Cancer therapy using Anti-pd-1 or Anti-pd-l1 antibodies

Abstract

The present invention relates to improving treatment for cancer with PD-1 / PD-L1 antibody. In particular, the invention is based on the finding that patients with a TGFβ-specific T cell response are more likely to show a positive outcome in cancer treatment with PD-1 / PD-L1 antibody. Therefore, the invention provides for the treatment of that patient group with PD-1 / PD-L1 antibody as well as actively promoting a TGFβ-specific T cell response to improvement treatment with PD-1 / PD-L1 antibody.

Description

[0001] THERAPY

[0002] Field of the Invention

[0003] The present invention is concerned with identifying cancer patients for which treatment with PD-1 / PD-L1 antibodies will be particularly effective. The present invention is further concerned with ways to increase the effectiveness of PD-1 / PD-L1 antibody treatment for a given group of cancer patients. The present invention is also concerned with compositions and methods for treating cancer.

[0004] Background of the Invention

[0005] The discovery of the immune check points, CTLA-4 and PD-1, and the subsequent introduction of immune checkpoint inhibitors (ICIs) have revolutionized the treatment and prognosis of several cancers (Robert, C., Nat. Commun. 11, (2020)). However, not all cancers are sensitive to ICI, for example, pancreatic cancer (PC) remains highly refractory to ICI. Several trials that investigated the ICI in patients with advanced pancreatic cancer failed to demonstrate clinically relevant efficacy (Brahmer, J. R. et al., N. Engl. J. Med. 366, 2455- 2465 (2012); O’Reilly, E. M. et al., JAMA Oncol. 5, 1431-1438 (2019); and Royal, R. E. et al., J. Immunother. 33, 828-833 (2010)).

[0006] Recently, a phase II trial in patients with refractory metastatic pancreatic cancer (CheckPAC) investigated a PD-1 antibody Nivolumab with, or without, Ipilimumab, combined with radiotherapy (Chen, I. M. et al.,. J. Clin. Oncol. 71, (2022)). A clinical benefit rate of 37.2% was observed in the combination group, however only 14% of patients achieved a partial response. There is therefore an ongoing need for improved approaches for treating cancers that are refractory to PD-1 / PD-L1 antibody treatment and particularly for pancreatic cancer. of the Invention

[0007] The present invention provides a way to improve treatment with PD-1 / PD-L1 antibodies, particularly for cancer types that are refractory to treatment with such antibodies. The invention is based on the finding that the presence or absence of a TGF-beta specific T cell response is indicative of how effective treatment with PD-1 / PD-L1 antibody will be and also, if such a response is present, the magnitude of the response is also indicative of how effective treatment will be. The present invention therefore identifies a patient group for which treatment with PD-1 / PD-L1 antibody will be particularly effective. It also allows for the use of TGFbeta or immunogenic peptides from TGFbeta to stimulate, or boost, a TGF- beta T cell response and hence increase the likely efficacy of PD-1 / PD-L1 antibody treatment for cancer.

[0008] The invention is particularly effective for cancers that are refractory to treatment with PD-1 / PD-L1 antibody such as pancreatic cancer. A PD-1 / PD-L1 antibody which may be employed in the invention is Nivolumab. Hence, in an embodiment the antibody is Nivolumab and the cancer is pancreatic cancer. A particularly useful indicator is the presence, and size if present, of a T cell response against the TGFbeta- 15 peptide sequence of SEQ ID NO: 28.

[0009] Accordingly, the present invention provides a PD-1 / PD-L1 antibody for use in a method of treating a cancer in a patient, wherein the method comprises administering the PD- 1 / PD-L1 antibody to said patient, wherein the patient has been previously identified as having a TGFbeta-specific T cell response.

[0010] The present invention further provides a method of treating cancer in a patient, the method comprising administering a PD-1 / PD-L1 antibody to said patient, wherein the patient has been previously identified as having a TGFbeta-specific T cell response.

[0011] The present invention also provides a method of stratifying a patient with cancer into one of at least two treatment groups, the method comprising: i. performing an assay on a sample previously obtained from the patient to detect the presence or absence of a TGFb-specific T cell response; ii. allocating the patient to a first treatment group if a TGFb-specific response is present or stratifying the patient into a second treatment group if a TGFb-specific response is absent; wherein if the patient is allocated to the first treatment group they are to be administered with a PD-l / PD-Ll antibody.

[0012] The present invention also provides a method of stratifying a patient with cancer into one of at least two treatment groups, the method comprising: i. performing an assay on a sample previously obtained from the patient to detect the level of a TGFb-specific T cell response, if present; ii. allocating the patient to a first treatment group if the TGFb-specific T cell response is at least a threshold value for a TGFb-specific T cell and stratifying the patient into a second treatment group if a TGFb-specific response is below the threshold value; wherein if the patient is allocated to the first treatment group they are to be administered with a PD-l / PD-Ll antibody.

[0013] The present invention also provides a PD-1 / PD-L1 antibody for use in a method of treating cancer, wherein the method comprises:

[0014] (i) administering to a patient an immunogenic fragment of human transforming growth factor (TGFb) which comprises or consists of a sequence of at least 9 consecutive amino acids of SEQ ID NO: 1; and

[0015] (ii) administering the PD-1 / PD-L1 antibody.

[0016] The present invention further provides an immunogenic fragment of human transforming growth factor b (TGFb) for use in a method of treating a cancer in a patient, the method comprising

[0017] (i) administering to the patient the immunogenic fragment of human transforming growth factor b (TGFb), wherein the immunogenic fragment comprises or consists of a peptide sequence of at least 9 consecutive amino acids of SEQ ID NO: 1; and

[0018] (ii) administering to the patient a PD-1 / PD-L1 antibody.

[0019] Brief Description of the Figures

[0020] Figure 1: TGFb-15 specific responses in pancreatic cancer (PC) patients. A. Peripheral blood mononuclear cells (PBMCs) from pancreatic cancer patients were stimulated once in vitro with TGFb-15 peptide and IL-2, and incubated for 14 days before plating in interferon-gamma enzyme-linked immunosorbent spots (IFNy ELISPOTs) at a concentration of 2 xlO5cells / well with overnight incubation. The experiments were performed in triplicates with negative control wells left unstimulated. The responses were characterized in 32 samples isolated at baseline (left) and in 31 samples isolated after 4 series of treatment (right). B. Representative pictures of a response at baseline (top) and a response after 4 series of treatment. C. Normalized counts of the data from A comparing the amplitude of response in patients with clinical benefit and response in patients with progressive disease. Normalization was performed by subtracting the mean spot count of the control wells from the mean spot count of peptide stimulated wells. Error bars in A and C depict standard error of the mean and statistics were performed using a paired T-test. Statistics were made using Mann- Whitney test.

[0021] Figure 2. The amplitude of TGFb-15 specific immune responses fluctuates over time. A. Normalized counts at baseline were compared to counts after 4 series of treatment (i.e. 8 weeks after initial treatment) for patients with clinical benefit (left) and patients with progressive disease (right). Statistics made using Wilcoxon matched-pairs signed rank test. B. Representative pictures of baseline response and response after 4 series in a patient with clinical benefit (top), and of a response in a patient with progressive disease (bottom). C. Normalized TGFb-15 specific immune responses analyzed over time (longer than 2 years) in three patients with clinical benefit. Normalization of spot counts was performed as described previously.

[0022] Figure 3. Differences in decline of the TGFb-15 specific immune responses in patients with lasting clinical benefit vs. patients without lasting clinical benefit.

[0023] Comparison of TGFb-15 specific immune response at baseline and after four series of treatment in patients with lasting clinical benefit (A) and without lasting clinical benefit (B). Lasting clinical benefit defined as a Partial Response (PR) or Stable Disease (SD) lasting longer than 6 months. Statistics made using Wilcoxon matched-pairs signed rank test.

[0024] Figure 4. Strong TGFb-15 specific responses before treatment initiation predict superior survival. A. Kaplan-Meier curve displaying overall survival in patients with a response above or below the median response amplitude to TGFb-15. B. Kaplan-Meier curve displaying progression free survival in patients with a response above or below the median response amplitude to TGFb-15. Time-to-event analyses were performed using the log-rank test.

[0025] Figure 5. Survival analysis in patients based on the amplitude of the TGFb-15 specific response. A. Analysis of overall survival in patients based on the normalized TGF|3- 15 responses. Patients were divided in to four groups depending on the amplitude of the TGFb- 15 specific response. B. Analysis of progression free survival as in A. Statistics performed using the log-rank test.

[0026] Figure 6. Correlation between TGFb-15 specific responses and responses to tetanus epitope. A. Normalized responses to TGFb-15 and tetanus responses were plotted and correlation analyzed using simple linear regression. B. Normalized responses to TGFb-15 and influenza responses were plotted and correlation analyzed using simple linear regression.

[0027] Figure 7: The amplitude of TGFb-15 specific responses is not coupled to the amplitude of responses to tetanus peptide. A. PBMCs were tested for response to the tetanus epitope “tetanus-long” using in vitro IFNg ELISPOT assays. The amplitude of the normalized tetanus-long specific response was compared between patients with a response above or below the median TGFb-15 specific immune response. Responses against tetanus- long were not analyzed in baseline samples in five patients from the group with a response above the median and in one patient from the group with a response below the median, but in samples acquired within two weeks of baseline. B. Representative pictures of a patient with an absent TGFb-specific immune response (top) but an intact tetanus specific immune response (bottom). C. Same analysis as in A with a short influenza virus derived epitope “Cl 8 A2 Flu” instead of tetanus derived epitope. D. Representative pictures of a patient with an absent TGFb-specific immune response (top) but an intact influenza virus specific immune response (bottom). Error bars in A and C depict standard error of the mean. Statistics made using Mann- Whitney test.

[0028] Figure 8: Clostridium tetani and Influenza specific responses are not associated with survival. A. Kaplan-Meier curve displaying overall survival in patients with a response above or below the median response amplitude to Tetanus-Long. B. Kaplan-Meier curve displaying progression free survival in patients with a response above or below the median response amplitude to Tetanus-Long. C. Kaplan-Meier curve displaying overall survival in patients with a response above or below the median response amplitude to C 18 A2 Flu. D. Kaplan-Meier curve displaying progression free survival in patients with a response above or below the median response amplitude to Cl 8 A2 Flu. Time-to-event analyses were performed using the log-rank test.

[0029] Figure 9. Repeated in vitro stimulations of both patient and healthy donor PBMCs with TGFb-15 peptide increase the amplitude of the TGFb-15 specific immune response. A. PBMCs from 16 PC patients with a weak TGFb-15 response were cultured in vitro and tested for response to TGFb-15 after one in vitro stimulation with TGFb-15 peptide or after three in vitro stimulations with TGF|3-15 peptide with normalized responses shown in a heatmap (top) and representative responses after 1 stimulation and 3 stimulations (bottom). Some cultures only received two in vitro stimulations denoted by a black star next to spot count. B. The ability of repeated stimulations to enhance the TGFb-15 specific immune response was analyzed in PBMCs from 7 healthy donors as described in A using 5-6 xlO5cells / well with normalized results (top) and representative responses (bottom). C. The ability of repeated stimulations to enhance the TGFb-15 specific immune response was analyzed in PBMCs from 15 healthy donors as described in A using 2 xlO5cells / well with normalized results (top) and representative responses (bottom).

[0030] Figure 10. Increased distribution free resampling (DFR) responses after repeated stimulations. A. Comparison of DFR responses and DFR2x responses after 1 or repeated stimulations in PC patients. B. Comparison as in A but in Healthy donors. Statistical analysis was done using the DFR and DFR2x method.

[0031] Figure 11. Strong TGFb-33 specific responses before treatment initiation predict superior survival. A. Kaplan-Meier curve displaying overall survival in patients with a response above or below the median response amplitude to TGFb-33. B. Kaplan-Meier curve displaying progression free survival in patients with a response above or below the median response amplitude to TGFb-33. Time-to-event analyses were performed using the log-rank test.

[0032] Figure 12. T-cell responses against TGFb-33 in patients with pancreatic cancer receiving radiotherapy and treatment with anti-CTLA-4 and anti-PD-1. Peripheral blood mononuclear cells (PBMCs) from pancreatic cancer patients were stimulated once in vitro with TGFb-33 peptide and IL-2, and incubated for 14 days before plating in interferon-gamma enzyme-linked immunosorbent spots (IFNy ELISPOTs) at a concentration of 2 xlO5cells / well with overnight incubation. The experiments were performed in duplicates or triplicates with negative control wells left unstimulated. The responses were characterized in 31 samples isolated at baseline (left) and in 28 samples isolated after 4 series of treatment (right). The figure displays the normalized response amplitude to TGFb-33 which was calculated by subtracting the mean number of spots from negative control wells from the mean number of spots from the peptide stimulated wells.

[0033] Brief Description of the Sequences

[0034] SEQ ID NO: 1 is the amino acid sequence of the full-length precursor of human TGFb-1 (also referred to as the TGFb-1 pre-protein).

[0035] SEQ ID NO: 2 is the amino acid sequence of the signal peptide of human TGFb-1.

[0036] SEQ ID NO: 3 is the amino acid sequence of the LAP peptide of human TGFb-1.

[0037] SEQ ID NO: 4 is the amino acid sequence of mature human TGFb-1.

[0038] SEQ ID NOs: 5-64 are each an amino acid sequence of a polypeptide fragment derived from human TGFb-1.

[0039] SEQ ID NO: 65 is the amino acid sequence of the LAP sub-region comprising a high frequency of immunogenic sequences.

[0040] SEQ ID NO: 66 is the amino acid sequence of the minimal epitope sequence within the TGFb-15 peptide sequence (SEQ ID NO: 28). SEQ ID NO: 66 is also referred to herein as “TGFb-15-15short”. SEQ ID NO: 67 is the amino acid sequence of TGFb-A2-01.

[0041] Detailed Description of the Invention

[0042] It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0043] Definitions

[0044] Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular (“a”, “an”, and “the”) will also include the plural and vice versa unless the content clearly dictates otherwise. Thus, for example, reference to “a polypeptide” includes “polypeptides”, and the like. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0045] In instances where the terms “comprising” and “comprises” are used, also provided is something “consisting essentially of’ or “consisting of’ what is set out.

[0046] A “polypeptide” is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The term “polypeptide” thus includes short peptide sequences and also longer polypeptides and proteins. As used herein, the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including both D or L optical isomers, and amino acid analogs and peptidomimetics.

[0047] The terms “patient” and “subject” are used interchangeably and typically refer to a human.

[0048] By “immunogenic” herein it is meant that a polypeptide is capable of eliciting an immune response to the TGFb protein, in particular TGFb-1 protein, typically when said protein is present in or on cells expressing the TGFb-1 protein. In other words, the polypeptide may be described as immunogenic to TGFb. The polypeptide may alternatively be described as an immunogenic fragment of TGFb. The immune response may refer to a T cell response, and so the polypeptide may be described as an immunogenic fragment of TGFb comprising a T cell epitope. The immune response may be detected in at least one individual (or in sample taken from the individual) after administration of the polypeptide to said individual (or said sample).

[0049] Reference herein to TGF-b, T-GF-beta, and the like corresponds to reference to TGF- p. However, to avoid the use of Greek symbols and aid reproducibility of the text, the former nomenclature has been used.

[0050] TGFbetal-specifc T cell responses

[0051] The present invention is based on the finding that patients displaying a T cell response specific for TGFbeta represent a patient group for which therapy with PD-1 / PD-L1 antibody is likely to be particularly effective. Furthermore, it has also been found that the magnitude of the T cell response specific for TGFbeta indicates how effective treatment with PD-1 / PD-L1 antibody for cancer is likely to be. That means it is possible to determine the presence or absence of a T cell response specific for TGFbeta, or to quantify it, for a given patient to determine how effective treatment with PD-1 / PD-L1 antibody is likely to be. It may be therefore used as a way to determine whether or not to treat a patient with PD-1 / PD-L1 antibody. The finding that the T cell response specific for TGFbeta response indicates how effective treatment with PD-1 / PD-L1 antibody will be also means for those patients lacking such a response, or with only a weak response, the patients may be treated to trigger or increase such a response to help improve the effectiveness of treatment with PD-1 / PD-L1 antibody. The T cell response specific for TGFbeta may refer to that against TGFbetal. The invention is particularly useful in the context for cancers that are refractory to treatment with PD-1 / PD-L1 antibody. In an especially preferred embodiment, the cancer is pancreatic cancer and the antibody is Nivolumab.

[0052] Particularly, whether or not a TGFbeta response is present, or the size of the response, is measured at “baseline” that is before treatment has begun. It may also be measured during the course of treatment as well. In a further embodiment, whether or not a TGFbeta response is present may be measured at baseline and then after a treatment intended to increase the T cell response for TGFbeta response to check that the response occurred or increased.

[0053] In some embodiments, whether or not a patient has a T cell response specific for TGFbeta at all is measured. It may be that the result for a test sample for the patient is compared to that for a negative control and if there is not a significant increase in the result for the test sample versus the negative control it may be said that a response is lacking. It may be that rather than determining simply the presence or absence of a response, the size of the response is measured, as the present inventors have found that patients with a higher level of response show more positive results with PD-1 / PD-L1 antibody.

[0054] Whether or not a patient has a TGFbeta-specific T cell response, or the size of the response, may be measured by any suitable method. Whether or not such a response is present may be measured by determining whether or not T cells isolated from the patient show a response when exposed to TGFbeta-1 or a TGFbeta-1 peptide. Any of the TGFbeta peptide sequences set out herein may be employed, with an especially preferred peptide of interest being the TGFbeta- 15 peptide sequence of (SEQ ID NO: 28) or TGFbeta short peptide sequence of (SEQ ID NO:66). A further preferred peptide of interest is the TGFbeta- 33 peptide sequence of SEQ ID NO: 55.

[0055] A patient sample for assessment may be one obtained from blood. Peripheral blood mononuclear cells (PBMCs) represent a preferred sample used herein. In another embodiment, the cells are purified T cells, for example T cells purified from the blood of the patient. One method for measuring whether or not a subject displays a TGFbeta-specific T cell response is to use peripheral blood mononuclear cells PBMCs isolated from the subject and assay whether or not they show a response to TGFbeta-1 or a TGFbeta-1 peptide. One suitable assay is to incubate the PBMCs with TGFbeta-1 or a TGFbeta-1 peptide, then determine whether or not the cells present are activated by the TGFbeta-1 or a TGFbeta-1 peptide. A possible method for determining whether or not activated T cells show a response to TGFbeta-1 is an ELISPOT assay. The assay employed in the Examples of the present application may be used to determine whether or not a response is present and if it is, to quantify it.

[0056] In one embodiments, an assay comprises: (a) incubating PBMCs isolated from the patient with TGFbeta-1 or a TGFbeta-1 peptide for from 7 to 14 days; (b) plating a known number of cells into plates coated with a primary IFN-y specific antibody, incubating the plate overnight, then removing the cells; (c) detecting the presence of bound IFN-y using secondary antibody, streptavidin- ALP, and enzyme substrate with washing of wells with PBS before and between each step, meaning that the location of an activated cell secreting IFN- y can be identified as a spot; and (d) counting the number of spots and hence the number of cells specific for TGFbeta-1. In step (a) IL-2 may also be included in the incubation step. The assay may comprise controls, such as also performing a negative control sample where no TGFbeta-1 or a TGFbeta-1 peptide is included in the incubation step of (a). In one embodiment a subject is defined as lacking a T cell response for TGFbeta if it gives the same, or similar, result to a negative control. In one embodiment the absence of a response in an ELISPOT assay may be said to be no more than five times the number of spots in comparison to the equivalent negative control. As well as, or alternatively, an assay may further comprise a positive control, for example a sample including T cells that will display a response against TGFbeta.

[0057] In another embodiment, the size of any TGFbeta T cell response is measured. In further embodiment, a method may be used which allows the number of T cells producing a TGFbeta-specific response to be enumerated. A way of doing so is to employ a spot-based assays. An ELISpot assay represents an assay used herein. The assay employed in the Examples of the present application may be, for instance, employed.

[0058] The invention may comprise producing, or comparing to, the value obtained against an expected value. It may be that a patient is selected for treatment on the basis of having a baseline T cell response specific for TGFbeta- 1 that is the same or greater than a threshold value. One way of setting a threshold value is in terms of the median value. In some embodiments, the median value may be the median baseline value seen in cancer sufferers, particularly cancer sufferers with the same type of cancer. The median may be calculated from cancer suffers of the same cancer type and gender. It may be calculated from representative samples of such cancer sufferers. The expected median value for a given value may have already been calculated and can be used. In further embodiments, another way of setting a threshold value is in terms of the 75th percentile value. The 75th percentile value may be the 75th percentile value of the baseline value seen in cancer sufferers, particularly cancer sufferers with the same type of cancer. The 75th percentile may be calculated from cancer suffers of the same cancer type and gender. It may be calculated from representative samples of such cancer sufferers. The expected 75th percentile value for a given value may have already been calculated and can be used.

[0059] The invention may involve working out in which percentile the value for a patient falls within the value for a population of patients with the same condition. The invention may be used to give an indication of the likely efficacy of treatment with PD-1 / PD-L1 antibody. In some embodiments, a “threshold value” may be applied to decide whether or not the subject is given PD-1 / PD-L1 antibody. In another embodiment, it may be that if the value is below the threshold value then the patient is selected for treatment to increase the TGFbeta-specific T cell response to help improve the likely efficacy of PD-1 / PD-L1 antibody. A TGFbeta- 1 peptide, or encoding sequence, may be administered to the subject to try and stimulate such a response.

[0060] Patients

[0061] The invention may be applied to any suitable mammalian subject, though the patient is human. The patient may be male or female. In one embodiment the patient is male.

[0062] The patient will have cancer. In one embodiment, the cancer is selected from pancreatic cancer, melanoma, lung cancer, malignant pleural mesothelioma, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, urothelial carcinoma, colon cancer, esophageal squamous cell carcinoma, liver cancer, gastric cancer, and esophageal or gastroesophageal junction (GEJ) cancer. In one embodiment, the patient may have Unresectable or Metastatic Melanoma. In another embodiment the patient may have Metastatic Non-Small Cell Lung Cancer. In another embodiment, the patient may have Malignant Pleural Mesothelioma. In another embodiment, the patient may have advanced Renal Cell Carcinoma. In another embodiment, the patient may have classical Hodgkin Lymphoma. In another embodiment, the patient may have Squamous Cell Carcinoma of the Head and Neck. In another embodiment, the patient may have Urothelial Carcinoma. In a further embodiment, the patient may have Microsatellite Instability-High or Mismatch Repair Deficient Metastatic Colorectal Cancer. In a further embodiment, the patient may have Hepatocellular Carcinoma. In a further embodiment, the patient may have Esophageal Cancer. In a further embodiment, the patient may have Gastric Cancer, Gastroesophageal Junction Cancer, and Esophageal Adenocarcinoma. In one embodiment, the cancer is metastatic cancer. In another embodiment, the cancer is metastatic cancer with two or less metastases.

[0063] In one embodiment, the cancer is a cancer that is refractory to treatment with PD- 1 / PD-L1 antibody. An example of such a refractory cancer is pancreatic cancer. In an especially preferred embodiment, the subject may therefore have pancreatic cancer. In one embodiment, the pancreatic cancer is metastatic cancer. In further embodiment, the cancer is refractory metastatic pancreatic cancer (mPC).

[0064] PD-1 / PD-L1 antibodies and additional cancer treatments

[0065] A PD-1 / PD-L1 antibody is typically one that blocks the interaction between PD-1 and PD-L1. Hence, in one embodiment the antibody binds PD-1. In another embodiment, the antibody binds PD-L1. Examples of PD-1 antibodies include Nivolumab, Pembrolizumab, Cemiplimab, and Dostarlimab. Examples of PD-L1 antibodies include Atezolizumab, Avclumab, and Durvalumab.

[0066] An especially preferred PD-1 antibody of interest is Nivolumab. Nivolumab is sold under the brand name Opdivo®. It may also be sold under the names ONO-4538, BMS- 936558, or MDX1106. Nivolumab may be used to treat a variety of cancers.

[0067] It may be that the patient is administered only PD-1 / PD-L1 antibody. PD-1 / PD-L1 antibody though is sometimes given in combination with other therapies or as a further line of treatment after treatment with another therapy. In one embodiment, it may be that in any of the embodiments set out herein where a subject is given PD-1 / PD-L1 antibody that they are also given a second cancer therapy as well. It may be that the patient is given both PD-l / PD- L1 antibody and a CTLA-4 antibody. Preferably the CTLA-4 antibody is Ipilimumab. It may be that the patient is given both PD-1 / PD-L1 antibody and radiotherapy. It may be that the subject is given PD-1 / PD-L1 antibody, CTLA-4 antibody, and radiotherapy. A preferred CTLA-4 antibody is Ipilimumab. A form of radiotherapy is stereotactic body radiotherapy (SBRT). In such embodiments, Nivolumab is a PD-1 antibody of interest. Hence, it may be that Nivolumab is administered alone. It may be that Nivolumab is administered with a CTLA-4 antibody (e.g., Ipilimumab). It may be in either case that radiotherapy is administered. Hence, in one embodiment Nivolumab, Ipilimumab, and SBRT are administered. In one embodiment, radiotherapy is provided in the form of stereotactic body radiotherapy (SBRT) at about 15 Gy on a single site of disease. In a further embodiment, SBRT is provided on day 1 of a 14-day treatment cycle. In one embodiment, Nivolumab is administered at a dose of about 3 mg / kg (up to a maximum of about 240 mg). In one embodiment, Nivolumab is administered intravenously. In a further embodiment, Nivolumab is administered on day 1 (± 3 days) of each 14-day treatment cycle. In one embodiment, Ipilimumab is administered at a dose of about 1 mg / kg. In one embodiment, Ipilimumab is administered intravenously. In a further embodiment, Ipilimumab is administered on day 1 of a 14-day treatment cycle, and subsequently once every 6 weeks (± 3 days) thereafter.

[0068] It may be that a CTLA-4 antibody is given, or that the patient is already being treated with such an antibody. An example of a CTLA-4 antibodies is Ipilimumab sold under the brand name Yervoy®. A further example of a preferred CTLA-4 antibody is Tremelimumab.

[0069] In those patients where it is decided to stimulate a TGFbeta-specific T cell response as a way to try and increase the likely efficacy of PD-1 / PD-L1 antibody, a TGFbeta-1 peptide as described further herein may be administered to the patient to stimulate such a response. In one embodiment, a TGFbeta-1 peptide is administered at a dose of about 200 pg. The TGFbeta-1 peptide may be administered in emulsion with an adjuvant. In one embodiment, a TGFbeta-1 peptide is administered at a dose of about 200 pg, in emulsion with about 500 pl Montanide ISA-51. The peptide-adjuvant emulsion may be administered on day 1 of the first 6 14-day treatment cycles and subsequently once every 4 weeks (± 3 days) thereafter.

[0070] In the case where an additional agent or therapy are to be given to the patient as well as PD-1 / PD-L1 antibody it may be that they are given simultaneously, separately or sequentially. Two drugs may be given in the same composition or in separate compositions. In embodiments where a TGFbeta-1 peptide is given to try and stimulate a TGFbeta-specific T cell response, it may be given prior to the PD-1 / PD-L1 antibody. In one embodiment the TGFbeta peptide may be given, the stimulation of a T cell response against TGFbeta may be confirmed, and then PD-1 / PD-L1 antibody is given or PD-1 / PD-L1 antibody is given once the value of the response increases over a threshold. In another embodiment, the TGFbeta- Ipeptide and the PD-1 / PD-L1 antibody may be given at the same time.

[0071] TGFbeta-1 epitopes and peptides

[0072] The TGFbeta is typically TGFbeta-1. It may be that TGFbetal sequences are used in terms of whether or not a T cell response against TGFbeta is present in a patient. Alternatively, the sequences discussed below may be used to stimulate such a response.

[0073] The sequence of the full-length human TGFbeta-1 pre-protein (NP000651.3) (SEQ ID NO: 1) is provided below:

[0074] 10 20 30 40 50

[0075] MPPSGLRLLL LLLPLLWLLV LTPGRPAAGL STCKTIDMEL VKRKRIEAIR 60 70 80 90 100

[0076] GQILSKLRLA SPPSQGEVPP GPLPEAVLAL YNSTRDRVAG ESAEPEPEPE 110 120 130 140 150

[0077] ADYYAKEVTR VLMVETHNEI YDKFKQSTHS IYMFFNTSEL REAVPEPVLL 160 170 180 190 200

[0078] SRAELRLLRL KLKVEQHVEL YQKYSNNSWR YLSNRLLAPS DSPEWLSFDV 210 220 230 240 250

[0079] TGVVRQWLSR GGEIEGFRLS AHCSCDSRDN TLQVDINGFT TGRRGDLATI 260 270 280 290 300

[0080] HGMNRPFLLL MATPLERAQH LQSSRHRRAL DTNYCFSSTE KNCCVRQLYI 310 320 330 340 350

[0081] DFRKDLGWKW IHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA 360 370 380 390

[0082] SAAPCCVPQA LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS

[0083] Table 1 below sets out various TGFbeta-1 related sequences including specific

[0084] TGFbeta-1 peptides which may be used in the present invention. In Table 1 below, “Start pos” and “End pos” indicate the positions within full length human TGFbeta-1 pre -protein (SEQ ID NO: 1) unless otherwise indicated.

[0085] Table 1

[0086]

[0087] The presence, or size, of a T cell response specific for TGFbeta may be determined using TGFbeta-1 itself or using one of the TGFbeta-1 peptide sequences herein. The TGFbeta- 1 peptide sequences discussed herein may also be used to bring about, or increase the size of a T cell response specific for TGFbeta-1 and so improve the treatment for cancer. Thus, the sequences discussed below may be relevant for detecting / measuring TGFbeta- specificT cell response, but also in seeking to stimulate / increase such a response. In an especially preferred embodiment, a peptide sequence is that of the TGFb-15 peptide of SEQ ID NO: 28. In a further particularly preferred embodiment a peptide is that of SEQ ID NO: 66. In a further particularly preferred embodiment a peptide is that of SEQ ID NO: 55.

[0088] A preferred TGFbeta-1 peptide sequence for employing in the invention is an immunogenic fragment of human TGFbeta-1 (SEQ ID NO: 1) which comprises or consists of a sequence of at least 9 consecutive amino acids of SEQ ID NO: 1. The sequence of at least 9 consecutive amino acids of SEQ ID NO: 1 may, for instance, correspond to a sequence of at least 9 consecutive amino acids of the signal peptide (SP) domain of TGFbeta-1, for example a sequence of at least 9 consecutive amino acids of SEQ ID NO: 2. It may correspond to a sequence of at least 9 amino acids of the latency-associated peptide (LAP) domain of TGFb- 1, for example at least 9 consecutive amino acids of SEQ ID NO: 3. It may correspond to a sequence of at least 9 consecutive amino acids located within the LAP sub-region bounded by amino acid positions 121 and 160 of SEQ ID NO: 1, for example a sequence of at least 9 consecutive amino acids of SEQ ID NO: 65. It may correspond to a sequence of at least 9 consecutive amino acids of the mature TGFbl polypeptide, for example a sequence of at least 9 consecutive amino acids of SEQ ID NO: 4.

[0089] The polypeptide may comprise or consist of up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 consecutive amino acids of SEQ ID NO: 1. The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 2 and 5-67. The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, 63, 5, 7-9, 43-45, 13-15, 24- 26, 29-31, 50-52, 56-58, 64, 65, 2, 66, 67 or 5. In some embodiments, polypeptides comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, 63, 66, 67 or 5.

[0090] The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 28, 66, 29-31, 67, 5-9, 42-45, 12-15, 55-58, 23-26, 49-52, 63, 64, 65 or 2. In some embodiments, polypeptides comprise or consist of the amino acid sequence of SEQ ID NOs: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63.

[0091] The polypeptide may have a maximum length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 amino acids. The C terminal amino acid of the polypeptide may be replaced with the corresponding amide. The polypeptide may be isolated.

[0092] In some embodiments, polypeptides comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, or 63. In further embodiments, polypeptides comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63. Longer polypeptide fragments of SEQ ID NO: 1 which incorporate these sequences may also be employed.

[0093] The polypeptide may comprise an HLA-A2 restricted epitope. In one embodiment, the HL A- A2 -restricted epitope comprises or consists of the amino acid sequence of SEQ ID NO: 66. In another embodiment, peptides which comprise an HLA-A2 restricted epitope consisting of the amino acid sequence of SEQ ID NO: 66 are peptides which comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 28-31 or 65. Alternatively, the HL A- A2 -restricted epitope comprises or consists of the amino acid sequence of SEQ ID NO: 67. In some embodiments, peptides which comprise a HLA-A2 restricted epitope consisting of the amino acid sequence of SEQ ID NO: 67 are peptides which comprise or consist of the amino acid sequences of any one of SEQ ID NOs: 5, 8, 9 or 2.

[0094] In any polypeptide described herein, the amino acid sequence may be modified by one, two, three, four, or five (that is up to five) additions, deletions or substitutions, provided that a polypeptide having the modified sequence exhibits the same or increased immunogenicity to TGFbl, as compared to a polypeptide having the unmodified sequence. By “the same” it is to be understood that the polypeptide of the modified sequence does not exhibit significantly reduced immunogenicity to TGFbl as compared to polypeptide of the unmodified sequence. Any comparison of immunogenicity between sequences is to be conducted using the same assay. Unless otherwise specified, modifications to a polypeptide sequence are conservative amino acid substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table 2 below. Where amino acids have similar polarity, this can be determined by reference to the hydropathy scale for amino acid side chains in Table 3.

[0095] Table 2- Chemical properties of amino acids

[0096] Table 3 - Hydropathy scale

[0097] Side Chain Hydropathy

[0098] He 4.5 Vai 4.2 Leu 3.8 Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly -0.4 Thr -0.7 Ser -0.8 Trp -0.9 Tyr -1.3 Pro -1.6 His -3.2

[0099] Glu -3.5 Gin -3.5 Asp -3.5 Asn -3.5 Lys -3.9 Arg -4.5

[0100] In any polypeptide disclosed herein, any one or more of the following modifications may be made to improve physiochemical properties (e.g. stability), provided that the polypeptide exhibits the same or increased immunogenicity to TGFbl, as compared to a polypeptide having the unmodified sequence: (i) replacement of the C terminal amino acid with the corresponding amide (may increase resistance to carboxypeptidases); (ii) replacement of the N terminal amino acid with the corresponding acylated amino acid (may increase resistance to aminopeptidases); (iii) replacement of one or more amino acids with the corresponding methylated amino acids (may improve proteolytic resistance); and / or (iv) replacement of one or more amino acids with the corresponding amino acid in D- configuration (may improve proteolytic resistance).

[0101] Preferred peptides that may be employed are set out in WO 2020 / 245264 Al (PCT / EP2020 / 065472) the entirety of which is incorporated by reference and the TGFbl sequences disclosed therein are also specifically incorporated by reference, both in relation to potential epitopes the T cell response may be against, but also as sequences used to stimulate such a response. In any of the embodiments set out herein which use a peptide to stimulate a response, a sequence encoding such a peptide may be administered instead, as may be a composition comprising a peptide or encoding sequence.

[0102] Compositions comprising polypeptides

[0103] The present invention provides for use of TGFbl peptides set out herein to increase, or bring about, a TGFbeta-specific T cell response, where the subject is also administered with a PD-1 / PD-L1 antibody. The TGFbetal peptide may be provided in the form of a pharmaceutical composition for use in such methods. The composition may also comprise a PD-1 / PD-L1 antibody. The present invention also provides a PD-1 / PD-L1 antibody for use as set out herein and the PD-1 / PD-L1 antibody may be formulated as it is currently sold, but any suitable means of formulation may be employed.

[0104] In some embodiments, a pharmaceutical composition comprises at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient. In some embodiments, a pharmaceutical composition comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight different polypeptides and at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient. In some embodiments, a pharmaceutical composition comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight different encoding polynucleotides of the invention and at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0105] The carrier, preservative and excipient must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to a subject to which the composition is administered. Typically, all components and the final composition are sterile and pyrogen free. The composition may be a pharmaceutical composition. The composition may comprise an adjuvant. Adjuvants are any substance whose admixture into the composition increases or otherwise modifies the immune response elicited by the composition. Adjuvants, broadly defined, are substances which promote immune responses. Adjuvants may also have a depot effect, in that they also result in a slow and sustained release of an active agent from the administration site. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) at pages 61-63.

[0106] Adjuvants may be selected from the group consisting of: A1K(SO4)2, AlNa(SO4)2, A1NH4 (SO4), silica, alum, Al(0H)3, Ca3 (PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl- nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N- acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(l ’2’-dipalmitoyl-sn -glycero-3- hydroxphosphoryloxy)-ethylamine (CGP 19835 A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2% squalene / Tween-80.RTM. emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund’s Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A, ALUN (see 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51 and QS-21. Various saponin extracts have also been suggested to be useful as adjuvants in immunogenic compositions. Granulocyte -macrophage colony stimulating factor (GM-CSF) may also be used as an adjuvant.

[0107] Adjuvants to be used with the invention in embodiments for stimulate a T cell response include oil / surfactant based adjuvants such as Montanide adjuvants (available from Seppic, Belgium), for example, Montanide ISA-51. Other adjuvants are bacterial DNA based adjuvants, such as adjuvants including CpG oligonucleotide sequences. Yet other adjuvants are viral dsRNA based adjuvants, such as poly I:C. GM-CSF and Imidazoquinolines are also examples of adjuvants.

[0108] In one embodiment, the adjuvant is a Montanide ISA adjuvant. In further embodiment, the Montanide ISA adjuvant is Montanide ISA 51 or Montanide ISA 720.

[0109] In Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) at pages 61-63 it is also noted that, when an antigen of interest is of low molecular weight, or is poorly immunogenic, coupling to an immunogenic carrier is recommended. A polypeptide of the invention may be therefore coupled to a carrier. A carrier may be present independently of an adjuvant. The function of a carrier can be, for example, to increase the molecular weight of a polypeptide fragment in order to increase activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life. Furthermore, a carrier may aid in presenting the polypeptide or fragment thereof to T-cells. Thus, in the composition, the polypeptide may be associated with a carrier such as those set out below. The carrier may be any suitable carrier known to a person skilled in the art, for example a protein or an antigen presenting cell, such as a dendritic cell (DC). Carrier proteins include keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. Alternatively^ the carrier protein may be tetanus toxoid or diphtheria toxoid. Alternatively, the carrier may be a dextran such as sepharose. The carrier must be physiologically acceptable to humans and safe.

[0110] If the composition comprises an excipient, it must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient. These excipients and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0111] Formulation of a suitable composition can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan. Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers. Some ampoules or multi-dose containers may contain a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. In one embodiment of a composition, the active ingredient is provided in dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to administration of the reconstituted composition. The composition may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the adjuvants, excipients and auxiliary substances described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides. Other compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Alternatively, the active ingredients of the composition may be encapsulated, adsorbed to, or associated with, particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyomithine, spermine, spermidine, as well as conjugates of these molecules.

[0112] Compositions and methods for improving the use ofPD-l / PD-Ll antibody

[0113] The present invention provides PD-1 / PD-L1 antibody for use in a method of treating a cancer in a patient, wherein the method comprises administering PD-1 / PD-L1 antibody to said patient and wherein the patient has been previously identified as having TGFbeta-1 T cell response. The present invention provides PD-1 / PD-L1 antibody for use in a method of treating a cancer in a patient, wherein the method comprising administering PD-1 / PD-L1 antibody to said patient and wherein the value of any baseline TGFbeta-1 specific T cell response in the patient has been measured and determined to be the same or greater than a threshold value. Preferably, the threshold value may be the median response value. In another embodiment, the threshold value may be 75th percentile for patients with the same cancer. Various threshold values are discussed elsewhere herein and may be employed. By the same cancer is meant if, for instance the invention is applied to pancreatic cancer, the median value is that for pancreatic cancer patients in general.

[0114] Preferably the patient may have already been, will be, or is also being treated with a CTLA-4 antibody and / or radiotherapy. The CTLA-4 antibody is Ipilimumab. The method of treatment may therefore further comprise administration of CTLA-4 (e.g. Ipilimumab) and / or radiotherapy. The present invention also provides a CTLA-4 antibody (e.g., Ipilimumab) for use in a method of treating cancer, wherein the patient is administered with PD-1 / PD-L1 antibody and / or radiotherapy, wherein the patient has been previously identified as having a TGFbeta-specific T cell response or has been identified as having a TGFbeta-specific T cell response equal or more than a threshold value.

[0115] In any of the embodiments discussed above the measurement for a TGFbeta-specific T cell response may have already been performed. Alternatively, it may be that such measurement forms part of the method referred to itself.

[0116] The present invention further provides a method of stratifying a patient with cancer into one of at least two treatment groups, the method comprising: (i) performing an assay on a sample previously obtained from the patient to detect the presence or absence of a TGFbeta-specific T cell response; (ii) allocating the patient to a first treatment group if a TGFbeta-specific response is present or allocating the patient into a second treatment group if TGFbeta-specific response is absent. In some embodiments, if the patient is allocated to the first treatment group they are to be administered with a PD-1 / PD-L1 antibody. In some embodiments, if the patient is allocated to the second treatment group, they are to be administered with a TGFbeta-1 peptide to increase, or bring about, a TGFbeta-1 -specific T cell response. In some embodiments, the patients allocated to the second treatment group are to be administered with a PD-1 / PD-L1 antibody simultaneously, separately, or sequentially with a TGFbeta-1 peptide.

[0117] The present invention further provides a method of stratifying a patient into one of at least two treatment groups, the method comprising: (i) performing an assay on a sample previously obtained from the patient to detect the level of a TGFbeta-specific T cell response, if present; (ii) allocating the patient to a first treatment group if the TGFbeta-specific T cell response is at least a threshold value or allocating the patient into a second treatment group if a TGFbeta-specific response is below the threshold value. In some embodiments, if the patient is allocated to the first treatment group they are to be administered with a PD-l / PD- L1 antibody. In some embodiments, if the patient is allocated to the second treatment group, they are to be administered with a TGF-betal peptide to increase, or bring about, a TGFbeta- specific T cell response. In some embodiments, the patients allocated to the second treatment group are to be administered with a PD-1 / PD-L1 antibody simultaneously, separately, or sequentially with a TGFbetal peptide.

[0118] Any of the threshold values discussed herein may be, for instance, used as the dividing line for stratification.

[0119] The value for the TGFbeta-specific T cell response seen for a given subject may be used to give the patient an indication of how successful treatment with PD-1 / PD-L1 antibody is likely to be. For example, the present invention provides a method of providing a patient with an indication of how effective treatment with PD-1 / PD-L1 antibody is likely to be for that specific patient comprising detecting in a sample from the patient the presence or absence of a TGFbeta-specific T cell response and, if present, the strength of the TGFbeta- specific T cell response, and then determining the likelihood of efficacy of PD-1 / PD-L1 antibody for that subject from that measurement. Such a method may comprise working out where the response falls in percentile terms. It may comprise determining the magnitude of the response, if present, compared to the median value expected. Such a method, for example, may be used to determine a treatment plan. It may be used to decide on whether to stimulate a TGFbeta-specific T cell response as described herein.

[0120] The present invention also provides an immunogenic fragment of human TGFbeta for use in a method of treating cancer, wherein the method comprises administering the immunogenic fragment of human TGFbeta to a cancer patient that has previously been identified as lacking a TGFbeta-specific T cell response or have a TGFbeta-specific T cell response less than a threshold value, wherein the patient is also administered with a PD- 1 / PD-L1 antibody. In some embodiments, immunogenic fragment of human TGFbeta is a TGFbeta- 1 peptide. In one embodiment, the method may comprise first administering the TGFbeta- 1 peptide before administering PD-1 / PD-L1 antibody. In one embodiment, the method may comprise checking that a TGFbeta-specific T cell response has resulted from administering the peptide or that the response is at least a threshold value and if that has occurred then administering the PD-1 / PD-L1 antibody. The present invention further provides PD-1 / PD-L1 antibody for use in such methods. In some embodiments, a CTLA-4 antibody (e.g. Ipilimumab) is provided for use in such methods. In some embodiments, radiotherapy is provided for use in such methods.

[0121] The present invention also provides use of an immunogenic TGFbeta- 1 peptide for improving cancer treatment with PD-1 / PD-L1 antibody. Any of the methods set out herein may be used to improve PD-1 / PD-L1 antibody. The improvement may be in terms of increasing overall survival time (OS). Additionally or alternatively the improvement may be in terms of increasing the length of Progression Free Survival (PFS). In one embodiment, the treatment increases the chance of the patient showing clinical benefit from the treatment for six or more months from the start of the treatment given.

[0122] In embodiments where a TGFbeta- 1 peptide is administered to stimulate or boost a response, it may be administered once. In embodiments TGFbeta-1 peptide may be administered repeatedly. For instance, it may be administered one to five times, such as two, three, four or five times. It may be administered repeatedly until a response is seen or rises to the desired level.

[0123] Where a product for use in a method of treatment is set out herein, the present invention also provides the method itself. The present invention also provides use of what is set out in the manufacture of a medicament to treat the stated condition.

[0124] The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

[0125] EXAMPLES

[0126] The following is a description of various methods and materials used in the studies. They are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for.

[0127] Example 1

[0128] Introduction

[0129] We have investigated why not all patients show good improvement with antibodies against PD-1 / PD-L1 and as described herein have identified the presence or absence, and in particular the size, of a TGFbeta-specific T cell response in a given subject as indicative of how effective treatment will be. That finding helps predict how effective antibody treatment will be for a particular patient and also whether to promote a TGFbeta-specific T cell response prior to therapy with the antibody, with that combination therapy promoting the efficiency of the antibody therapy. Materials and methods

[0130] Patients and donors

[0131] Buffy coats from healthy donors were attained anonymously from the blood bank at Rigshospitalet, Copenhagen, Denmark. The usage of anonymized biological material does not require approval from an ethics committee according to Danish Law. Access to buffy coats from pancreatic cancer patients was given from the Department of Oncology, Copenhagen University Hospital, Herlev, Denmark. All patients gave informed consent, in agreement with the Helsinki Declaration, before use of buffy coats.

[0132] Peptide

[0133] The sequence for the specific peptide TGFp-15 is: REAVPEPVLLSRAELRLLRL (SEQ ID NO: 28). The peptide was acquired at a high purity (>90%) from Schafer (Copenhagen, Denmark) and was dissolved in DMSO at a concentration of 10 mM.

[0134] In vitro cultures and Enzyme-Linked Immunospot assay (ELISPOT)

[0135] Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved, as previously described (Holmstrom, M. O. & Andersen, M. H. Healthy, Cancers (Basel). 12, (2020)). PBMCs received prior in vitro stimulation with the TGFp-15 epitope. After incubation for 9-10 days, the cells were counted using a Countess II Automated Cell Counter (Thermo-Fisher) and the occurrence of T cells specific to TGFp-15 was assessed with an interferon-g (IFN-g) ELISPOT assay. Cells were plated out in triplicates with a concentration of 200,000 cells per well and stimulated with peptide for a final concentration of 5 pM in the well. PVDF membrane plates (Merck, Germany) coated with primary IFN-y specific antibody (Mabtech, Sweden) were used for the ELISPOT assays. After incubation overnight the cells were poured off, and, following manufacturer’s protocol (Mabtech, Sweden), the wells were coated with secondary antibody, streptavidin- ALP, and enzyme substrate with washing of wells with PBS before and between each step. The plates were counted using the ImmunoSpot S6 Ultimate Analyzer (CTL Analyzers, Shaker Heights, OH, USA) when dry. The normalized mean spots were defined as the mean number of spots in peptide-stimulated wells subtracted by the mean number of spots in negative control wells. In the repeated stimulations assays the in vitro cultures received 2 pl of 10 mM TGFp-15 on day 0 and then at day 1 they received 120 U / mL of IL-2 (Novartis, Switzerland). That was repeated every 7 days once or twice and after the final stimulation incubation was continued for 9-10 days after which they were set up in ELISPOT assays as described above.

[0136] Statistics

[0137] Statistical analysis of paired observations was performed using two tailed paired T- test and when sample size was small nonparametric Wilcoxon matched pairs signed rank test. For unpaired observations two tailed nonparametric Mann- Whitney test was used. These tests were done using the Graphpad Prism Version 9.

[0138] Survival analysis was performed with the statistical software R using the survminer package for analysis. All variables that were found to be associated with survival and progression free survival in the original trial (Chen, I. M. et al, 2022, supra) were tested for association with these in our cohort of patients tested for immune responses. This univariate analysis was performed using the log-rank analysis. All statistically significant parameters were included in a Cox proportional hazards model to analyze the independent association of immune responses to overall survival and progression free survival. Kaplan-Meier curves were made using Graphpad Prism Version 9. Analysis of the ELISPOT data also used the distribution free resampling (DFR) method and the more conservative DFR2x method.

[0139] Results

[0140] Patient characteristics

[0141] We scrutinized samples from patients with metastatic pancreatric cancer (PC) enrolled in the CheckPAC trial and showed spontaneous immune responses to the TGFbeta-15 epitope. CheckPAC was a phase II, single-center trial in patients with refractory metastatic PC conducted at Copenhagen University Hospital, Herlev, Denmark. The trial included 84 patients that received stereotactic body radiation therapy (SBRT) combined with Immune Checkpoint Inhibitor (ICI). The trial included two arms: arm A received single agent nivolumab, and arm B received nivolumab combined with Ipilimumab. Among the 84 patients included, we analyzed immune responses to the TGFb-15 epitope in 32 patients. The patient characteristics are displayed in Table 4. Of the 32 patients analyzed, 7 (22%) achieved a partial response (PR), 14 (44%) had stable disease (SD), and 11 (34%) displayed progressive disease (PD). The achievement of either PR or SD was defined as clinical benefit from treatment. PD was defined as no clinical benefit from therapy. Within a median followup time of 219 days (range 65 - 1511), two patients remained alive (6%), and of these, one (3%) showed no sign of disease. Table 4

[0142] Patients with clinical benefit harbored T cells specific to TGFb.

[0143] We analyzed TGFb- 15 -specific responses in the samples from the patients, finding that T cells isolated both at baseline and after four series of treatment displayed responses to TGFb-15 (Figure 1A-B). The patients with clinical benefit had significantly higher baseline immune-response amplitudes than patients with progressive disease (PD), whereas we found no difference in response amplitude after treatment (8 weeks) (Figure 1C). The data indicated that a TGFb-specific immune response present before initiating treatment influences the clinical response to SRBT / ICI. Our data therefore indicates that TGFbeta-specific T cells may be important in the clinical response to therapy. Of note, we found that patients with clinical benefit displayed a significant drop in the TGFp-specific response amplitude 8 weeks after the start of treatment (Figure 2A-B). Furthermore, patients with lasting clinical benefit to therapy, defined as a PR or SD lasting longer than 6 months, displayed a significantly lower TGFp-specific response 8 weeks after treatment, compared to patients without a lasting clinical benefit, (Figure 3A-B).

[0144] Next, we analyzed serial PBMC samples from three patients with a follow-up time longer than 2 years. We analyzed TGFb- 15 -specific immune responses to assess temporal variations in the response. Interestingly, we found that the response amplitude fluctuated over time (Figure 2C). T-cell responses specific to TGFb-15 were independently associated with improved survival Our data strongly suggested that an intact TGFb-specific immune response could impact the response to therapy. Therefore, we investigated whether the baseline TGFb- specific immune response was associated with survival in our patient cohort. We stratified patients based on whether they showed responses above or below the median normalized TGFb-15 -specific immune response. Interestingly, we found that patients with a response amplitude above the median had significantly longer overall survival (OS) than those with responses below the median (univariate Cox-regression, hazard ratio [HR]: 0.171, p=2.54 xlO'4; Figure 4 A and Table 5 below).

[0145] Table 5

[0146] Next, we investigated whether the TGFb-15-specific immune response was independently associated with overall survival (OS). We performed univariate survival analysis on clinical parameters that could be associated with survival, with a special focus on parameters that were included in the survival analysis in the original CheckPAC trial report. A univariate Cox-regression analysis showed that <2 metastases was associated with a lower OS (HR= 2.64, p=0.03), but no other parameters showed statistical significance (Table 5). A multivariate analysis that included the metastases parameter and the TGFb-15-specific immune response showed that only the TGFb-15-specific immune response was independently associated with OS (HR: 0.18) for patients with a response above the median amplitude (p= 8x1 O'4; Table 5). Of note, the univariate analysis showed borderline significance for reduced survival among patients who received single-agent nivolumab (HR: 2.04, p=0.055). Hence, we included this parameter in another multivariate analysis, together with metastases and the TGFb- 15 -specific immune response. In the latter model, neither the metastases variable nor the treatment variable were associated with OS; however, a TGFb- 15-specific response above the median remained associated with improved survival (HR: 0.19, p=0.0032; data not shown). The same analyses were performed for Progression Free Survival (PFS). In the univariate analysis, the TGFb- 15 -specific response was associated with a prolonged PFS (HR: 0.227, p=0.0015; Figure 4B and Table 6 below). Other significant parameters from the univariate analysis were: nivolumab treatment (HR: 2.86, p=0.007), male sex (HR: 0.406, p=0.029), and >36 g / L albumin in peripheral blood (HR: 4.68, p=0.038). When these significant parameters were included in the multivariate analysis, together with the TGFb-15-specific immune response, only the TGFb-15response above the median remained independently associated with PFS (HR: 0.322, p=0.023; Table 6 below). Interestingly, the patients with a TGFb-15 response above the 75th percentile had the highest OS and PFS of examined samples (Figure 5A-B). These results further indicate that the level of TGFb-15-specific T-cells in patients with pancreatic cancer is important in achieving a clinical response to SBRT / ICI treatment.

[0147] Table 6

[0148] Association between TGFb-15-specific response and survival was not due to general immune dysfunction in non-responding patients

[0149] We investigated whether patients with poor survival might harbor a dysfunctional immune system, and thus, the low response amplitude observed in these patients could be caused by general T-cell dysfunction. We analyzed the PBMC (peripheral blood mononuclear cell) response amplitude when challenged with two broadly immunogenic epitopes - one derived from Clostridium tetani (Slingluff, C. L. et al.. J. Immunother. Cancer 9, (2021)) (tetanus-long) and the other derived from influenza virus (Cl 8 A2 Flu). The latter was a nonamer epitope restricted to HLA-A2; consequently only samples with HLA-A2+were analyzed for responses against the influenza epitope. We analyzed 23 patient samples for responses against tetanus and 16 for responses against influenza. Samples from patients with strong and weak TGFb-15 specific immune responses had similar response amplitudes to both tetanus and influenza epitopes (Figure 7A-D). Additionally, we showed that the TGFb- 15-response amplitude was not correlated with the tetanus response (^=0.05) nor the influenza response (r2=0.15) (Figure 6A-B). Perhaps more importantly, the response amplitude to the tetanus and influenza epitopes was not associated with either OS or PFS (Figure 8A-D).

[0150] Repeated antigen stimulations with a TGFb-15 peptide led to T-cell responses in PBMCs that did not display a response after one in vitro stimulation

[0151] The above results strongly indicates that a measurable TGFb-15 -specific immune response is important in the clinical response to SBRT / ICI treatment in patients with pancreatic cancer. Thus, we reasoned that, in pancreatic cancer, inducing a TGFb-15 response with therapeutic peptide vaccines might be effective, when combined with SRBT / ICI. We investigated whether a TGFb-15 specific immune response could be enhanced in PBMCs from healthy individuals and patients with pancreatic cancer by repeatedly stimulating with the epitope. We chose to work with PBMC samples from earlier experiments that showed weak or absent responses to TGFb-15 after one in vitro stimulation. T-cell responses were analyzed after one in vitro stimulation and after one or two additional in vitro stimulations. We showed that repeated antigen stimulations increased the TGFb-15-specific immune response in PBMCs from both patients with PC (Figure 9 A) and healthy individuals (Figure 9B-C). Of tested pancreatic cancer patients, 27% had a DFR2x response after 1 stimulation which increased to 66% after repeated stimulations (Figure 10A). With the tested healthy donors, 9% had a DFR2x response after 1 stimulation which increased to 71% after repeated stimulations (Figure 10B). These data showed that repeated antigen stimulations with the TGFp-15 peptide increased the numbers of TGFbeta-specific T cells in PBMCs.

[0152] Discussion

[0153] We have here investigated relationships between the TGFb-15-specific immune response and clinical outcomes. Interestingly, compared to patients who did not show clinical benefit to treatment, patients with clinical benefit had a significantly stronger TGFb-15- specific immune response before treatment initiation and a significantly stronger decline in the TGFb-15 -specific T cell response after initiation of treatment. The latter phenomenon could be explained by migration of TGFbeta-specific T-cells to the tumor after initiating ICI therapy. These results were consistent with the striking observation that patients with a TGFb-15 -specific immune response above the median had significantly longer Progression- Free survival (PFS) and Overall Survival (OS), compared to patients with a response below the median. We also investigated whether the identified difference in OS between patients with strong and weak TGFb-15-specific immune responses might be attributed to differences in the general immune status of the patients. We compared the spontaneous immune responses in these two groups against two highly immunogenic epitopes derived from common pathogens - influenza virus and C. tetani (Slinghuf et al., 2021, supra). We found no difference between groups in the response amplitude to neither influenza nor tetanus. Additionally, there was no correlation between the response amplitude to TGFb-15 and the response to tetanus or influenza epitopes. Interestingly, patients with a strong pathogen specific immune response did not show either improved OS or improved PFS after ICI treatment. This implied that the strength of the TGFb-15 -specific immune response did not depend on the general immune constitution; instead, it reflected the number of anti-regulatory TGFbeta-specific T cells.

[0154] These findings indicate that TGFbeta-specific anti-regulatory T cells played a role in the response to ICI therapy in pancreatic cancer. Given the many effects of TGFbeta in pancreatic cancer, it is worthwhile to consider the potential immunomodulating and tumorsuppressive effects that TGFbeta-specific T cells could have in patients. In pancreatic cancer, approximately 95% of patients harbor an activating mutation in the KRAS-gene (Prior, I. A., et al, Cancer Res. ’ll, 2457-2467 (2012). These mutations induce the production of TGFb in transformed cells (Zdanov, S. et al., Cancer Immunol. Res. 4, 354-365 (2016) and Cheng, H. et al., Cancer Lett. 446, 103-111 (2019)). Hence, TGFbeta-specific T cells would act directly on the transformed cells. However, several other prevalent immunosuppressive cells would also be targeted.

[0155] Pancreatic cancer is characterized by a highly desmplastic stroma and cancer associated fibroblasts (CAFs) are the main culprits behind this feature (Kalluri, R., Nat. Rev. Cancer 16, 582-598 (2016) and Kobayashi, H. et al., Nat. Rev. Gastroenterol. Hepatol. 16, 282-295 (2019)). CAFs are thought to originate from both bone marrow-derived mesenchymal stem cells and pancreatic stellate cells (Moir, J. A. G., et al. Surg. Oncol. 24, 232-238 (2015)). Through secretion of TGFb, pancreatic cancer-derived cell lines can activate CAF, which increases the deposition of extracellular matrix proteins and enhances fibrosis (Lohr, M. et al, Cancer Res. 61, 550-555 (2001) and Principe, D. R. et al. Cancer Res. 76, 2525 (2016)). Furthermore, activated CAFs also secrete TGFb, which acts in an autocrine fashion thus resulting in additional secretion of TGFb and notably intratumoral TGFb expression was shown to correlate with tissue fibrosis. Another study demonstrated that pancreatic cancer patients with high levels of fibrosis in combination with the CAF- marker aSMA, showed inferior survival (Sadozai, H. et al., Front. Immunol. 12, 1-15 (2021)). That finding underscored the importance of CAFs in pancreatic cancer. The immunomodulatory effects of CAFs have been studied extensively, and CAF populations are similar among different cancers (Kieffer, Y. et al., Cancer Discov. 10, 1330-1351 (2020)). Of note, one group of CAFs is characterized by increased TGFb signaling - so called myofibroblastic CAFs (myCAFs). Interestingly, a subset of myCAFs was associated with a lack of response to ICI in several cancers (Ohlund D., JEM (2017) doi:10.1084 / jem.20162024).

[0156] The composition of immune cells in pancreatic cancer is heterogeneous (Steele, N. G. et al.,. Nat. cancer 1, 1097 (2020)) but generally, most are of myeloid origin (Steele, N. G. et al.,. Nat. cancer 1, 1097 (2020); Vayrynen, S. A. et al., Clin. Cancer Res. 27, 1069-1081 (2021); and Elyada, E. et al., Cancer Discov. 9, 1102-1123 (2019)). These myeloid cells include TAMs (Tumor Associated Macrophages), MDSCs (Myeloid-derived suppressor cells), and neutrophils, all expressing TGFp, which modulates the TME (tumor microenvironment). Neutrophils have been shown to secrete high amounts of TGFb in the pancreatic cancer TME, which attracts and activates CAFs (Aoyagi, Y. et al.,. Br. J. Cancer 200491791, 1316-1326 (2004)). Additionally, local TGFb converts myeloid cells into M2 macrophages, TAMs, and MDSCs, which are elevated in pancreatic cancer (Clark, C. E. et al.,. Cancer Res. 67, 9518-9527 (2007), and they negatively impact both PFS and OS in patients with pancreatic cancer (Ino, Y. et al., Br. J. Cancer 2013 1084 108, 914-923 (2013); Sadozai, H. et al (2021) supra,' Tsujikawa, T. et al., Cell Rep. 19, 203-217 (2017)). These cells express TGFp (Zhu, L., et al,. Cell Biol. Int. (2017) doi:10.1002 / cbin,10788) and, apart from their own immunosuppressive properties, they also mediate the conversion of naive T- cells into Tregs (Huang, B. et al., Cancer Res. 66, 1123-1131 (2006); Siret, C. et al., Front. Immunol. 10, 3070 (2020)). Hence, the emergence of both MDSCs and Tregs in the TME of pancreatic cancer depends on TGFb. Moreover, the level of CD8+T-cells in the TME is inversely correlated with the levels of myeloid cells and Tregs (Clark, C. E. et al., (2007) supra,' Steele, N. G. et al., Nat. cancer 1, 1097 (2020); and Siret, C. et a / ., (2020) supra). Thus, TGFb is an attractive target for enhancing the effect of cancer immune therapy in pancreatic cancer.

[0157] We have shown that the levels of T cells specific for TGFb correlate with disease outcome both in terms of PFS and OS. We also consider that therapeutic cancer vaccines of TGFp-derived epitopes represent potential future treatment modality in combination with ICI treatments. Moreover, repeated peptide vaccinations could enhance the TGFbeta-specific immune response in patients. The rationale for repeated vaccinations is to increase the numbers of TGFbeta-specific T cells that migrate to the TME; these cells will attack TGFbeta-expressing cells and release Thl cytokines; thus, TGFb signaling will be reduced, and the TME will be converted to an immunopermissive environment that favors the killing of transformed cells by tumor-specific T-cells.

[0158] In the present study, we mimicked therapeutic vaccinations by performing repeated antigen stimulations in PBMCs that displayed a weak / absent response to TGFb-15. We found that repeated stimulations induced a strong TGFb-15-specific immune response in almost all cultures. This finding supported the idea that repeated vaccinations with TGFb derived peptides can induce a TGFb-specific immune response. Moreover, these findings indicate that TGFb- 15 -specific T cells were not terminally exhausted or absent in samples that did not respond.

[0159] Conclusion

[0160] PBMC samples from patients with pancreatic cancer treated with ICI and SBRT displayed immune responses to the TGFb-15 epitope. Patients with clinical benefit from treatment had stronger TGFb- 15 -specific T-cell responses than patients with progressive disease. Furthermore, a strong TGFb-15-specific T cell response before treatment initiation was independently associated with prolonged progression free survival and overall survival. We also showed that low-level TGFb-15-specific responses observed in some patients was not due to a general dysfunction of the immune system, as these patients retained a normal T- cell response to common pathogen-derived epitopes. Furthermore, we showed that TGFb- 15-specific immune responses could be induced / enhanced by repeated antigen stimulations. Consequently, administering therapeutic cancer vaccinations to deliver repeated TGFb- 15- antigen stimulations in patients may induce a specific T-cell response that is likely to lead to a clinical response.

[0161] Example 2

[0162] Materials and methods

[0163] All materials and methods were as for Example 1. The sequence for the peptide TGFb-33 is: FCLGPCPYIWSLDTQYSKVL (SEQ ID NO: 55). Results

[0164] T-cell responses specific to TGFb-33 were independently associated with improved survival To further investigate whether the baseline TGFb-33-specific immune response was associated with improved survival in a similar patient cohort to that of Example 1 , patients were stratified based on whether they showed responses above or below to or equal to the median normalized TGFb-33-specific immune response at baseline. We analyzed TGFb-33- specific responses in the samples from the patients, finding that T cells isolated both at baseline and after four series of treatment displayed responses to TGFb-33 (Figure 12).

[0165] Additionally, we tested clinical parameters that could impact survival in patients for their association to survival. Hence, the following parameters were tested in univariate Coxregression analysis for association to overall survival (n = 33 for baseline values and n = 28 for follow up values):

[0166] Treatment arm

[0167] Sex

[0168] - Age

[0169] Performance status

[0170] Weight loss > 5 %

[0171] Number of metastatic sites > 1 or < 1

[0172] Whipple procedure

[0173] Biliary stent

[0174] Tumor marker CA-119-9 above or below median

[0175] Neutrophil-to-lymhocyte ratio (NLR) > 5

[0176] Bilirubin > 25 pmol / L

[0177] - Albumin < 36 g / L

[0178] C-reactive protein > 10

[0179] Modified Glasgow prognostic score (mGPS) Number of prior lines of therapy Response to TGFb33 at baseline > median Response to TGFb33 at follow-up > median

[0180] Interestingly, it was found that patients with a TGFb-33 -response amplitude above the median at baseline had significantly longer overall survival (OS) compared to patients with responses below or equal to the median (univariate Cox-regression, hazard ratio [HR]: 6.85, p=3.00 xlO'4; Figure 11A and Table 7 below). The only other parameter with a statistically significant association to overall survival was the number of metastatic sites, as patients with <1 metastases displayed an inferior OS (HR= 2.5, p=0.0368) (Table 7). Only variables with a statistically significant association to overall survival in the univariate analysis are displayed in Table 7.

[0181] Incorporating the TGFb-33-response and the number of metastatic sites in a multivariate Cox-regression model showed that the amplitude of the TGFb-33-specific immune response at baseline was independently associated with OS (HR: 6.1, p= l.lxlO'3; for patients with a response below or equal to the median amplitude) (Table 7).

[0182] Table 7

[0183] The same analyses were performed for Progression Free Survival (PFS). In the univariate analysis, the TGFb-33-specific response was associated with PFS (HR: 6.85, p=3.00 xlO'4for patients with a response below or equal to the median amplitude; Figure 1 IB and Table 8 below). Other significant parameters from the univariate analysis were: male sex (HR: 0.38, p=0.0188), and <1 metastases (HR: 2.5, p=0.0312). A multivariate analysis in which these statistically significant parameters were incorporated showed that the TGFb-33- specific immune response was independently associated with OS (HR: 2.9, p=0.0141 for patients with a response below or equal to the median amplitude, Table 8 below). Only variables with a statistically significant association to progression free survival in the univariate analysis are displayed in Table 8.

[0184] Table 8 Example 3

[0185] "Non-immunogenicity" of pancreatic cancer (PC) with high prevalence of immunosuppressive cells and typically a scarcity of tumor-infiltrating effector lymphocytes is considered as one of the reasons for lacking responsiveness to single-agent immunotherapies. Considering the emerging role of the tumor micro environment, the combination of checkpoint blocking antibodies with immunomodulation of the tumor microenvironment could lead to better responses in tumor historically resistant to radiation and checkpoint blocking antibody approaches as single modalities. For example, the data from the Phase 2 study CheckPAC (NCT02866383) in patients with resistant metastatic PC, showed durable clinical benefit in a small subgroup of patients after adding stereotactic body radiotherapy (SBRT) of 15 Gy to a combination of nivolumab and ipilimumab (presented at ASCO GI 2022, San Francisco, see abstract by Chen et al., “Randomized phase 2 study of nivolumab with or without ipilimumab in combination with stereotactic body radiotherapy in patients with refractory metastatic pancreatic cancer (CHECKPAC)”, 2022 ASCO Gastrointestinal Cancers Symposium).

[0186] We have now found that the TGF|3-15 immune response is corelated to clinical benefit, supporting the rationale for combining of TGF|3-15 peptide vaccine with CheckPAC strategy (SBRT of 15 Gy in combination with nivolumab and ipilimumab). Accordingly, an interventional Phase I study CheckVAC (NCT05721846) is being carried out to evaluate the safety and tolerability of nivolumab with ipilimumab combined with TGF|3-15 peptide vaccine and SBRT for refractory PC. The study will measure adverse events, overall response rate, overall survival, progression free survival, duration of response, best overall response and disease control rate.

[0187] The following inclusion criteria are being used:

[0188] • Signed informed consent o Subjects must have signed and dated an IRB / IEC approved written informed consent form in accordance with regulatory and institutional guidelines. This must be obtained before the performance of any protocol related procedures that are not part of normal subject care o Subjects must be willing and able to comply with scheduled visits, treatment schedule, laboratory testing, and other requirements of the study • Histological or cytological confirmation of advanced pancreatic carcinoma prior to entering this study

[0189] • Prior therapy requirements: o There is no upper limit on the number of prior chemotherapy regimens received. Participants must have received and progressed during or after at least 1 line of systemic chemotherapy in the metastatic setting (gemcitabine or 5-FU based regimens). o Notes:

[0190] ■ If a participant received adjuvant / neoadjuvant systemic combinational therapy, and progressed within 6 months, the adjuvant / neoadjuvant treatment will be considered as 1 line of systemic treatment.

[0191] ■ In general, discontinuation of 1 drug in a multi-drug regimen and continuation of other drug(s), is considered part of the same line of treatment. Restarting the same regimen after a drug holiday or maintenance chemotherapy can also be considered part of the same line of treatment. Switching from IV (5-FU) to an oral formulation (capecitabine) of the same drug is also considered part of the same line of treatment

[0192] ■ Minimum time from first systemic therapy for recurrent / metastatic adenocarcinoma of pancreas to progression should be at least 3 months

[0193] • Age 18 years and older

[0194] • ECOG Performance Status (PS) 0-1

[0195] • All participants will be required to undergo mandatory pre- and on-treatment biopsies at acceptable clinical risk as judged by the investigator. An archival pre -treatment sample is not acceptable.

[0196] • Participants must have normal organ and marrow function as defined below: o Absolute neutrophil count (ANC) > 1.5 x 109 / L o Platelet count > 75 x 109 / L o Serum bilirubin < 1.5 x upper limit of normal (ULN) o AST / ALT < 5 x ULN o Serum creatinine < 1.5 x ULN or CrCl > 40 mL / min (using the Cockcroft- Gault formula) • Women of childbearing potential (WOCBP) must use method(s) of contraception as indicated in Appendix 3. For a teratogenic study drug and / or when there is insufficient information to assess teratogenicity (preclinical studies have not been done), a highly effective method(s) of contraception (failure rate of less than 1% per year) is required. The individual methods of contraception and duration should be determined in consultation with the investigator. WOCBP must follow instructions for birth control when the half-life of the investigational drug is greater than 24 hours, contraception should be continued for a period of 30 days plus the time required for the investigational drug to undergo five half-lives. The half-life of nivolumab and ipilimumab is up to 25 days and 18 days, respectively. WOCBP should therefore use an adequate method to avoid pregnancy during the treatment and for 23 weeks (30 days plus the time required for nivolumab to undergo five half-lives) after the last dose of investigational drug

[0197] • Men who are sexually active with WOCBP must use any contraceptive method with a failure rate of less than 1% per year. The investigator shall review contraception methods and the time period that contraception must be followed. Men that are sexually active with WOCBP must follow instructions for birth control when the halflife of the investigational drug is greater than 24 hours, contraception should be continued for a period of 90 days plus the time required for the investigational drug to undergo five half-lives. The half-life of nivolumab is up to 25 days. Men who are sexually active with WOCBP must continue contraception during the treatment and for 31 weeks (90 days plus the time required for nivolumab to undergo five half-lives) after the last dose of investigational drug. Women who are not of childbearing potential (i.e. who are postmenopausal or surgically sterile as well as azoospermic men do not require contraception

[0198] • Subjects must have signed and dated a BIOP AC approved written informed consent form in accordance with regulatory and institutional guidelines.

[0199] The following exclusion criteria are being used:

[0200] • Any serious or uncontrolled medical disorder that, in the opinion of the investigator, may increase the risk associated with study participation or study drug administration, impair the ability of the subject to receive protocol therapy, or interfere with the interpretation of study results • Prior treatment with an anti-PD-1, anti-PD-Ll, anti-PD-L2, anti-CD137, or anti- CTLA-4 antibody, or any other antibody or drug specifically targeting T-cell costimulation or checkpoint pathways

[0201] • Participants with active, known or suspected autoimmune disease. Participants are permitted to enroll with vitiligo, type I diabetes mellitus, residual hypothyroidism due to autoimmune condition only requiring hormone replacement, psoriasis not requiring systemic treatment, or conditions not expected to recur in the absence of an external trigger.

[0202] • Current or prior use of immunosuppressive medication within 14 days before the first dose of nivolumab, ipilimumab and radiation in combination with TGF|3-15 peptide vaccine. The following are exceptions to this criterion: o Intranasal, inhaled, or topical steroids; or local steroid injections (e.g. intraarticular injection) o Systemic corticosteroids at physiologic doses not to exceed 10 mg / day of prednisone or equivalent o Steroids as premedication for hypersensitivity reactions (e.g. CT scan premedication)

[0203] • Participants should be excluded if they have known history of testing positive for human immunodeficiency virus (HIV) or known acquired immunodeficiency syndrome (AIDS)

[0204] • Allergies and Adverse Drug Reaction o History of allergy to study drug components o History of severe hypersensitivity reaction to any monoclonal antibody

[0205] • WOCBP who are pregnant or breastfeeding

[0206] The following dosage regimens are being used:

[0207] It is expected that administration of the TGFb-15 peptide vaccine will enhance the clinical benefit of SBRT, nivolumab and ipilimumab for the treatment of refractory PC.

[0208] * * * * *

[0209] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.

[0210] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

Claims

CLAIMS1. A PD-1 / PD-L1 antibody for use in a method of treating a cancer in a patient, wherein the method comprises administering the PD-1 / PD-L1 antibody to said patient, wherein the patient has been previously identified as having a TGFbeta-specific T cell response.

2. The PD-1 / PD-L1 antibody for the use of claim 1, wherein the TGFbeta-specific T cell response is against a peptide sequence having the amino acid sequence of SEQ ID NOs: 28, 55, 66, 29-31, 67, 5-9, 42-45, 12-15, 56-58, 23-26, 49-52, 63, 64, 65 or 2, preferably wherein the TGFbeta-specific T cell response is against a peptide sequence having the amino acid sequence of SEQ ID No: 28, 55 or 66, more preferably SEQ ID NO: 28.

3. The PD-1 / PD-L1 antibody for the use of claim 1 or 2, wherein the patient displays a baseline TGFbeta-specific T cell response prior to the treatment that is at least the median baseline value of the TGFbeta-specific T cell response seen cancer patients with the same type of cancer, preferably wherein the patient displays a baseline TGF|3-specific T cell response prior to the treatment that is at least the 75th percentile of the baseline value of the TGFp-specific T cell response in cancer patients with the same type of cancer.

4. The PD-1 / PD-L1 antibody for the use of any one of the preceding claims, wherein the PD-1 / PD-L1 antibody is a PD-1 antibody.

5. A PD-1 / PD-L1 antibody for the use of any one of the preceding claims, wherein the PD-1 / PD-L1 antibody is Nivolumab.

6. A PD-1 / PD-L1 antibody for the use of claim 5, wherein the patient is also being administered with a CTLA-4 antibody, preferably Ipilimumab.

7. A PD-1 / PD-L1 antibody for the use of any one of the preceding claims wherein the patient is also being administered with radiotherapy.

8. The PD-1 / PD-L1 antibody for the use of any one of the preceding claims wherein the cancer is pancreatic cancer and preferably is metastatic pancreatic cancer.

9. A method of treating cancer in a patient, the method comprising administering a PD- 1 / PD-L1 antibody to said patient, wherein the patient has been previously identified as having a TGFbeta-specific T cell response.

10. The method of claim 9, wherein the TGFbeta-specific T cell response is against a peptide sequence having the amino acid sequence of SEQ ID NOs: 28, 55, 66, 29-31, 67, 5-9, 42-45, 12-15, 56-58, 23-26, 49-52, 63, 64, 65 or 2, preferably wherein the TGFbeta-specific T cell response is against a peptide sequence having the amino acid sequence of SEQ ID No: 28, 55 or 66, more preferably SEQ ID NO: 28.

11. The method of claim 9 or 10, wherein the patient displays a baseline TGFbeta-specific T cell response prior to the treatment that is at least the median baseline value of the TGFbeta-specific T cell response seen in the cancer patients with the same type of cancer, preferably wherein the patient displays a baseline TGFbeta-specific T cell response prior to the treatment that is at least the 75thpercentile of the baseline value of the TGFbeta-specific T cell response seen in the cancer patients with the same type of cancer.

12. The method of any one of claims 9 to 11, wherein the PD-1 / PD-L1 antibody is Nivolumab.

13. The method of claim 12, wherein the patient is also being administered with a CTLA- 4 antibody, preferably where the CTLA-4 antibody is Ipilimumab.

14. The method of any one of claims 9 to 13, wherein the patient is also being administered with radiotherapy.

15. The method of any one of claims 9 to 14, wherein the cancer is pancreatic cancer and preferably is metastatic pancreatic cancer.

16. A method of stratifying a patient with cancer into one of at least two treatment groups, the method comprising:i. performing an assay on a sample previously obtained from the patient to detect the presence or absence of a TGFb-specific T cell response; ii. allocating the patient to a first treatment group if a TGFb-specific response is present or stratifying the patient into a second treatment group if a TGFb-specific response is absent; wherein if the patient is allocated to the first treatment group they are to be administered with a PD-l / PD-Ll antibody.

17. A method of stratifying a patient with cancer into one of at least two treatment groups, the method comprising: i. performing an assay on a sample previously obtained from the patient to detect the level of a TGFb-specific T cell response, if present; ii. allocating the patient to a first treatment group if the TGFb-specific T cell response is at least a threshold value for a TGFb-specific T cell and stratifying the patient into a second treatment group if a TGFb-specific response is below the threshold value; wherein if the patient is allocated to the first treatment group they are to be administered with a PD-l / PD-Ll antibody.

18. The method of claim 17, wherein the threshold value is the median baseline TGFb- specific T cell response for cancer patients with the same type of cancer and preferably wherein the threshold value is 75% of the baseline TGFb-specific T cell response in cancer patients with the same cancer type.

19. The method of any one of claims 16 to 18, wherein:(a) the PD-1 / PD-L1 antibody is Nivolumab, preferably wherein the patient is also being treated radiotherapy and / or the CTLA4 antibody is Ipilimumab and preferably with both; and / or(b) the cancer is pancreatic cancer, preferably wherein the cancer is metastatic pancreatic cancer.

20. The method of any one of claims 16 to 19, wherein the TGFbeta-specific T cell response is against a peptide sequence having the amino acid sequence of SEQ ID NOs: 28, 55, 66, 29-31, 67, 5-9, 42-45, 12-15, 56-58, 23-26, 49-52, 63, 64, 65 or 2, preferably against apeptide sequence having the amino acid sequence of SEQ ID No: 28 or 55, more preferably SEQ ID NO: 28.

21. A PD-1 / PD-L1 antibody for use in a method of treating cancer, wherein the method comprises:(i) administering to a patient an immunogenic fragment of human transforming growth factor (TGFb) which comprises or consists of a sequence of at least 9 consecutive amino acids of SEQ ID NO: 1; and(ii) administering the PD-1 / PD-L1 antibody.

22. An immunogenic fragment of human transforming growth factor b (TGFb) for use in a method of treating a cancer in a patient, the method comprising(i) administering to the patient the immunogenic fragment of human transforming growth factor b (TGFb), wherein the immunogenic fragment comprises or consists of a peptide sequence of at least 9 consecutive amino acids of SEQ ID NO: 1; and(ii) administering to the patient a PD-1 / PD-L1 antibody.

23. The PD-1 / PD-L1 antibody for the use of claim 21 or an immunogenic fragment of human transforming growth factor (TGFb) for the use of claim 22, wherein the method comprises first identifying the patient as one who either lacks a response a TGFbeta-specific T cell response or who has a TGFbeta-specific T cell response below a threshold value.

24. The PD-1 / PD-L1 antibody for the use of claim 21 or 23 or the immunogenic fragment of human transforming growth factor (TGFb) for the use of claim 22 or 23, wherein:(i) the cancer is pancreatic cancer, preferably metastatic pancreatic cancer;(ii) the method further comprises administering radiotherapy; and / or(iii) the method further comprises administering Ipilimumab.

25. The immunogenic fragment of human transforming growth factor (TGFb) for the use of any one of claims 22 to 24, wherein:(i) the immunogenic fragment of human TGF|3 comprises the amino acid sequence of SEQ ID NOs: 28, 55, 66, 29-31, 67, 5-9, 42-45, 12-15, 56-58, 23-26, 49-52, 63,64, 65 or 2, preferably wherein the immunogenic fragment of human TGF|3 comprises the amino acid sequence of SEQ ID NOs 28 or 55, more preferably SEQ ID NO: 28;(ii) the immunogenic fragment of TGF|3 is administered to the patient prior to or simultaneously with other treatments; (iii) the immunogenic fragment of TGF|3 is administered to the patient repeatedly; and / or(iv) the immunogenic fragment of TGF|3 is administered to the patient repeatedly until the patient has a TGF|3-specific T cell response or has a TGF|3-specific T cell response at least the threshold value.

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