants thereof refer to the numbering system for heavy chain variable domains or light chain variable domains set forth in the above-mentioned Kabat et al literature. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which correspond to shortening or insertion of FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat numbering) following residue 52 of H2 and an insert residue (e.g., residues 82a, 82b, 82c, etc. according to Kabat numbering) following heavy chain FR residue 82. The Kabat numbering of residues of a given antibody can be determined by aligning the antibody sequences with regions of homology of the "standard" Kabat numbering sequences.

The term "package insert" is used to refer to instructions generally included in commercial packages of therapeutic products that contain information concerning the indication, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

As used herein, "in combination with … …" refers to a treatment regimen that includes administration of a PD-1 axis binding antagonist (e.g., alemtuzumab) and an additional therapeutic agent in addition to one treatment regimen. Thus, "in combination with … …" refers to the administration of one mode of treatment to a patient before, during, or after another mode of treatment.

A drug administered "concurrently" with one or more other drugs is administered within the same treatment cycle, on the same day, and optionally at the same time as the treatment with the one or more other drugs. For example, for cancer therapy administered every 3 weeks, the concurrently administered drugs are each administered on day 1 of the 3 week cycle.

The term "detection" includes any means of detection, including direct detection and indirect detection.

The term "biomarker" as used herein refers to an indicator that is detectable in a sample, e.g., predictive, diagnostic, and/or prognostic, e.g., ctDNA, PD-L1, or tissue tumor mutational burden (tTMB). In some aspects, the biomarker is the presence or level of ctDNA in a biological sample obtained from the patient. Biomarkers can be used as indicators of specific subtypes of a disease or disorder (e.g., cancer) characterized by certain molecular, pathological, histological, and/or clinical features. In some aspects, the biomarker may serve as an indicator of the likelihood of a therapeutic benefit. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), polynucleotide copy number alterations (e.g., DNA copy number), polypeptides, and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and/or glycolipid-based molecular markers.

The "amount" or "level" of a biomarker (e.g., ctDNA) associated with an increased clinical benefit of a patient is a detectable level in a biological sample. These may be measured by methods known to those skilled in the art and disclosed herein. The expression level or amount of the biomarker assessed can be used to determine the response to treatment.

In general, the terms "level of expression" or "expression level" are used interchangeably and generally refer to the amount of a biomarker in a biological sample. "expression" generally refers to the process of converting information (e.g., genetic code and/or epigenetic information) into structures that are present and run in a cell. Thus, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide). Transcribed polynucleotides, translated polypeptides, or fragments of a polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide) are also considered to have been expressed, whether they originate from transcripts generated by alternatively spliced or degraded transcripts, or from post-translational processing of the polypeptide (e.g., by proteolysis). "expressed genes" include those transcribed into polynucleotides such as mRNA and then translated into polypeptides, and also those transcribed into RNA but not translated into polypeptides (e.g., transfer RNA and ribosomal RNA).

"increased expression," "increased expression level," "increased level," "elevated expression level," or "elevated level" refers to increased expression or increased level of a biomarker in a patient relative to a control such as one or more individuals not having cancer (e.g., urothelial cancer) or an internal control (e.g., a housekeeping biomarker).

"reduced expression," "reduced expression level," "reduced expression level," or "reduced level" refers to reduced expression or reduced level of a biomarker in a patient relative to a control such as one or more individuals or internal controls (e.g., housekeeping biomarkers) that do not have cancer (e.g., urothelial cancer). In some embodiments, reduced expression is little or no expression.

The term "housekeeping biomarker" refers to a biomarker or a set of biomarkers (e.g., polynucleotides and/or polypeptides) that are typically similarly present in all cell types. In some embodiments, the housekeeping biomarker is a "housekeeping gene". "housekeeping gene" refers herein to a gene or set of genes encoding proteins whose activity is essential for maintaining cellular function, and housekeeping genes are typically found similarly in all cell types.

As used herein, the term "sample" refers to a composition obtained or derived from a subject patient that contains cells and/or other molecular entities to be characterized and/or identified, e.g., based on physical, biochemical, chemical, and/or physiological characteristics. For example, the phrase "disease sample" and variations thereof refers to any sample obtained from a subject patient that is expected or known to contain the cells and/or molecular entities to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate and tissue culture media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof. In some aspects, the sample is a blood sample, a plasma sample, a serum sample, a urine sample, a cerebrospinal fluid (CSF) sample, a nasal swab sample, a saliva sample, a fecal sample, or a vaginal secretion sample.

"tissue sample" or "cell sample" refers to a collection of similar cells obtained from the tissue of a patient. The source of the tissue or cell sample may be solid tissue from fresh, frozen and/or preserved organs, tissue samples, biopsies and/or aspirates; blood or any blood component, such as plasma; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells of the patient at any time during pregnancy or development. The tissue sample may also be a primary or cultured cell or cell line. Optionally, the tissue or cell sample is obtained from a diseased tissue/organ. For example, a "tumor sample" is a tissue sample obtained from a tumor (e.g., a liver tumor) or other cancerous tissue. The tissue sample may comprise a mixed population of cell types (e.g., tumor cells and non-tumor cells, cancer cells and non-cancer cells). The tissue sample may comprise compounds that are not naturally mixed with the tissue in the natural environment, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

As used herein, "tumor-infiltrating immune cells" refers to any immune cells present in a tumor or sample thereof. Tumor infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells, other tumor stromal cells (e.g., fibroblasts), or any combination thereof. Such tumor-infiltrating immune cells can be, for example, T lymphocytes (such as cd8+ T lymphocytes and/or cd4+ T lymphocytes), B lymphocytes, or other myeloid lineage cells, including granulocytes (e.g., neutrophils, eosinophils, and basophils), monocytes, macrophages, dendritic cells (e.g., finger dendritic cells), tissue cells, and natural killer cells.

As used herein, "tumor cells" refers to any tumor cells present in a tumor or sample thereof. Using methods known in the art and/or described herein, tumor cells can be distinguished from other cells that may be present in a tumor sample, such as stromal cells and tumor-infiltrating immune cells.

As used herein, "reference level", "reference sample", "reference cell", "reference tissue", "control sample", "control cell" or "control tissue" refers to a level, sample, cell, tissue or standard for comparison purposes. In one example, the reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased portion (e.g., tissue or cells) of the body of the same subject. For example, the reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue can be healthy and/or non-diseased cells or tissues adjacent to a diseased cell or tissue (e.g., cells or tissues adjacent to a tumor). In another example, the reference sample is obtained from untreated body tissue and/or cells of the same patient. In yet another example, the reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased portion (e.g., tissue or cells) of the individual's body that is not the patient. In yet another embodiment, the reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from untreated tissue and/or cells that are not part of the patient's individual body.

For purposes herein, a "slice" of a tissue sample refers to a single portion or piece of the tissue sample, e.g., a slice of tissue or cells excised from the tissue sample (e.g., a tumor sample). It is to be understood that multiple portions of a tissue sample may be obtained and analyzed, provided that it is understood that the same portion of the tissue sample may be analyzed at the morphological and molecular level, or may be analyzed for polypeptides (e.g., by immunohistochemistry) and/or polynucleotides (e.g., by in situ hybridization).

"correlating" or "correlating" refers to comparing the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol in any manner. For example, the results of the first analysis or scheme may be used when performing the second scheme and/or the results of the first analysis or scheme may be used to determine whether the second analysis or scheme should be performed. With respect to embodiments of polypeptide assays or protocols, the results of polypeptide expression assays or protocols can be used to determine whether a particular therapeutic protocol should be administered. With respect to embodiments of polynucleotide assays or protocols, the results of polynucleotide expression assays or protocols can be used to determine whether a particular therapeutic protocol should be administered.

The phrase "based on" as used herein refers to information about one or more biomarkers used to inform information provided on a treatment decision, package insert, or marketing/promotion guide, etc.

As used herein, the terms "mutation load", "tumor mutation load score", "TMB score", "tissue tumor mutation load score" and "tTMB score" are each used interchangeably to refer to the level (e.g., number) of variation (e.g., one or more variations, such as one or more somatic variations) per preselected unit (e.g., per megabase) in a predetermined set of genes (e.g., in a coding region of the predetermined set of genes) detected in a tumor tissue sample (e.g., a Formalin Fixed and Paraffin Embedded (FFPE) tumor sample, an archive tumor sample, a fresh tumor sample, or a frozen tumor sample). For example, tTMB scores may be measured based on whole genomes or exomes, or on subsets of genomes or exomes. In certain embodiments, tTMB scores measured based on a subset of the genome or exome may be extrapolated to determine the whole genome or exome mutation load. In some embodiments, tTMB score refers to the level of accumulated somatic mutations in a patient. tTMB scores may refer to accumulated somatic mutations in patients with cancer (e.g., urothelial cancer). In some embodiments, tTMB score refers to mutations accumulated in the whole genome of a patient. In some embodiments, tTMB score refers to mutations accumulated within a particular tissue sample (e.g., a tumor tissue sample biopsy, e.g., a urothelial cancer tumor sample) collected from a patient.

The terms "somatic variant," "somatic mutation," or "somatic variation" refer to genetic variation that occurs in somatic tissue (e.g., cells outside the germ line). Examples of genetic variations include, but are not limited to, point mutations (e.g., single nucleotide exchange for another nucleotide (e.g., silent mutations, missense mutations, and nonsense mutations)), insertions and deletions (e.g., addition and/or removal of one or more nucleotides (e.g., indels)), amplifications, gene replication, copy Number Alterations (CNAs), rearrangements, and splice variants. The presence of a particular mutation may be associated with a disease state (e.g., cancer, e.g., urothelial cancer).

The term "patient-specific variant" refers to a variant (e.g., a somatic variant) that is present in a tumor in a given patient. Patient-specific variants can be detected in ctDNA, for example, using a personalized ctDNA multiplex polymerase chain reaction (mPCR) method. It will be appreciated that a given patient-specific variant may be specific to the patient or may be present in a tumor in an individual other than the patient.

As used herein, the term "reference tTMB score" refers to one tTMB score for which another tTMB score is compared, e.g., for diagnostic, predictive, prognostic, and/or therapeutic determinations. For example, the reference tTMB score may be a reference sample, tTMB scores in a reference population, and/or a predetermined value. In some cases, the reference tTMB score is a cut-off value that significantly distinguishes patients in the reference population that have been treated with a first subset of PD-1 axis binding antagonist therapies from patients in the same reference population that have not received therapy or have been treated with a second subset of non-PD-1 axis binding antagonist therapies based on a significant difference between patient responsiveness to treatment with PD-1 axis binding antagonist therapies in the absence of therapy or patient responsiveness to treatment with non-PD-1 axis binding antagonist therapies (these responsiveness being at or above the cut-off value and/or below the cut-off value). In some cases, the responsiveness of the patient to treatment with the PD-1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the patient to treatment with the non-PD-1 axis binding antagonist therapy in the absence of therapy, or to a level at or above the cutoff value. In some cases, the responsiveness of the patient to treatment with the non-PD-1 axis binding antagonist therapy in the absence of therapy or to treatment with the PD-1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the patient to treatment with the PD-L1 axis binding antagonist therapy, below the cutoff value.

It will be appreciated by those skilled in the art that the value of the reference tTMB score may vary depending on the type of cancer, the method used to measure the tTMB score, and/or the statistical method used to generate the tTMB score.

The term "equivalent TMB value" refers to a value corresponding to the tTMB score, which can be calculated by dividing the somatic variant count by the number of bases sequenced. In some cases, whole exome is sequenced. In other cases, the number of bases sequenced is about 1.1Mb (e.g., about 1.125 Mb), e.g., as defined byRated by the panel). It will be appreciated that tTMB scores generally correlate linearly with the size of the genomic region sequenced. Such equivalent tTMB values indicate the degree of equivalence of tumor mutational burden when compared to tTMB scores and are used interchangeably in the methods described herein, e.g., to predict a cancer patient's response to a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., alemtuzumab). For example, in some cases, the equivalent tTMB value is a normalized tTMB value, which can be calculated by dividing the count of somatic variants (e.g., somatic mutations) by the number of bases sequenced. For example, the equivalent tTMB value can be expressed as the number of somatic mutations counted over a defined number of sequenced bases (e.g., about 1.1Mb (e.g., about 1.125 Mb), e.g., as determined by Rated by the panel). For example, a tTMB score of about 25 (as determined as the number of somatic mutations counted over about 1.1 Mb) corresponds to an equivalent tTMB value of about 23 mutations/Mb. It is to be understood that tTMB score (e.g., expressed as TMB score of the number of somatic mutations counted over a defined number of sequenced bases) as described herein (e.g., about 1.1Mb (e.g., about 1.125 Mb), such as by->Genetic package rated)), encompasses equivalent tTMB values obtained using different methods (e.g., whole exome sequencing or whole genome sequencing). For example, for a genome package of whole exomeThe target region may be about 50Mb and the sample from which about 500 individual cell mutations are detected is an equivalent tTMB value to a tTMB score of about 10 mutations/Mb. In some cases, the number of somatic mutations (e.g., about 1.1Mb (e.g., about 1.125 Mb), as counted over a specified number of sequenced bases in a subset of a genome or exome (e.g., a predetermined set of genes), e.g., as by->The genetic package assessments) the tTMB score as determined by whole exome sequencing is less than about 30% (e.g., less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1% or less). See, for example, chalmers et al Genome Medicine9:34,2017.

Methods and compositions for treatment of urothelial cancer

Provided herein are methods, compositions, and uses for novel adjuvant therapy and/or adjuvant therapy of urothelial cancer (e.g., MIUC) in a patient in need thereof. The methods, compositions, and uses may involve administering a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., alemtuzumab) to a patient based on the presence and/or level of ctDNA in a biological sample obtained from the patient. In some cases, the methods, compositions, and uses can involve determining whether ctDNA is present in a biological sample obtained from a patient (in other words, the biological sample is ctDNA positive or ctDNA negative). In other cases, the methods, compositions, and uses may involve determining the level of ctDNA in a biological sample, which may be compared to a reference ctDNA level.

In one aspect, provided herein is a method of treating urothelial cancer (e.g., MIUC) in a patient in need thereof, the method comprising administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy, and wherein the patient has been identified as likely to benefit from the treatment regimen based on the presence of ctDNA in a biological sample obtained from the patient.

In another aspect, provided herein is a method of treating urothelial cancer (e.g., MIUC) in a patient in need thereof, the method comprising: (a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and (b) administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy.

In another aspect, provided herein is a PD-1 axis binding antagonist or a pharmaceutical composition comprising a PD-1 axis binding antagonist for use in treating urothelial cancer (e.g., MIUC) in a patient in need thereof, wherein the treatment comprises administering an effective amount of a treatment regimen comprising the PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy, and wherein the patient has been identified as likely to benefit from the treatment regimen based on the presence of ctDNA in a biological sample obtained from the patient.

In another aspect, provided herein is a PD-1 axis binding antagonist or a pharmaceutical composition comprising a PD-1 axis binding antagonist for use in treating urothelial cancer (e.g., MIUC) in a patient in need thereof, the treatment comprising: (a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and (b) administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy.

In another aspect, provided herein is a method of treating urothelial cancer (e.g., MIUC) in a patient in need thereof, the method comprising administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy, and wherein a level of ctDNA in a biological sample obtained from the patient that is equal to or greater than a reference level of ctDNA indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist.

In another aspect, provided herein is a method of treating urothelial cancer (e.g., MIUC) in a patient in need thereof, the method comprising: (a) Determining the level of ctDNA in a biological sample obtained from the patient, wherein a level of ctDNA in the biological sample that is equal to or higher than a reference level of ctDNA indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and (b) administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist based on the level of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy.

In another aspect, provided herein is a PD-1 axis binding antagonist or a pharmaceutical composition comprising a PD-1 axis binding antagonist for use in treating urothelial cancer (e.g., MIUC) in a patient in need thereof, wherein the treatment comprises administering an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy, and wherein a level of ctDNA in a biological sample obtained from the patient that is equal to or greater than a reference level of ctDNA indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist.

In another aspect, provided herein is a PD-1 axis binding antagonist or a pharmaceutical composition comprising a PD-1 axis binding antagonist for use in treating urothelial cancer (e.g., MIUC) in a patient in need thereof, the treatment comprising: (a) Determining the level of ctDNA in a biological sample obtained from the patient, wherein a level of ctDNA in the biological sample that is equal to or higher than a reference level of ctDNA indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and (b) administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist based on the level of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy.

In another aspect, provided herein is a method of identifying a patient having urothelial cancer (e.g., MIUC) who is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist, the method comprising determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample identifies the patient as a patient likely to benefit from treatment with a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy. In some cases, the method further comprises administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist.

In another aspect, provided herein is a method of identifying a patient having urothelial cancer (e.g., MIUC) who is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist, the method comprising determining the level of ctDNA in a biological sample obtained from the patient, wherein a level of ctDNA in the biological sample that is equal to or greater than a reference level of ctDNA indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy. In some cases, the method further comprises administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist.

In another aspect, provided herein is a method of selecting a therapy for a patient having urothelial cancer (e.g., MIUC), the method comprising (a) determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and (b) selecting a treatment regimen comprising a PD-1 axis binding antagonist based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy. In some cases, the method further comprises administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist.

In another aspect, provided herein is a method of selecting a therapy for a patient having urothelial cancer (e.g., MIUC), the method comprising (a) determining the level of ctDNA in a biological sample obtained from the patient, wherein a level of ctDNA in the biological sample equal to or greater than a reference level of ctDNA indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and (b) selecting a treatment regimen comprising a PD-1 axis binding antagonist based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy. In some cases, the method further comprises administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist.

In some cases, the biological sample is obtained prior to or concurrent with administration of the first dose of the therapeutic regimen. In some cases, the biological sample is obtained on cycle 1 day 1 (C1D 1) of the treatment regimen. In some cases, the biological sample is obtained within about 60 weeks (e.g., within about 60 weeks, about 55 weeks, about 50 weeks, about 45 weeks, about 40 weeks, about 35 weeks, about 30 weeks, about 25 weeks, about 20 weeks, about 19 weeks, about 18 weeks, about 17 weeks, about 16 weeks, about 15 weeks, about 14 weeks, about 13 weeks, about 12 weeks, about 11 weeks, about 10 weeks, about 9 weeks, about 8 weeks, about 7 weeks, about 6 weeks, about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, or about 1 week) from surgical excision. In some cases, the biological sample is obtained within about 30 weeks from surgical excision. In some cases, the biological sample is obtained within about 20 weeks from surgical excision.

In some cases, the biological sample is obtained from about 2 to about 20 weeks after surgical excision (e.g., about 2 to about 20 weeks, about 2 to about 19 weeks, about 2 to about 18 weeks, about 2 to about 17 weeks, about 2 to about 16 weeks, about 2 to about 15 weeks, about 2 to about 14 weeks, about 2 to about 13 weeks, about 2 to about 12 weeks, about 2 to about 11 weeks, about 2 to about 10 weeks, about 2 to about 9 weeks, about 2 to about 8 weeks, about 2 to about 7 weeks, about 2 to about 6 weeks, about 2 to about 5 weeks, about 2 to about 4 weeks, about 2 to about 3 weeks, about 4 to about 20 weeks, about 4 to about 19 weeks, about 4 to about 18 weeks, about 4 to about 17 weeks, about 4 to about 16 weeks, about 4 to about 15 weeks, about 4 to about 14 weeks, about 4 to about 13 weeks, about 4 to about 12 weeks, about 4 to about 11 weeks, about 4 to about 10 weeks, about 4 to about 9 weeks, about 4 to about 8 weeks, about 4 to about 7 weeks, about 4 to about 6 weeks, about 4 to about 5 weeks, about 20 to about 20 weeks about 6 to about 19 weeks, about 6 to about 18 weeks, about 6 to about 17 weeks, about 6 to about 16 weeks, about 6 to about 15 weeks, about 6 to about 14 weeks, about 6 to about 13 weeks, about 6 to about 12 weeks, about 6 to about 11 weeks, about 6 to about 10 weeks, about 6 to about 9 weeks, about 6 to about 8 weeks, about 6 to about 7 weeks, about 8 to about 20 weeks, about 8 to about 19 weeks, about 8 to about 18 weeks, about 8 to about 17 weeks, about 6 to about 16 weeks, about 6 to about 15 weeks, about 6 to about 14 weeks, about 8 to about 13 weeks, about 8 to about 12 weeks, about 8 to about 11 weeks, about 8 to about 10 weeks, about 8 to about 9 weeks, about 10 to about 20 weeks, about 10 to about 19 weeks, about 10 to about 18 weeks, about 10 to about 17 weeks, about 10 to about 16 weeks, about 10 to about 15 weeks, about 10 to about 14 weeks, about 10 to about 13 weeks, about 10 to about 12 weeks, about 10 to about 13 weeks, about 10 to about 11 weeks, about 12 to about 20 weeks, about 12 to about 19 weeks, about 12 to about 18 weeks, about 12 to about 17 weeks, about 12 to about 16 weeks, about 12 to about 15 weeks, about 12 to about 14 weeks, about 12 to about 13 weeks, about 14 to about 20 weeks, about 14 to about 19 weeks, about 14 to about 18 weeks, about 14 to about 17 weeks, about 14 to about 16 weeks, about 14 to about 15 weeks, about 16 to about 20 weeks, about 16 to about 19 weeks, about 16 to about 18 weeks, about 16 to about 17 weeks, about 18 to about 20 weeks, or about 18 to about 19 weeks).

It will be appreciated that ctDNA may be detected in any suitable biological sample. In some cases, the biological sample is a blood sample, a plasma sample, a serum sample, a urine sample, a CSF sample, a nasal swab sample, a saliva sample, a fecal sample, or a vaginal secretion sample. In some cases, the biological sample is a blood sample, a plasma sample, or a serum sample. In some cases, the biological sample is a plasma sample.

In another aspect, provided herein is a method of monitoring the response of a patient having urothelial cancer (e.g., MIUC), the patient having been administered at least a first dose of a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant or adjuvant therapy, and wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen, the method comprising determining whether ctDNA is present in a biological sample obtained from the patient at a time point after the first dose of the treatment regimen, thereby monitoring the response of the patient. In some cases, the absence of ctDNA in a biological sample obtained from a patient at a point in time after administration of a first dose of a therapeutic regimen indicates that the patient is responsive to the therapeutic regimen. In some embodiments, the treatment regimen is neoadjuvant therapy. In other embodiments, the treatment regimen is adjuvant therapy.

In another aspect, provided herein is a method of monitoring the response of a patient having urothelial cancer (e.g., MIUC), the patient having been administered at least a first dose of a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant or adjuvant therapy, and wherein a level of ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen, the method comprising determining the level of ctDNA in a biological sample obtained from the patient at a time point after the first dose of the treatment regimen, thereby monitoring the response of the patient. In some cases, a decrease in the level of ctDNA in a biological sample obtained from a patient at a point in time after administration of a first dose of a treatment regimen relative to the level of ctDNA in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen indicates that the patient is responsive to the treatment regimen. In some embodiments, the treatment regimen is neoadjuvant therapy. In other embodiments, the treatment regimen is adjuvant therapy.

In another aspect, provided herein is a PD-1 axis binding antagonist or a pharmaceutical composition comprising a PD-1 axis binding antagonist for use in treating a patient having urothelial cancer (e.g., MIUC) that has been administered at least a first dose of a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant or adjuvant, wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen. In some embodiments, the treatment regimen is neoadjuvant therapy. In other embodiments, the treatment regimen is adjuvant therapy.

In another aspect, provided herein is a method of identifying a patient having urothelial cancer (e.g., MIUC) who may benefit from a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant therapy or adjuvant therapy and the patient has been administered at least a first dose of the treatment regimen, and wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen, the method comprising: determining whether ctDNA is present in a biological sample obtained from the patient at a point in time after administration of the first dose of the treatment regimen, wherein the absence of ctDNA in the biological sample at the point in time after administration of the treatment regimen identifies the patient as likely to benefit from treatment with a treatment regimen comprising a PD-1 axis binding antagonist. In some embodiments, the treatment regimen is neoadjuvant therapy. In other embodiments, the treatment regimen is adjuvant therapy.

In another aspect, provided herein is a method of identifying a patient having urothelial cancer (e.g., MIUC) who may benefit from a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant or adjuvant therapy and the patient has been administered at least a first dose of the treatment regimen, and wherein a level of ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen, the method comprising: determining a ctDNA level in a biological sample obtained from the patient at a time point after administration of the first dose of the treatment regimen, wherein a decrease in the ctDNA level in the biological sample at the time point after administration of the treatment regimen relative to the ctDNA level in the biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen identifies the patient as likely to benefit from treatment with a treatment regimen comprising a PD-1 axis binding antagonist. In some embodiments, the treatment regimen is neoadjuvant therapy. In other embodiments, the treatment regimen is adjuvant therapy.

Any suitable point in time after the first dose of the treatment regimen is administered may be used. For example, in some cases, the time point after the first dose of the treatment regimen is cycle 2 day 1 (C2D 1, cycle 3 day 1 (C3D 1), cycle 4 day 1 (C4D 1), cycle 5 day 1 (C5D 1), cycle 6 day 1 (C6D 1), cycle 7 day 1 (C7D 1), cycle 8 day 1 (C8D 1), cycle 9 day 1 (C9D 1), cycle 10 day 1 (C10D 1), cycle 11 day 1 (C11D 1), cycle 12 day 1 (C12D 1), or at a later cycle of the treatment regimen.

In some cases, the biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen and/or the biological sample obtained from the patient at a point in time after administration of the first dose of the treatment regimen is a blood sample, a plasma sample, a serum sample, a urine sample, a CSF sample, a nasal swab sample, a saliva sample, a stool sample, or a vaginal secretion sample. For example, in some cases, the biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen and/or the biological sample obtained from the patient at a point in time after administration of the first dose of the treatment regimen is a plasma sample.

In some cases, the benefit is in terms of improved disease-free survival (DFS), improved total survival (OS), improved disease-specific survival, or improved distant metastasis-free survival. In some cases, the benefit is in terms of improved DFS. In some cases, the benefit is in terms of improved OS. In some cases, the improvement is relative to observation or relative to adjuvant therapy with placebo.

The presence and/or level of ctDNA in a biological sample may be determined using any suitable method, for example, any method known in the art or described in section V below. For example, the presence and/or level of ctDNA is determined by a Polymerase Chain Reaction (PCR) -based method, a hybridization capture-based method, a methylation-based method, or a fragment histology method.

In some cases, the presence and/or level of ctDNA is determined by a personalized ctDNA multiplex polymerase chain reaction (mPCR) method. In some cases, the personalized ctDNA mPCR method comprises: (a) (i) sequencing DNA obtained from a tumor sample obtained from a patient to produce tumor sequence reads; (ii) Sequencing DNA obtained from a normal tissue sample (e.g., buffy coat) obtained from the patient to produce a normal sequence read; (b) Identifying one or more patient-specific variants by calling a somatic variant identified from the tumor sequence read and excluding germline variants and/or potent unclonable hematopoietic (CHIP) variants, wherein the germline variants or CHIP variants are identified from the normal sequence read or from a publicly available database; (c) Designing a mPCR assay for a patient that detects a set of patient-specific variants; and (d) analyzing a biological sample obtained from the patient using the mPCR assay to determine whether ctDNA is present in the biological sample. In some cases, the sequencing is WES or WGS. In some cases, the sequencing is WES. In some cases, the patient-specific variant is a single nucleotide variant (SNV) or short indels (insertions or deletions of bases). In some cases, the set of patient-specific variants includes at least 1 patient-specific variant. In some cases, the set of patient-specific variants includes at least 2 patient-specific variants. In some cases, the set of patient-specific variants includes at least 8 patient-specific variants. In some cases, the set of patient-specific variants includes 2 to 200 patient-specific variants. In some cases, the set of patient-specific variants includes 8 to 50 patient-specific variants. In some cases, the set of patient-specific variants includes 8 to 32 patient-specific variants. In some cases, the set of patient-specific variants includes 16 patient-specific variants. In some cases, analyzing a biological sample obtained from a patient using a mPCR assay includes sequencing amplicons produced by the mPCR assay to identify patient-specific variants in the biological sample. In some cases, the personalized ctDNA mPCR method isctDNA test or ArcherDx Personalized Cancer Monitoring (PCM) ^TM ) And (5) testing. In some cases, the presence of at least one patient-specific variant in a biological sample identifies the presence of ctDNA in the biological sample. In some cases, the presence of two patient-specific variants in the biological sample identifies the presence of ctDNA in the biological sample.

In some cases, about 2 to about 200 patient-specific variants are detected in the biological sample, e.g., about 2 to about 200, about 2 to about 175, about 2 to about 150, about 2 to about 125, about 2 to about 100, about 2 to about 75, about 2 to about 50, about 2 to about 48, about 2 to about 46, about 2 to about 44, about 2 to about 42, about 2 to about 40, about 2 to about 38, about 2 to about 36, about 2 to about 34, about 2 to about 32, about 2 to about 30, about 2 to about 28, about 2 to about 26, about 2 to about 24, about 2 to about 22, about 2 to about 20, about 2 to about 18, about 2 to about 16, about 2 to about 14, about 2 to about 12, about 2 to about 10, about 2 to about 8, about 2 to about 6, about 2 to about 4, about 4 to about 32, about 4 to about 30, about 4 to about 28, about 4 to about 26, about 4 to about 24, about 4 to about 22 about 4 to about 20, about 4 to about 18, about 4 to about 16, about 4 to about 14, about 4 to about 12, about 4 to about 10, about 4 to about 8, about 4 to about 6, about 6 to about 32, about 6 to about 30, about 6 to about 28, about 6 to about 26, about 6 to about 24, about 6 to about 22, about 6 to about 20, about 6 to about 18, about 6 to about 16, about 6 to about 14, about 6 to about 12, about 6 to about 10, about 6 to about 8, about 8 to about 32, about 8 to about 30, about 8 to about 28, about 8 to about 26, about 8 to about 24, about 8 to about 22, about 8 to about 20, about 8 to about 18, about 8 to about 16, about 8 to about 14, about 8 to about 12, about 8 to about 10, about 32 to about 10, about 10 to about 30, about 10 to about 28, about 10 to about 26, about 10 to about 24, about 10 to about 22, about 10 to about 20, about 10 to about 18, about 10 to about 16, about 10 to about 14, about 10 to about 12, about 12 to about 32, about 12 to about 30, about 12 to about 28, about 12 to about 26, about 12 to about 24, about 12 to about 22, about 12 to about 20, about 12 to about 18, about 12 to about 16, about 12 to about 14, about 14 to about 32, about 14 to about 30, about 14 to about 28, about 14 to about 26, about 14 to about 24, about 14 to about 22, about 14 to about 20, about 14 to about 18, about 14 to about 16, about 16 to about 32, about 16 to about 30, about 16 to about 28, about 16 to about 26 about 16 to about 24, about 16 to about 22, about 16 to about 20, about 16 to about 18, about 18 to about 32, about 18 to about 30, about 18 to about 28, about 18 to about 26, about 18 to about 24, about 18 to about 22, about 18 to about 20, about 20 to about 32, about 20 to about 30, about 20 to about 28, about 20 to about 26, about 20 to about 24, about 20 to about 22, about 22 to about 32, about 22 to about 30, about 22 to about 28, about 22 to about 26, about 22 to about 24, about 24 to about 32, about 24 to about 30, about 24 to about 28, about 24 to about 26, about 26 to about 32, about 26 to about 30, about 26 to about 28, about 28 to about 32, about 28 to about 30, or about 30 to about 32 patient-specific variants. In some cases, about 2 to about 16 patient-specific variants are detected in the biological sample.

In some cases, the average allele frequency of a given patient-specific variant in the biological sample is from about 0.0001% to about 99%, for example, about 0.0001%, about 0.0002%, about 0.0003%, about 0.0004%, about 0.0005%, about 0.0006%, about 0.0007%, about 0.0008%, about 0.0009%, about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35% >. About 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some cases, the average allele frequency of a given patient-specific variant in the biological sample is from about 0.001% to about 99%.

The biological sample may have any suitable volume. For example, in some cases, the biological sample has a volume of about 0.02mL to about 80mL (e.g., about 0.02mL, about 0.3mL, about 0.4mL, about 0.5mL, about 0.6mL, about 0.7mL, about 0.8mL, about 0.9mL, about 1mL, about 2mL, about 3mL, about 4mL, about 5mL, about 6mL, about 7mL, about 8mL, about 9mL, about 10mL, about 12mL, about 14mL, about 16mL, about 18mL, about 20mL, about 22mL, about 24mL, about 26mL, about 28mL, about 30mL, about 32mL, about 34mL, about 36mL, about 38mL, about 40mL, about 45mL, about 50mL, about 55mL, about 60mL, about 65mL, about 70mL, about 75mL, or about 80 mL).

For example, in some cases, the biological sample has a volume of about 1mL to about 20mL (e.g., about 2mL to about 20mL, about 2mL to about 18mL, about 2mL to about 16mL, about 2mL to about 14mL, about 2mL to about 12mL, about 2mL to about 10mL, about 2mL to about 8mL, about 2mL to about 6mL, about 2mL to about 4mL, about 4mL to about 20mL, about 4mL to about 18mL, about 4mL to about 16mL, about 4mL to about 14mL, about 4mL to about 12mL, about 4mL to about 10mL, about 4mL to about 8mL, about 4mL to about 6mL, about 6mL to about 20mL, about 6mL to about 18mL, about 6mL to about 16mL, about 6mL to about 14mL, about 6mL to about 12mL, about 6mL to about 8mL, about 8mL to about 20mL, about 8mL to about 18mL, about 8mL to about 16mL, about 16mL to about 12mL, about 14mL to about 10mL, about 14mL to about 12mL, about 14mL to about 18mL, about 10mL to about 12mL, about 14mL to about 18mL to about 12mL, about 10mL to about 12mL to about 18mL, about 12mL to about 10mL to about 12mL, about 12mL to about 10mL, about 12mL to about 18mL to about 12 mL. In some cases, the biological sample has a volume of about 1mL, about 2mL, about 3mL, about 4mL, about 5mL, about 6mL, about 7mL, about 8mL, about 9mL, about 10mL, about 11mL, about 12mL, about 13mL, about 14mL, about 15mL, about 16mL, about 17mL, about 18mL, about 19mL, or about 20 mL. In some cases, the biological sample has a volume of about 2 to about 10 mL. In some cases, the biological sample has a volume of about 2 to about 8 mL.

The biological sample may contain any suitable amount of cfDNA (e.g., ctDNA). For example, a biological sample may contain cfDNA (e.g., ctDNA) of about 2ng to about 200ng (e.g., about 2ng, about 5ng, about 10ng, about 15ng, about 20ng, about 25ng, about 30ng, about 35ng, about 40ng, about 45ng, about 50ng, about 55ng, about 60ng, about 65ng, about 70ng, about 80ng, about 85ng, about 90ng, about 95ng, about 100ng, about 105ng, about 110ng, about 115ng, about 120ng, about 125ng, about 130ng, about 135ng, about 140ng, about 145ng, about 150ng, about 155ng, about 160ng, about 165ng, about 170ng, about 175ng, about 180ng, about 185ng, about 190ng, about 195ng, or about 200 ng). In some cases, the biological sample may contain about 10 to about 70ng cfDNA (e.g., ctDNA).

In the case where ctDNA levels are determined, ctDNA levels may be expressed, for example, as Variant Allele Frequencies (VAFs) or in terms of mutations/mL.

In the case where ctDNA levels are determined, any suitable ctDNA reference level may be used. For example, the reference level of ctDNA may be (1) the level of ctDNA in a biological sample obtained from a patient prior to or concurrent with administration of a treatment regimen comprising a PD-1 axis binding antagonist; (2) ctDNA levels from a reference population; (3) a preset level of ctDNA; or (4) ctDNA levels in biological samples obtained from the patient at a second time point before or after the first time point.

In some cases, the urothelial cancer is MIUC. In some cases, the MIUC is a Myometrial Invasive Bladder Cancer (MIBC) or a myometrial invasive urinary tract urothelial cancer (myometrial invasive UTUC). In some cases, MIUC is histologically confirmed and/or wherein the patient has an eastern tumor cooperative group (ECOG) physical status of less than or equal to 2.

In some cases, the patient has been previously treated with neoadjuvant chemotherapy. In some cases, the patient's MIUC is ypT2-4a or ypN + and M0 at the time of surgical resection. In some cases, patients have not received prior neoadjuvant chemotherapy.

In other cases, the patient is not suitable for cisplatin or has refused cisplatin-based adjuvant chemotherapy. In some cases, the MIUC of the patient is pT3-4a or pN+ and M0 at the time of surgical excision.

In some cases, the patient has undergone surgical resection and lymph node cleaning. In some cases, the surgical resection is a cystectomy or a nephroureterectomy.

In some cases, the patient has no evidence of residual disease or metastasis as assessed by post-operative radiological imaging.

In some cases, a tumor sample obtained from a patient has been determined to have a tumor mutation load (tTMB) score equal to or higher than a reference tissue tTMB score. In some cases, the reference tTMB score is a pre-specified tTMB score. In some cases, the pre-specified tTMB score is between about 8 and about 30 mut/Mb. In some cases, the pre-specified tTMB fraction is about 10 mutations per megabase (mut/Mb).

In some cases, the tumor sample is from a surgical resection.

In some cases, the patient has an increased expression level of one or more genes selected from PD-L1, IFNG, and CXCL9 in a biological sample obtained from the patient relative to a reference expression level of the one or more genes.

In some cases, the patient has increased expression levels of two or more genes selected from PD-L1, IFNG, and CXCL9 in a biological sample obtained from the patient relative to reference expression levels of the two or more genes. For example, in some cases, a patient may have increased expression levels of PD-L1 and IFNG, PD-L1 and CXCL9, or IFNG and CXCL9 relative to reference expression levels of the two or more genes.

In some cases, the patient has increased expression levels of PD-L1, IFNG, and CXCL9 in a biological sample obtained from the patient relative to reference expression levels of PD-L1, IFNG, and CXCL 9.

In some cases involving determining the expression level of PD-L1, IFNG, and/or CXCL9, an expression level that is higher than a reference expression level, or an increased or increased expression or amount, may refer to an overall increase in any of the levels or amounts of a biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA) or cell) that is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more than a reference expression level, a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue, as compared to the levels or amounts of any of the biomarkers (e.g., protein, nucleic acid (e.g., gene or mRNA) or cell) described herein and/or detected by methods known in the art. In certain embodiments, elevated expression or amount refers to an increase in the expression level/amount of a biomarker (e.g., one or more of PD-L1, IFNG, and/or CXCL 9) in a sample, wherein the increase is at least about 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 100x, 1000x, or any of the expression levels/amounts of the respective biomarkers in the reference expression level, the reference sample, the reference cell, the reference tissue, the control sample, the control cell, or the control tissue. In some embodiments, elevated expression or amount refers to an increase in the expression level/amount of a biomarker (e.g., PD-L1, IFNG, and/or CXCL 9) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20, about 30-fold, about 50, about 100-fold, or about 100-fold compared to a reference expression level, a reference sample, a reference cell, a reference tissue, a control sample, a control cell, a control tissue, or an internal control (e.g., a housekeeping gene).

In some cases, the expression level of PD-L1, IFNG, and/or CXCL9 is an mRNA expression level. In other cases, the expression level of PD-L1, IFNG, and/or CXCL9 may be a protein expression level.

In some cases, the expression level of the pan F-TBRS signature may be determined in a biological sample obtained from the patient. The expression level of the pan F-TBRS trait can be determined, for example, as described in U.S. patent application publication No. 2020/0263261, which is incorporated herein by reference in its entirety. In other examples, the expression level of any of the features described in U.S. patent application publication No. 2020/0263261 can be determined, including 22 gene (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH PXD2A, TAGLN, TGFBI, TNS1, and/or TPM 1) or 6 gene (ACTA 2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR 2) features, including any combination of the genes described in U.S. patent application publication No. 2020/0263261. In another example, the signature may be a pan F-TBRS signature, including one or more genes selected from ACTA2, ACTG2, TAGLN, TNS1, CNN1, TPM1, CTGF, PXDC1, ADAM12, FSTL3, TGFBI, and ADAM 19.

In some cases, the patient has a reduced expression level of one or more pan F-TBRS genes selected from ACTA2, ACTG2, TAGLN, TNS1, CNN1, TPM1, CTGF, PXDC1, ADAM12, FSTL3, TGFBI, and ADAM19 in a biological sample obtained from the patient relative to a reference expression level of the one or more pan F-TBRS genes.

In some cases, the patient has reduced expression levels of the at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all twelve pan-F-TBRS genes in a biological sample obtained from the patient relative to reference expression levels of the at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all twelve pan-F-TBRS genes.

In examples involving pan-TBRS characteristics, lower than the reference expression level, or reduced (reduced) expression or amount may refer to a reduction in the expression level/amount of a biomarker (e.g., one or more of protein, nucleic acid (e.g., gene or mRNA) detected by standard methods known in the art, such as those described herein, as compared to the reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the expression level/amount of the biomarker in the sample (e.g., one or more of ACTA2, ACTG2, TAGLN, TNS1, CNN1, TPM1, CTGF, PXDC1, ADAM12, FSTL3, fbi, and/or ADAM 19), wherein the reduction is at least about any one of 0.9x, 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, 0.1x, 0.05x, or 0.01x the reduced (reduced) expression or amount refers to an expression level/amount of the biomarker (e.g., ACTA2, ACTG2, TAGLN, TNS1, CNN1, TPM1, CTGF, PXDC1, ADAM12, FSTL3, TGFBI, and/or ADAM 19) that is greater than about 1.1 times the expression level/amount of the biomarker in the reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene), about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold or greater overall increase.

In some cases, the expression level of one or more pan F-TBRS genes is mRNA expression level. In other cases, the expression level of one or more of the pan F-TBRS genes is a protein expression level.

In some cases, the biological sample obtained from the patient is a tumor sample.

In some cases, the tumor of the patient has a basal squamous subtype. In some cases, basal squamous cell subtypes can be assessed by cancer genomic profile (TCGA) classification. TCGA classification can be performed, for example, as described in Robertson et al Cell 171 (3): 540-556, e25, 2017.

In some cases, the patient has increased expression levels of one or more genes selected from CD44, KRT6A, KRT, KRT14, COL17A1, DSC3, GSDMC, TGM1, and PI3 relative to a reference expression level of the one or more genes.

Any suitable PD-1 axis binding antagonist may be used, including any PD-1 axis binding antagonist known in the art or described in section IV below. In some cases, the PD-1 axis binding antagonist is selected from the group consisting of: PD-L1 binding antagonists, PD-1 binding antagonists and PD-L2 binding antagonists. In some cases, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some cases, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some cases, the anti-PD-L1 antibody is alemtuzumab, devaluzumab, avilamab, or MDX-1105. In other cases, the PD-1 axis binding antagonist is PD-1 binding. In some cases, the PD-1 binding antagonist is an anti-PD-1 antibody. In some cases, the anti-PD-1 antibody is na Wu Shankang, pamil mab, MEDI-0680, swamp mab, cimetidine Li Shan antibody, karilimab, singdi Li Shan antibody, tirelimab, terlipressin Li Shan antibody, or multi-tarolimab.

In preferred embodiments, the PD-1 axis binding antagonist is alemtuzumab.

For example, in one aspect, provided herein is a method of treating MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, the method comprising: (a) determining whether the patient is ctDNA positive; and (b) administering to the patient an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen is adjuvant therapy.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for treating MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the treatment comprises administering an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen is adjuvant therapy, and wherein the patient is ctDNA positive.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for treating MIUC (e.g., MIBC or myometrial invasive UTUC) in a patient in need thereof, the treatment comprising: (a) determining whether the patient is ctDNA positive; and (b) administering to the patient an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen is adjuvant therapy.

In any of the foregoing aspects, the patient may be determined to be ctDNA positive after surgical resection (e.g., cystectomy).

For example, in one aspect, provided herein is a method of adjunctive therapy for MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to be ctDNA positive following surgical resection, the method comprising administering to the patient an effective amount of a therapeutic regimen comprising alemtuzumab.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for adjunctive treatment of MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to be ctDNA positive following surgical excision, and wherein the treatment comprises administration of an effective amount of a treatment regimen comprising alemtuzumab.

In one aspect, provided herein is a method of adjunctive therapy for MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to be ctDNA positive following surgical resection, the method comprising administering to the patient an effective amount of a therapeutic regimen comprising alemtuzumab, wherein the therapeutic regimen comprises administering to the patient alemtuzumab intravenously (e.g., by infusion) at a dose of 1200mg on day 1 of each 21-day cycle.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for adjunctive treatment of MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to be ctDNA positive following surgical resection, and wherein the treatment comprises administration of an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen comprises intravenous (e.g., by infusion) administration of alemtuzumab to the patient at a dose of 1200mg on day 1 of each 21-day cycle.

In one aspect, provided herein is a method of adjunctive treatment of MIBC in a patient in need thereof, wherein the patient has been determined to be ctDNA positive following surgical resection, the method comprising administering to the patient an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen comprises intravenous (e.g., by infusion) administration of alemtuzumab to the patient at a dose of 1680mg on day 1 of each 21-day cycle.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for adjunctive treatment of MIBC in a patient in need thereof, wherein the patient has been determined to be ctDNA positive after surgical excision, and wherein the treatment comprises administering an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen comprises intravenous (e.g., by infusion) administration of alemtuzumab to the patient at a dose of 1200mg on day 1 of each 21-day cycle.

In some examples of any of the above aspects, the treatment regimen comprises up to 16 cycles. In other examples, the treatment regimen includes more than 16 cycles.

In one aspect, provided herein is a method of adjunctive therapy for MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to be ctDNA positive following surgical resection, the method comprising administering to the patient an effective amount of a therapeutic regimen comprising alemtuzumab, wherein the therapeutic regimen comprises administering to the patient alemtuzumab intravenously (e.g., by infusion) at a dose of 1680mg on day 1 of each 28-day cycle.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for adjunctive treatment of MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to be ctDNA positive following surgical resection, and wherein the treatment comprises administration of an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen comprises intravenous (e.g., by infusion) administration of alemtuzumab to the patient at a dose of 1680mg on day 1 of each 28-day cycle.

In one aspect, provided herein is a method of adjunctive treatment of MIBC in a patient in need thereof, wherein the patient has been determined to be ctDNA positive following surgical resection, the method comprising administering to the patient an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen comprises intravenous (e.g., by infusion) administration of alemtuzumab to the patient at a dose of 1680mg on day 1 of each 28-day cycle.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for adjunctive treatment of MIBC in a patient in need thereof, wherein the patient has been determined to be ctDNA positive after surgical excision, and wherein the treatment comprises administering an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen comprises intravenous (e.g., by infusion) administration of alemtuzumab to the patient at a dose of 1680mg on day 1 of each 28-day cycle.

In some examples of any of the above aspects, the treatment regimen comprises up to 12 cycles. In other examples, the treatment regimen includes more than 12 cycles.

In any of the foregoing aspects, the ctDNA status of the patient can be determined in any suitable sample, such as a blood sample, a plasma sample, a serum sample, a urine sample, a CSF sample, a nasal swab sample, a saliva sample, a fecal sample, or a vaginal secretion sample. In some cases, the sample is a plasma sample.

For example, in one aspect, provided herein is a method of treating MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, the method comprising: (a) Determining whether a plasma sample obtained from the patient is ctDNA positive, wherein a ctDNA positive plasma sample indicates that the patient is likely to benefit from a treatment regimen comprising alemtuzumab; and (b) administering to the patient an effective amount of a treatment regimen comprising alemtuzumab based on the ctDNA positive plasma sample, wherein the treatment regimen is adjuvant therapy.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for treating MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the treatment comprises administering an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen is adjuvant therapy, and wherein the patient is identified as likely to benefit from the treatment regimen based on a plasma sample obtained from the patient being ctDNA positive.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for treating MIUC (e.g., MIBC or myometrial invasive UTUC) in a patient in need thereof, the treatment comprising: (a) Determining whether a plasma sample obtained from the patient is ctDNA positive, wherein a ctDNA positive plasma sample indicates that the patient is likely to benefit from a treatment regimen comprising alemtuzumab; and (b) administering to the patient an effective amount of a treatment regimen comprising alemtuzumab based on the ctDNA positive plasma sample, wherein the treatment regimen is adjuvant therapy.

In one aspect, provided herein is a method of adjunctive treatment of MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to have a ctDNA positive plasma sample following a cystectomy, the method comprising administering to the patient an effective amount of a treatment regimen comprising alemtuzumab.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for adjunctive treatment of MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to have a ctDNA positive plasma sample following cystectomy, and wherein the treatment comprises administration of an effective amount of a therapeutic regimen comprising alemtuzumab.

In one aspect, provided herein is a method of adjunctive treatment of MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to have a ctDNA positive plasma sample following cystectomy, the method comprising administering to the patient an effective amount of a therapeutic regimen comprising alemtuzumab, wherein the therapeutic regimen comprises intravenous (e.g., by infusion) administration of alemtuzumab to the patient at a dose of 1200mg on day 1 of each 21-day cycle.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for adjunctive treatment of MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to have a ctDNA positive plasma sample following cystectomy, and wherein the treatment comprises administering an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen comprises administering the alemtuzumab intravenously (e.g., by infusion) to the patient at a dose of 1200mg on day 1 of each 21-day cycle.

In one aspect, provided herein is a method of adjunctive therapy for MIBC in a patient in need thereof, wherein the patient has been determined to have a ctDNA positive plasma sample following a cystectomy, the method comprising administering to the patient an effective amount of a therapeutic regimen comprising alemtuzumab, wherein the therapeutic regimen comprises administering alemtuzumab intravenously (e.g., by infusion) to the patient at a dose of 1680mg on day 1 of each 21-day cycle.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for adjunctive treatment of MIBC in a patient in need thereof, wherein the patient has been determined to have a ctDNA positive plasma sample following a cystectomy, and wherein the treatment comprises administration of an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen comprises intravenous (e.g., by infusion) administration of alemtuzumab to the patient at a dose of 1200mg on day 1 of each 21-day cycle.

In some examples of any of the above aspects, the treatment regimen comprises up to 16 cycles. In other examples, the treatment regimen includes more than 16 cycles.

In one aspect, provided herein is a method of adjunctive treatment of MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to have a ctDNA positive plasma sample following a cystectomy, the method comprising administering to the patient an effective amount of a therapeutic regimen comprising alemtuzumab, wherein the therapeutic regimen comprises intravenous (e.g., by infusion) administration of alemtuzumab to the patient at a dose of 1680mg on day 1 of each 28-day cycle.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for adjunctive treatment of MIUC (e.g., MIBC or muscle invasive UTUC) in a patient in need thereof, wherein the patient has been determined to have a ctDNA positive plasma sample following cystectomy, and wherein the treatment comprises administering an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen comprises administering alemtuzumab to the patient intravenously (e.g., by infusion) at a dose of 1680mg on day 1 of each 28-day cycle.

In one aspect, provided herein is a method of adjunctive therapy for MIBC in a patient in need thereof, wherein the patient has been determined to have a ctDNA positive plasma sample following a cystectomy, the method comprising administering to the patient an effective amount of a therapeutic regimen comprising alemtuzumab, wherein the therapeutic regimen comprises administering alemtuzumab intravenously (e.g., by infusion) to the patient at a dose of 1680mg on day 1 of each 28-day cycle.

In another aspect, provided herein is an alemtuzumab or a pharmaceutical composition comprising alemtuzumab for adjunctive treatment of MIBC in a patient in need thereof, wherein the patient has been determined to have a ctDNA positive plasma sample following a cystectomy, and wherein the treatment comprises administration of an effective amount of a treatment regimen comprising alemtuzumab, wherein the treatment regimen comprises intravenous (e.g., by infusion) administration of alemtuzumab to the patient at a dose of 1680mg on day 1 of each 28-day cycle.

In some examples of any of the above aspects, the treatment regimen comprises up to 12 cycles. In other examples, the treatment regimen includes more than 12 cycles.

In any of the foregoing aspects, ctDNA positivity can be measured using personalized mPCR (e.g., natera Assay), wherein plasma samples assessed as having 2 or more mutations as assessed by the personalized mPCR assay are considered ctDNA positive.

In any of the foregoing aspects, ctDNA positivity can be determined using a food and drug administration approved test.

In some examples, the PD-1 axis binding antagonist is administered as monotherapy. In other examples, the PD-1 axis binding antagonist is administered in combination with an effective amount of one or more additional therapeutic agents.

In some cases, the method, PD-1 axis binding antagonist for use, pharmaceutical composition for use, or use further comprises administering an additional therapeutic agent to the patient. In some cases, the additional therapeutic agent is selected from the group consisting of: immunotherapeutic agents, cytotoxic agents, growth inhibitory agents, radiotherapeutic agents, anti-angiogenic agents, and combinations thereof.

In any of the foregoing examples, each dosing cycle may have any suitable length, for example, about 7 days, about 14 days, about 21 days, about 28 days, or longer. In some cases, each dosing cycle is about 14 days. In some cases, each dosing cycle was about 21 days. In some cases, each dosing cycle is about 28 days (e.g., 28 days ± 3 days).

The patient is preferably a human.

As a general proposal, a therapeutically effective amount of a PD-1 axis binding antagonist (e.g., alemtuzumab) administered to a human will be in the range of about 0.01 to about 50mg/kg patient body weight, whether by one or more administrations.

In some exemplary embodiments, the PD-1 axis binding antagonist is administered at a dose of about 0.01 to about 45mg/kg, about 0.01 to about 40mg/kg, about 0.01 to about 35mg/kg, about 0.01 to about 30mg/kg, about 0.01 to about 25mg/kg, about 0.01 to about 20mg/kg, about 0.01 to about 15mg/kg, about 0.01 to about 10mg/kg, about 0.01 to about 5mg/kg, or about 0.01 to about 1mg/kg, e.g., daily, weekly, biweekly, every three weeks, or every four weeks.

In one instance, the PD-1 axis binding antagonist is administered to a human at a dose of about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, about 1400mg, or about 1500 mg. In some cases, the PD-1 axis binding antagonist may be administered at a dose of about 1000mg to about 1400mg every three weeks (e.g., about 1100mg to about 1300mg every three weeks, e.g., about 1150mg to about 1250mg every three weeks).

In some cases, a total of 1 to 50 doses of the PD-1 axis binding antagonist are administered to a patient, e.g., 1 to 50 doses, 1 to 45 doses, 1 to 40 doses, 1 to 35 doses, 1 to 30 doses, 1 to 25 doses, 1 to 20 doses, 1 to 15 doses, 1 to 10 doses, 1 to 5 doses, 2 to 50 doses, 2 to 45 doses, 2 to 40 doses, 2 to 35 doses, 2 to 30 doses, 2 to 25 doses, 2 to 20 doses, 2 to 15 doses, 2 to 10 doses, 2 to 5 doses, 3 to 50 doses, 3 to 45 doses, 3 to 40 doses, 3 to 35 doses, 3 to 30 doses, 3 to 25 doses, 3 to 20 doses, 3 to 15 doses, 3 to 10 doses, 3 to 5 doses, 4 to 50 doses, 4 to 45 doses, 4 to 40 doses, 4 to 35 doses, 4 to 30 doses, 4 to 25 doses, 4 to 20 doses, 4 to 15 doses, 4 to 10 doses, 4 to 5 doses, 5 to 50 doses, 5 to 45 doses, 5 to 40 doses, 5 to 35 doses, 5 to 30 doses, 5 to 25 doses, 5 to 20 doses, 5 to 15 doses, 5 to 10 doses, 10 to 50 doses, 10 to 45 doses, 10 to 40 doses, 10 to 35 doses, 10 to 30 doses, 10 to 25 doses, 10 to 20 doses, 10 to 15 doses, 15 to 50 doses, 15 to 45 doses, 15 to 40 doses, 15 to 35 doses, 15 to 30 doses, 15 to 25 doses, 20 to 50 doses, 20 to 45 doses, 20 to 40 doses, 20 to 35 doses, 20 to 30 doses, 20 to 25 doses, 25 to 50 doses, 25 to 45 doses, 25 to 40 doses, 25 to 35 doses, 25 to 30 doses, 30 to 50 doses, 30 to 45 doses, 30 to 40 doses, 30 to 35 doses, 35 to 50 doses, 35 to 45 doses, 35 to 40 doses, 40 to 50 doses, 40 to 45 doses, or 45 to 50 doses. In certain cases, the dose may be administered intravenously. In some cases, a total of 16 doses of the PD-1 axis binding antagonist are administered to the patient. In other cases, a total of 12 doses of the PD-1 axis binding antagonist are administered to the patient.

In some cases, the alemtuzumab is administered intravenously to the patient at a dose of about 840mg every 2 weeks, at a dose of about 1200mg every 3 weeks, or at a dose of about 1680mg every 4 weeks. In some cases, the alemtuzumab is administered intravenously to the patient at a dose of 840mg every 2 weeks. In some cases, the alemtuzumab is administered intravenously to the patient at a dose of 1200mg every 3 weeks. In some cases, the alemtuzumab is administered on day 1 of each 21-day (±3-day) cycle for 16 cycles or one year, whichever occurs first. In some cases, the alemtuzumab is administered intravenously to the patient at a dose of 1680mg every 4 weeks. In some cases, the alemtuzumab is administered on day 1 of each 28-day (±3-day) cycle for 12 cycles or one year, whichever occurs first.

The PD-1 axis binding antagonist and/or any additional therapeutic agent may be administered in any suitable manner known in the art. For example, the PD-1 axis binding antagonist and/or any additional therapeutic agent may be administered sequentially (on different days) or simultaneously (on the same day or within the same treatment cycle). In some cases, the PD-1 axis binding antagonist is administered prior to the additional therapeutic agent. In some cases, the PD-1 axis binding antagonist is administered after the additional therapeutic agent. In some cases, the PD-1 axis binding antagonist and/or the additional therapeutic agent may be administered on the same day. In some cases, the PD-1 axis binding antagonist may be administered prior to the additional therapeutic agent administered on the same day. For example, a PD-1 axis binding antagonist may be administered prior to chemotherapy on the same day. In another example, a PD-1 axis binding antagonist may be administered on the same day prior to chemotherapy and another drug (e.g., bevacizumab). In other cases, the PD-1 axis binding antagonist may be administered after additional therapeutic agents administered on the same day. In other cases, the PD-1 axis binding antagonist is administered at the same time as the additional therapeutic agent. In some cases, the PD-1 axis binding antagonist is in a separate composition from the additional therapeutic agent. In some cases, the PD-1 axis binding antagonist is in the same composition as the additional therapeutic agent. In some cases, the PD-1 axis binding antagonist is administered via an intravenous line separate from any other therapeutic administered to the patient on the same day.

The PD-1 axis binding antagonist and any additional therapeutic agent may be administered by the same route of administration or by different routes of administration. In some cases, the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some cases, the additional therapeutic agent is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

In preferred embodiments, the PD-1 axis binding antagonist is administered intravenously. In one example, the alemtuzumab can be administered intravenously over 60 minutes; if the first infusion can be tolerated, all subsequent infusions can be delivered over 30 minutes. In some examples, the PD-1 axis binding antagonist is not administered as an intravenous bolus or bolus injection.

Also provided herein are methods for treating urothelial cancer in a patient comprising administering to the patient a treatment regimen comprising an effective amount of a PD-1 axis binding antagonist (e.g., alemtuzumab) in combination with another anticancer agent or cancer therapy. For example, a PD-1 axis binding antagonist may be administered in combination with: additional chemotherapeutics or chemotherapeutics (see definition above); targeted therapies or targeted therapeutic agents; immunotherapy or immunotherapeutic agents, e.g., monoclonal antibodies; one or more cytotoxic agents (see definition above); or a combination thereof. For example, the PD-1 axis binding antagonist can be administered in combination with bevacizumab, paclitaxel, protein-bound paclitaxel (e.g., albumin-bound paclitaxel), carboplatin, cisplatin, pemetrexed, gemcitabine, etoposide, cobicitinib (cobimeinib), vemurafenib, or a combination thereof.

For example, when administered with chemotherapy with or without bevacizumab, alemtuzumab may be administered prior to chemotherapy and bevacizumab at a dose of 1200mg every 3 weeks. In another example, after completion of 4 to 6 cycles of chemotherapy, and if bevacizumab is discontinued, atrazumab may be administered at a dose of 840mg every 2 weeks, 1200mg every 3 weeks, or 1680mg every four weeks. In another example, alemtuzumab can be administered at a dose of 840mg followed by 100mg/m ² Protein-bound paclitaxel (e.g., albumin-bound paclitaxel); for each 28 day cycle, alemtuzumab was administered on days 1 and 15, and protein-bound paclitaxel was administered on days 1, 8, and 15. In another example, when administered with carboplatin and etoposide, atraumatin may be administered at a dose of 1200mg every 3 weeks prior to chemotherapy. In yet another example, after completion of 4 cycles of carboplatin and etoposide, atraumatic bead mab may be administered at a dose of 840mg every 2 weeks, 1200mg every 3 weeks, or 1680mg every 4 weeks. In another example, after a 28-day period of cobicitinib and vemurafenib is completed, the abzhuzumab may be administered orally once daily at a dose of 840mg every 2 weeks with cobicitinib (21 days for administration, 7 days for withdrawal) at a dose of 60mg and at A dose of 720mg is administered orally twice daily with vemurafenib.

In some cases, the treatment may further include additional therapies. Any suitable additional therapy known in the art or described herein may be used. The additional therapy may be radiation therapy, surgery, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, gamma radiation, or a combination of the above.

In some cases, the additional therapy is administration of side-effect limiting agents (e.g., agents intended to reduce the occurrence and/or severity of therapeutic side-effects, e.g., anti-nausea agents, corticosteroids (e.g., prednisone or equivalent, e.g., at a dose of 1 to 2 mg/kg/day), hormone replacement drugs, etc.).

Evaluation of PD-L1 expression

The expression of PD-L1 in a patient treated according to any of the methods and compositions described herein for use can be assessed. The methods and compositions for use may include determining the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from a patient. In other examples, the PD-L1 expression level in a biological sample (e.g., a tumor sample) obtained from the patient has been determined prior to initiation of the treatment or after initiation of the treatment. PD-L1 expression may be determined using any suitable method. PD-L1 expression can be determined, for example, as described in U.S. patent application Ser. Nos. 15/787,988 and 15/790,680. Any suitable tumor sample may be used, for example, formalin Fixed and Paraffin Embedded (FFPE) tumor samples, archived tumor samples, fresh tumor samples, or frozen tumor samples.

For example, PD-L1 expression can be determined from the percentage of tumor samples occupied by tumor-infiltrating immune cells expressing detectable levels of PD-L1 expression, as the percentage of tumor-infiltrating immune cells expressing detectable levels of PD-L1 expression in a tumor sample, and/or as the percentage of tumor cells expressing detectable levels of PD-L1 expression in a tumor sample. It will be appreciated that in any of the foregoing examples, the percentage of tumor sample occupied by tumor-infiltrating immune cells can be the percentage of tumor area covered by tumor-infiltrating immune cells in a section of tumor sample obtained from a patient, e.g., as assessed by IHC using an anti-PD-L1 antibody (e.g., SP142 antibody). Any suitable anti-PD-L1 antibody may be used, including, for example, SP142 (Ventana), SP263 (Ventana), 22C3 (Dako), 28-8 (Dako), E1L3N (Cell Signaling Technology), 4059 (ProSci, inc.), H5H1 (Advanced Cell Diagnostics), and 9a11. In some examples, the anti-PD-L1 antibody is SP142. In other examples, the anti-PD-L1 antibody is SP263.

In some examples, a tumor sample obtained from a patient has a detectable level of PD-L1 expression in less than 1% of tumor cells in the tumor sample, in 1% or more of tumor cells in the tumor sample, in 1% to less than 5% of tumor cells in the tumor sample, in 5% or more of tumor cells in the tumor sample, in 5% to less than 50% of tumor cells in the tumor sample, or in 50% or more of tumor cells in the tumor sample.

In some examples, a tumor sample obtained from a patient has a detectable level of PD-L1 expression in tumor-infiltrating immune cells that occupy less than 1% of the tumor sample, greater than 1% of the tumor sample, 1% to less than 5% of the tumor sample, greater than 5% of the tumor sample, 5% to less than 10% of the tumor sample, or greater than 10% of the tumor sample.

In some examples, tumor samples may be scored for PD-L1 positives in tumor-infiltrating immune cells and/or tumor cells according to the diagnostic assessment criteria shown in table a and/or table B, respectively.

TABLE A infiltrating Immune Cell (IC) IHC diagnostic criteria

TABLE B Tumor Cell (TC) IHC diagnostic criteria

PD-1 axis binding antagonists

PD-1 axis binding antagonists may include PD-L1 binding antagonists, PD-1 binding antagonists, and PD-L2 binding antagonists. Any suitable PD-1 axis binding antagonist may be used.

PD-L1 binding antagonists

In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. In other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In still other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. The PD-L1 binding antagonist may be, but is not limited to, an antibody, antigen binding fragment thereof, immunoadhesin, fusion protein, oligopeptide or small molecule. In some cases, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 (e.g., GS-4224, INCB086550, MAX-10181, INCB090244, CA-170, or ABSK 041). In some cases, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 and VISTA. In some cases, the PD-L1 binding antagonist is CA-170 (also known as AUPM-170). In some cases, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 and TIM 3. In some cases, the small molecule is a compound described in WO 2015/033301 and WO 2015/033299.

In some cases, the PD-L1 binding antagonist is an anti-PD-L1 antibody. Various anti-PD-L1 antibodies are contemplated and described herein. In any case herein, the isolated anti-PD-L1 antibody can bind to human PD-L1 (e.g., human PD-L1 shown in UniProtKB/Swiss-Prot accession No. Q9NZQ7-1, or a variant thereof). In some cases, the anti-PD-L1 antibody is capable of inhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1. In some cases, the anti-PD-L1 antibody is a monoclonal antibody. In some cases, the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, fab '-SH, fv, scFv, and (Fab') 2 fragments. In some cases, the anti-PD-L1 antibody is a humanized antibody. In some cases, the anti-PD-L1 antibody is a human antibody. Exemplary anti-PD-L1 antibodies include alemtuzumab, MDX-1105, MEDI4736 (Devaluzumab), MSB0010718C (Avmumab), SHR-1316, CS1001, en Wo Lishan antibody, TQB2450, ZKAB001, LP-002, CX-072, IMC-001, KL-A167, APL-502, ke Xili mab, lodendmab, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, RC98, PDL-GEX, KD036, KY1003, YBL-007, and HS-636. Examples of anti-PD-L1 antibodies and methods for their preparation that can be used in the methods of the invention are described in international patent application publication No. WO 2010/077634 and U.S. patent No. 8,217,149, each of which is incorporated herein by reference in its entirety.

In some cases, the anti-PD-L1 antibody comprises:

(a) Sequences of HVR-H1, HVR-H2 and HVR-H3 of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4) and RHWPGGFDY (SEQ ID NO: 5), respectively, and

(b) HVR-L1, HVR-L2 and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7) and QQYLYHPAT (SEQ ID NO: 8), respectively.

In one embodiment, the anti-PD-L1 antibody comprises:

(a) A heavy chain variable region (VH) comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR RHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 9), and

(b) A light chain variable region (VL) comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQG TKVEIKR (SEQ ID NO: 10).

In some cases, the anti-PD-L1 antibody comprises (a) a VH comprising an amino acid sequence that hybridizes with SEQ ID NO:9 (e.g., an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity), or comprising SEQ ID NO: 9; (b) VL comprising a sequence identical to SEQ ID NO:10 (SEQ ID NO): 10; or (c) a VH as described in (a) and a VL as described in (b).

In one embodiment, the anti-PD-L1 antibody comprises alemtuzumab, which comprises:

(a) The following heavy chain amino acid sequences:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1) and

(b) The following light chain amino acid sequences:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:2)。

in some cases, the anti-PD-L1 antibody is avilamab (American Chemical Abstracts (CAS) accession number 1537032-82-8). Avermectin, also known as MSB0010718C, is a human monoclonal IgG1 anti-PD-L1 antibody (Merck KGaA), a pyroxene company.

In some cases, the anti-PD-L1 antibody is Dewaruzumab (CAS registry number 1428935-60-7). Dewaruzumab, also known as MEDI4736, is an Fc-optimized human monoclonal IgG1 kappa anti-PD-L1 antibody (MedImmune, african) described in WO 2011/066389 and US 2013/034559.

In some cases, the anti-PD-L1 antibody is MDX-1105 (BAIMEISHIGULAR Co., ltd. (Bristol Myers Squibb)). MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody as described in WO 2007/005874.

In some cases, the anti-PD-L1 antibody is LY3300054 (elli Lilly).

In some cases, the anti-PD-L1 antibody is STI-A1014 (Sorrento). STI-A1014 is a human anti-PD-L1 antibody.

In some cases, the anti-PD-L1 antibody is KN035 (Suzhou corning jerry corporation (Suzhou Alphamab)). KN035 is a single domain antibody (dAB) generated from a camelid phage display library.

In some cases, the anti-PD-L1 antibody comprises a cleavable moiety or linker that, when cleaved (e.g., by a protease in the tumor microenvironment), activates the antibody antigen binding domain to bind its antigen, e.g., by removing a non-binding spatial portion. In some cases, the anti-PD-L1 antibody is CX-072 (CytomX Therapeutics).

In some cases, the anti-PD-L1 antibody comprises six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and/or a heavy chain variable domain and a light chain variable domain from the anti-PD-L1 antibodies described in the following patents: US 20160108123, WO 2016/000619, WO 2012/145493, US patent No. 9,205,148, WO 2013/181634 or WO 2016/061142.

In a further specific aspect, the anti-PD-L1 antibody has reduced or minimal effector function. In a still further specific aspect, minimal effector function results from an "Fc mutation of a null effector" or a glycosylation free mutation. In a further aspect, the null effector Fc mutation is an N297A or D265A/N297A substitution in the constant region. In a further aspect, the null effector Fc mutation is an N297A substitution in the constant region. In some cases, the isolated anti-PD-L1 antibody is aglycosylated. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid (most typically serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used). Glycosylation sites can be conveniently removed from antibodies by altering the amino acid sequence to remove one of the tripeptide sequences described above (for an N-linked glycosylation site). Variations may be made by substitution of an asparagine, serine or threonine residue within a glycosylation site to another amino acid residue (e.g., glycine, alanine or conservative substitutions).

PD-1 binding antagonists

In some cases, the PD-1 axis binding antagonist is a PD-1 binding antagonist. For example, in some cases, a PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand binding partners. In some cases, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In other cases, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In still other cases, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. The PD-1 binding antagonist may be, but is not limited to, an antibody, antigen binding fragment thereof, immunoadhesin, fusion protein, oligopeptide or small molecule. In some cases, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). For example, in some cases, the PD-1 binding antagonist is an Fc fusion protein. In some cases, the PD-1 binding antagonist is AMP-224.AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor as described in WO 2010/027827 and WO 2011/066342. In some cases, the PD-1 binding antagonist is a peptide or a small molecule compound. In some cases, the PD-1 binding antagonist is AUNP-12 (Pierre Fabre)/Aurigene. See, for example, WO 2012/168944, WO 2015/036927, WO 2015/044900, WO 2015/033303, WO 2013/144704, WO 2013/132317 and WO 2011/161699. In some cases, the PD-1 binding antagonist is a small molecule that inhibits PD-1.

In some cases, the PD-1 binding antagonist is an anti-PD-1 antibody. A variety of anti-PD-1 antibodies may be utilized in the methods and uses disclosed herein. In any of the cases herein, the PD-1 antibody can bind to human PD-1 or a variant thereof. In some casesIn some cases, the anti-PD-1 antibody is a monoclonal antibody. In some cases, the anti-PD-1 antibody is an antibody fragment selected from the group consisting of: fab, fab '-SH, fv, scFv and (Fab') ₂ Fragments. In some cases, the anti-PD-1 antibody is a humanized antibody. In other cases, the anti-PD-1 antibody is a human antibody. Exemplary anti-PD-1 antagonist antibodies include Na Wu Shankang, palbociclizumab, MEDI-0680, PDR001 (Stidazumab), REGN2810 (Simipu Li Shan antibody), BGB-108, paruo Li Shan, carilizumab, xindi Li Shan antibody, tirilizumab, teripu Li Shan antibody, dutarizumab, ralfordin Li Shan antibody, sashan Li Shan antibody, pe An Puli mab, CS1003, HLX10, SCT-I10A, sapalizumab, butelimumab, jenomab, BI 754091, silimumab, YBL-006, BAT1306, HX008, bragg Li Shan antibody, AMG 404, CX-188, JTX-4014, A, sym021, LZM009, F520, SG001, ENUM 244C8, ENUM D4, STI-1110, AK-103 and hAb21.

In some cases, the anti-PD-1 antibody is nivolumab (CAS registry number 946414-94-4). Nawuzumab (Bai Shi Gui Bao/Daye pharmaceutical (Ono)), also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558 andis an anti-PD-1 antibody as described in WO 2006/121168.

In some cases, the anti-PD-1 antibody is palbociclizumab (CAS registry number 1374853-91-4). Parbolizumab (Merck), also known as MK-3475, merck 3475, pembrolizumab, SCH-900475 andis an anti-PD-1 antibody described in WO 2009/114335.

In some cases, the anti-PD-1 antibody is MEDI-0680 (AMP-514; ashikan). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.

In some cases, the anti-PD-1 antibody is PDR001 (CAS registry number 1859072-53-9; north). PDR001 is a humanized IgG4 anti-PD-1 antibody that blocks the binding of PD-L1 and PD-L2 to PD-1.

In some cases, the anti-PD-1 antibody is REGN2810 (Regeneron). REGN2810 is a human anti-PD-1 antibody.

In some cases, the anti-PD-1 antibody is BGB-108 (BeiGene, baiji).

In some cases, the anti-PD-1 antibody is BGB-A317 (Baiji Shenzhou).

In some cases, the anti-PD-1 antibody is JS-001 (Shanghai Junychia). JS-001 is a humanized anti-PD-1 antibody.

In some cases, the anti-PD-1 antibody is STI-A1110 (Soronto Corp.). STI-A1110 is a human anti-PD-1 antibody.

In some cases, the anti-PD-1 antibody is INCSHR-1210 (Incyte). INCSHR-1210 is a human IgG4 anti-PD-1 antibody.

In some cases, the anti-PD-1 antibody is PF-06801591 (gabbro).

In some cases, the anti-PD-1 antibody is TSR-042 (also known as ANB011; tesaro/AnaptysBio).

In some cases, the anti-PD-1 antibody is AM0001 (ARMO Biosciences).

In some cases, the anti-PD-1 antibody is ENUM 244C8 (Enumeral Biomedical Holdings). ENUM 244C8 is an anti-PD-1 antibody that inhibits the function of PD-1 without preventing the binding of PD-L1 to PD-1.

In some cases, the anti-PD-1 antibody is ENUM 388D4 (Enumeral Biomedical Holdings). ENUM 388D4 is an anti-PD-1 antibody that competitively inhibits the binding of PD-L1 to PD-1.

In some cases, the anti-PD-1 antibody comprises six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and/or a heavy chain variable domain and a light chain variable domain from the anti-PD-1 antibodies described in the following patents: WO 2015/112800, WO 2015/112805, WO 2015/112900, US 20150210769, WO2016/089873, WO 2015/035606, WO 2015/085847, WO 2014/206107, WO 2012/145493, US 9,205,148, WO 2015/119930, WO 2015/119923, WO 2016/032927, WO 2014/179664, WO 2016/106160 and WO 2014/194302.

In a further specific aspect, the anti-PD-1 antibody has reduced or minimal effector function. In a still further specific aspect, minimal effector function results from an "Fc mutation of a null effector" or a glycosylation free mutation. In a further aspect, the null effector Fc mutation is an N297A or D265A/N297A substitution in the constant region. In some cases, the isolated anti-PD-1 antibody is aglycosylated.

PD-L2 binding antagonists

In some cases, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some cases, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its ligand binding partner. In a specific aspect, the PD-L2 binding ligand partner is PD-1. The PD-L2 binding antagonist may be, but is not limited to, an antibody, antigen binding fragment thereof, immunoadhesin, fusion protein, oligopeptide or small molecule.

In some cases, the PD-L2 binding antagonist is an anti-PD-L2 antibody. In any of the cases herein, the anti-PD-L2 antibody can bind to human PD-L2 or a variant thereof. In some cases, the anti-PD-L2 antibody is a monoclonal antibody. In some cases, the anti-PD-L2 antibody is an antibody fragment selected from the group consisting of: fab, fab '-SH, fv, scFv and (Fab') ₂ Fragments. In some cases, the anti-PD-L2 antibody is a humanized antibody. In other cases, the anti-PD-L2 antibody is a human antibody. In a further specific aspect, the anti-PD-L2 antibody has reduced or minimal effector function. In a still further specific aspect, minimal effector function results from an "Fc mutation of a null effector" or a glycosylation free mutation. In a further aspect, the null effector Fc mutation is an N297A or D265A/N297A substitution in the constant region. In some cases, the isolated anti-PD-L2 antibody is aglycosylated.

V. detection and assessment of ctDNA

Provided herein are methods for treating urothelial cancer in a patient comprising administering to the patient a treatment regimen comprising a PD-1 axis binding antagonist (e.g., alemtuzumab) that involves determining the presence and/or level of ctDNA in a biological sample obtained from the patient. Related compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture for use are also provided. Any of the methods, compositions for use, kits, or articles of manufacture described herein may relate to any suitable method for detecting ctDNA. In some examples, ctDNA can be detected using a targeting method (e.g., PCR-based methods, cancer personalized profiling by deep sequencing (CAPP-Seq) or Integrated Digital Error Suppression (iDES) CAPP-Seq, TAM-Seq, safe-Seq, or duplex sequencing). In other examples, ctDNA may be detected using non-targeting methods (e.g., digital karyotyping, personalized analysis of rearranged ends (park), or by detecting DNA methylation and/or methylolation in ctDNA).

Any suitable biological sample may be used to detect ctDNA. In some examples, ctDNA can be assessed in blood, serum, or plasma. In certain examples, any of the methods disclosed herein may involve detecting ctDNA in plasma. In other examples, ctDNA may be assessed in a non-blood sample (e.g., cerebrospinal fluid, saliva, sputum, pleural effusion, urine, stool, or semen).

ctDNA may be extracted from a biological sample using any suitable method. For example, blood may be collected in EDTA tubes and/or cell stabilizing tubes (e.g., steck tubes). Blood may be processed within a suitable time after collection from the patient (e.g., within about 2 hours for EDTA tubes or about 4 days for cell-stabilizing tubes (e.g., steck tubes).

As a non-limiting example, ctDNA may be extracted as described in Reinert et al JAMA Oncol.5 (8): 1124-1131, 2019. Briefly, blood samples can be obtained by double centrifugation of blood at room temperature within 2 hours after collection in EDTA tubes, and plasma is centrifuged first at 3000g for 10 minutes and then at 30000g for 10 minutes. Plasma can be aliquoted into 5mL cryotubes and stored at-80 ℃. cfDNA can useThe circulating nucleic acid kit (Qiagen) was extracted and eluted into a DNA suspension (Sigma). The cfDNA sample may, for example, use QUANT-iT ^TM High sensitivity dsDNA assay kit (Invitrogen)) Or using fluorometers (e.g. QUBIT ^TM Fluorometer). Other methods for extracting ctDNA are known in the art.

In some examples, ctDNA may be detected using PCR-based methods, hybridization capture-based methods, methylation-based methods, or fragment histology methods.

In some examples, ctDNA may be detected using a PCR-based method, such as digital PCR (dPCR) (e.g., digital droplet PCR (ddPCR) or BEAMing dPCR). For example, a PCR-based method may involve detecting one or more mutations associated with a cancer (e.g., urothelial cancer), such as by sequencing (e.g., next generation sequencing) or mass spectrometry. PCR-based methods can be targeted or non-targeted. The PCR-based method may involve detecting somatic variants in a set of cancer-associated genes, e.g., a set comprising 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, or more genes. Exemplary PCR-based methods include personalized ctDNA multiplex polymerase chain reaction (mPCR) methods, TAM-SEQ ^TM And Safe-Seq.

In certain examples, a personalized ctDNA multiplex polymerase chain reaction mPCR method can be used to detect ctDNA. In some cases, the personalized ctDNA mPCR method comprises one or more (e.g., 1, 2, 3, or all 4) of the following steps: (a) (i) sequencing DNA obtained from a tumor sample obtained from a patient to produce tumor sequence reads; (ii) Sequencing DNA obtained from a normal tissue sample obtained from the patient to produce a normal sequence read; (b) Identifying one or more patient-specific variants by calling a somatic variant identified from the tumor sequence read and excluding germline variants and/or CHIP variants, wherein the germline variants or CHIP variants are identified from the normal sequence read or from a publicly available database; (c) Designing a mPCR assay for a patient that detects a set of patient-specific variants; and (d) analyzing a biological sample obtained from the patient using the mPCR assay to determine whether ctDNA is present in the biological sample. In some cases, the sequencing is WES or WGS. In some cases, the sequencing is WES. In some cases, the patient-specific variant is SNV or a short indel. In some cases, the patient-specific variant is SNV. In some cases, the set of patient-specific variants includes at least 2 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or more) patient-specific variants. In some cases, the set of patient-specific variants includes at least 1 patient-specific variant. In some cases, the set of patient-specific variants includes at least 2 patient-specific variants. In some cases, the set of patient-specific variants includes at least 8 patient-specific variants. In some cases, the set of patient-specific variants includes 2 to 200 patient-specific variants. In some cases, the set of patient-specific variants includes 8 to 50 patient-specific variants. In some cases, the set of patient-specific variants includes 8 to 32 patient-specific variants. In some cases, the set of patient-specific variants includes 16 patient-specific variants. In some cases, analyzing a biological sample obtained from a patient using a mPCR assay includes sequencing amplicons produced by the mPCR assay to identify patient-specific variants in the biological sample. In some cases, the presence of at least one patient-specific variant in the biological sample identifies the presence of ctDNA in the biological sample. In some cases, the presence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more patient-specific variants in the biological sample identifies the presence of ctDNA in the biological sample. In certain instances, the presence of 2 patient-specific variants in the biological sample identifies the presence of ctDNA in the biological sample. In certain instances, the presence of 0 or 1 patient-specific variant in the biological sample is indicative of the absence of ctDNA in the biological sample.

In some cases, the personalized ctDNA mPCR method is NateractDNA test or ArcherDx Personalized Cancer Monitoring (PCM) ^TM ) And (5) testing. In some examples, the personalized ctDNA mPCR method may be as described in one or more of U.S. patent nos. 10,538,814, 10,557,172, 10,590,482, and/or 10,597,708.

In other examples, ctDNA may be detected using hybridization-based capture methods, for example, by deep sequencing (CAPP-Seq) (see, e.g., newman et al, nat. Med.20 (5): 548-554, 2014) or Integrated Digital Error Suppression (iDES) CAPP-Seq (see, e.g., newman et al, nat. Biotechnol.34 (5): 547-555, 2016) cancer personalized profiling.

In other examples, ctDNA may be detected using methylation or fragment histology methods (e.g., a Guardant LUNAR assay, a GRAIL assay, a Freenome assay, or cell-free methylated DNA immunoprecipitation and high throughput sequencing (cfMeDIP-seq)), see, e.g., nuzzo et al Nature Med.26:1041-1043, 2020). Methylation-based methods can include, for example, whole genome bisulfite sequencing methods or targeted methylation assays. In some examples, methylation methods include targeted methylation assays, such as GRAIL assays (see, e.g., liu et al Annals Oncol.31 (6): 745-759, 2020). In some examples, methylation-based methods may also provide tissue source information (see, e.g., liu et al, supra; and Guo et al Nat. Genet.49 (4): 635-642, 2017).

Evaluation of TMB

Provided herein are methods for treating urothelial cancer (e.g., MIUC) in a patient, comprising administering to the patient a treatment regimen comprising a PD-1 axis binding antagonist (e.g., alemtuzumab) that involves determining tTMB score in a sample obtained from the patient. Any of the methods, compositions for use, kits, or articles of manufacture described hereinOne may involve any suitable method for determining tTMB scores. For example, tTMB scoring can be performed using whole-exome sequencing, whole-genome sequencing, or by using targeted genetic packages (panels) (e.g.,genetic package). For example, in some cases, WES can be used to design personalized mPCR assays to detect ctDNA and determine tTMB scores for patients. In some aspects, tTMB scores may be determined as disclosed in international patent application publication No. PCT/US2017/055669, the disclosure of which is incorporated herein by reference in its entirety. In other aspects, the bTMB score may be determined in a renewal sample obtained from the patient. Any suitable method may be used to determine the bTMB score of the patient. For example, in some aspects, the bTMB score may be determined as described in international patent application publication No. PCT/US2018/043074, the disclosure of which is incorporated herein by reference in its entirety.

In some aspects, a tumor sample obtained from a patient has been determined to have a tissue tTMB score equal to or higher than a reference tTMB score. Any suitable reference tTMB score may be used.

In some cases, the reference tTMB score is a tTMB score in a reference population of individuals having urothelial cancer, wherein the population of individuals consists of a predetermined first subset of individuals who have been treated with PD-1 axis binding antagonist therapy and (i) a second subset of individuals who have not been treated or (ii) have been treated with non-PD-L1 axis binding antagonist therapy that does not include a PD-L1 axis binding antagonist. In some cases, the reference tTMB score significantly separates the first subset of individuals from the second subset of individuals based on a significant difference in responsiveness to treatment with the PD-L1 axis binding antagonist therapy relative to responsiveness to (i) the absence of treatment or (ii) treatment with the non-PD-L1 axis binding antagonist therapy. Responsiveness may be in terms of improved ORR, CR rate, pCR rate, PR rate, improved survival (e.g., DFS, DSS, no distant metastasis survival, PFS and/or OS), improved DOR, improved time to function and QoL deterioration, and/or ctDNA clearance. Improvements (e.g., in terms of remission rate (e.g., ORR, CR, and/or PR), survival (e.g., DFS, DSS, no distant metastasis survival, PFS, and/or OS), DOR, time to improved function and QoL deterioration, and/or ctDNA clearance) may be relative to a suitable reference, such as observation or reference treatment (e.g., treatment that does not include a PD-1 axis binding antagonist (e.g., treatment with a placebo)). In some cases, improvement (e.g., in terms of remission rate (e.g., ORR, CR, and/or PR), survival (e.g., DFS, DSS, distant metastasis free survival, PFS, and/or OS), or DOR) may be relative to observations.

In some cases, the reference tTMB score is a pre-specified tTMB score. In some cases, the reference tTMB score is between about 5 and about 100 mutations per Mb (mut/Mb), e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53 about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100mut/Mb. For example, in some cases, the reference tTMB score is between about 8 and about 30mut/Mb (e.g., about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 mut/Mb). In some cases, the reference tTMB score is between about 10 and about 20mut/Mb (e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mut/Mb). In particular cases, the reference tTMB score may be 10mut/Mb, 16mut/Mb, or 20mut/Mb. In a particular case, the reference tTMB score may be 10mut/Mb. The reference tTMB score may be a tTMB value equivalent to any of the previously pre-assigned tTMB scores.

In some cases, a tumor sample from a patient has a tTMB score of greater than or equal to about 5 mut/Mb. For example, in some cases, tTMB scores from tumor samples are between about 5 and about 100mut/Mb (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53 about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 mut/Mb). In some cases, a tumor sample from a patient has a tTMB score of greater than or equal to about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 mut/Mb. For example, in some cases, a tumor sample from a patient has a tTMB score of greater than or equal to about 10mut/Mb. In some embodiments, the reference tTMB score is 10mut/Mb. In some cases, tTMB score from tumor samples is between about 10 and 100 mut/Mb. In some cases, tTMB score from tumor samples is between about 10 and 20mut/Mb. In some cases, a tumor sample from a patient has a tTMB score of greater than or equal to about 16mut/Mb. In some cases, a tumor sample from a patient has a tTMB score greater than or equal to about 16mut/Mb, and the reference tTMB score is 16mut/Mb. In other cases, a tumor sample from a patient has a tTMB score of greater than or equal to about 20mut/Mb. In some cases, a tumor sample from a patient has a tTMB score greater than or equal to about 20mut/Mb, and the reference tTMB score is about 20mut/Mb.

In some cases, the tTMB score or reference tTMB score is expressed as the number of somatic mutations counted in a specified number of sequenced bases. For example, in some cases, the specified number of sequenced bases is between about 100kb to about 10 Mb. In some cases, the specified number of sequenced bases is about 1.1Mb (e.g., about 1.125 Mb), e.g., as defined byRated by the panel). In some cases, the tTMB score or the reference tTMB score is an equivalent TMB value. In some cases, the equivalent TMB value is determined by WES. In other cases, the equivalent TMB value is determined by WGS.

In some cases, the test simultaneously sequences a coding region of about 300 genes (e.g., of at least about 300 to about 400 genes of different sets, e.g., about 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 genes) that covers at least about 0.05Mb to about 10Mb (e.g., 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Mb), wherein the typical median depth of exon coverage is at least about 500x (e.g., 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x, or 1,000x). In other cases, the test is performed simultaneously on about 400 genes, about 425 genes, about 450 genes, about 475 genes, about 500 genes, about 525 genes, about 550 genes, about 575 genes, about 600 genes, about 625 genes, about 650 genes, about 675 genes, about 700 genes, about 725 genes, about 750 genes, about 775 genes, about 800 genes, about 825 genes, about 850 genes, about 875 genes, about 900 genes, about 925 genes, about 950 genes, about 975 genes, about 1000 genes The coding region of the gene or more than 1000 genes was sequenced. In some cases, the set of genes isThe genome of the genetic package (see, e.g., frampton et al Nat. Biotechnol.31:1023-31,2013, incorporated herein by reference in its entirety). In some cases, the group of genes is +.>CDx Gene set of the Gene pack. In some embodiments, the testing is performed on the genome of the individual at greater than about 10Mb, e.g., greater than about 10Mb, greater than about 15Mb, greater than about 20Mb, greater than about 25Mb, greater than about 30Mb, greater than about 35Mb, greater than about 40Mb, greater than about 45Mb, greater than about 50Mb, greater than about 55Mb, greater than about 60Mb, greater than about 65Mb, greater than about 70Mb, greater than about 75Mb, greater than about 80Mb, greater than about 85Mb, greater than about 90Mb, greater than about 95Mb, greater than about 100Mb, greater than about 200Mb, greater than about 300Mb, greater than about 400Mb, greater than about 500Mb, greater than about 600Mb, greater than about 700Mb, greater than about 800Mb, greater than about 900 Gb, greater than about 1Gb, greater than about 2Gb, greater than about 3 or about 3.3Gb. In some cases, the test simultaneously sequences the coding region of 315 cancer-associated genes and introns from 28 genes that are normally rearranged or altered in cancer, to a median depth of coverage of typically greater than 500-fold. In some cases, each overlaid sequencing read represents a unique DNA fragment to enable highly sensitive and specific detection of genomic changes that occur at low frequencies due to tumor heterogeneity, low tumor purity, and small tissue samples. In other cases, the presence and/or level of somatic mutation is determined by WES. In some cases, the presence and/or level of somatic mutation is determined by WGS.

The tTMB score of a patient may be determined based on the number of somatic changes in a tumor sample obtained from the patient. In some cases, the somatic cell changes to silent mutations (e.g., synonymous changes). In other cases, the somatic cells are changed to non-synonymous SNVs. In other cases, the somatic cell changes to a passenger mutation (e.g., no detectable effect on cloning suitability change). In some cases, somatic changes are not significant Variants (VUS), e.g., changes whose pathogenicity is neither confirmed nor excluded. In some cases, somatic alterations have not been identified as being associated with a cancer phenotype.

In some cases, somatic alterations are not related or known to be associated with effects on cell division, growth, or survival. In other cases, somatic alterations are associated with effects on cell division, growth, or survival.

In some cases, the number of somatic alterations excludes functional alterations between subgenomic intervals.

In some cases, the functional change is an alteration that has an effect on cell division, growth, or survival (e.g., promotes cell division, growth, or survival) as compared to a reference sequence (e.g., a wild-type or unmutated sequence). In some cases, the functional change is identified by including the functional change in a database of functional changes (e.g., a COSIC database) (see Forbes et al nucleic acids Res.43 (D1): D805-D811,2015, the entire contents of which are incorporated herein by reference). In other cases, the functional change is a change with a known functional state (e.g., occurs as a known somatic change in the COSMIC database). In some cases, the functional change is a change that may have a functional state (e.g., a truncation of a tumor suppressor gene). In some cases, the functional change is a driver gene mutation (e.g., a change that provides a selective advantage for cloning in its microenvironment, such as by increasing cell survival or proliferation). In other cases, the functional change is a change that can cause clonal expansion. In some cases, the change in functionality is capable of causing a change in one, two, three, four, five, or all six of the following: (a) self-sufficiency of the growth signal; (b) reduced, e.g., insensitivity to counter growth signals; (c) reduced apoptosis; (d) increased replication potential; (e) sustained angiogenesis; or (f) tissue invasion or metastasis.

In some cases, the functional change is not a passenger mutation (e.g., is not a change with no detectable effect on the cloning suitability of the cell). In some cases, the functional change is not a Variant of Unknown Significance (VUS) (e.g., a change whose pathogenicity is neither confirmed nor excluded).

In certain instances, multiple (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) functional alterations in the preselected tumor genes in the predetermined set of genes are excluded. In some cases, all functional changes in a preselected gene (e.g., a tumor gene) in the predetermined set of genes are excluded. In some cases, multiple functional changes in multiple pre-selected genes (e.g., tumor genes) in the predetermined set of genes are excluded. In some cases, all functional changes in all genes (e.g., tumor genes) in the predetermined set of genes are excluded.

In some cases, the number of somatic alterations excludes germline mutations between subgenomic regions.

In certain instances, the germline is altered to a SNP, base substitution, insertion, deletion, indel, or silent mutation (e.g., synonymous mutation).

In some cases, the germ line alteration is excluded using a method that does not compare to the matched normal sequence. In other cases, germ line changes are excluded by methods that include the use of algorithms. In some cases, the germ line changes are identified by including them in a database of germ line changes (e.g., dbSNP database) (see Shermy et al Nucleic Acids Res.29 (1): 308-311,2001, which is incorporated herein by reference in its entirety). In other cases, germ line changes are identified by including them in two or more counts of the ExAC database (see Exome Aggregation Consortium et al bioRxiv preprint, 10 month 30 2015, incorporated herein by reference in its entirety). In some cases, germ line changes are identified by including them in a 1000 genome project database (McVean et al Nature 491,56-65,2012, incorporated herein by reference in its entirety). In some cases, germline changes are identified by including them in an ESP database (exon variant server, NHLBI GO Exome Sequencing Project (ESP), seattle, WA).

VII pharmaceutical composition and formulation

Also provided herein are pharmaceutical compositions and formulations comprising a PD-1 axis binding antagonist (e.g., alemtuzumab) and optionally a pharmaceutically acceptable carrier.

The pharmaceutical compositions and formulations described herein may be prepared in the form of lyophilized formulations or aqueous solutions by mixing an active ingredient (e.g., a PD-1 axis binding antagonist) of the desired purity with one or more optional pharmaceutically acceptable carriers (see, e.g., remington's Pharmaceutical Sciences, 16 th edition, osol, a. Code (1980)).

An exemplary preparation of alemtuzumab comprises glacial acetic acid, L-histidine, polysorbate 20 and sucrose, at a pH of 5.8. For example, alemtuzumab can be provided in a 20mL vial containing 1200mg of alemtuzumab formulated in glacial acetic acid (16.5 mg), L-histidine (62 mg), polysorbate 20 (8 mg), and sucrose (821.6 mg), at a pH of 5.8. In another example, alemtuzumab can be provided in a 14mL vial containing 840mg of alemtuzumab formulated in glacial acetic acid (11.5 mg), L-histidine (43.4 mg), polysorbate 20 (5.6 mg), and sucrose (575.1 mg), at a pH of 5.8.

Products or kits

In another aspect, provided herein is an article of manufacture or kit comprising a PD-1 axis binding antagonist (e.g., alemtuzumab). In some cases, the article of manufacture or kit further comprises a package insert comprising instructions for using the PD-1 axis binding antagonist to treat or delay progression of urothelial cancer in a patient. In some cases, the article of manufacture or kit further comprises a package insert comprising instructions for using the PD-1 axis binding antagonist in combination with one or more additional therapeutic agents to treat or delay progression of urothelial cancer in a patient. Any PD-1 axis binding antagonist and/or additional therapeutic agent described herein may be included in the article of manufacture or kit.

In some cases, the PD-1 axis binding antagonist and any additional therapeutic agent are in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The container may be formed from a variety of materials, for example glass, plastic (such as polyvinyl chloride or polyolefin) or metal alloys (such as stainless steel or hastelloy). In some cases, the container contains the formulation and a label on or associated with the container may indicate instructions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some cases, the article of manufacture further comprises one or more other agents (e.g., additional chemotherapeutic or antineoplastic agents). Suitable containers for one or more medicaments include, for example, bottles, vials, bags, and syringes.

Any of the articles of manufacture or kits may include instructions for administering a PD-1 axis binding antagonist and/or any additional therapeutic agent to a patient according to the methods described herein, e.g., any of the methods detailed in section II above.

In another aspect, provided herein is an article of manufacture comprising a PD-1 axis binding antagonist and instructions for administering the PD-1 axis binding antagonist for treating urothelial cancer in a patient in need thereof, wherein the treatment comprises administering an effective amount of a treatment regimen comprising the PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy, and wherein the patient has been identified as likely to benefit from the treatment regimen based on the presence of ctDNA in a biological sample obtained from the patient.

In another aspect, provided herein is an article of manufacture comprising a PD-1 axis binding antagonist and instructions for administering the PD-1 axis binding antagonist for treating urothelial cancer in a patient in need thereof, the treatment comprising: (a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and (b) administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy.

In another aspect, provided herein is an article of manufacture comprising a PD-1 axis binding antagonist and instructions for administering the PD-1 axis binding antagonist for treating a patient having urothelial cancer, the patient having been administered at least a first dose of a treatment regimen comprising the PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant therapy or adjuvant therapy, wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen. In some embodiments, the treatment regimen is neoadjuvant therapy. In other embodiments, the treatment regimen is adjuvant therapy.

Examples

Example 1: clinical outcome in ctDNA positive urothelial cancer patients treated with adjuvant immunotherapy

Historically, despite the development of tumor stage, radiological imaging, and tissue-based prognostic biomarkers, it has been difficult to determine after surgery which patients have residual disease and which have healed. Thus, many surgically cured patients are unnecessarily exposed to the toxicity of adjuvant therapy, while other patients with residual disease may not receive additional therapy until disease progression is detected by imaging (an opportunity to timely receive adjuvant therapy for cure purposes may be missed). These limitations can be overcome by making possible early identification of patients carrying minimal residual lesions (MRD) and at highest risk of radiological recurrence, detection of ctDNA shortly after surgical excision. Whether the MRD status as assessed by ctDNA can identify which patients are likely to benefit from adjuvant therapy and which patients can be free of additional therapy has not been studied in a randomized setting.

This example describes the results from IMvigor010 (NCT 02450331), a global, phase III, open-label, randomized trial with alemtuzumab as adjuvant therapy for patients with high risk of Myometrial Invasive Urothelial Cancer (MIUC) of the bladder or upper urinary tract. IMvigor010 showed no significant Disease Free Survival (DFS) benefit nor total survival (OS) benefit in the unselected patients. Thus, this is an ideal setting for studying the following problems: whether MRD (+) patients according to ctDNA (which have a high likelihood of recurrence) can obtain clinical benefit from adjuvant treatment with immune checkpoint inhibition (e.g., with PD-1 axis binding antagonists such as alemtuzumab).

A. Target and endpoint

i. Main efficacy goal

The primary efficacy objective of this study was to evaluate the efficacy of adjuvant therapy with alemtuzumab in MIUC based on DFS (defined by local (pelvic) or urinary tract recurrence, distant UC metastasis or death from any cause), as compared to observations.

Secondary efficacy targets

The secondary efficacy objective of this study was to evaluate the efficacy of adjuvant treatment with atrazumab in MIUC based on OS (defined by time from randomization to death for any reason), compared to observations.

Exploratory efficacy goals

The prospective exploratory goal of this study was to evaluate the utility of ctDNA to identify patients who might benefit from the treatment with atuzumab. ctDNA was measured at the beginning of therapy (C1D 1) and at week 9 (C3D 1).

B. Study design

IMvigor010 is a global, phase III, open-label, randomized, control study aimed at assessing efficacy and safety of adjuvant therapy with atuzumab in patients with MIUC at 809 sites at high risk of recurrence after resection, as compared to observations. The primary endpoint was DFS as assessed by the investigator, which was defined as the time from random grouping to recurrence or death of invasive urothelial cancer.

Patient 1:1 was randomly assigned to either the atuzumab arm or the observation arm. Treatment (or patient experience observations) with alemtuzumab (1200 mg every 3 weeks) for 1 year or until UC recurs or unacceptable toxicity. Disease recurrence was assessed at baseline and once every 12 weeks for 3 years, once every 24 weeks from 4 to 5 years and at 6 years. Disease recurrence assessment of patients in the observation arm followed the same schedule as in the atuzumab arm. There were 809 patients in the study (406 receiving alemtuzumab and 403 receiving observations). ctDNA C1D1 biomarker evaluable population (BEP, 72% of intent-to-treat (ITT) population) included 581 patients.

No intersection is allowed between the arm of the atuzumab and the viewing arm.

Tumor tissue is collected from surgical resection samples, preferably Formalin Fixed Paraffin Embedded (FFPE) tissue blocks (n=138), followed by archiving unstained FFPE tissue sections (n=443). The PD-L1 expression was evaluated centrally using the VENTANA SP IHC assay. Tumors are classified as expressing PD-L1 (IC 2/3 status) when they have a coverage of 5% or more of the tumor area with PD-L1 expressing tumor infiltrating immune cells.

C. Materials and methods

i. Patient(s)

A total of 809 patients were enrolled in the group IMvigor010 study (406 received and 403 received observations). The ctDNA C1D1 BEP (72% of ITT population) included 581 patients.

inclusion criteria

Inclusion criteria requires that patients be at high risk at the time of pathological staging (either pT3-T4a or n+ for patients not treated with neoadjuvant chemotherapy or pT2-T4a or n+) for patients treated with neoadjuvant chemotherapy. The patient needs to have undergone surgical resection (cystectomy or nephroureterectomy) and lymph node dissection, and no evidence of residual disease or metastasis, as confirmed by post-operative radiological imaging negativity.

Study treatment

Treatment (or patient experience observations) with alemtuzumab (1200 mg every 3 weeks) for 1 year or until UC recurs or unacceptable toxicity. Disease recurrence was assessed at baseline and once every 12 weeks for 3 years, once every 24 weeks from 4 to 5 years and at 6 years. Disease recurrence assessment of patients in the observation arm followed the same schedule as in the atuzumab arm. No intersection is allowed between the arm of the atuzumab and the viewing arm.

Metaphase analysis

Median follow-up time was 21.9 months (calculated using the reverse Kaplan-Meier method) ranging from 16 to 45 months. At data expiration, OS follow-up in ITT population is incomplete and ongoing. Mid-term analysis did not reach the median OS; 118 patients (29.1%) in the arm of alemtuzumab and 124 patients (30.8%) in the arm of observation died. 33.3% and 29.6% of relapsed patients in the alemtuzumab arm and the observation arm, respectively, received subsequent cancer treatment. Subsequent cancer therapies include chemotherapy in 25.6% and 24.3% respectively and immunotherapy in 8.6% and 20.3% respectively, and represent the expected treatment pattern for first-line advanced disease.

v. blood collection and treatment

Cycle 1 day 1 (C1D 1) plasma time points were collected at a median time point of 79 days after surgical excision (MIBC patients with IQR of 65 to 92 days), independent of ctDNA levels (fig. 16A to 16D). The time-to-collection analysis was performed only on patients with MIBC, as patients with upper urinary tract UC typically underwent two surgeries. Peripheral Blood Mononuclear Cells (PBMCs) were collected in three 8.5mL ACD tubes at the beginning of C1D1 and outer Zhou Xiejiang in two 6mL EDTA tubes at the beginning of C1D1 and C3D 1. Plasma was separated from the cell pellet within 30 minutes after collection and aliquoted for storage at-80 ℃. Note that the Natera assay used in this study has been validated against frozen plasma for blood samples collected with rotational sedimentation K2-EDTA within 2 hours after collection, but the clinical version of the assay utilized stable cfDNA and allowed for room temperature transport of Streck collection tubes within 7 days. The analysis used a total of 1076 plasma samples from 591 patients (581 from C1D1, 495 from C3D 1), median of plasma used of 3.7mL (IQR 3.2-4.2 mL), and 21.5ng cfDNA per patient (IQR 13.2-34.2 ng). To identify ctDNA in patient plasma, cfDNA extraction and library preparation steps are performed (see, e.g., reinert et al JAMA oncocol.5 (8): 1124-31 (2019)).

Tumor tissue treatment

Tumor tissue is collected from surgical resection samples, preferably Formalin Fixed Paraffin Embedded (FFPE) tissue blocks (n=138), followed by archiving unstained FFPE tissue sections (n=443). UsingGenomic DNA was extracted using the DNA FFPE tissue kit. The PD-L1 expression was evaluated centrally using the VENTANA SP IHC assay. Tumors are classified as expressing PD-L1 (IC 2/3 status) when they have a coverage of 5% or more of the tumor area with PD-L1 expressing tumor infiltrating immune cells. />

Whole exome sequencing of tumor tissue and matched normal DNA

Genomic DNA (gDNA) with a median of 500ng was used for whole exome sequencing workflow both from tumor and normal sources. Library preparation based on Illumina adaptors was performed on the gDNA. Targeted exome capture was then performed using a custom capture probe set targeting about 19,500 genes. These targeting libraries are found in NovaSeq ^TM Sequencing was performed at 2X 100bp on the platform to achieve an average target coverage on deduplication of 180X and related matched normal samples of tumor tissue at 50X. A FastQ file was prepared using bcl2FastQ2 and quality checked using FastQC. Reads were mapped to human reference genome hg19 using a Burrows-Wheeler alignment tool (v.0.7.12) and checked for quality using Picard and multi qc.

Somatic variant callsctDNA assay design

Somatic variant calls were made using the consensus variant call method developed by Natera using tumor tissue input and matched normal sequencing data. Variations previously reported as germline in the public dataset (1000 Genome project, exAC, ESP, dbSNP) were filtered out, and other collections were also filtered out. Normal WES data from paired tumors and matches were first analyzed for quality index and sample consistency and then processed through bioinformatics channels that allow identification of putative clonal somatic single nucleotide variants. Matched normal sequencing was performed to computationally remove putative germline mutations and potential indeterminate clonal hematopoietic mutations. In the candidate pool of tumor DNA specific putative cloned variants per patient, PCR amplicons were designed based on optimized design parameters using a prioritized list of variants, ensuring uniqueness of human genome, amplicon efficiency and primer interactions. Tumor Mutation Burden (TMB) was calculated as the total number of somatic mutations per megabase of the captured exome, and TMB positive patients were patients with ≡10 mutations/Mb (average of ctDNA BEP).

Following plasma cfDNA extraction and library preparation, aliquots of cfDNA library were subjected to multiple targeted PCR followed by amplicon-based sequencing on Illumina platform and reaching each amplicon>100,000 times the average next generation sequencing depth. A patient is considered ctDNA positive when at least 2 or more mutations in the patient's plasma are observed (Coombes et al Clin Cancer Res.25 (14): 4255-4263 (2019)). ctDNA (+) samples also reported the average number of tumor molecules per mL of plasma (sample MTM/mL), which is the average across all variants of tumor molecules per mL of plasma that met QC requirements. NateraAnalytical studies of the assay, as previously published, have been demonstrated to have a variant allele frequency of 0.01%>Sensitivity and high specificity of 95% (Coombes et al Clin Cancer Res.25 (14): 4255-4263 (2019)). />The turnaround time of the assay is (i) 2 to 3 weeks for the first plasma sample, including tissue WES, assay design, and plasma ctDNA analysis/reporting, and (ii) one week for all subsequent plasma treatments and ctDNA analysis/reporting.

RNA treatment

Macroscopic dissection of tumor areas of formalin-fixed paraffin-embedded (FFPE) tissue was performed using hematoxylin and eosin (H & E) as guidance. RNA was extracted using a high purity FFPET RNA isolation kit (Roche) and quantified and quality assessed by a Qubit and Agilent bioanalyzer (Agilent Bioanalyzer). Random primers were used to prime first strand cDNA synthesis from total RNA, and then dUTP was used to replace dTTP in the premix (master mix) to generate second strand cDNA to facilitate preservation of strand information. The library was enriched for mRNA fractions by positive selection using a mixture of biotinylated oligonucleotides corresponding to the coding regions of the genome. Library was sequenced using Illumina sequencing method.

x.RNA-seq data generation and processing

UsingThe RNA Access technology (Illumina) generates a full transcriptome map. The RNA-seq reads are first aligned with the ribosomal RNA sequence to remove the ribosomal reads. The remaining reads were aligned with the ginseng genome (NCBI building 38) using GSNAP (Wu and Nacu. Bioinformation.26 (7): 873-881 (2010); wu et al Methods Mol biol.1418:283-334 (2016)) version 2013-10-10, allowing a maximum of two matches per 75 base sequence (parameters:' -M2-N10-B2-i 1-N1-w 200000-E1-pair max-rna = 200000-clip-overlap). To quantify gene expression levels, the number of reads mapped to each RefSeq gene exon was calculated using the functions provided by the R/Bioconductor package genomics alignments. The original counts were gene length adjusted using a number of transcripts per million reads (TPM) normalization followed by log2 conversion. The raw data and the processed data may be obtained according to a data sharing protocol for patients with available RNA-seq data for n=728 bits. All assays in this study used n=573 patients with RNA-seq and ctDNA data.

Unsupervised mRNA expression clustering

The TCGA subtype was assigned according to the method described previously (Robertson et al cell.171 (3): 540-556.e25 (2017)). Briefly, the RNA expression data of the samples were normalized using M-value normalized pruning averages and transformed using voom, resulting in log per million reads with associated exact weights ₂ Counting. The top 25% most variant genes ranked by standard deviation were selected across all considered samples. Log of 4660 genes ₂ The normalized expression was centered on the median before consensus clustering, and the samples were divided into five clusters. Expression cluster analysis consensus hierarchical clustering method by using distance matrix of 1-CCompleted by the method, element C _ij Represents the Spearman correlation between samples i and j across 4660 genes in R. Consensus matrix M _K K=5 is the number of clusters calculated by iterative standard hierarchical clustering (k×500) times using the average linking option and 80% resampling in sample space. Clustering summarises Robertson et al Cell 171 (3): five different clusters reported in e25 (2017), as shown by the features shown on the heatmap.

xii Gene enrichment analysis (GSEA)

GSEA ranks all genes in the dataset based on differential expression. GSEA was performed and then competition tests were performed using the CAMERA enrichment method (Wu and Smyth.nucleic Acids Res.40 (17): e133 (2012)) to assess whether genes in a given set are top ranked in terms of differential expression relative to genes not in that set. The Hallmark gene set collection from the molecular characterization database (Subramannian et al Proc Natl Acad Sci U S A102 (43): 15545-15550 (2005)) was used to identify the enriched pathways. Including pathways with P values <0.05 after adjustment.

Statistical analysis

ctDNA statistical analysis planning (ctDNA SAP) was planned and finalized before blinding the clinical data analyzed for the primary trial. The main objective of ctDNA studies was to provide evidence that 1) in ctDNA positive patients of C1D1, atrazumab provided improved DFS compared to the observation arm, 2) the presence of ctDNA in plasma of C1D1 was associated with DFS reduction, 3) the presence of ctDNA in plasma of C3D1 was associated with reduced DFS, and 4 a) clearance of ctDNA in plasma was associated with increased DFS at C3D1 and 4 b) clearance occurred at a higher rate in the atrazumab arm compared to the observation arm. Clearance was defined in this study as ctDNA (+) from C1 to ctDNA (-) of C3, and was assessed only in ctDNA (+) patients of C1. Principal analysis used univariate method and classification ctDNA (ctdna+/-). Secondary targets include ctDNA as a continuous variable (average tumor molecules per mL of sample of plasma), as well as multivariate methods adjusted for known risk factors. Secondary endpoints included OS, and secondary biomarkers included clinical and pathological risk factors, PD-L1, TMB, and molecular genetic features from RNAseq. Based on the hierarchical study design, formal testing of OS as a secondary endpoint is not allowed in IMvigor 010. Analysis planning requires significant assessment of the primary analysis at a p-value <0.05 level. Bonferroni correction was applied to p-values of 4 pre-specified primary targets (5 hypotheses total).

The univariate Cox proportional hazards model was used to estimate the risk ratio (HR) of recurrence or death. For completeness (tables 1, 2 and 7) we provide additional estimates for: 1) The stratified Cox model was tuned for lymph node status, PD-L1 status, tumor stage, past neoadjuvant chemotherapy, and number of resected lymph nodes using the same stratification factors as described for IMvigor010 primary clinical analysis (lymph node status, PD-L1 status, and tumor stage), and 2) multivariate Cox regression analysis. All Cox models use a "precision" approach to handling binding event times. The DFS and OS were compared between treatment groups using a log rank test, and the Kaplan-Meier method was applied to the DFS and OS, with 95% CI constructed by Greenwood's formula.

Dfs and OS: ctDNA (+) and ctDNA (-), directed against the atuzumab arm and the observation arm

C1D 1ctDNA status based on C1D1 BEP. C3D1ctDNA status based on C1/C3 BEP.

* A univariate Cox proportional hazards model was pre-specified in the ctDNA statistical analysis program.The hierarchical Cox proportional hazards model was used for IMvigor010 primary analysis. The layering factors are: lymph node status (+or-), PD-L1 status (IC 0/1 or IC 2/3) and tumor stage (.ltoreq.pT2 or pT3/4). / >Multivariate Cox proportional hazards regression analysis was pre-specified in ctDNA statistical analysis plans. The layering factors are: lymph node status (+or-), PD-L1 status (IC 0/1 or IC 2/3), tumor stage (+.pT2 or pT3/4), previous neoadjuvant chemotherapy (yes or no) and lymph node number<10 or more than 10).

Dfs and OS: based on C1D1 ctDNA status, alemtuzumab and observations

* A univariate Cox proportional hazards model was pre-specified in the ctDNA statistical analysis program.The hierarchical Cox proportional hazards model was used for IMvigor010 primary analysis. The layering factors are: lymph node status (+or-), PD-L1 status (IC 0/1 or IC 2/3) and tumor stage (.ltoreq.pT2 or pT3/4). />Multivariate Cox proportional hazards regression analysis was pre-specified in ctDNA statistical analysis plans. The layering factors are: lymph node status (+or-), PD-L1 status (IC 0/1 or IC 2/3), tumor stage (+.pT2 or pT3/4), previous neoadjuvant chemotherapy (yes or no) and lymph node number<10 or more than 10).

Descriptive statistics are used to summarize clinical features, including mean, median, and range of continuous variables, and frequency and percentage of classified variables. Comparison of ctDNA clearance between arms was assessed using Fisher's exact test (two-sided). correlation between ctDNA positivity and baseline prognostic factors was measured using Kruskal-Wallis rank sum test for numerical variables and Fisher accurate test (double sided) for classification variables. The correlation between C1D1 collection time (in days) and ctDNA status was measured using the Wilcoxon test (double sided). All statistical analyses were performed in R.

xiv. ABACUS test design

This clinical trial is not intended to be performed simultaneously with IMvigor 010. The clinical aspects of ABACUS have been previously published (Powles et al Nat Med.25 (11): 1706-1714 (2019)). This ctDNA analysis is exploratory. The test method is briefly described as follows: the present study was an open-label, international, multicenter phase II trial that assessed the efficacy of two cycles (1200 mg q3 w) of preoperative alemtuzumab in patients with histologically confirmed (T2-T4 a) urothelial bladder cancer awaiting planning of a cystectomy. The end points of the design and inclusion criteria were published (Powles et al Nat Med.25 (11): 1706-1714 (2019)). In short, qualification criteria include rejection or inability to perform cisplatin-based neoadjuvant chemotherapy, no evidence of advanced disease, ECOG physical status of 0 or 1, and MIBC patients with sufficient end organ function. The main exclusion criteria included contraindications of the use of immune checkpoint inhibitors once and immunotherapy or cystectomy. All patients provided written informed consent, including the exploratory biomarker endpoint described herein. The study was approved by the institutional review board and ethics board of each participating center and was conducted in accordance with the principles of good clinical practice, regulations of the declaration of helsinki and other applicable local regulations (NCT 02662309). The study was sponsored by university of marie king, london. The litz experimental cancer center clinical trial group (Barts Experimental Cancer Center Clinical Trials Group) is fully responsible for the management of the trial and the daily running of the trial, and the trial is supervised by the Independent Data Monitoring Committee (IDMC). The newly emerging security data is periodically reviewed by the IDMC.

D. Results

IMvigor010 ctDNA biomarker evaluable population

A total of 809 patients were enrolled in the group IMvigor010 study (406 in the atuzumab arm; 403 in the observation arm) with a median follow-up time of 21.9 months. ctDNA C1D1 BEP (72% of ITT population) included 581 patients with a median follow-up time of 23.0 months (fig. 1A). Baseline characteristics of ctDNA BEP populations were comparable and balanced well between arms (table 3), and survival results were as described for DFS (hr=0.88 (0.70-1.11); p=0.2720) (fig. 1B) and OS (hr=0.89 (0.66-1.21)) (fig. 1C).

TABLE 3 comparison of baseline characteristics in period 1 day 1 ctDNA Biomarkers Evaluable Populations (BEPs)

/>

* Immunohistochemical assay according to VENTANA SP 142.Data were missing for eighty-five patients. />Data were missing for one hundred nine patients.

In C1D1, 37% (214/581) of the patients were found to be ctDNA (+). ctDNA positively identified patients at higher risk of disease recurrence compared to ctDNA (-) (observation arm DFS hr=6.3 (4.45-8.92); p < 0.0001) and shorter OS (observation arm hr=8.0 (4.92-12.99)) (fig. 2B and 2D). In the C1D1 ctDNA (+) population, 116 patients in the arm were abzhuzumab and 98 patients in the arm were observed, and baseline characteristics including immune biomarkers were balanced between the arms (table 4). The analysis was repeated using a multivariate method and the results were similar (table 1). The post-operative C1D1 collection time (median 79 days) was independent of higher ctDNA positive rate or higher ctDNA levels (fig. 16A to 16D). There was no difference between the time of collection for ctDNA negative patients and ctDNA positive patients (Wilcoxon p=0.18, double sided). ctDNA positivity of C1D1 was median 4.3 months (ranging from 0.7 to 32.3 months) prior to radiological imaging clinical recurrence (fig. 3).

TABLE 4 balance of baseline characteristics between arms within ctDNA (+) population

/>

/>

* Immunohistochemical assay according to VENTANA SP 142. NA: not usable.

ctDNA positivity of c1d1 was associated with improved DFS compared to observations following treatment with atuzumab

Patients with ctDNA (+) at C1D1 had improved DFS after adjuvant treatment with atuzumab compared to the observed patients (hr=0.58 (0.43-0.79); p=0.0024, median DFS 4.4 and 5.9 months) (fig. 4A). Similarly, this ctDNA (+) patient population had OS (hr=0.59 (0.41-0.86), median OS 15.8 and 25.8 months) improved with atrazumab compared to observations (fig. 4B). For patients that were ctDNA negative (ctDNA (-)), there was no difference in DFS or OS between the two groups (hr=1.14 (0.81-1.62) and hr=1.31 (0.77-2.23) (median was not reached by both populations), respectively). The analysis was repeated using a multivariate method and the results were similar (table 2).

To assess whether other important baseline clinical factors drive these results, exploratory analysis of baseline characteristics including lymph node status, tumor staging, past neoadjuvant chemotherapy, PD-L1 status, and number of resected lymph nodes was performed. Biomarkers can evaluate the subset with improved results after treatment with alemtuzumab not found by univariate analysis among others (fig. 5A-5C). Furthermore, adjustments were made for these clinical features in the multivariate analysis of DFS and OS, confirming that ctDNA can independently identify patients with improved outcome for alemtuzumab (tables 1, 2 and 6). Finally, subgroups in the ctDNA (+) population showed no evidence that a single clinical feature was driving improved outcome in ctDNA (+) patients (fig. 6A, 6B, 7A and 7B).

To support the discovery using binary cut-off values for ctDNA, consecutive ctDNA indices were also evaluated as secondary exploratory targets. Higher thresholds for sample MTM/mL (average number of tumor molecules per mL of plasma) did not identify groups at substantially higher risk of recurrence or death (fig. 13A-13F), indicating that any presence of ctDNA was more relevant than the total burden of ctDNA when identifying high risk patients.

improved DFS in ctDNA (+)/TMB (+) patients and ctDNA (+)/PD-L1 (+) patients

High TMB (tmb+) was unable to predict DFS benefit from atuzumab (hr=0.84 (0.55-1.28)) across all patients in biomarker study (regardless of ctDNA status) (fig. 8A, 9A and 9B). However, ctDNA (+)/TMB (+) patients showed improved DFS risk ratio (hr=0.34 (0.19-0.6)) compared to ctDNA (+)/TMB (-) (hr=0.72 (0.50-1.04)) (fig. 8B). Similar findings were observed when OS was measured in the same population (ctDNA (+)/TMB (+) hr=0.47 (0.22-0.99) and ctDNA (+)/TMB (-) hr=0.63 (0.4-0.97)) (fig. 8C and 8D), and also when the multivariate method was used.

Likewise, PD-L1 high (PD-l1+) status did not boost DFS benefit (hr=1.09 (0.76-1.56)) in biomarker study populations (regardless of ctDNA status) (fig. 8E, 10A, and 10B). However, ctDNA (+)/PD-L1 (+) shows an improved DFS hazard ratio (hr=0.52 (0.33-0.82)) compared to ctDNA (+)/PD-L1 (-) (hr=0.70 (0.46-1.06)) (fig. 8F). When OS was measured in the same population, similar findings were observed (ctDNA (+)/PD-L1 (+) hr=0.46 (0.26-0.82) and ctDNA (+)/PD-L1 (-) hr=0.79 (0.48-1.30)) (fig. 8G and 8H). The multivariate approach gives similar results.

To assess the change in ctDNA status in response to treatment, patients with plasma samples from both C1D1 and C3D1 (485 patients, 60% ITT) were studied. The C1D1/C3D1 BEP was analyzed for imbalance between the arm of the alemtuzumab and the observation group and clinical factors. The baseline characteristics were typically well balanced, with no imbalance found (table 5).

Table 5 comparison of baseline characteristics in C1/C3 BEP

/>

* The patient had ctDNA samples at C1 and C3.Immunohistochemical assay according to VENTANA SP 142.

In C3D1, 38.4% (186/485) patients were found to be ctDNA (+) and these patients were at higher risk of disease progression and recurrence compared to ctDNA (-) (observation arm DFS hr=8.65 (5.67-13.18); p < 0.0001) (fig. 11A to 11D). C3D1 ctDNA positivity is also a negative prognostic factor for OS (observation arm OS hr= 12.74 (6.26-25.93); p < 0.0001). The results were similar when using the multivariable method (table 1).

A change in ctDNA status from baseline (C1D 1) to the time point of treatment (C3D 1); ctDNA clearance associated with improved DFS

ctDNA clearance assessed in patients who were ctDNA (+) at C1D1 and defined as achieving a ctDNA (-) state by C3D1 was quantified and compared between treatment arms. Clearance occurs in patients with subsequent ctDNA (-) to C3D1, while non-clearance occurs in patients with C3D1 that retain ctDNA (+). Clearance was observed in 18.2% (18/99) of patients in the arm of alemtuzumab, in contrast to 3.8% (3/79) in the arm observed (p=0.0204) (fig. 12A). Compared to patients who remained ctDNA positive, patients who cleared ctDNA in the arm of atuzumab had excellent DFS and OS (DFS hr=0.26 (0.12-0.56), p=0.0014, median DFS 5.7 months and unrealized, and OS hr=0.14 (0.03-0.59)) (fig. 12B to 12E and table 6). Similar findings were observed when using the univariate approach (table 7). In general, patients who were ctDNA (-) or cleared ctDNA at both time points had longer DFS than patients who were ctDNA (+) or became ctDNA (+) at both time points (fig. 12A to 12E).

TABLE 6 median DFS and OS for the arm of Ab and the arm of view based on the change in ctDNA status from baseline (C1D 1) to the time point of treatment (C3D 1)

ctDNA kinetics from C1D1 to C3D1, including patients with ctDNA clearance at C1D1 (Pos > Neg) and to C3D1 (Pos > Pos), patients with ctDNA clearance at C1D1 (Pos > Pos), patients with ctDNA (-) and with ctDNA (-) maintained at C1D1 (Neg > Neg), and patients with ctDNA (-) at C1D1 and with ctDNA (+) changed to ctDNA at C3D1 (Neg > Pos), median DFS and OS in the arm for alemtuzumab, and median DFS and OS in the arm of observation.

Dfs and OS: ctDNA clearance and non-clearance of the atuzumab arm and the observation arm

Based on analysis of patients with a C1D1 ctDNA (+) status. * A univariate Cox proportional hazards model was pre-specified in the ctDNA statistical analysis program.The hierarchical Cox proportional hazards model was used for IMvigor010 primary analysis. The layering factors are: lymph node status (+or-), PD-L1 status (IC 0/1 or IC 2/3) and tumor stage (.ltoreq.pT2 or pT3/4). />Multivariate Cox proportional hazards regression analysis was pre-specified in ctDNA statistical analysis plans. LayeringThe factors are: lymph node status (+or-), PD-L1 status (IC 0/1 or IC 2/3), tumor stage (+.pT2 or pT3/4), previous neoadjuvant chemotherapy (yes or no) and lymph node number <10 or more than 10).

Comparing patients with reduced ctDNA levels to patients with elevated ctDNA levels, patients with reduced ctDNA levels in the arm of atuzumab were found to be more frequent (44.4% versus 19.0% in observation). The decrease in ctDNA correlates with improved results (fig. 14A to 14E). The DFS/OS improvement in patients who reduced ctDNA but maintained ctDNA (+) was not as pronounced as that achieved by clearance of ctDNA (fig. 15A-15D).

v. ABACUS ctDNA study supports correlation of ctDNA with clinical outcome under New helper settings

To support the findings of the above work, we explored ctDNA data from a prospective phase II study of neoadjuvant alemtuzumab prior to cystectomy of myometrial invasive urothelial carcinoma (fig. 17A-17C). The clinical characteristics of the patient and efficacy end points of the study have been previously published (Powles et al Nat Med.25 (11): 1706-1714 (2019)). Briefly, 2 cycles of 3 weeks of alemtuzumab were administered followed by cystectomy. The study reached a major endpoint of complete remission of the pathology, ctDNA analysis was exploratory. 40/96 patients had plasma samples available for ctDNA analysis at baseline (pre-neoadjuvant treatment). Samples were taken before and after neoadjuvant alemtuzumab (pre-cystectomy). The same ctDNA method was used in both studies, but no analysis performed simultaneously with IMvigor010 was specified in advance, so the results should be interpreted carefully. At baseline, 62.5% (25/40) of the patient became ctDNA (+), which correlated with poor outcome (fig. 17A-17C). In patients achieving complete pathology relief (pCR) or Major Pathology Relief (MPR), alemtuzumab was correlated with a decrease in ctDNA levels (fig. 12F-12G). Clearance was assessed in patients (n=17) who were ctDNA (+) at baseline and plasma was available after neoadjuvant treatment. In 3/17 (18%) patients, alemtuzumab was associated with ctDNA clearance (fig. 12H). Non-responsive patients did not show significant changes in ctDNA levels. The results of these new helper settings further support a link between ctDNA kinetics and clinical response to alemtuzumab. Thus, these data indicate that ctDNA positives can be used as predictive therapeutic markers for the alemtuzumab response in a new helper setting.

vi. transcription correlation positive for ctDNA, biomarkers in ctDNA (+) population for response to alemtuzumab

To explore the underlying mechanism of the findings described above, exploratory transcriptional analysis was performed on tumors in IMvigor 010. Gene expression profiles correlated with C1D1 ctDNA positivity and clinical recurrence. A linear model was first applied to identify genes differentially expressed between ctDNA (+) and ctDNA (-) patients, and then a pathway enrichment analysis was performed using the Hallmark gene set from MSigDB (Subramannian et al Proc Natl Acad Sci U S A102 (43): 15545-15550 (2005)). Compared to ctDNA (-) patients, tumors from ctDNA (+) are rich in cell cycle and keratin genes (fig. 18A-18B), which may represent a more aggressive cancer phenotype. Of the ctDNA (+) patient population of the atuzumab arms, non-relapsing patients were further enriched for interferon-induced genes, while relapse was associated with angiogenesis and transforming growth factor- β signaling (fig. 18C). Next, PD-L1 and TMB were explored, which have previously been demonstrated to select for responses to immune checkpoint inhibitors across a range of cancers under metastatic settings. Their role in the auxiliary setting is not yet defined. Neither TMB nor PD-L1 in this study identified a subset that benefited from alemtuzumab in the entire patient population (BEP). However, in ctDNA (+) patient populations, TMB (+) and PD-L1 (+) were enriched for improved clinical outcome achieved with atuzumab (fig. 6A, 6B, 7A, 7B, 8D, 8F, 8H and 19A-19D), which was not observed in ctDNA negative patients (fig. 6A, 6B, 7A, 7B, 9A, 9B, 10A, 10B and 20A-20C). In the ctDNA (+) population, the tGE (CD 274, IFNG, CXCL 9) features previously demonstrated to identify responses to alemtuzumab at the transfer setting were also enriched for improved results achieved with alemtuzumab (fig. 18D). Resistance of metastatic urothelial cancer to immunotherapy is associated with high expression of the F-TBRS (pan-fibroblast TGF-beta response) signature. Here, we demonstrated under the helper setting that alemtuzumab was also associated with worse outcome in patients with high F-TBRS (fig. 18E) and high angiogenic features (fig. 18F) in ctDNA (+). These data underscores that predictive biomarkers of response should be interpreted in the context of MRD in a post-operative setting.

Correlation of TCGA subtype and recurrence in ctdna (-) population

TCGA studies of urothelial cancer have identified a subset of molecules with different clinical characteristics (Robertson et al cell.171 (3): 540-556.e25 (2017)). However, it is not clear how these subtypes affect the clinical outcome of random data. Hierarchical clustering summarises the biological features in the TCGA subgroup (fig. 21A), with ctDNA (+) and ctDNA (-) patients in these subgroup-wide BEPs similarly distributed (fig. 22A). In patients not selecting ctDNA, TCGA classification did not identify a subset of patients with improved results achieved with alemtuzumab (fig. 6A, 6B, 7A and 7B). However, in the ctDNA (+) population, the clinical outcome of the basal-squamous subgroup appears to be improved, which subgroup is partially enriched for biomarkers of established response to immunotherapy (FIGS. 6A, 6B, 7A, 7B, 21B-21E and 22A to 22H) (Robertson et al Cell 171 (3): 540-556.e25 (2017)). These findings were not observed in ctDNA (-) patients (fig. 6A, 6B, 7A, 7B, 9A, 9B, 10A, 10B, 20A to 20C and 21B to 21E). These data indicate that TCGA analysis can be used to better predict the outcome of ctDNA (+) patients after surgery.

Due to the subset recurrence of ctDNA (-) patients (30.6% in observations), baseline clinical parameters and molecular characteristics of ctDNA (-) patients in the observation arm were next explored (fig. 21F to 21I). The expression of extracellular matrix (ECM), matrix and tgfβ -induced genes from tumors of recurrent ctDNA (-) patients was increased (fig. 21F to 21G), which may be against any pre-existing immunity. Luminal infiltration TCGA subtype was also most prominent in recurrent ctDNA (-) patients (fig. 21H). While surgery may have been successful in non-recurrent ctDNA (-) patients, gene expression analysis additionally showed increased expression of Interferon (IFN) -induced genes in these patients (fig. 21G), suggesting that pre-existing immunity may also be relevant to preventing recurrence. Finally, the anatomical location of recurrence differs between ctDNA (-) patients and ctDNA (+) patients, where ctDNA (-) recurrence is associated with local recurrence and ctDNA (+) is associated with distant recurrence (fig. 21I). These data underscore that molecular features derived from tumors may affect the relationship between ctDNA status and recurrence.

Discussion of viii

This example demonstrates prospective exploratory analysis of DFS and OS in ctDNA-classified patients for IMvigor010, a phase III trial for assessing PD-L1 inhibitors as adjuvant treatment and post-operative observations in patients with high risk of relapse. Compared to ctDNA (-) patients, the risk of recurrence was increased 6-fold and the risk of death was increased 8-fold in post-operative ctDNA (+) patients. This suggests that ctDNA positivity after surgery may be an indicator of substitution for MRD. In this high risk post-operative ctDNA (+) population, patients receiving alemtuzumab were found to have a reduced recurrence rate of about 42% and a reduced mortality rate of 41% compared to the observations. Furthermore, treatment with two cycles of atuzumab resulted in clearance of ctDNA in 18% of ctDNA (+) patients, whereas 3.8% was observed in the arm. Patients with ctDNA clearance on the arm of atuzumab had persistent DFS compared to patients without clearance. These findings suggest the effect of atuzumab on outcome in ctDNA (+) patients and suggest that ctDNA clearance may be an alternative indicator of therapeutic response. No differences in clinical outcome using alemtuzumab were detected in ctDNA (-) patients, meaning that these lower risk patients (63% of ITT) could be free of adjuvant alemtuzumab treatment. These findings are clinically relevant and the selection of high risk group patients who may benefit from intervention using validated blood tests is of broad appeal in post-operative settings.

Initiating personalized therapy based on the identification of MRD, rather than treating unselected patients or waiting for radiological recurrence, would be a significant change in post-operative cancer treatment. This example reveals substantial improvement in clinical outcome in ctDNA (+) patients treated with auxiliary atuzumab. These individuals are likely to have molecular residual disease after surgery. In addition, a parallel new helper alemtuzumab study (ABACUS study) in UC was also presented, which also showed that ctDNA (+) patients had poor prognosis. With this new helper setting, a decrease in ctDNA levels correlates with the response, supporting findings of helper studies.

Protein and transcriptome biomarker analysis gave the biology behind the positive response to ctDNA and to alemtuzumab, emphasizing the relevance of immunity to the matrix environment. The relationship between tumor-based biomarkers and ctDNA underscores that predictive biomarkers of response should be interpreted in the context of MRD, improving our understanding of disease and response to treatment.

Tissue-based TMB and PD-L1 biomarkers have been previously shown to be useful in predicting responses to immune checkpoint inhibitors, particularly at metastatic settings. In IMvigor010, these tissue-based biomarkers did not identify patients who benefited from alemtuzumab. However, in the ctDNA (+) population, TMB (+) or PD-L1 (+) had improved results achieved with atlizumab compared to TMB (-) or PD-L1 (-). Without wishing to be bound by theory, with the aid of settings, predictive biomarkers of efficacy may be most suitable for patients with post-operative MRD. A portion of the post-operative patients will be in a fully relieved state, so the tissue biomarker status will become insignificant as there is no residual tumor. However, in ctDNA (+) patients, TMB and PD-L1 may provide correlation with efficacy of checkpoint inhibition due to the effect of immunotherapy on residual tumors. PD-L1, TMB and basal squamous transcriptomics features demonstrated that it was possible to enrich ctDNA (+) population for improved results achieved with atuzumab. The multi-platform approach may be the best approach for selecting patients in the future. The principle of identification of treatable postoperative populations by blood drawing is an attractive intervention.

Many studies have evaluated the role of adjuvant therapy in MIUC, but have not demonstrated significant survival benefits. IMvigor010 is such a study; however, improvements in DFS and OS were observed in ctDNA (+) patients treated with atuzumab compared to observations. These findings indicate that personalized immunotherapy may be the best method for treating post-MRD (+) UC. While other adjuvant studies may be positive for DFS benefit in unselected patients, it may be desirable to employ personalized approaches to select MRD (+) patients for immunotherapy to demonstrate OS benefit, as well as to determine MRD (-) patients at lower risk and less likely to benefit from unnecessary therapy. Sequential detection ("monitoring" or "monitoring") can increase the sensitivity of ctDNA detection at the auxiliary setting, which is being explored in prospective assays.

Taken together, this phase III trial shows that post-operative ctDNA testing can identify ctDNA (+) patients who may be at high risk of relapse and death due to MRD. ctDNA (+) patients have elevated ctDNA clearance in the treatment arm and improved outcome when TMB and PD-L1 immune biomarkers are also positive. These new findings indicate that ctDNA is a marker for MRD and response to alemtuzumab and correlate ctDNA with tumor biology. Based on overall data, intervention with additional atuzumab may improve outcome in selected post-operative MIUC patients, supporting atuzumab as an important neoadjuvant treatment option.

Example 2: IMvigor011: phase III, double blind, multicenter, randomized study of alemtuzumab (anti-PD-L1 antibody) and placebo as adjuvant therapy in patients with high risk myolayer invasive bladder cancer who were ctDNA positive following cystectomy

This example describes IMvigor011, a phase III, randomized, placebo-controlled, double-blind study aimed at assessing efficacy and safety in patients with MIBC who were ctDNA positive after cystectomy and at high risk of relapse, adjunctive treatment with alemtuzumab compared to placebo.

A. Target and endpoint

i. Main efficacy goal

The main therapeutic goal of this study was to evaluate alemtuzumab based on the following endpoints

Efficacy compared to placebo:

● Disease Free Survival (DFS) assessed by Independent Review Facility (IRF) in ctDNA positive patients (primary analysis population) within 20 weeks after cystectomy, defined as the time from randomization to the first occurrence of a DFS event defined as any one of the following:

local (pelvic) recurrence of Urothelial Carcinoma (UC) (including soft tissue and regional lymph nodes)

Urinary tract recurrence of UC (including all pathological stages and stages)

Distal transfer of UC

Death due to any cause

Secondary efficacy targets

The secondary efficacy objective of this study was to evaluate the efficacy of alemtuzumab compared to placebo based on the following endpoints:

● Total survival (OS) in ctDNA positive patients (the main analysis population) within 20 weeks after cystectomy, defined as the time from randomization to death for any reason

● DFS for IRF assessment in all randomized patients

● DFS assessed by researchers in a primary analysis population

● Investigator assessed DFS in all randomized patients

● Disease-specific survival, assessed by researchers in a primary analysis population, is defined as the time from randomization to death by UC, based on the assessment of death by the researchers

● The non-distant metastasis survival assessed by researchers in a primary analysis population is defined as the time from randomization to diagnosis of distant (i.e., non-local) metastasis or death for any reason

● The time to functional and quality of life (QoL) deterioration in all randomized populations of the primary analysis population is defined as the time from randomization to the date when the patient's score first drops by ≡10 minutes from baseline in European cancer research and treatment organization (EORTC) quality of life questionnaire-core 30 (QLQ-C30) body function scale, role function scale and Global Health (GHS)/QoL scale (alone)

● ctDNA clearance in the main analysis population, defined as the proportion of patients who were ctDNA positive at baseline and ctDNA negative at either cycle 3, day 1 or cycle 5, day 1

B. Study design

This is a global phase III, randomized, placebo-controlled, double-blind study aimed at assessing efficacy and safety in patients with MIBC who were ctDNA positive after cystectomy and at high risk of relapse with adjuvant treatment with alemtuzumab compared to placebo (see figure 23).

Patients with histologically confirmed invasive urothelial carcinoma of the bladder myolayer (also known as Transitional Cell Carcinoma (TCC)) with an age of > 18 years and an ECOG physical state of < 2 are eligible. Patients with bladder as the primary affected site need to have undergone radical cystectomy and lymph node cleaning. Patients who have received prior NAC are eligible, but need to have a tumor stage of ypT2-4a or ypN + and M0 at the time of pathological examination of the cystectomy specimen. Patients not receiving prior NAC need to be non-conforming or refused treatment with cisplatin-based adjuvant chemotherapy and require tumor staging of pT3-4a or pn+ and M0.

The present study requires tumor tissue specimens and blood collection from qualified patients to prospectively detect the presence of post-operative ctDNA, screening for qualification to enter the monitoring and treatment phases, and for continuing ctDNA clearance analysis or for continuing ctDNA monitoring during the study. Tumor specimens from surgical excision (i.e., radical cystectomy or lymph node sweeping) of patients who have provided informed consent were collected and evaluated for PD-L1 expression by Immunohistochemistry (IHC). Tumor specimens were also subjected to Whole Exome Sequencing (WES). Blood samples were collected to determine normal DNA and ctDNA in the patient's blood. Only patients whose tumors had a sufficient number of viable tumors to undergo WES and that could evaluate PD-L1 expression (as confirmed by the central pathology laboratory prior to patient entry into the group study) were eligible. Tumor specimens from patients were sequenced against matched normal DNA to create a set of multiplex polymerase chain reaction (mPCR) assays for detecting the first 16 clonal mutations specific to tumor tissue of each patient.

All eligible patients with personalized mPCR assays, regardless of plasma ctDNA status, were entered into the monitoring phase of the group study, provided they agreed to participate in the monitoring phase and were free of residual disease as assessed by IRF. Patients may enter the group for a monitoring period of at least 6 weeks but no more than 14 weeks from the day of cystectomy.

Patients in the group monitoring period underwent blood collection for plasma ctDNA testing and monitoring imaging for tumor recurrence. Blood was collected every 6 weeks from the day of entry to week 36 or from the day of cystectomy to week 36, whichever occurred first. After the latest blood collection 36 weeks before from the cystectomy, the blood collection followed a subsequent monitoring imaging schedule. The monitoring period of monitoring imaging was performed every 12 weeks from the day of group entry, until week 84 or until 21 months from the day of cystectomy, whichever occurs first. In the event of a recurrence of the disease assessed by the investigator, the patient stopped the monitoring period.

Patient blood samples collected during the monitoring period were evaluated for the presence of up to 16 mutations identified from the primary tumor. Plasma samples assessed as having 2 or more mutations were considered ctDNA positive. Patients entered the treatment phase of the study and randomized to treatment at the time when the first plasma sample was ctDNA positive, provided that there was no evidence of disease recurrence as assessed by IRF at imaging within 28 days prior to initiation of treatment, and provided that they had agreed to participate in the treatment phase. Only patients positive for ctDNA will enter the treatment period. Patients who are ctDNA negative will continue to undergo monitoring until they are ctDNA positive, ctDNA negative, or have imaging recurrence assessed by the investigator at 21 months from their cystectomy.

During screening, tumor tissue specimens from patients were also subjected to prospective detection of PD-L1 expression by the central laboratory, and PD-L1 status (IHC scores of IC0/1 versus IC 2/3) was used as one of the stratification factors.

Patients entering the treatment period were randomized to one of the following arms at a 2:1 ratio:

● Arm a (experimental arm): infusion of 1680mg IV of alemtuzumab every 4 weeks (Q4W) on day 1 of each 28 day cycle

● Arm B (control arm): placebo IV infusion on day 1Q 4W of each 28 day cycle

Patients with both treatment arms will receive either atractylizumab (1680 mg fixed dose) or 12 cycles or up to 1 year (first come) of treatment matching placebo. Treatment will be administered by IV infusion on day 1 of each 28 day cycle.

In the event of an IRF assessed disease recurrence event, unacceptable toxicity, withdrawal consent or study termination, the alemtuzumab/placebo is discontinued.

Randomization is layered by the following factors:

● Lymph node status (positive and negative)

● Tumor stage after cystectomy (.ltoreq.pT2 and pT3/pT4)

● PD-L1 IHC State (IHC score for IC0/1 and IC 2/3)

PD-L1 expression (IC 2/3, corresponding to the presence of distinguishable PD-L1 staining of any intensity in tumor-infiltrating immune cells covering > 5% of the tumor area occupied by tumor cells, associated intratumoral and continuous peri-tumor stroma) was assessed by a central laboratory assay using VENTANA PD-L1 (SP 142).

● Time from cystectomy to first ctDNA positive sample (.ltoreq.20 weeks and >20 weeks)

Randomization occurred within 14 days after patient plasma samples were confirmed to be ctDNA positive. Study drug administration was started within 4 calendar days after randomization.

All patients entering the treatment period underwent a planned tumor recurrence assessment at baseline and every 9 weeks (±7 days) during the first year after randomization. Once every 9 weeks (±7 days) at 2 years after the treatment/placebo period is completed; once every 12 weeks (±10 days) at 3 rd year; once every 24 weeks (±10 days) at 4 to 5 years; and disease state assessment for tumor recurrence was performed at year 6 (about 48 weeks after the last evaluation of year 5).

Patients that remained ctDNA negative for 21 months from the day of cystectomy were not randomized to treatment and withdrawn from the study.

C. Materials and methods

i. Inclusion criteria

The patient needs to meet the following criteria regarding study entry:

inclusion criteria for monitoring period

● Histologically confirmed bladder MIUC (also referred to as TCC)

Patients with cancer exhibiting mixed histology need to have dominant transitional cell patterns

● TNM classification at the time of pathological examination of the surgical excision specimens (based on AJCC cancer stage manual, 7 th edition; edge et al, 2010) is as follows:

For patients treated with prior NAC: tumor stage is ypT2-4a or ypN + and M0

For patients who did not receive the prior NAC: tumor stage was pT3-4a or pN+ and M0

● Surgical excision of MIUC of bladder

Radical cystectomy may be performed by open, laparoscopic or robotic methods. Cystectomy needs to include bilateral lymph node cleansing, the scope of which is at the discretion of the attending surgeon, but the optimal scope should extend proximally from at least the middle of the common iliac artery to the Cooper ligament, distally, bilaterally to the genital femoral nerve, and downwardly to the obturator nerve. The method of diversion of urine flow for patients undergoing cystectomy is at the discretion of the surgeon and is selected by the patient.

Patients with surgical incision negative (i.e. R0 resection) or carcinoma in situ at the distal ureter or ureter edge are eligible.

Patients with positive R2 (defined as identification of a tumor at the perivesical fat border of the ink marks surrounding the vesical resection specimen) or positive R1 (defined as identification of microscopic disease at the tumor border) were excluded except for carcinoma in situ at the distal ureter or ureter border.

● Patients who have not received prior platinum-based NAC, who have rejected or failed ("unsuitable") cisplatin-based adjuvant chemotherapy

Patients who have received at least three cycles of platinum-containing regimen are considered to have received past NAC.

Non-compliance with cisplatin treatment conditions may be defined by any of the following criteria:

■ Impaired renal function (glomerular filtration rate (GFR) <60 mL/min); GFR should be assessed by direct measurement (i.e. creatinine clearance or ethylenediamine tetraacetate) or, if not available, by serum/plasma creatinine calculation (Cockcroft Gault formula)

■ The hearing loss (measured by audiometry) at two consecutive frequencies was 25dB

■ Grade 2 or more peripheral neuropathy (i.e., sensory changes or abnormalities, including stinging sensation)

■ ECOG physical stamina is 2

● The availability of surgical tumor specimens suitable (e.g., of sufficient quality and quantity) for determining ctDNA status and for exploratory biomarker studies assessed by central laboratory testing. Representative Formalin Fixed Paraffin Embedded (FFPE) tumor blocks were submitted along with associated pathology reports; if available, two FFPE tumor masses are recommended. Fewer than 20 (but not fewer than 16) patients with baseline available slides may still meet the conditions of the study after approval by the medical inspector.

● Tumor tissue specimens were submitted within 10 weeks after cystectomy for ctDNA assay development.

● ctDNA assays were developed based on tumor tissue specimens and matched to normal DNA in blood.

● Post-operative blood samples were submitted for screening to identify somatic mutations in tumor tissue and plasma preparation to determine ctDNA status

● Tumor PD-L1 expression according to IHC and MIUC diagnosis as recorded by focused testing of representative tumor tissue specimens

● The absence of residual lesions and the absence of metastases, as confirmed by negative baseline Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) scans of pelvis, abdomen and chest at no more than 4 weeks prior to group entry.

Confirmation of assessment of the disease-free state by independent central radiological examination of the imaging data

Imaging of the upper urinary tract is required, and may include one or more of the following: intravenous pyelography (IVP), CT urography, renal ultrasound and retrograde pyelography, ureteroscopy or MRI urography. However, if the imaging of the abdomen and pelvis covers the upper urinary tract, there is no need to separately image the upper urinary tract by one of these modes. Imaging must be completed no more than 4 weeks prior to group entry

● Complete recovery within 14 weeks after cystectomy and incorporation into group

The circle must last at least 6 weeks since the operation

Additional inclusion criteria for treatment period

Patients entering the monitoring period need to meet the following criteria to randomize into the treatment period of the study:

● Plasma samples were evaluated as ctDNA positive, defined as the presence of two or more mutations based on patient-personalized ctDNA mPCR assays.

● ECOG physical ability state is less than or equal to 2

● The absence of residual lesions and the absence of metastases, as confirmed by negative baseline CT or MRI scans of pelvis, abdomen and chest no more than 4 weeks prior to randomization.

Confirmation of no pathology was assessed by independent central radiological review of the imaging data.

The upper urinary tract needs to be imaged, which may include one or more of the following: IVP, CT urography, renal ultrasound, retrograde pyelography, ureteroscopy or MRI urography. However, if the imaging of the abdomen and pelvis covers the upper urinary tract, there is no need to separately image the upper urinary tract by one of these modes. Imaging must be completed no more than 4 weeks prior to grouping.

Exclusion criteria

Patients meeting any of the following conditions will be excluded from the study:

● History of severe allergic, anaphylactic or other hypersensitivity reactions to chimeric or humanized antibodies or fusion proteins

● Hypersensitivity to any component of a biopharmaceutical or alemtuzumab formulation produced in chinese hamster ovary cells is known

● Any approved anti-cancer therapy, including chemotherapy or hormonal therapy, was received within 3 weeks prior to study entry into the group.

Allow hormone replacement therapy or oral contraceptives.

● Adjuvant chemotherapy or radiotherapy for UC after cystectomy

Patients who had received primary chemo-radiation to preserve the bladder prior to cystectomy were eligible and would be treated the same as patients who had received prior NAC treatment.

Patients in the monitoring period who met any of the following additional medication-related criteria were excluded from the treatment period:

● Prior treatment with CD137 agonists or immune checkpoint blocking therapies, including anti-CD 40, anti-CTLA-4, anti-PD-1 and anti-PD-L1 therapeutic antibodies

● Treatment with systemic immunostimulants (including but not limited to IFN or IL-2) within 6 weeks prior to cycle 1, day 1, or within 5 half-lives of the drug, whichever is longer

Study treatment

The study drug (IMP) of this study was alemtuzumab. The placebo is identical in appearance to the alemtuzumab and will contain the same excipients but no alemtuzumab drug product. The alemtuzumab/placebo will be administered by IV infusion at a fixed dose of 1680mg on day 1 of each 28 day (±3 day) cycle for 12 cycles or 1 year, whichever is first. This dose level is equivalent to an average weight-based dose of about 20 mg/kg.

Statistical analysis

The primary efficacy endpoint was DFS assessed by IRF, defined as the time from randomization to the first occurrence of DFS events. DFS was analyzed in a primary analysis population, defined as randomized patients with ctDNA positive samples obtained within 20 weeks after cystectomy. The data for patients without DFS events is deleted at the last date that the patient was rated as alive and free of recurrence. Data for patients not undergoing post-baseline disease assessment was deleted on the randomization date.

DFS between treatment arms was compared using a hierarchical log rank test. Original falseThe device and alternative hypotheses may be represented by the survival function S in arm A (Abstrazumab) and arm B (placebo), respectively _A (t) and S _B (t) to express:

H ₀ ：S _A (t)＝S _B (t) and H ₁ ：S _A (t)≠S _B (t)

HR，λ _A /λ _B Wherein lambda is _A And lambda (lambda) _B Representing the risk of DFS events in arm a and arm B, respectively, will be estimated using a hierarchical Cox regression model that has the same hierarchical variables used for the hierarchical log rank test and provides 95% CI. Results of the non-hierarchical analysis will also be provided. HR (HR)<1 indicates that atrazumab is beneficial for therapeutic benefit. The stratification factors of the primary analysis population will include lymph node status, tumor staging after cystectomy, PD-L1 IHC status, and time from cystectomy to first ctDNA positive sample; however, if necessary, layering factors may be combined for analysis purposes to minimize the size of the small layer units.

The type 1 error (. Alpha.) of this study was 0.05 (double sided). The primary endpoint of DFS assessed for IRF of the primary analysis population and the key secondary endpoint of OS and DFS assessed for IRF of all randomized populations controls type 1 errors. To control type 1 errors in IRF assessed DFS and OS endpoints at α=0.05 (double sided), the treatment arms were compared in a hierarchical manner as follows: step 1: DFS assessed for IRF of the main analysis population was assessed at α=0.05 (double sided). Step 2: if the DFS analysis results assessed for IRF of the main analysis population were statistically significant, α=0.05 was passed to OS analysis for the main analysis population. If the DFS results assessed for IRF of the main analysis population are not statistically significant, no formal treatment comparisons of OS are made. Step 3: if OS results in the main analysis population were statistically significant in the mid-term or final OS analysis, α=0.05 was passed to the analysis of DFS assessed for IRF in all randomized populations. If the OS for the primary analysis population results is not statistically significant in either the mid-term or final analysis, no formal treatment comparisons will be made for IRF rated DFS in all randomized populations.

Estimating median DFS per treatment arm using Kaplan-Meier method; a Kaplan-Meier curve was generated. 95% CI of median DFS for each treatment arm was constructed using the Brookmeyer-Crowley method. The DFS rate was estimated by Kaplan-Meier method at different time points per treatment arm (i.e. every 6 months after randomization) and 95% CI was calculated using the grid Lin Wude formula. The 95% CI of the ratio difference between the two arms was estimated using a normal approximation method.

Additional analyses were performed on the DFS endpoint assessed for both IRFs described above, including analysis at selected time points and subgroup analysis.

If both the DFS and OS analysis results for IRF assessment of the primary analysis population are statistically significant, the DFS for IRF assessment is formally analyzed in all randomized populations (i.e., all patients randomized to treatment, regardless of the length of time between cystectomy and ctDNA positive status). In this case, a nominal amount of α (i.e., 0.0001) is assigned to each OS metaphase analysis to maintain a family I error control of the IRF rated DFS in all randomized populations (Haybittle-Peto boundaries). This class I error control method considers non-blind study results prior to analysis of IRF-assessed DFS in all randomized populations, as the primary analysis population is included in the analysis of all randomized populations.

The secondary efficacy endpoint was OS, defined as the time from randomization to death for any reason. OS was analyzed in a primary analysis population, defined as randomized patients with ctDNA positive samples obtained within 20 weeks after cystectomy. The method of comparing OS between treatment arms is the same as that used to compare treatment to the primary efficacy endpoint of IRF assessed DFS. The statistical significance limits for the mid-term and final OS analysis will be determined based on the Lan-DeMets implementation of the function used for O' Brien-Fleming. OS was also analyzed as a exploratory analysis in all randomized populations, using the same method as OS in the main analysis population.

ctDNA clearance was analyzed in the main analysis population, defined as the proportion of patients who were ctDNA positive at baseline and ctDNA negative at cycle 3, day 1 or cycle 5, day 1. The proportion of patients with ctDNA clearance and their 95% CI per treatment arm were calculated using the Clopper Pearson method. The CI for the difference in ratio between the two arms is determined using a normal approximation of the binomial distribution. The ratio was compared between the two arms using the layered Cochran-Mantel-Haenszel test.

Other embodiments

Some embodiments of the techniques described herein may be defined in accordance with any of the following numbered embodiments:

1. a method of treating urothelial cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy, and wherein the patient has been identified as likely to benefit from the treatment regimen based on the presence of circulating tumor DNA (ctDNA) in a biological sample obtained from the patient.

2. A method of treating urothelial cancer in a patient in need thereof, the method comprising:

(a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and

(b) Administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy.

3. A method of identifying a patient having urothelial cancer who is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist, the method comprising determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample identifies the patient as a patient likely to benefit from treatment with a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy.

4. A method of selecting a therapy for a patient having urothelial cancer, the method comprising

(a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and

(b) Selecting a treatment regimen comprising a PD-1 axis binding antagonist for the patient based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy.

5. The method according to embodiment 3 or 4, further comprising administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist.

6. The method according to any one of embodiments 1-5, wherein the biological sample is obtained prior to or concurrently with administration of the first dose of the therapeutic regimen.

7. The method according to embodiment 6, wherein the biological sample is obtained on cycle 1 day 1 (C1D 1) of the treatment regimen.

8. The method according to any one of embodiments 1-7, wherein the biological sample is obtained within about 30 weeks from surgical excision.

9. The method according to embodiment 8, wherein the biological sample is obtained within about 20 weeks from surgical excision.

10. The method according to embodiment 8 or 9, wherein the biological sample is obtained from about 2 to about 20 weeks from surgical excision.

11. The method according to any one of embodiments 1 to 10, wherein the biological sample is a blood sample, a plasma sample, a serum sample, a urine sample, a cerebrospinal fluid (CSF) sample, a nasal swab sample, a saliva sample, a fecal sample, or a vaginal secretion sample.

12. The method of embodiment 11, wherein the biological sample is a plasma sample.

13. A method of monitoring the response of a patient having urothelial cancer, the patient having been administered at least a first dose of a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant or adjuvant therapy, and wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen, the method comprising determining whether ctDNA is present in a biological sample obtained from the patient at a time point after the first dose of the treatment regimen, thereby monitoring the response of the patient.

14. The method of embodiment 13, wherein the absence of ctDNA in the biological sample obtained from the patient at a time point after administration of the first dose of the treatment regimen indicates that the patient is responsive to the treatment regimen.

15. A method of identifying a patient having urothelial cancer who is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant or adjuvant therapy and the patient has been administered at least a first dose of the treatment regimen, and wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen, the method comprising:

determining whether ctDNA is present in a biological sample obtained from the patient at a point in time after administration of the first dose of the treatment regimen, wherein the absence of ctDNA in the biological sample at the point in time after administration of the treatment regimen identifies the patient as likely to benefit from treatment with a treatment regimen comprising a PD-1 axis binding antagonist.

16. The method according to any one of embodiments 13-15, wherein the treatment regimen is adjuvant therapy.

17. The method of any one of embodiments 13-16, wherein the point in time after administration of the first dose of the treatment regimen is cycle 3 day 1 (C3D 1) or cycle 5 day 1 (C5D 1) of the treatment regimen.

18. The method according to any one of embodiments 13 to 17, wherein the biological sample obtained from the patient before or simultaneously with said first dose of the treatment regimen and/or the biological sample obtained from the patient at a point in time after administration of the first dose of the treatment regimen is a blood sample, a plasma sample, a serum sample, a urine sample, a CSF sample, a nasal swab sample, a saliva sample, a stool sample or a vaginal secretion sample.

19. The method of embodiment 18, wherein the biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen and/or the biological sample obtained from the patient at a point in time after administration of the first dose of the treatment regimen is a plasma sample.

20. The method of any one of embodiments 1-12 and 15-19, wherein the benefit is in terms of improved disease-free survival (DFS), improved total survival (OS), improved disease-specific survival, or improved distant metastasis-free survival.

21. The method of embodiment 20 wherein the benefit is in terms of improved DFS.

22. The method of embodiment 20 wherein the benefit is in terms of improved OS.

23. The method of any one of embodiments 20-22, wherein improvement is relative to observation or relative to adjuvant therapy with placebo.

24. The method of any one of embodiments 1 to 23, wherein the presence of ctDNA is determined by a Polymerase Chain Reaction (PCR) -based method, a hybridization capture-based method, a methylation-based method, or a fragment histology method.

25. The method of embodiment 24, wherein the presence of ctDNA is determined by a personalized ctDNA multiplex polymerase chain reaction (mPCR) method.

26. The method of embodiment 25, wherein the personalized ctDNA mPCR method comprises:

(a)

(i) Sequencing DNA obtained from a tumor sample obtained from the patient to produce tumor sequence reads; and

(ii) Sequencing DNA obtained from a normal tissue sample obtained from the patient to produce a normal sequence read;

(b) Identifying one or more patient-specific variants by calling a somatic variant identified from the tumor sequence read and excluding a germline variant or a potent unclonable hematopoietic (CHIP) variant, wherein the germline variant or CHIP variant is identified from the normal sequence read or from a publicly available database;

(c) Designing a mPCR assay for a patient that detects a set of patient-specific variants; and

(d) The mPCR assay is used to analyze a biological sample obtained from the patient to determine whether ctDNA is present in the biological sample.

27. The method of embodiment 26, wherein the sequencing is Whole Exome Sequencing (WES) or Whole Genome Sequencing (WGS).

28. The method of embodiment 27, wherein the sequencing is WES.

29. The method according to any one of embodiments 26 to 28, wherein the patient-specific variant is a Single Nucleotide Variant (SNV) or a short indel.

30. The method according to any one of embodiments 26-29, wherein the set of patient-specific variants comprises at least 2 patient-specific variants.

31. The method of embodiment 30, wherein the set of patient-specific variants comprises 2 to 200 patient-specific variants.

32. The method of embodiment 31, wherein the set of patient-specific variants comprises 16 patient-specific variants.

33. The method according to any one of embodiments 26-32, wherein analyzing a biological sample obtained from the patient using the mPCR assay comprises sequencing amplicons produced by the mPCR assay to identify patient-specific variants in the biological sample.

34. The method of any one of embodiments 25 to 33, wherein the personalized ctDNA mPCR method isctDNA test or ArcherDx Personalized Cancer Monitoring (PCM) ^TM ) And (5) testing.

35. The method according to any one of embodiments 25 to 34, wherein the presence of at least one patient-specific variant in the biological sample identifies the presence of ctDNA in the biological sample.

36. The method of embodiment 35, wherein the presence of two patient-specific variants in the biological sample identifies the presence of ctDNA in the biological sample.

37. The method according to any one of embodiments 1-36, wherein the urothelial carcinoma is Myometrial Invasive Urothelial Carcinoma (MIUC).

38. The method of embodiment 37, wherein the MIUC is Myometrial Invasive Bladder Cancer (MIBC) or myometrial invasive urinary tract urothelial cancer (myometrial invasive UTUC).

39. The method of embodiment 37 or 38, wherein the MIUC is histologically confirmed, and/or wherein the patient has an eastern tumor cooperative group (ECOG) physical status of less than or equal to 2.

40. The method according to any one of embodiments 37-39, wherein the patient has been previously treated with neoadjuvant chemotherapy.

41. The method of embodiment 40, wherein the patient's MIUC is ypT2-4a or ypN + and M0 at the time of surgical excision.

42. The method of any one of embodiments 37-41, wherein the patient did not receive prior neoadjuvant chemotherapy.

43. The method of embodiment 42, wherein the patient is not suitable for cisplatin or has been denied cisplatin-based adjuvant chemotherapy.

44. The method of embodiment 42 or 43, wherein the patient's MIUC is pT3-4a or pN+ and M0 at the time of surgical excision.

45. The method of any one of embodiments 1-41, wherein the patient has undergone surgical resection and lymph node cleaning.

46. The method of embodiment 45, wherein the surgical resection is a cystectomy or a nephroureterectomy.

47. The method according to any one of embodiments 1 to 46, wherein the patient has no evidence of residual disease or metastasis as assessed by post-operative radiological imaging.

48. The method of any one of embodiments 1-47, wherein a tumor sample obtained from the patient has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that account for about 1% or more of the tumor sample.

49. The method of embodiment 48, wherein the tumor sample has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that comprise about 1% or more to less than 5% of the tumor sample.

50. The method of embodiment 48, wherein the tumor sample has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that comprise about 5% or more of the tumor sample.

51. The method of embodiment 50, wherein the tumor sample has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that comprise about 5% or more to less than 10% of the tumor sample.

52. The method of embodiment 48 or 50, wherein a tumor sample obtained from the patient has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that account for about 10% or more of the tumor sample.

53. The method of any one of embodiments 1-47, wherein a tumor sample obtained from the patient has been determined to have a detectable level of PD-L1 expression in less than 1% of tumor-infiltrating immune cells that comprise the tumor sample.

54. The method of any one of embodiments 1-53, wherein a tumor sample obtained from the patient has been determined to have a reference tissue tumor mutation load (tTMB) score equal to or higher than the tTMB score.

55. The method of embodiment 54 wherein the reference tTMB score is a pre-specified tTMB score.

56. The method of embodiment 55, wherein the pre-specified tTMB score is between about 8 and about 30 mut/Mb.

57. The method of embodiment 56, wherein the pre-specified tTMB score is about 10 mutations per megabase (mut/Mb).

58. The method of any one of embodiments 48 to 57, wherein the tumor sample is from a surgical resection.

59. The method of any one of embodiments 1-58, wherein the patient has an increased expression level of one or more genes selected from PD-L1, IFNG, and CXCL9 in a biological sample obtained from the patient relative to a reference expression level of the one or more genes.

60. The method of embodiment 59, wherein the patient has increased expression levels of two or more genes selected from PD-L1, IFNG, and CXCL9 in a biological sample obtained from the patient relative to reference expression levels of the two or more genes.

61. The method of embodiment 60, wherein the patient has increased expression levels of PD-L1, IFNG and CXCL9 in a biological sample obtained from the patient relative to reference expression levels of PD-L1, IFNG and CXCL 9.

62. The method of any one of embodiments 59 to 61, wherein the expression level of PD-L1, IFNG and/or CXCL9 is mRNA expression level.

63. The method of any one of embodiments 1-62, wherein the patient has a reduced expression level of one or more pan F-TBRS genes selected from ACTA2, ACTG2, TAGLN, TNS1, CNN1, TPM1, CTGF, PXDC1, ADAM12, FSTL3, TGFBI, and ADAM19 in a biological sample obtained from the patient relative to a reference expression level of the one or more pan F-TBRS genes.

64. The method of embodiment 63, wherein the patient has reduced expression levels of the at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all twelve pan-F-TBRS genes in a biological sample obtained from the patient relative to reference expression levels of the at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all twelve pan-F-TBRS genes.

65. The method of embodiment 63 or 64, wherein the expression level of the one or more pan F-TBRS genes is mRNA expression level.

66. The method of any one of embodiments 59 to 65, wherein the biological sample obtained from the patient is a tumor sample.

67. The method according to any one of embodiments 1-66, wherein the tumor of the patient has a basal-squamous subtype.

68. The method of embodiment 67, wherein the patient has an increased expression level of one or more genes selected from the group consisting of CD44, KRT6A, KRT, KRT14, COL17A1, DSC3, GSDMC, TGM1, and PI3 relative to a reference expression level of the one or more genes.

69. The method of any one of embodiments 1-68, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist.

70. The method of embodiment 69, wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

71. The method of embodiment 70, wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.

72. The method of embodiment 71, wherein the anti-PD-L1 antibody is alemtuzumab, devaluzumab, avilamab, or MDX-1105.

73. The method of embodiment 72, wherein the anti-PD-L1 antibody is alemtuzumab.

74. The method of example 73, wherein the alemtuzumab is administered intravenously at a dose of 840mg every two weeks.

75. The method of example 73, wherein the alemtuzumab is administered intravenously at a dose of 1200mg every three weeks.

76. The method of example 73, wherein the alemtuzumab is administered intravenously at a dose of 1680mg every four weeks.

77. The method of embodiment 76, wherein the alemtuzumab is administered on day 1 of each 28-day (±3-day) cycle for 12 cycles or one year, whichever occurs first.

78. The method of embodiment 69, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist

79. The method of embodiment 78, wherein the PD-1 binding antagonist is an anti-PD-1 antibody.

80. The method of embodiment 79, wherein the anti-PD-1 antibody is sodium Wu Shankang, palbociclib, MEDI-0680, swabber, cimiput Li Shan, karilib, singal Li Shan, tirelib, terlipl Li Shan, or multi-tarolib.

81. The method of any one of embodiments 1-80, further comprising administering an additional therapeutic agent to the patient.

82. The method of embodiment 81, wherein the additional therapeutic agent is selected from the group consisting of: immunotherapeutic agents, cytotoxic agents, growth inhibitory agents, radiotherapeutic agents, anti-angiogenic agents, and combinations thereof.

83. A PD-1 axis binding antagonist for use in treating urothelial cancer in a patient in need thereof, wherein the treatment comprises administering an effective amount of a treatment regimen comprising the PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy, and wherein the patient has been identified as likely to benefit from the treatment regimen based on the presence of ctDNA in a biological sample obtained from the patient.

84. A PD-1 axis binding antagonist for use in the treatment of urothelial cancer in a patient in need thereof, the treatment comprising:

(a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and

(b) Administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy.

85. A PD-1 axis binding antagonist for use in treating a patient having urothelial cancer, the patient having been administered at least a first dose of a treatment regimen comprising the PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant therapy or adjuvant therapy, wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen.

86. A pharmaceutical composition comprising a PD-1 axis binding antagonist for use in treating urothelial cancer in a patient in need thereof, wherein the treatment comprises administering an effective amount of a treatment regimen comprising the PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy, and wherein the patient has been identified as likely to benefit from the treatment regimen based on the presence of ctDNA in a biological sample obtained from the patient.

87. A pharmaceutical composition comprising a PD-1 axis binding antagonist for use in the treatment of urothelial cancer in a patient in need thereof, the treatment comprising:

(a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and

(b) Administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy.

88. A pharmaceutical composition comprising a PD-1 axis binding antagonist for use in treating a patient having urothelial cancer, the patient having been administered at least a first dose of a treatment regimen comprising the PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant therapy or adjuvant therapy, wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen.

89. Use of a PD-1 axis binding antagonist in the manufacture of a medicament for treating urothelial cancer in a patient in need thereof, wherein the treatment comprises administering an effective amount of a treatment regimen comprising the PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy, and wherein the patient has been identified as likely to benefit from the treatment regimen based on the presence of ctDNA in a biological sample obtained from the patient.

90. Use of a PD-1 axis binding antagonist in the manufacture of a medicament for treating urothelial cancer in a patient in need thereof, the treatment comprising:

(a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and

(b) Administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy.

91. Use of a PD-1 axis binding antagonist in the manufacture of a medicament for treating a patient having urothelial cancer, the patient having been administered at least a first dose of a treatment regimen comprising the PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant therapy or adjuvant therapy, wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen.

92. An article of manufacture comprising a PD-1 axis binding antagonist and instructions for administering the PD-1 axis binding antagonist for treating urothelial cancer in a patient in need thereof, wherein the treatment comprises administering an effective amount of a treatment regimen comprising the PD-1 axis binding antagonist, wherein the treatment regimen is adjuvant therapy, and wherein the patient has been identified as likely to benefit from the treatment regimen based on the presence of ctDNA in a biological sample obtained from the patient.

93. An article of manufacture comprising a PD-1 axis binding antagonist and instructions for administering the PD-1 axis binding antagonist for treating urothelial cancer in a patient in need thereof, the treatment comprising:

(a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and

(b) Administering to the patient an effective amount of a treatment regimen comprising a PD-1 axis binding antagonist based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy.

94. An article of manufacture comprising a PD-1 axis binding antagonist and instructions for administering the PD-1 axis binding antagonist for treating a patient having urothelial cancer, the patient having been administered at least a first dose of a treatment regimen comprising the PD-1 axis binding antagonist, wherein the treatment regimen is neoadjuvant therapy or adjuvant therapy, wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen.

Although the invention has been described in considerable detail by way of illustration and example for the purpose of clarity of understanding, such illustration and example should not be construed to limit the scope of the invention.

Sequence listing

<110> GeneTek company (Genentech, inc.)

<120> methods and compositions for neoadjuvant and adjuvant urothelial cancer therapy

<130> 50474-242WO3

<150> US 63/210,950

<151> 2021-06-15

<150> US 63/120,643

<151> 2020-12-02

<160> 10

<170> patent in version 3.5

<210> 1

<211> 447

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 2

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 3

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 3

Gly Phe Thr Phe Ser Asp Ser Trp Ile His

1 5 10

<210> 4

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 4

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

Lys Gly

<210> 5

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 5

Arg His Trp Pro Gly Gly Phe Asp Tyr

1 5

<210> 6

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 6

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 7

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 7

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210> 8

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 8

Gln Gln Tyr Leu Tyr His Pro Ala Thr

1 5

<210> 9

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 10

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

Claims (83)

1. A method of treating Myometrial Invasive Urothelial Cancer (MIUC) in a patient in need thereof, said method comprising administering to said patient an effective amount of a treatment regimen comprising an anti-PD-L1 antibody, wherein said anti-PD-L1 antibody comprises (a) hypervariable region (HVR) -H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively, wherein said treatment regimen is adjuvant therapy, and wherein said patient has been identified as likely to benefit from said treatment regimen based on the presence of circulating tumor DNA (ctDNA) in a biological sample obtained from said patient.

2. A method of treating MIUC in a patient in need thereof, said method comprising:

(a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody; and

(b) Based on the presence of ctDNA in the biological sample, administering to the patient an effective amount of a treatment regimen comprising an anti-PD-L1 antibody, wherein the treatment regimen is adjuvant therapy, and wherein the anti-PD-L1 antibody comprises (a) HVR-H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively.

3. A method of identifying a patient having MIUC who is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody, the method comprising determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample identifies the patient as likely to benefit from treatment with a treatment regimen comprising an anti-PD-L1 antibody, wherein the treatment regimen is adjuvant therapy, and wherein the anti-PD-L1 antibody comprises (a) HVR-H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively.

4. A method of selecting a therapy for a patient suffering from MIUC, said method comprising

(a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody; and

(b) Selecting a treatment regimen comprising an anti-PD-L1 antibody based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy, and wherein the anti-PD-L1 antibody comprises (a) a sequence of GFTFSDSWIH (SEQ ID NO:

3) AWISPYGGSTYYADSVKG (SEQ ID NO: 4) And RHWPGGFDY (SEQ ID NO: 5) HVR-H1, HVR-H2, and HVR-H3 sequences of (b) are RASQDVSTAVA (SEQ ID NO: 6) SASFLYS (SEQ ID NO: 7) And QQYLYHPAT (SEQ ID NO: 8) HVR-L1, HVR-L2 and HVR-L3 sequences of (c).

5. The method of claim 3 or 4, further comprising administering to the patient an effective amount of a treatment regimen comprising the anti-PD-L1 antibody.

6. The method of any one of claims 1 to 5, wherein the biological sample is obtained prior to or concurrently with administration of the first dose of the therapeutic regimen.

7. The method of claim 6, wherein the biological sample is obtained on cycle 1 day 1 (C1D 1) of the treatment regimen.

8. The method of any one of claims 1-7, wherein the biological sample is obtained within about 30 weeks from surgical excision.

9. The method of claim 8, wherein the biological sample is obtained within about 20 weeks from surgical excision.

10. The method of claim 8 or 9, wherein the biological sample is obtained from about 2 to about 20 weeks after surgical excision.

11. The method of any one of claims 1 to 10, wherein the biological sample is a blood sample, a plasma sample, a serum sample, a urine sample, a cerebrospinal fluid (CSF) sample, a nasal swab sample, a saliva sample, a fecal sample, or a vaginal secretion sample.

12. The method of claim 11, wherein the biological sample is a plasma sample.

13. A method of monitoring a response of a patient having MIUC, said patient having been administered at least a first dose of a treatment regimen comprising an anti-PD-L1 antibody, wherein said treatment regimen is neoadjuvant therapy or adjuvant therapy, and wherein ctDNA is present in a biological sample obtained from said patient prior to or concurrently with said first dose of said treatment regimen, said method comprising determining whether ctDNA is present in a biological sample obtained from said patient at a time point after administration of said first dose of said treatment regimen, thereby monitoring a response of said patient, wherein said anti-PD-L1 antibody comprises (a) HVR-H1, HVR-L2 and HVR-L3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4) and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), sasfys (SEQ ID NO: 7) and QQYLYHPAT (SEQ ID NO: 8), respectively.

14. The method of claim 13, wherein the absence of ctDNA in the biological sample obtained from the patient at a point in time after administration of the first dose of the treatment regimen indicates that the patient is responsive to the treatment regimen.

15. A method of identifying a patient having MIUC who is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody, wherein the treatment regimen is neoadjuvant or adjuvant therapy and the patient has been administered at least a first dose of the treatment regimen, and wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen, the method comprising:

determining whether ctDNA is present in a biological sample obtained from the patient at a point in time after administration of the first dose of the treatment regimen, wherein the absence of ctDNA in the biological sample at a point in time after administration of the treatment regimen identifies the patient as likely to benefit from treatment with a treatment regimen comprising an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody comprises (a) HVR-H1, HVR-H2 and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4) and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) RASQDVSTAVA (SEQ ID NO:

6) SASFLYS (SEQ ID NO: 7) And QQYLYHPAT (SEQ ID NO:

8) HVR-L1, HVR-L2 and HVR-L3 sequences of (c).

16. The method of any one of claims 13 to 15, wherein the treatment regimen is adjuvant therapy.

17. The method of any one of claims 13 to 16, wherein the point in time after administration of the first dose of the therapeutic regimen is cycle 3 day 1 (C3D 1) or cycle 5 day 1 (C5D 1) of the therapeutic regimen.

18. The method of any one of claims 13 to 17, wherein the biological sample obtained from the patient prior to or concurrently with a first dose of the treatment regimen and/or the biological sample obtained from the patient at a point in time after administration of the first dose of the treatment regimen is a blood sample, a plasma sample, a serum sample, a urine sample, a CSF sample, a nasal swab sample, a saliva sample, a fecal sample, or a vaginal secretion sample.

19. The method of claim 18, wherein the biological sample obtained from the patient before or concurrently with a first dose of the treatment regimen and/or the biological sample obtained from the patient at a point in time after administration of the first dose of the treatment regimen is a plasma sample.

20. The method of any one of claims 1 to 12 and 15 to 19, wherein the benefit is in terms of improved disease-free survival (DFS), improved total survival (OS), improved disease-specific survival, or improved distant metastasis-free survival.

21. The method of claim 20, wherein the benefit is in terms of improved DFS.

22. The method of claim 20, wherein the benefit is in terms of improved OS.

23. The method of any one of claims 20 to 22, wherein improvement is relative to observation or relative to adjuvant therapy with placebo.

24. The method of any one of claims 1 to 23, wherein the presence of ctDNA is determined by a Polymerase Chain Reaction (PCR) -based method, a hybrid capture-based method, a methylation-based method, or a fragment histology method.

25. The method of claim 24, wherein the presence of ctDNA is determined by a personalized ctDNA multiplex polymerase chain reaction (mPCR) method.

26. The method of claim 25, wherein the personalized ctDNA mPCR method comprises:

(a)

(i) Sequencing DNA obtained from a tumor sample obtained from the patient to produce tumor sequence reads; and

(ii) Sequencing DNA obtained from a normal tissue sample obtained from the patient to produce a normal sequence read;

(b) Identifying one or more patient-specific variants by calling a somatic variant identified from the tumor sequence reads and excluding germline variants or potent unclonable hematopoietic (CHIP) variants, wherein the germline variants or CHIP variants are identified from the normal sequence reads or from a publicly available database;

(c) Designing a mPCR assay for the patient that detects a set of patient-specific variants; and

(d) Analyzing a biological sample obtained from the patient using the mPCR assay to determine whether ctDNA is present in the biological sample.

27. The method of claim 26, wherein the sequencing is Whole Exome Sequencing (WES) or Whole Genome Sequencing (WGS).

28. The method of claim 27, wherein the sequencing is WES.

29. The method of any one of claims 26 to 28, wherein the patient-specific variant is a Single Nucleotide Variant (SNV) or a short indel.

30. The method of any one of claims 26-29, wherein the set of patient-specific variants comprises at least 2 patient-specific variants.

31. The method of claim 30, wherein the set of patient-specific variants comprises 2 to 200 patient-specific variants.

32. The method of claim 31, wherein the set of patient-specific variants comprises 16 patient-specific variants.

33. The method of any one of claims 26-32, wherein analyzing the biological sample obtained from the patient using the mPCR assay comprises sequencing amplicons produced by the mPCR assay to identify patient-specific variants in the biological sample.

34. The method of any one of claims 25 to 33, wherein the personalized ctDNA mPCR method isctDNA test or ArcherDx Personalized Cancer Monitoring (PCM) ^TM ) And (5) testing.

35. The method of any one of claims 25 to 34, wherein the presence of at least one patient-specific variant in the biological sample identifies the presence of ctDNA in the biological sample.

36. The method of claim 35, wherein the presence of two patient-specific variants in the biological sample identifies the presence of ctDNA in the biological sample.

37. The method of any one of claims 1 to 36, wherein the MIUC is a Myometrial Invasive Bladder Cancer (MIBC) or a myometrial invasive urinary tract urothelial cancer (myometrial invasive UTUC).

38. The method of claim 37, wherein the MIUC is histologically confirmed, and/or wherein the patient has an eastern tumor cooperative group (ECOG) physical status of less than or equal to 2.

39. The method of any one of claims 1 to 12 and 16 to 38, wherein the patient has been previously treated with neoadjuvant chemotherapy.

40. The method of claim 39, wherein the patient's MIUC is ypT2-4a or ypN + and M0 at the time of surgical excision.

41. The method of any one of claims 1 to 40, wherein the patient did not receive anamnesis adjuvant chemotherapy.

42. The method of claim 41, wherein the patient is not suitable for cisplatin or has refused cisplatin-based adjuvant chemotherapy.

43. The method of claim 41 or 42, wherein the patient's MIUC is pT3-4a or pn+ and M0 at the time of surgical excision.

44. The method of any one of claims 1 to 12 and 16 to 43, wherein the patient has undergone surgical resection and lymph node cleaning.

45. The method of claim 44, wherein the surgical resection is a cystectomy or a nephroureterectomy.

46. The method of any one of claims 1 to 45, wherein the patient has no evidence of residual disease or metastasis assessed by post-operative radiological imaging.

47. The method of any one of claims 1 to 46, wherein a tumor sample obtained from the patient has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that account for about 1% or more of the tumor sample.

48. The method of claim 47, wherein the tumor sample has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that comprise about 1% or more to less than 5% of the tumor sample.

49. The method of claim 47, wherein the tumor sample has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that comprise about 5% or more of the tumor sample.

50. The method of claim 49, wherein the tumor sample has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that comprise about 5% or more to less than 10% of the tumor sample.

51. The method of claim 47 or 49, wherein the tumor sample obtained from the patient has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that account for about 10% or more of the tumor sample.

52. The method of any one of claims 1 to 46, wherein a tumor sample obtained from the patient has been determined to have a detectable level of PD-L1 expression in less than 1% of tumor-infiltrating immune cells that comprise the tumor sample.

53. The method of any one of claims 1-52, wherein a tumor sample obtained from the patient has been determined to have a reference tissue tumor mutation load (tTMB) score equal to or higher than a tTMB score.

54. The method of claim 53, wherein the reference tTMB score is a pre-specified tTMB score.

55. The method of claim 54, wherein the pre-specified tTMB score is between about 8 and about 30 mut/Mb.

56. The method of claim 55, wherein the pre-specified tTMB score is about 10 mutations per megabase (mut/Mb).

57. The method of any one of claims 47-56, wherein the tumor sample is from a surgical resection.

58. The method of any one of claims 1 to 57, wherein the patient has an increased expression level of one or more genes selected from PD-L1, IFNG, and CXCL9 in a biological sample obtained from the patient relative to a reference expression level of the one or more genes.

59. The method of claim 58, wherein the patient has increased expression levels of two or more genes selected from PD-L1, IFNG, and CXCL9 in the biological sample obtained from the patient relative to reference expression levels of the two or more genes.

60. The method of claim 59, wherein the patient has increased expression levels of PD-L1, IFNG, and CXCL9 in a biological sample obtained from the patient relative to reference expression levels of PD-L1, IFNG, and CXCL 9.

61. The method of any one of claims 58 to 60, wherein the expression level of PD-L1, IFNG and/or CXCL9 is mRNA expression level.

62. The method of any one of claims 1-61, wherein the patient has a reduced expression level of one or more pan F-TBRS genes selected from ACTA2, ACTG2, TAGLN, TNS1, CNN1, TPM1, CTGF, PXDC1, ADAM12, FSTL3, TGFBI, and ADAM19 in a biological sample obtained from the patient relative to a reference expression level of the one or more pan F-TBRS genes.

63. The method of claim 62, wherein the patient has reduced expression levels of the at least two, at least three, at least four, at least five, at least six, at least eight, at least ten, at least eleven, or all twelve pan-F-TBRS genes in the biological sample obtained from the patient relative to reference expression levels of at least two, at least three, at least four, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all twelve of the pan-F-TBRS genes.

64. The method of claim 62 or 63, wherein said expression level of said one or more pan F-TBRS genes is mRNA expression level.

65. The method of any one of claims 58 to 64, wherein the biological sample obtained from the patient is a tumor sample.

66. The method of any one of claims 1-65, wherein the tumor of the patient has a basal-squamous subtype.

67. The method of claim 66, wherein the patient has increased expression levels of one or more genes selected from CD44, KRT6A, KRT, KRT14, COL17A1, DSC3, GSDMC, TGM1, and PI3 relative to a reference expression level of the one or more genes.

68. The method of any one of claims 1-67, wherein the anti-PD-L1 antibody is alemtuzumab.

69. The method of claim 68, wherein the alemtuzumab is administered intravenously at a dose of 840mg every two weeks.

70. The method of claim 68, wherein the alemtuzumab is administered intravenously at a dose of 1200mg every three weeks.

71. The method of claim 68, wherein the alemtuzumab is administered intravenously every four weeks at a dose of 1680 mg.

72. The method of claim 71, wherein the alemtuzumab is administered on day 1 of each 28-day (±3-day) cycle for 12 cycles or one year, whichever occurs first.

73. The method of any one of claims 1-72, further comprising administering an additional therapeutic agent to the patient.

74. The method of claim 73, wherein the additional therapeutic agent is selected from the group consisting of: immunotherapeutic agents, cytotoxic agents, growth inhibitory agents, radiotherapeutic agents, anti-angiogenic agents, and combinations thereof.

75. An anti-PD-L1 antibody or a pharmaceutical composition comprising an anti-PD-L1 antibody for use in treating MIUC in a patient in need thereof, wherein said treatment comprises administration of an effective amount of a therapeutic regimen comprising an anti-PD-L1 antibody comprising (a) hypervariable region (HVR) -H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively, wherein said therapeutic regimen is adjuvant therapy, and wherein said patient has been identified as likely to benefit from said therapeutic regimen based on the presence of ctDNA in a biological sample obtained from said patient.

76. An anti-PD-L1 antibody or a pharmaceutical composition comprising an anti-PD-L1 antibody for use in treating MIUC in a patient in need thereof, said treatment comprising:

(a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody; and

(b) Administering to the patient an effective amount of a treatment regimen comprising an anti-PD-L1 antibody based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy, and wherein the anti-PD-L1 antibody comprises (a) hypervariable region (HVR) -H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively.

77. An anti-PD-L1 antibody or a pharmaceutical composition comprising an anti-PD-L1 antibody for use in treating a patient suffering from MIUC, said patient having been administered at least a first dose of a treatment regimen comprising an anti-PD-L1 antibody, wherein said anti-PD-L1 antibody comprises (a) hypervariable region (HVR) -H1, HVR-H2 and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4) and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2 and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7) and QQYLYHPAT (SEQ ID NO: 8), respectively, wherein said treatment regimen is neoadjuvant or adjuvant therapy, and wherein ctDNA is present in a biological sample obtained from said patient prior to or concurrent with said first dose of said treatment regimen.

78. Use of an anti-PD-L1 antibody in the manufacture of a medicament for treating MIUC in a patient in need thereof, wherein said treatment comprises administering an effective amount of a treatment regimen comprising an anti-PD-L1 antibody, wherein said anti-PD-L1 antibody comprises (a) hypervariable region (HVR) -H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively, wherein said treatment regimen is adjuvant therapy, and wherein said patient has been identified as likely to benefit from said treatment regimen based on the presence of ctDNA in a biological sample obtained from said patient.

79. Use of an anti-PD-L1 antibody in the manufacture of a medicament for treating MIUC in a patient in need thereof, said treatment comprising:

(a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody; and

(b) Administering to the patient an effective amount of a treatment regimen comprising an anti-PD-L1 antibody based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy, and wherein the anti-PD-L1 antibody comprises (a) hypervariable region (HVR) -H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively.

80. Use of an anti-PD-L1 antibody in the manufacture of a medicament for treating a patient suffering from MIUC, said patient having been administered at least a first dose of a treatment regimen comprising an anti-PD-L1 antibody, wherein said anti-PD-L1 antibody comprises (a) hypervariable region (HVR) -H1, HVR-H2 and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4) and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2 and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7) and QQYLYHPAT (SEQ ID NO: 8), respectively, wherein said treatment regimen is neoadjuvant or adjuvant therapy, and wherein ctDNA is present in a biological sample obtained from said patient prior to or concurrent with said first dose of said treatment regimen.

81. An article of manufacture comprising an anti-PD-L1 antibody and instructions for administering the anti-PD-L1 antibody for treating MIUC in a patient in need thereof, wherein the treatment comprises administering an effective amount of a treatment regimen comprising an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody comprises (a) hypervariable region (HVR) -H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively, wherein the treatment regimen is adjuvant therapy, and wherein the patient has been identified as likely to benefit from the treatment regimen based on the presence of ctDNA in a biological sample obtained from the patient.

82. An article of manufacture comprising an anti-PD-L1 antibody and instructions for administering the anti-PD-L1 antibody for treating MIUC in a patient in need thereof, said treatment comprising:

(a) Determining whether ctDNA is present in a biological sample obtained from the patient, wherein the presence of ctDNA in the biological sample indicates that the patient is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody; and

(b) Administering to the patient an effective amount of a treatment regimen comprising an anti-PD-L1 antibody based on the presence of ctDNA in the biological sample, wherein the treatment regimen is adjuvant therapy, and wherein the anti-PD-L1 antibody comprises (a) hypervariable region (HVR) -H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively.

83. An article of manufacture comprising an anti-PD-L1 antibody and instructions for administering the anti-PD-L1 antibody for treating a patient having MIUC, the patient having been administered at least a first dose of a treatment regimen comprising an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody comprises (a) hypervariable region (HVR) -H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively, and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively, wherein the treatment regimen is neoadjuvant therapy or adjuvant therapy, and wherein ctDNA is present in a biological sample obtained from the patient prior to or concurrent with the first dose of the treatment regimen.