Use of HAL in a method for predicting bladder cancer immunotherapy response

Abstract

The disclosure relates to methods of predicting the likelihood that a subject having bladder cancer responds to immunotherapy, such as BCG therapy.

Description

[0001] Methods

[0002] Technical field

[0003] The disclosure relates to methods of predicting the likelihood that a subject having bladder cancer responds to immunotherapy, such as to BCG therapy.

[0004] Background

[0005] Bladder cancer is any of several types of cancer arising from the tissues of the urinary bladder. The detection and diagnosis of bladder cancer typically involves visual inspection of the inside of the bladder by cystoscopy, which allows the physician to take biopsy samples for pathology assessment of suspicious lesions and to undertake minor remedial work.

[0006] If cystoscopy confirms suspected bladder cancer, for removal of tumors and establishing pathological classification, a transurethral resection (TUR) is typically carried out, i.e. a cystoscopy procedure where lesions are resected using a resection device which is inserted into the bladder through a working channel of the cystoscope and the resected tissue is examined by histopathology for classification and staging.

[0007] The treatment of bladder cancer depends on how deeply the tumor invades into the bladder wall and muscles surrounding the bladder: bladder cancer can be nonmuscle invasive (NMIBC) or muscle invasive (MIBC). In the case of NMIBC, the TUR is in itself the initial surgical treatment, but the disease may in addition be treated with intravesical chemotherapy or immunotherapy. For patients with MIBC, the standard treatment is radical cystectomy, i.e., removal of the bladder, with adjuvant chemotherapy and radiation.

[0008] Since the use of white light in TUR to identify lesions can lead to missing those that are present but not readily visible, such as flat and obscure carcinoma in situ, photodynamic diagnosis / detection (PDD) is often used in such procedures. PDD improves visualization of lesions, increases lesion detection, improves resection quality and thereby reduces recurrence. In general, PDD involves the administration of a photosensitizer or a precursor thereof (which collectively may be referred to herein as a "photosensitizing agent") to an area of interest. The photosensitizer or precursor thereof is taken up into the cells, where a precursor of a photosensitizer is converted into a photosensitizer. Upon exposure of the area of interest to light of a suitable wavelength, the photosensitizer is activated (i.e. excited) and, upon relaxation to its ground state, fluorescence occurs and is detected.

[0009] Hexyl 5-ALA ester (hexaminolevulinate, HAL) and its salts are known precursors of photosensitizers. HAL preferably penetrates rapidly proliferating cells, e.g. tumor cells, where it is converted to porphyrins (e.g. protoporphyrin IX, “PpIX”), which are photosensitizers. Upon exposure of the cells to blue light, PpIX is activated and, upon relaxation to its ground state, PpIX emits red light and thus enables specific and accurate visualization of the tumor. Hexvix® (Photocure ASA, Norway), marketed in the US and Canada as Cysview®, is a commercially available approved drug that comprises HAL and is used in combination with blue light in for the detection of bladder cancer in cystoscopy (“HAL-guided cystoscopy”) and TUR procedures (“HAL-guided TUR”).

[0010] In patients with NMIBC, HAL-guided cystoscopy and TUR has increased detection of both papillary lesions and flat carcinoma in situ. HAL-guided TUR of bladder cancer in patients with NMIBC has further reduced the rate of residual tumors after such procedures and has led to superior recurrence free survival (RFS) rates and prolonged RFS intervals compared to white light TUR alone (see Rink M, et al. Eur Urol 4(64), 2013, 624). Existing European guidelines on NMIBC and several expert groups consensus statements recommend the use of HAL-guided TUR in various settings of management of NMIBC and some even recommend its use in all NMIBC patients at initial TUR (see Witjes JA, et al., Eur Urol 1 (66), 2014, 863).

[0011] HAL and its salts have also been suggested for use in photodynamic therapy (PDT) of bladder cancer (see for instance US 2005 / 0031541 , WO 2017 / 103285, WO 2017 / 103283, WO 2017 / 103280 or Bader et al., Urol. Oncol. Seminars and Original Investigations 31 , 2013, 1178-1183). Similar to PDD, PDT involves the administration of a “photosensitizing agent” to an area of interest followed by activation of the photosensitizer by light of a suitable wavelength to elicit a PDT effect. The therapeutic anti-tumor effect of PDT is, inter alia, based on a phototoxic reaction: the photosensitizer or precursor thereof is taken up into the cells, where a precursor of a photosensitizer is converted into a photosensitizer. Upon exposure of the area of interest to photoactivating light, the photosensitizer is activated, i.e. excited from a ground singlet state to an excited singlet state. It then undergoes intersystem crossing to a longer-lived excited triplet state. One of the few chemical species present in tissue with a ground triplet state is molecular oxygen. When the activated photosensitizer and an oxygen molecule are in proximity, an energy transfer can take place that allows the activated photosensitizer to relax to its ground singlet state and create an excited singlet state oxygen molecule. Singlet oxygen is a very aggressive chemical species and will rapidly react with any nearby biomolecules. Ultimately, these reactions will kill cells, i.e. cancer cells.

[0012] Recently, a study using an orthotopic model of bladder cancer in rats has shown a treatment effect of HAL PDD which seems to be based on immune activation / modulation of a tumor-specific immune response by modulation of the tumor microenvironment, as evident by tumor infiltration of CD3+ and CD8+ T cells, rather than the direct killing of tumor cells (Lamy et al., Biomedicines 10(3), 2022, 548). Similar observations were made in humans - a retrospective study in patients with NMIBC has shown that HAL-guided TUR may influence the immune cell composition and tumor microenvironment (Elbaek et al., Eur Urol Open Sci. 58, 2023, 37-46).

[0013] Although NMIBC lesions can be thoroughly removed by HAL-guided TUR, and HAL- guided TUR favorably affects recurrence rate, tumors may recur and even progress to Ml BC in a limited number of cases. It is therefore necessary to consider adjuvant therapy, i.e. adjuvant (intravesical) chemotherapy and / or immunotherapy. Currently, the two most common adjuvant intravesical therapies used for this purpose are Bacillus Calmette-Guerin (BCG) immunotherapy and mitomycin chemotherapy.

[0014] According to guidelines for the treatment of NMIBC, in patients with TaT1 tumors at intermediate or high risk of recurrence and intermediate or high risk of progression, one immediate post-TUR instillation of chemotherapy like mitomycin should be followed by a minimum of one year of intravesical BCG immunotherapy, or by further instillations of chemotherapy. In patients with high-risk bladder tumors such as carcinoma in situ, intravesical BCG therapy for longer than one year is indicated.

[0015] BCG is a vaccine against tuberculosis that is prepared from attenuated (weakened) live bovine tuberculosis bacillus, Mycobacterium bovis, that has lost its virulence in humans. The exact mechanism by which BCG prevents recurrence is unknown but it is thought that BCG acts via activation of both the innate and the adaptive immune systems, thereby promoting lasting antitumor effects.

[0016] BCG treatment is not without challenges: due to the high frequency of BCG-induced cystitis and allergic reactions some patients cannot tolerate an induction course or a maintenance therapy with BCG. As with any immunotherapy, not all patients respond to BCG and some patients are deemed BCG-non responders. T cell exhaustion was proposed as one of the contributing factors to BCG unresponsiveness, i.e. stimulation of the immune system by BCG treatment may lead in some patients to dysfunctional T cells in the tumor microenvironment (Strandgaard et al., Eur Urol 82 (2022), 646- 656). The standard and currently recommended treatment following BCG failure is removal of the bladder through cystectomy.

[0017] Given that it had been beneficial to explore other alternative adjuvant or bladder sparing treatment options (such as checkpoint inhibitor immunotherapy) early on in patients who turn out to be BCG-non responders during the course of their treatment and global shortages of BCG due the growing use and need for this product around the world, there is a need for a method to predict if a patient is likely to respond to BGC or if he / she likely is a non-responder, before said patient is started on BCG treatment.

[0018] Summary

[0019] The inventors propose that a bladder cancer patient’s immune response to HAL can be used as a prediction for said patient’s response to immunotherapy, such as BCG therapy. Hence, by using HAL, it can be predicted if said patient is likely to respond to immunotherapy or if said patient is likely to be a non-responder.

[0020] While on a group level bladder cancer patients having undergone HAL-guided blue light cystoscopy show signs of immune activation, different patients within such group react differently to HAL and while some may show various degrees of activation of their immune system (as can be determined by analyzing one or more biomarkers which are indicators of the state of the patient’s immune system and said biomarkers indicate an immune activation), others show only moderate immune activation or no activation or may even show signs of immune exhaustion, including T cell exhaustion (Elbaek et al., previously mentioned herein).

[0021] HAL in the form of Hexvix has an excellent safety profile and there are very few side effects related to an instillation into the bladder of Hexvix.

[0022] Thus, in some embodiments, the disclosure relates to a method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state, and b) thereby predicting the likelihood.

[0023] In some embodiments, the disclosure relates to a method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state, wherein the state of the subject’s immune system is predictive of their response to immunotherapy and b) thereby predicting the likelihood.

[0024] In some other embodiments, the disclosure relates to a method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state; and b) predicting that the subject is likely to respond to immunotherapy, if said state is determined to be immune activation.

[0025] In some embodiments, the disclosure relates to a method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in the sample which are indicators of said state; and b) predicting that the subject is likely to respond to immunotherapy, if said state is determined to be immune activation.

[0026] In some alternative embodiments, the disclosure relates to a method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state; and b) predicting that the subject is unlikely to respond to immunotherapy, if said state is determined to be immune exhaustion.

[0027] In some embodiments, the disclosure relates to a method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in the sample which are indicators of said state; and b) predicting that the subject is unlikely to respond to immunotherapy, if said state is determined to be immune exhaustion.

[0028] In yet some other embodiments, the disclosure relates to a method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state; and b) predicting that the subject is likely to respond to immunotherapy, if said state is determined to be immune activation or predicting that the subject is unlikely to respond to immunotherapy, if said state is determined to be immune exhaustion. In some embodiments, the disclosure relates to a method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in the sample which are indicators of said state; and b) predicting that the subject is likely to respond to immunotherapy, if said state is determined to be immune activation or predicting that the subject is unlikely to respond to immunotherapy, if said state is determined to be immune exhaustion.

[0029] In some embodiments, any of the aforementioned methods further comprises selecting a treatment based on the prediction, i.e. selecting an immunotherapy treatment if the subject is likely to respond to immunotherapy or selecting a nonimmunotherapy treatment, if the subject is unlikely to respond to immunotherapy. In some other embodiments, any the aforementioned methods further comprises selecting a treatment based on the prediction, i.e. selecting an immunotherapy treatment if the subject is likely to respond to immunotherapy or selecting a nonimmunotherapy treatment, if the subject is unlikely to respond to immunotherapy and treating the subject with the selected treatment.

[0030] In yet some other embodiments, the disclosure relates to a method of selecting a treatment for a subject having bladder cancer, the method comprising using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state, and depending on the state, selecting a treatment which is an immunotherapy treatment or selecting a treatment which is a nonimmunotherapy treatment.

[0031] In some embodiments, the disclosure relates to a method of selecting a treatment for a subject having bladder cancer, the method comprising using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in the sample which are indicators of said state, and depending on the state, selecting a treatment which is an immunotherapy treatment or selecting a treatment which is a non-immunotherapy treatment.

[0032] In some embodiments, said state is immune activation and the selected treatment is an immunotherapy treatment. In some other embodiments, said state is immune exhaustion and the selected treatment is a non-immunotherapy treatment.

[0033] In some embodiments, the method includes the step of treating the subject with the selected treatment.

[0034] In yet some other embodiments, the disclosure relates to a method of treating a subject having bladder cancer with an immunotherapeutic, wherein said immunotherapeutic is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0035] In some embodiments, the disclosure relates to a method of treating a subject having bladder cancer with an immunotherapeutic, wherein said immunotherapeutic is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0036] In yet some other embodiments, the disclosure relates to an immunotherapeutic for use in the treatment of a subject having bladder cancer, wherein said immunotherapeutic is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0037] In some embodiments, the disclosure relates to an immunotherapeutic for use in the treatment of a subject having bladder cancer, wherein said immunotherapeutic is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0038] In yet some other embodiments, the disclosure relates to the use of an immunotherapeutic for the manufacture of medicament for use in immunotherapy treatment of a subject having bladder cancer, wherein said medicament is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0039] In yet some other embodiments, the disclosure relates to the use of an immunotherapeutic for the manufacture of medicament for use in immunotherapy treatment of a subject having bladder cancer, wherein said medicament is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0040] Figures

[0041] Figure 1 shows the normalized protein expression of a group of biomarkers as described in the Example for each patient in the group of BCG responders (N=21) and in the group of BCG non-responders (N=16) and the median NPX value for the biomarker group calculated separately for BCG responders and BCG non- responders.

[0042] Detailed description

[0043] In some embodiments, the subject is one having non-muscle invasive bladder cancer (NMIBC). In some embodiments, the subject is one that has never had a BCG treatment prior to said instillation of HAL or a pharmaceutically acceptable salt thereof.

[0044] In some embodiments, the instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder is followed by exposing the inside of the bladder to light, preferably blue light, such as blue light and white light. In some embodiments, the inside of the bladder is not exposed to light after instillation of HAL or a pharmaceutically acceptable salt.

[0045] In some embodiments, the instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder is carried out in connection with a cystoscopy or a TUR. In some embodiments, the inside of the bladder is exposed to white light during such cystoscopy or TUR.

[0046] In some embodiments, the cystoscopy is a HAL-guided cystoscopy. In some embodiments, the TUR is a HAL-guided TUR. In some embodiments, the HAL- guided cystoscopy is carried out in the surveillance setting post TUR. In some embodiments, the inside of the bladder is exposed to blue light during such HAL- guided cystoscopy or HAL-guided TUR.

[0047] In some embodiments, the immunotherapy is an immune checkpoint inhibitor therapy and the immunotherapeutic is an immune checkpoint inhibitor, such as a PD-1 or a PD-L1 checkpoint inhibitor such as an anti-PD-1 or an anti-PD-L1 antibody. In some embodiments, the antibody is selected from avelumab, nivolumab, pembrolizumab, atezolizumab and durvalumab.

[0048] In some embodiments, the immunotherapy includes administration of more than one immune checkpoint inhibitor, i.e. two or more different immune checkpoint inhibitors.

[0049] In some other embodiments, the immunotherapy is an intravesical immunotherapy and the immunotherapeutic is e.g. BCG, nadofaragene firadenovec or nogapendekin alfa inbakicept. In yet some other embodiments, the immunotherapeutic is BCG.

[0050] In yet some other embodiments, the immunotherapy is a combination of the above- mentioned immunotherapies, such as a combination of an immune checkpoint inhibitor therapy and an intravesical immunotherapy, e.g. a combination of BCG and an immune checkpoint inhibitor.

[0051] The sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder is a biological sample. In some embodiments, the sample is a blood sample. In some other embodiments, the sample is a urine sample. In yet some other embodiments, the sample is a tissue sample from the inside of the bladder, such as a sample from a tumor in the bladder or a sample of the tissue in the vicinity of a tumor.

[0052] A tissue sample may be obtained during cystoscopy, e.g. during surveillance of the subject performed, e.g., with the aim to determine if a bladder cancer has progressed or returned. In some embodiments, the cystoscopy is performed with white light. In some other embodiments, the cystoscopy is a HAL-guided cystoscopy.

[0053] In some embodiments, a biological sample is obtained from the subject prior to receiving an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder, hereinafter a “subject reference sample”. In some of such embodiments, the subject reference sample serves as a reference for comparing the state of the subject’s immune system prior to having received an instillation of HAL or a pharmaceutically acceptable salt thereof with the state after such instillation to determine the changes in the state of the subject’s immune system elicited by such instillation. In some embodiment, the subject reference sample is a blood sample, a urine sample or a tissue sample. In some embodiments, several subject reference samples are obtained.

[0054] In some other embodiments, a biological sample of a healthy individual (or samples of several healthy individuals) serves as a reference and the state of the subject’s immune system after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder is compared to such reference to determine the changes in the state of the subject’s immune system elicited by instillation of HAL. In some embodiments, samples of several healthy individuals are used as a reference.

[0055] In yet some other embodiments, a biological sample of a bladder cancer patient (or samples of several bladder cancer patients), e.g. a patient having the particular type of bladder cancer which also the subject in question has, serves as a reference and the state of the subject’s immune system after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder is compared to such reference to determine the changes in the state of the subject’s immune system elicited by instillation of HAL. In some embodiments, samples of several bladder cancer patients, such as several patients having NMIBC, are used as a reference.

[0056] In some embodiments, said bladder cancer patients have not had an instillation of HAL or a pharmaceutically acceptable salt thereof into their bladder.

[0057] In some other embodiments, said bladder cancer patients have had an instillation of HAL or a pharmaceutically acceptable salt thereof into their bladder.

[0058] In some embodiments, the sample of the subject is obtained 16 weeks or less after the subject had an instillation of HAL or a pharmaceutically acceptable salt thereof, such as 4 days to 16 weeks, or 1 week to 12 weeks or 10 days to 8 weeks.

[0059] In some embodiments, several samples of the subject are obtained, e.g. a blood sample and a urine sample or a blood sample and a tissue sample from the inside of the bladder or a urine sample and a tissue sample form the inside of the bladder or a blood sample, a urine sample and a tissue sample from the inside of the bladder. HAL or a pharmaceutically acceptable salt of HAL is instilled into the bladder as a pharmaceutical composition.

[0060] As used herein, the term “HAL” denotes hexyl 5-ALA ester (n-hexyl aminolevulinate, i.e. n-hexyl 5-amino-4-oxo-pentanoate) and the term “pharmaceutically acceptable salt” denotes a salt that is suitable for and fulfils the requirements related to for instance safety, bioavailability and tolerability.

[0061] A “pharmaceutical composition” means a composition in any form suitable for instillation into the bladder.

[0062] Wavelengths of light that are generally considered suitable to activate the photosensitizer PpIX in vivo following the instillation into the bladder of HAL or a pharmaceutically acceptable salt thereof include: white light, i.e. visible light having wavelengths of from about 350 to about 700 nm; blue light, i.e. light having a wavelength of from about 360 to about 450 nm; and red light, i.e. light having a wavelength of from about 600 to about 670 nm. Preferably, the bladder is exposed to blue light which is used to activate PpIX following the instillation into the bladder of HAL or a pharmaceutically acceptable salt into the bladder.

[0063] The composition comprising HAL or a pharmaceutically acceptable salt thereof may be instilled into the subject’s bladder through a catheter. Preferably, the composition is retained in the bladder for a predetermined period of time. The subject’s bladder will preferably be empty at the time of instillation and, if necessary, may be evacuated prior to instillation of the composition.

[0064] Once instilled into the subject’s bladder, the composition may be retained in the bladder for a predetermined period of time. A suitable period of time may readily be determined by those skilled in the art. The composition may, for example, be left in the bladder for a period of from about 10 minutes to about 3 hours, preferably from about 20 minutes to about 2 hours, more preferably from about 30 minutes to 1 hour. Advantageously, the composition may be retained in the subject’s bladder for no less than about 30 minutes or no less than about 1 hour. At the end of this period, the bladder is evacuated. If the subject cannot retain the composition for a suitable period of time, for example for about 10 minutes, or for about 20 minutes, or for about 30 minutes, or for about 1 hour, the instillation procedure may be repeated.

[0065] In some embodiments, the composition is instilled into the patient’s bladder through a catheter and is left in the bladder for about 30 minutes or for about 1 hour. The bladder is then evacuated.

[0066] The synthesis of HAL is known in the art. It may, for example, be prepared as described in WO 96 / 28412, the entire contents of which are incorporated herein by reference. For example, HAL may be prepared by reaction of 5-ALA with hexanol in the presence of a catalyst, such as an acid. Alternatively, HAL is commercially available, e.g. in the form of Hexvix® (available from Photocure ASA) or Cysview® (available from Photocure Inc.).

[0067] HAL may be employed in the form of a pharmaceutically acceptable salt. Such salts are preferably acid addition salts with pharmaceutically acceptable organic or inorganic acids. Suitable acids include, for example, hydrochloric, nitric, hydrobromic, phosphoric, sulfuric, sulfonic acid and sulfonic acid derivatives. Such salts of ALA- esters are described in WO 2005 / 092838, the entire contents of which are incorporated herein by reference. A preferred acid addition salt of HAL for use in the invention is the hydrochloride salt. Synthetic procedures for salt formation are conventional in the art and are for instance described in WO 2005 / 092838.

[0068] The pharmaceutical composition contains HAL or a pharmaceutically acceptable salt thereof in a concentration in the range of 0.1 to 5% by weight of the total weight of the composition, or the equivalent concentration of a pharmaceutically acceptable salt of HAL, preferably 0.15 to 3.5%, and most preferably 0.17%, which corresponds to e.g., 0.2% HAL hydrochloride (8 mM).

[0069] The pharmaceutical composition may comprise pharmaceutically acceptable carriers or excipients. The composition is preferably a liquid composition, more preferably a suspension or even more preferably a solution of HAL in a liquid carrier. Preferred liquid carriers include water and aqueous solutions, for example aqueous buffers. In some embodiments, the pharmaceutical composition is an aqueous solution of HAL or a pharmaceutically acceptable salt thereof.

[0070] In some embodiments, the pharmaceutical composition is a solution of HAL, or a pharmaceutically acceptable salt thereof, in an aqueous buffer such as an aqueous phosphate buffer. In some embodiments, the aqueous phosphate buffer comprises disodium phosphate dihydrate, potassium dihydrogen phosphate, sodium chloride, hydrochloric acid, sodium hydroxide and water.

[0071] In some embodiments, the pharmaceutical composition is an aqueous solution of HAL hydrochloride. For example, the composition may be a solution of HAL hydrochloride in an aqueous buffer having a pH in the range of 4.5 to 7.5, preferably in the range of 5.7 to 7.2.

[0072] In some embodiments, HAL or the pharmaceutically acceptable salt thereof is provided in a lyophilized form, and is reconstituted in a liquid carrier, such as in water or an aqueous buffer, prior to use.

[0073] In some embodiments, the pharmaceutical composition is Hexvix®, i.e. a solution of HAL hydrochloride (2 mg / ml; 8 mM) in an aqueous buffer comprising disodium phosphate dihydrate, potassium dihydrogen phosphate, sodium chloride, hydrochloric acid, sodium hydroxide and water.

[0074] The amount of the pharmaceutical composition to be instilled into the subject’s bladder may vary according to the subject’s bladder volume. A suitable amount may readily be determined by those skilled in the art. In general, a volume of about 50 ml of the composition may be suitable.

[0075] In some embodiments, the instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder is carried out in connection with a cystoscopy, i.e. a HAL- guided cystoscopy or a HAL-guided TUR.

[0076] In the methods disclosed herein, one or more biomarkers which are indicators of the state of a subject’s immune system are analyzed in the sample(s) obtained from the subject. In some embodiments, the one or more biomarkers are related to the tumor microenvironment of the subject’s bladder cancer.

[0077] In some embodiments, the term “analyze” means that such biomarker is detected in the sample.

[0078] In some other embodiments, the term “analyze” means that such biomarker is measured, i.e. the level of such biomarker in the sample is determined. Depending on the type of measurement, the term “level of a biomarker” may mean measuring the level of its expression, e.g. by determining the amount, the concentration and the like, or the presence or absence of the biomarker in the sample.

[0079] In yet some other embodiments, the term “analyze” means that the measurement is compared to a measurement of the same biomarker in a reference. In some embodiments, the reference is known, e.g. is found in the literature, e.g. in the scientific or patent literature describing the level of the biomarker in healthy individuals or bladder cancer patients, e.g. patients having the particular type of bladder cancer which also the subject in question has. In some other embodiments, the reference is one or more samples obtained from one or more healthy individuals or one or more bladder cancer patients, e.g. patients having the particular type of bladder cancer which also the subject in question has. The biomarker in question is measured in the reference sample and the sample obtained from the subject and the measured levels are compared. In yet some other embodiments, the reference is one or more subject reference samples. The biomarker in question is measured in the subject reference sample and the sample obtained from the subject and the measured levels are compared.

[0080] In yet some other embodiments, the measurement is compared to a threshold value. In some embodiments, the threshold value reflects a typical or central value in a dataset that is generated by determining the biomarker in question in multiple reference samples obtained from healthy individuals or bladder cancer patients.

[0081] In some embodiments, the threshold value reflects a typical or central value in a dataset that is generated by determining the biomarker in question in multiple reference samples obtained from bladder cancer patients that have had an instillation of HAL or a pharmaceutically acceptable salt thereof into their bladder, e.g. patients having the particular type of bladder cancer which also the subject in question has.

[0082] “Immune activation” means that the one or more biomarkers / level of one or more biomarkers analyzed in the sample obtained from the subject indicate an activation of the subject’s immune system. In some embodiments, “immune activation” means that the comparison of one or more biomarkers / level of one or more biomarkers analyzed the sample obtained from subject with the reference values or ranges of the same one or more biomarkers / level of the same one or more biomarkers indicate an immune activation. By way of example, a high number and / or density of tumor infiltrating lymphocytes (TILs) measured in a tissue sample of the subject or the expression of proteins known to play a role in promoting tumor immunity measured in a sample of the subject may indicate immune activation, i.e. the state of the subject’s immune system is activated.

[0083] “Immune exhaustion” means that the one or more biomarkers / level of one or more biomarkers analyzed in the sample obtained from subject indicate an exhaustion of the subject’s immune system. In some embodiments, “immune exhaustion” means that the comparison of one or more biomarkers / level of one or more biomarkers analyzed the sample obtained from subject with the reference values or ranges of the same one or more biomarkers / level of the same one or more biomarkers indicate an immune exhaustion. By way of example, a high presence of regulatory T cells (Tregs) measured in a sample obtained from the subject or the upregulation of TIM-3 in a sample obtained from the subject compared to the subject’s reference sample or a threshold value or the expression of proteins known to play a role in the suppression of tumor immunity measured in a sample of the subject may indicate immune exhaustion, i.e. the state of the subject’s immune system is exhausted.

[0084] In some embodiments, the measurement of at least one of the one or more biomarkers or biomarker levels is higher than the reference value or range for the biomarker or biomarker level or threshold value. In some embodiments, the measurement of at least one of the one or more biomarkers or biomarker levels is lower than the reference value or range for the biomarker or biomarker level or threshold value. Both higher and lower levels or being on or above or on or below a threshold can be indicators for immune activation or immune exhaustion, depending on the biomarker in question.

[0085] Biomarkers which are indicators of the state of a subject’s immune system are known in the art.

[0086] In some embodiments, the biomarkers are immune cells, such as macrophages, dendritic cells and lymphocytes, including B cells and T cells, such as cytotoxic T cells, T helper cells, Tregs, and natural killer cells. The composition and / or quantity of certain subtypes of immune cells in a subject’s sample are indicators of the state of the subject’s immune system.

[0087] In the following paragraphs, the term “CD” means cluster of differentiation, “PD” means programed cell death protein, “PD-L” means programed cell death ligand, “IFN” means interferon, “TNF” means tumor necrosis factor, “TNFRS” means tumor necrosis factor receptor superfamily, “IL” means interleukin, “CXCL” means chemokine (C-X-C motif) ligand, CCL means chemokine (C-C motif) ligand.

[0088] Hence, in some embodiments, the biomarker is TILs. Increased presence or number of TILs / high TIL density is an indicator for immune activation. In some embodiments, the biomarker is T helper cells (CD4+ cells), especially Th1 (type 1 helper T cell) subtype, or the biomarker is cytotoxic T cells (CD8+ cells) or the biomarker is macrophages. Increased levels of either of these cells are indicators for immune activation. In some other embodiments, the biomarker is Tregs which may suppress immune response and hence increased presence of Tregs are an indicator for immune exhaustion.

[0089] A population level of Th1 cells, cytotoxic T cells, macrophages or Tregs reflects the percentage of such cells (or a sub-type / subset thereof) relative to other white blood cells (e.g., total T lymphocytes, CD4+T lymphocytes (for Th1 cell population level), or total PBMCs (peripheral blood mononuclear cells)) in a biological sample obtained from the subject. The population level of such cells can be measured in a number of ways, including flow cytometry (e.g., to quantify the percentage of such cells relative to other cells in a biological sample), scRNA-seq (single cell RNA sequencing) or SITE-seq (selective integration of targeted endonucleases sequencing). Alternatively, immunohistochemical staining and optionally subsequent quantification of the staining, e.g., with suitable antibodies that bind to surface molecules on such cells, may be used.

[0090] In some embodiments, the biomarker is a receptor or a receptor ligand. In some embodiments, the expression of the receptor or ligand is determined.

[0091] Thus, in some embodiments, the biomarker is an immune checkpoint receptor. In some embodiments, the biomarker is PD-1 , TIM-3 (T-cell immunoglobulin and mucindomain containing-3), CTLA4 (cytotoxic T-lymphocyte associated protein 4) and LAG3 (lymphocyte-activation gene 3). In some embodiments, upregulated expression of these immune checkpoint receptors indicates immune exhaustion.

[0092] In some other embodiments, the biomarker is CD70 or CD5. In some embodiments, upregulated expression of these receptors indicates immune exhaustion.

[0093] In some embodiments, the biomarker is a cytokine or chemokine, including interferons, such as IFN-y, interleukins, tumor necrosis factors, including TNF-a and chemokines such as CXCL9, CXCL10 or CXCLU . In some embodiments, elevated levels of such cytokines or chemokines indicate immune activation.

[0094] Cytokines, chemokines, receptor expression and receptor ligand expression can be measured, for example, by gene expression analysis methods known in the art, including qRT-PCR, microarray, Northern blotting, RNA sequencing (RNA-seq) and proximity extension assay, or, in tissue samples, by immunohistochemical staining and optionally subsequent quantification of the staining, e.g., with a suitable antibody used for staining.

[0095] In some embodiments, the method of the disclosure includes the analysis of cytotoxic T cells and whether this T cell population consists of exhausted T cells. T cell exhaustion is a broad term that has been used to describe the response of T cells to chronic antigen stimulation making the T cells less effective or unable to clear infections or prevent tumor growth, i.e. T cells having an altered response compared to normal T cells. Indicators for T cell exhaustion include reduced effector functions such as reduced cytokine production, increased chemokine expression, persistent high level of inhibitory receptors and reduced proliferation capacity keeping the activity of the T cells suppressed (see for instance Strandgaard et al., previously cited herein).

[0096] In some embodiments, one biomarker / biomarker level is measured. In some other embodiments, several biomarkers / biomarker levels are measured, such as 2 to 200 or 3 to 150 or 4 to 100 or 5 to 75 or 6 to 50 or 7 to 25 biomarkers / biomarker levels.

[0097] In some embodiments, several biomarkers are grouped by biological features.

[0098] In some embodiments, several biomarkers / biomarker levels are measured from a group of biomarkers consisting of proteins that are known to play a role in the promotion of tumor immunity. In some embodiments, at least two biomarkers / biomarker levels of CD244, CD27, CD40, CD5, CD70, CD83, CXCL1 , CXCL10, CXCL11 , CXCL13, CXCL9, ICOSLG (inducible T cell costimulatory ligand), IL18, IL6, IL7, KLRD1 (killer cell lectin-like receptor D1), TNFRSF4 and TNFRSF9 are measured. In some embodiments all the aforementioned biomarkers are measured. In some embodiments, elevated levels of one or more of such biomarkers indicate immune activation. In some other embodiments, an elevated level of the whole group indicates immune activation.

[0099] In some other embodiments, several biomarkers / biomarker levels are measured from a group of biomarkers consisting of proteins that are known to play a role in the suppression of tumor immunity. In some embodiments, at least two biomarkers / biomarker levels of CCL17, CCL19, CCL20, CD5, CSF-1 (colony stimulating factor 1), CXCL1 , CXCL5, CXCL11 , CXCL13, Gal-9 (galalectin 9), IL18, IL6, IL8, LAMP3 (lysosomal-associated membrane protein 3), LAP TGF-p (latency associated peptide of TGF-P), MCP-4 (monocyte chemoattractant protein), MIC-A / B major histocompatibility complex class I related chain A / B protein), MMP12 (macrophage metalloelastase), PD-L1 , PD-L2 and PD-1 are measured. In some embodiments all the aforementioned biomarkers are measured. In some embodiments, elevated levels of one or more of such biomarkers indicate immune exhaustion. In some other embodiments, an elevated level of the whole group indicates immune exhaustion. In yet some other embodiments, several biomarkers / biomarker levels are measured from a group of biomarkers consisting of proteins that are known to play a role in apoptosis and cell killing. In some embodiments, at least two biomarkers / biomarker levels of CASP-8 (caspase), TNFRSF12, TNFRSF21 , TRAIL (tumor necrosis factor- related apoptosis inducing ligand) and TWEAK (tumor necrosis factor-like weak inducer of apoptosis) are measured. In some embodiments all the aforementioned biomarkers are measured. In some embodiments, elevated levels of one or more of such biomarkers indicate immune activation. In some other embodiments, an elevated level of the whole group indicates immune activation.

[0100] The prediction of likelihood that the subject responds to immunotherapy may be based on the results of the measurements of one biomarker. In a preferred embodiment, the prediction of likelihood that the subject responds to immunotherapy is based on the results of the measurements of several biomarkers. If several biomarkers are measured, the prediction of likelihood may be based on the results of the measurement of all the several biomarkers or only a subgroup thereof. In some embodiments, the results of the measurement are weighted, i.e. some biomarkers are considered stronger indicators than others and the results of their measurement affect the outcome of the prediction more profoundly. Likewise, other biomarkers are considered weaker indicators and thus the results of their measurements affect the outcome of the predication to a lesser degree. In some embodiments, biomarker measurements and correlation with prediction of likelihood to respond to immunotherapy as described herein employs computer-implemented methods. In some embodiments, biomarker measurements and correlation with prediction of likelihood to respond to immunotherapy as described herein done for larger groups of subjects is used to train and validate classifiers and design Al tools. In some embodiment, such Al tools are used in the methods disclosed herein.

[0101] According to the methods of the disclosure, if the analysis of the one or more biomarkers in the sample(s) shows that the state of the subject’s immune system is activated after having been exposed to HAL or a pharmaceutically acceptable salt thereof, there is a likelihood that said subject would respond to immunotherapy. The likelihood may be higher the more pronounced the activation is. Correspondingly, if the analysis of the one or more biomarkers in the sample(s) shows that the state of the subject’s immune system is exhausted after having been exposed to HAL or a pharmaceutically acceptable salt thereof, there is a likelihood that said subject would not respond to immunotherapy. The likelihood may be higher the more pronounced the exhaustion is.

[0102] The methods disclosed herein may be combined with other methods that can aid the clinician to determine whether to treat a subject with immunotherapy or not. Such methods include genetic profiling or molecular profiling.

[0103] Example

[0104] Clinical data from 37 patients with NMIBC who underwent HAL-guided cystoscopy or HAL-guided TUR (“HAL-guided procedure”) within 100 days of their first BCG treatment were analyzed. The 100-day cutoff was chosen to reflect the expected temporal window of any biological influence of the HAL-guided procedure.

[0105] The HAL-guided procedure was carried out by instillation of a solution of Hexvix (HAL HCI) prepared as described by the manufacturer in the packaging leaflet into the bladder of the patient and subsequently exposing the inside of the bladder to blue light provided by a commercially available blue light cystoscope.

[0106] Urine samples of the patients were collected after the HAL-guided procedure but prior to first BCG treatment and again after BCG treatment. All of the patients were treated with at least 5 cycles of BCG. Patient medical records were consulted to determine if a patient had responded to BCG treatment (BCG responder) or not (BCG nonresponders). A patient was defined as BCG non-responder if recurrence of highgrade urothelial carcinoma or progression to muscle-invasive bladder cancer was detected within 2 years post-BCG. If that was not the case, a patient was defined as BCG responder. Of the 37 patients, 16 were BCG non-responders and 21 were BCG responders.

[0107] The following 18 biomarkers were chosen for determining the state of the patients’ immune system: CD244, CD27, CD40, CD5, CD70, CD83, CXCL1 , CXCL10, CXCL11 , CXCL13, CXCL9, ICOSLG, IL18, IL6, IL7, KLRD1 , TNFRSF4 and TNFRSF9. Protein expression of each biomarker was determined using the Olink Target 96 Immuno-Oncology Assay (Olink®, Olink Proteomics) as described below and data analysis was carried out for the group of said 18 biomarkers which is an Olink pre-designed panel of biomarkers known to play a role in the promotion of tumor immunity.

[0108] Urine samples were analyzed for the presence of the biomarkers using Olink, a high- throughput protein analysis technology platform that uses proximity extension assay technology to detect and quantify a large number of proteins in minimal sample volumes. Normalized protein expression (NPX) median-based intensity normalized data provided by Olink was used for data analysis.

[0109] For the 37 patients, a median NPX value of 2.0 for the biomarker group was calculated. Further, a median NPX value for the biomarker group was calculated separately for BCG responders and BCG non-responders and included in Figure 1 , together with the NPX value for the biomarker group of each patient. The NPX value for the biomarker group differ significantly for the BCG responder and BCG nonresponder group, as assessed by a Wilcoxon signed-rank test.

[0110] As can be seen in Figure 1 , of the 16 BCG non-responders, 10 patients (63%) had an NPX value below 2.0, while 6 patients (37%) had an NPX value of or above 2.0. Further, of the 21 BCG responders, 13 patients (62%) had an NPX value of or above 2.0, while 8 patients (38%) had an NPX value below 2.0.

[0111] The data thus show that the mean NPX value of the biomarker group consisting of proteins known to play a role in promoting tumor immunity analyzed in urine samples in this study can be used to define a threshold for distinguishing immune activation (at or above the threshold) vs. non-activation (below the threshold) in patients having had a HAL-guided procedure. The data further show a trend that patients having an NPX value below the threshold were more likely to not respond to BCG, while patients at or above the threshold were more likely to respond to BCG.

[0112] Embodiments A

[0113] A1. A method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state, and b) predicting the subject’s response to immunotherapy.

[0114] A2. The method of embodiment A1 , wherein the subject is likely to respond to immunotherapy, if said state is determined to be immune activation and / or wherein the subject is unlikely to respond to immunotherapy, if said state is determined to be immune exhaustion.

[0115] A3. The method according to any of the previous embodiments A, wherein the method further comprises selecting a treatment for the subject, such as an immunotherapy treatment, if the state of the subject’s immune system was determined to be immune activation or such as a non-immunotherapy treatment, if the state of the subject’s immune system is determined to be immune exhaustion.

[0116] A4. An immunotherapeutic for use in the treatment of a subject having bladder cancer, wherein said immunotherapeutic is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is determined to be immune activation.

[0117] A5. The method or the immunotherapeutic for use according to any of the previous embodiments A, wherein the instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder was followed by exposure of the inside of the bladder to light, preferably to blue light.

[0118] A6. The method or the immunotherapeutic for use according to any of the previous embodiments A, wherein the sample is a blood sample, a urine sample or a tissue sample and / or wherein several samples were obtained from the subject.

[0119] A7. The method or the immunotherapeutic for use according to any of the previous embodiments A, wherein the sample is obtained 4 days to 16 weeks after the subject had an instillation of HAL or a pharmaceutically acceptable salt thereof. A8. The method or the immunotherapeutic for use according to any of the previous embodiments A, wherein further one or more biological samples are obtained from the subject prior to having received an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder.

[0120] A9. The method or the immunotherapeutic for use according to any of the previous embodiments A, wherein HAL or a pharmaceutically acceptable salt thereof is instilled into the bladder in the form of a pharmaceutical composition, such as a solution of HAL, or a pharmaceutically acceptable salt thereof, in an aqueous buffer.

[0121] A10. The method or the immunotherapeutic for use according to any of the previous embodiments A, wherein analyzing the one or more biomarkers in the biological sample comprises detecting the biomarker and / or measuring the level of the biomarker.

[0122] A11. The method or the immunotherapeutic for use according to embodiment A10, wherein analyzing the one or more biomarkers in the sample further comprises comparing its detection or measured level to the detection or measured level of the one or more biomarkers in a reference and wherein the reference is selected from the group consisting of biological samples of healthy individuals, biological samples of patients having bladder cancer and biological samples of the subject obtained prior to having received an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder.

[0123] A12. The method or the immunotherapeutic for use according to any of the previous embodiments A, wherein the one or more biomarkers are selected from the group consisting of immune cells, T cell exhaustion, receptor expression, receptor ligand expression, cytokines and chemokines.

[0124] A13. The method or the immunotherapeutic for use according to any of the previous embodiments B, wherein the one or more biomarkers are selected from the group consisting of T helper cells, cytotoxic T cells, Tregs, B cells, macrophages, such as M1 and M2 macrophages, PD-1 expression and PD-L1 expression. A14. The method or the immunotherapeutic for use according to any of the previous embodiments A, wherein several biomarkers are analyzed, such as 2 to 200 biomarkers.

[0125] A15. The method or the immunotherapeutic for use according to any of the previous embodiments A, wherein the immunotherapy or the immunotherapeutic is BCG.

[0126] Embodiments B

[0127] B1. A method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state, and b) thereby predicting the likelihood.

[0128] B2. A method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state, wherein the state of the subject’s immune system is predictive of their response to immunotherapy, and b) thereby predicting the likelihood.

[0129] B3. A method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state; and b) predicting that the subject is likely to respond to immunotherapy, if said state is determined to be immune activation or predicting that the subject is unlikely to respond to immunotherapy, if said state is determined to be immune exhaustion.

[0130] B4. A method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in the sample which are indicators of said state; and b) predicting that the subject is likely to respond to immunotherapy, if said state is determined to be immune activation or predicting that the subject is unlikely to respond to immunotherapy, if said state is determined to be immune exhaustion.

[0131] B5. The method according to any of the previous embodiments B, wherein the method further comprises selecting a treatment based on the prediction, such as selecting an immunotherapy treatment if the subject is likely to respond to immunotherapy or selecting a non-immunotherapy treatment, if the subject is unlikely to respond to immunotherapy.

[0132] B6. The method according to embodiment B5, wherein the method further comprises treating the subject with the selected treatment.

[0133] B7. A method of selecting a treatment for a subject having bladder cancer, the method comprising a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state and b) selecting a treatment which is an immunotherapy treatment, if said state is immune activation or selecting a treatment which is a non-immunotherapy treatment, if said state is immune exhaustion and optionally treating the subject with the selected treatment.

[0134] B8. A method of selecting a treatment for a subject having bladder cancer, the method comprising a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in the sample which are indicators of said state and b) selecting a treatment which is an immunotherapy treatment, if said state is immune activation or selecting a treatment which is a non-immunotherapy treatment, if said state is immune exhaustion and optionally treating the subject with the selected treatment.

[0135] B9. A method of treating a subject having bladder cancer with an immunotherapeutic, wherein said immunotherapeutic is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0136] B10. A method of treating a subject having bladder cancer with an immunotherapeutic, wherein said immunotherapeutic is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0137] B11. An immunotherapeutic for use in the treatment of a subject having bladder cancer, wherein said immunotherapeutic is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0138] B12. An immunotherapeutic for use in the treatment of a subject having bladder cancer, wherein said immunotherapeutic is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0139] B13. Use of an immunotherapeutic for the manufacture of medicament for use in immunotherapy treatment of a subject having bladder cancer, wherein said medicament is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0140] B14. Use of an immunotherapeutic for the manufacture of medicament for use in immunotherapy treatment of a subject having bladder cancer, wherein said medicament is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

[0141] B15. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein said subject has non-muscle invasive bladder cancer.

[0142] B16. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein said subject has never had a BCG treatment prior to the instillation of HAL or a pharmaceutically acceptable salt thereof into their bladder.

[0143] B17. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein after said instillation of HAL or a pharmaceutically acceptable salt thereof, the inside of the bladder is exposed to light, such as white light and / or blue light, preferably wherein said instillation is carried out in connection with a cystoscopy, such as a cystoscopy carried out in the surveillance setting post TUR or in connection with a TUR or wherein after said instillation of HAL or a pharmaceutically acceptable salt thereof, the inside of the bladder is not exposed to light.

[0144] B18. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein said immunotherapy is an immune checkpoint inhibitor therapy carried out with an immunotherapeutic which is one or more immune checkpoint inhibitors, such as a PD-1 or a PD-L1 checkpoint inhibitor such as an anti- PD-1 or an anti-PD-L1 antibody.

[0145] B19. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein said immunotherapy is an intravesical immunotherapy carried out with an immunotherapeutic which is BCG, nadofaragene firadenovec or nogapendekin alfa inbakicept, preferably BCG.

[0146] B20. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein said sample obtained from the subject is a biological sample, such as a blood sample, urine sample and / or tissue sample from the inside of the bladder.

[0147] B21. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein the sample of the subject is obtained 16 weeks or less after the subject had an instillation of HAL or a pharmaceutically acceptable salt thereof, such as 4 days to 16 weeks, or 1 week to 12 weeks or 10 days to 8 weeks.

[0148] B22. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein HAL or a pharmaceutically acceptable salt thereof is comprised in a liquid pharmaceutical composition, such as a solution of HAL or a pharmaceutically acceptable salt thereof in a liquid carrier, such as water or an aqueous buffer, such as an aqueous buffer, such as an aqueous phosphate buffer, such as an aqueous phosphate buffer comprising disodium phosphate dihydrate, potassium dihydrogen phosphate, sodium chloride, hydrochloric acid, sodium hydroxide and water. B23. The method, the immunotherapeutic for use or the use according to embodiment B22, wherein the liquid composition comprises HAL hydrochloride and / or wherein the liquid composition has a pH in the range of 4.5 to 7.5, preferably in the range of 5.7 to 7.2.

[0149] B24. The method, the immunotherapeutic for use or the use according to embodiments B22 or B23, wherein the liquid composition comprises 2mg / ml HAL hydrochloride.

[0150] B25. The method, the immunotherapeutic for use or the use according to embodiments B22 to B24, wherein a volume of about 50 ml of the liquid composition is instilled or is to be instilled into the bladder.

[0151] B26. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein the one or more biomarkers are related to the tumor microenvironment of the subject’s bladder cancer.

[0152] B27. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein the one or more biomarkers are measured in the sample, such as by determining the level of the one or more biomarkers, such as by determining its amount, concentration, presence or absence in the sample.

[0153] B28. The method, the immunotherapeutic for use or the use according to embodiments B27, wherein such measurement is compared to a measurement of the same biomarker in a reference.

[0154] B29. The method, the immunotherapeutic for use or the use according to embodiments B28, wherein the reference are one or more samples obtained from healthy individuals or obtained from one or more bladder cancer patients, such as patients having the particular type of bladder cancer which also the subject in question has.

[0155] B30. The method, the immunotherapeutic for use or the use according to embodiments B28, wherein the reference is a known reference, such as a reference found in the literature. B31. The method, the immunotherapeutic for use or the use according to embodiments B28, wherein the reference are one or more subject reference samples.

[0156] B32. The method, the immunotherapeutic for use or the use according to embodiments B27, wherein such measurement is compared to a threshold value.

[0157] B33. The method, the immunotherapeutic for use or the use according to embodiments B32, wherein the threshold value is generated by determining the one or more biomarkers in question in multiple reference samples obtained from healthy individuals or bladder cancer patients, such as patients having the particular type of bladder cancer which also the subject in question has.

[0158] B34. The method, the immunotherapeutic for use or the use according to embodiments B32, wherein the threshold value is generated by determining the one or more biomarkers in question in multiple reference samples obtained from bladder cancer patients that have had an instillation of HAL or a pharmaceutically acceptable salt thereof into their bladder, such as patients having the particular type of bladder cancer which also the subject in question has.

[0159] B35. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein the one or more biomarkers are immune cells, such as macrophages, dendritic cells and lymphocytes, including B cells and T cells, such as cytotoxic T cells, T helper cells, Tregs, and natural killer cells.

[0160] B36. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein the one or more biomarkers are receptors, such as immune checkpoint receptors and / or are receptor ligands.

[0161] B37. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein the one or more biomarkers are cytokines and / or chemokines. B38. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein several biomarkers are grouped by biological features.

[0162] B39. The method, the immunotherapeutic for use or the use according to embodiments B38, wherein one or more biomarkers, such as all biomarkers, from a group consisting of proteins that are known to play a role in the promotion of tumor immunity are measured.

[0163] B40. The method, the immunotherapeutic for use or the use according to embodiments B39, wherein said group includes CD244, CD27, CD40, CD5, CD70, CD83, CXCL1 , CXCL10, CXCL11 , CXCL13, CXCL9, ICOSLG (inducible T cell costimulatory ligand), IL18, IL6, IL7, KLRD1 (killer cell lectin-like receptor D1), TNFRSF4 and TNFRSF9.

[0164] B41. The method, the immunotherapeutic for use or the use according to embodiments B38, wherein one or more biomarkers, such as all biomarkers, from a group consisting of proteins that are known to play a role in the suppression of tumor immunity are measured.

[0165] B42. The method, the immunotherapeutic for use or the use according to embodiments B41 , wherein said group includes CCL17, CCL19, CCL20, CD5, CSF- 1 (colony stimulating factor 1), CXCL1 , CXCL5, CXCL11 , CXCL13, Gal-9 (galalectin 9), IL18, IL6, IL8, LAMP3 (lysosomal-associated membrane protein 3), LAP TGF-p (latency associated peptide of TGF-P), MCP-4 (monocyte chemoattractant protein), MIC-A / B major histocompatibility complex class I related chain A / B protein), MMP12 (macrophage metalloelastase), PD-L1 , PD-L2 and PD-1.

[0166] B43. The method, the immunotherapeutic for use or the use according to embodiments B38, wherein one or more biomarkers, such as all biomarkers, from a group consisting of proteins that are known to play a role in apoptosis and cell killing.

[0167] B44. The method, the immunotherapeutic for use or the use according to embodiments B43, wherein said group includes CASP-8 (caspase), TNFRSF12, TNFRSF21, TRAIL (tumor necrosis factor- related apoptosis inducing ligand) and TWEAK (tumor necrosis factor-like weak inducer of apoptosis).

[0168] B45. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein the prediction of likelihood that the subject responds to immunotherapy is based on the results of the measurements of one biomarker, preferably is based on the results of the measurements of several biomarkers, such as 2 to 200 or 3 to 150 or 4 to 100 or 5 to 75 or 6 to 50 or 7 to 25 biomarkers.

[0169] B46. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B, wherein the prediction of likelihood that the subject responds to immunotherapy is based on the results of the measurements of a subgroup of said several biomarkers.

[0170] B47. The method, the immunotherapeutic for use or the use according to any of the previous embodiments B27 to B46, wherein the results of the measurement of the one or more biomarkers are weighted.

[0171] B48. The method the immunotherapeutic for use or the use according to any of the previous embodiments B being combined with genetic profiling and / or molecular profiling.

Claims

Claims:

1. A method for predicting the likelihood that a subject having bladder cancer responds to immunotherapy, the method comprises a) using a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder for determining the state of the subject’s immune system by analyzing one or more biomarkers in the sample which are indicators of said state, wherein the state of the subject’s immune system is predictive of their response to immunotherapy and b) thereby predicting the likelihood.

2. The method of claim 1 , wherein the subject is likely to respond to immunotherapy, if said state is determined to be immune activation or is unlikely to respond to immunotherapy, if said state is determined to be immune exhaustion.

3. The method of any of the preceding claims, wherein said method further comprises selecting an immunotherapy treatment, if the subject is likely to respond to immunotherapy or selecting a non-immunotherapy treatment, if the subject is unlikely to respond to immunotherapy.

4. The method of claim 3, wherein said method further comprises treating the subject with the selected treatment.

5. An immunotherapeutic for use in the treatment of a subject having bladder cancer, wherein said immunotherapeutic is administered to said subject only after it has been predicted that the subject is likely to respond to such treatment and wherein the prediction is carried out by determining the state of the subject’s immune system, wherein said state is predictive of the subject’s response to immunotherapy, by analyzing one or more biomarkers in a biological sample obtained from the subject after having had an instillation of HAL or a pharmaceutically acceptable salt thereof into the bladder and said state is immune activation.

6. The method or the immunotherapeutic for use according to any of the previous claims, wherein HAL or a pharmaceutically acceptable salt thereof is comprised in a liquid pharmaceutical composition.

7. The method or the immunotherapeutic for use according to any of the previous claims, wherein after the instillation of HAL or a pharmaceutically acceptable salt thereof, the inside of the bladder is exposed to light, such as blue light and / or white light, preferably to blue light.

8. The method or the immunotherapeutic for use according to any of the previous claims, wherein said sample obtained from the subject is a biological sample, such as a blood sample, urine sample and / or tissue sample from the inside of the bladder.

9. The method or the immunotherapeutic for use according to any of the previous claims, wherein the sample of the subject is obtained 16 weeks or less after the subject had an instillation of HAL or a pharmaceutically acceptable salt thereof, such as 4 days to 16 weeks, or 1 week to 12 weeks or 10 days to 8 weeks.

10. The method or the immunotherapeutic for use according to any of the previous claims, wherein the one or more biomarkers are measured in the sample, such as by determining the level of the one or more biomarkers, such as by determining its amount, concentration, presence or absence in the sample.

11. The method or the immunotherapeutic for use according to claim 10, such measurement is compared to a measurement of the same biomarker in a reference.

12. The method or the immunotherapeutic for use according to claim 11 , wherein the reference are one or more samples obtained from healthy individuals or obtained from one or more bladder cancer patients, such as patients having the particular type of bladder cancer which also the subject in question has or are one or more subject reference samples or is a known reference.

13. The method or the immunotherapeutic for use according to claim 11 , wherein such measurement is compared to a threshold value.

14. The method or the immunotherapeutic for use according to claim 13, wherein the threshold value is generated by determining the one or more biomarkers in question in multiple reference samples obtained from healthy individuals or bladder cancer patients, such as patients having the particular type of bladder cancer which also thesubject in question has and / or such as patients that have had an instillation of HAL or a pharmaceutically acceptable salt thereof into their bladder.

15. The method or the immunotherapeutic for use according to any of the previous claims, wherein the one or more biomarkers are related to the tumor microenvironment of the subject’s bladder cancer.

16. The method or the immunotherapeutic for use according to any of the previous claims, wherein the one or more biomarkers are immune cells, such as macrophages, dendritic cells and lymphocytes, including B cells and T cells, such as cytotoxic T cells, T helper cells, Tregs, and natural killer cells.

17. The method or the immunotherapeutic for use according to any of the previous claims, wherein the one or more biomarkers are receptors, such as immune checkpoint receptors and / or are receptor ligands.

18. The method or the immunotherapeutic for use according to any of the previous claims, wherein the one or more biomarkers are cytokines and / or chemokines.

19. The method or the immunotherapeutic for use according to any of the previous claims, wherein several biomarkers are grouped by biological features.

20. The method or the immunotherapeutic for use according to any of the previous claims, wherein the prediction of likelihood that the subject responds to immunotherapy is based on the results of the measurements of one biomarker, preferably is based on the results of the measurements of several biomarkers, such as 2 to 200 or 3 to 150 or 4 to 100 or 5 to 75 or 6 to 50 or 7 to 25 biomarkers.

21. The method or the immunotherapeutic for use according to any of the previous claims being combined with genetic profiling and / or molecular profiling.

22. The method or the immunotherapeutic for use according to any of the previous claims, wherein the bladder cancer is non-muscle invasive bladder cancer.

23. The method or the immunotherapeutic for use according to any of the previous claims, wherein subject is one that has never had a BCG treatment prior to said instillation of HAL or a pharmaceutically acceptable salt thereof.

24. The method or the immunotherapeutic for use according to any of the previous claims, wherein said immunotherapy is an immune checkpoint inhibitor therapy carried out with an immunotherapeutic which is one or more immune checkpoint inhibitors, such as a PD-1 or a PD-L1 checkpoint inhibitor such as an anti-PD-1 or an anti-PD-L1 antibody.

25. The method or the immunotherapeutic for use according to any of the previous claims, wherein said immunotherapy is an intravesical immunotherapy.

26. The method or the immunotherapeutic for use according to claim 25, wherein said immunotherapy is carried with an immunotherapeutic which is BCG.

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