Cancer therapy using anti-PD-1 or anti-PD-L1 antibodies

Abstract

The present invention relates to improved treatment of cancer with a PD-1 / PD-L1 antibody. In particular, the present invention is based on the discovery that patients having a TGF [beta]-specific T cell response are more likely to exhibit positive results in cancer treatment with a PD-1 / PD-L1 antibody. Thus, the present invention provides for the treatment of the group of patients with a PD-1 / PD-L1 antibody and proactively promoting a TGF [beta]-specific T cell response to improve treatment with a PD-1 / PD-L1 antibody.

Description

Technical Field

[0001] The present invention relates to identifying cancer patients who are particularly responsive to treatment with PD-1 / PD-L1 antibodies for identification. The present invention also relates to methods for enhancing the effectiveness of PD-1 / PD-L1 antibody treatment for a given population of cancer patients. The present invention also relates to compositions and methods for treating cancer. Background Art

[0002] The discovery of immune checkpoints CTLA-4 and PD-1 and the subsequent introduction of immune checkpoint inhibitors (ICIs) have revolutionized the treatment and prognosis of multiple cancers (Robert, C., Nat. Commun. 11, (2020)). However, not all cancers are sensitive to ICIs. For example, pancreatic cancer (PC) remains highly refractory to ICIs. Several trials of ICIs in patients with advanced pancreatic cancer have 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)).

[0003] Recently, a phase II trial (CheckPAC) in patients with refractory metastatic pancreatic cancer studied the combination of the PD-1 antibody nivolumab with or without ipilimumab in combination 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 partial response. Thus, there is a continuing need for improved methods for treating cancers refractory to PD-1 / PD-L1 antibody treatment, particularly pancreatic cancer. Summary of the Invention

[0004] The present invention provides a method for improving PD-1 / PD-L1 antibody treatment, particularly for cancer types refractory to such antibody treatment. The present invention is based on the discovery that the presence or absence of a TGF-beta specific T cell response indicates the therapeutic effect of PD-1 / PD-L1 antibodies, and if such a response is present, the strength of the response can also indicate the therapeutic effect. Thus, the present invention identifies populations of patients who will be particularly responsive to PD-1 / PD-L1 antibody treatment. The present invention also allows for the use of TGFbeta or immunogenic peptides derived from TGFbeta to stimulate or enhance the TGF-beta specific T cell response, thereby increasing the potential efficacy of PD-1 / PD-L1 antibody treatment of cancer.

[0005] The present invention is particularly effective for treating refractory cancers (such as pancreatic cancer) with PD-1 / PD-L1 antibodies. The PD-1 / PD-L1 antibody that can be used in the present invention is nivolumab. Thus, in one embodiment, the antibody is nivolumab and the cancer is pancreatic cancer. A particularly useful indicator is the presence and magnitude (if present) of the T cell response against the TGFβ-15 peptide sequence of SEQ ID NO:28.

[0006] Accordingly, the present invention provides a PD-1 / PD-L1 antibody for use in a method of treating cancer in a patient, wherein the method comprises administering a PD-1 / PD-L1 antibody to the patient, wherein the patient has previously been identified as having a TGFβ-specific T cell response.

[0007] The present invention also provides a method of treating cancer in a patient, the method comprising administering a PD-1 / PD-L1 antibody to the patient, wherein the patient has previously been identified as having a TGFβ-specific T cell response.

[0008] The present invention also provides a method of stratifying cancer patients into one of at least two treatment groups, the method comprising:

[0009] i. assaying a sample previously obtained from the patient to detect the presence of a TGFβ-specific T cell response;

[0010] ii. if a TGFβ-specific response is present, assigning the patient to a first treatment group, or if a TGFβ-specific response is absent, stratifying the patient into a second treatment group; wherein if the patient is assigned to the first treatment group, a PD-1 / PD-L1 antibody is administered to them.

[0011] The present invention also provides a method of stratifying cancer patients into one of at least two treatment groups, the method comprising:

[0012] i. assaying a sample previously obtained from the patient to detect the level of a TGFβ-specific T cell response if present;

[0013] ii. if the TGFβ-specific T cell response is at least a threshold of TGFβ-specific T cells, assigning the patient to a first treatment group, and if the TGFβ-specific response is below the threshold, stratifying the patient into a second treatment group;

[0014] wherein if the patient is assigned to the first treatment group, a PD-1 / PD-L1 antibody is administered to them.

[0015] The present invention also provides a PD-1 / PD-L1 antibody for use in a method of treating cancer, wherein the method comprises:

[0016] (i) Administering to a patient an immunogenic fragment of human transforming growth factor (TGFβ) that comprises or consists of a sequence of at least 9 contiguous amino acids of SEQ ID NO:1; and

[0017] (ii) Administering a PD-1 / PD-L1 antibody.

[0018] The present invention also provides an immunogenic fragment of human transforming growth factor β (TGFβ) in a method for treating cancer in a patient, the method comprising:

[0019] (i) Administering to a patient an immunogenic fragment of human transforming growth factor β (TGFβ), wherein the immunogenic fragment comprises or consists of a peptide sequence of at least 9 contiguous amino acids of SEQ ID NO:1; and

[0020] (ii) Administering a PD-1 / PD-L1 antibody to the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1: TGFβ-15 specific responses in patients with pancreatic cancer (PC). A. Peripheral blood mononuclear cells (PBMCs) from pancreatic cancer patients were stimulated once in vitro with TGFβ-15 peptide and IL-2, incubated for 14 days before plating at a concentration of 2x10 5 cells / well, and incubated overnight. The experiments were performed in triplicate, and the negative control wells were not stimulated. Responses were characterized in 32 samples isolated at baseline (left) and 31 samples isolated after 4 cycles of treatment (right). B. Representative images of responses at baseline (upper) and after 4 cycles of treatment. C. Normalized counts from the data in A, comparing the magnitude of responses in patients with clinical benefit to those in patients with progressive disease. Normalization was performed by subtracting the mean spot count of the control wells from the mean number of spots in the peptide-stimulated wells. Error bars in A and C represent the standard error of the mean, and statistical analysis was performed using a paired T-test. Statistical analysis was performed using the Mann-Whitney test.

[0022] Figure 2: The magnitude of the TGFb-15 specific immune response fluctuates over time. A. For patients with clinical benefit (left) and patients with progressive disease (right), the normalized counts at baseline were compared to the counts after 4 cycles of treatment (i.e., 8 weeks after the initial treatment). Statistical analysis was performed using the Wilcoxon paired signed-rank test. B. Representative images of the baseline response and the response after 4 cycles in patients with clinical benefit (upper) and the response in patients with progressive disease (lower). C. The normalized TGFb-15 specific immune response was analyzed over time (over 2 years) in three patients with clinical benefit. Normalization of spot counts was performed as previously described.

[0023] Figure 3 : Difference in the decline of the TGFb-15 specific immune response between patients with sustained clinical benefit and those without. Comparison of the TGFb-15 specific immune response at baseline and after 4 cycles of treatment in patients with sustained clinical benefit (A) and those without sustained clinical benefit (B). Sustained clinical benefit was defined as partial response (PR) or stable disease (SD) lasting more than 6 months. Statistical analysis was performed using the Wilcoxon paired signed-rank test.

[0024] Figure 4: A strong TGFb-15 specific response before treatment predicts better survival. A. Kaplan-Meier curves show the overall survival of patients with a TGFb-15 response magnitude higher or lower than the median response magnitude. B. Kaplan-Meier curves show the progression-free survival of patients with a TGFb-15 response magnitude higher or lower than the median response magnitude. Time-to-event analysis was performed using the log-rank test.

[0025] Figure 5: Patient survival analysis based on the magnitude of the TGFb-15 specific response. A. Overall survival analysis of patients based on the normalized TGFβ-15 response. Patients were divided into four groups according to the magnitude of the TGFb-15 specific response. B. Progression-free survival analysis as in A. Statistical analysis was performed using the log-rank test.

[0026] Figure 6: Correlation between the TGFb-15 specific response and the tetanus epitope response. A. The normalized TGFb-15 response and the tetanus response were plotted and the correlation was analyzed using simple linear regression. B. The normalized TGFb-15 response and the influenza response were plotted and the correlation was analyzed using simple linear regression.

[0027] Figure 7: The magnitude of the TGFb-15 specific response is not correlated with the magnitude of the tetanus peptide response. A. PBMC responses to the tetanus epitope "tetanus-long" were tested using an in vitro IFNγ ELISPOT assay. The magnitude of the normalized tetanus-long specific response was compared among patients whose response was above the median TGFb-15 specific immune response. For five patients from the group with responses above the median and one patient with a response below the median, the tetanus-long response was not analyzed in the baseline sample but in samples obtained within two weeks after baseline. B. Representative pictures of patients lacking TGFb specific immune response (upper) but with intact tetanus specific immune response (lower). C. The same analysis as in A, using the short influenza virus-derived epitope "C18 A2 Flu" instead of the tetanus-derived epitope. D. Representative pictures of patients lacking TGFb specific immune response (top) but with intact influenza virus specific immune response (bottom). Error bars in A and C represent the standard error of the mean. Statistical analysis was performed using the Mann-Whitney test.

[0028] Figure 8: Clostridium tetani and influenza specific responses are not associated with survival. A. Kaplan-Meier curves showing overall survival of patients with a response magnitude to tetanus-Long above or below the median response. B. Kaplan-Meier curves showing progression-free survival of patients with a response magnitude to tetanus-Long above or below the median response. C. Kaplan-Meier curves showing overall survival of patients with a response magnitude to C18 A2 Flu above or below the median. D. Kaplan-Meier curves showing progression-free survival of patients with a response magnitude to C18 A2 Flu above or below the median. Time-to-event analysis was performed using the log-rank test.

[0029] Figure 9: Repeated in vitro stimulation of peripheral blood mononuclear cells (PBMC) from patients and healthy donors with TGFb-15 peptide increases the magnitude of the TGFb-15 specific immune response. A. Peripheral blood mononuclear cells (PBMC) from 16 PC patients with weak TGFb-15 responses were cultured in vitro and their responses to TGFb-15 were tested after one in vitro stimulation with TGFb-15 peptide or three in vitro stimulations with TGFβ-15 peptide; the normalized responses are shown in the heatmap (top), and representative responses after 1 stimulation and 3 stimulations are shown (bottom). Some cultures received only 2 in vitro stimulations, marked with black stars next to the spot counts. B. The ability of repeated stimulation to enhance the TGFb-15 specific immune response was analyzed in PBMC from 7 healthy donors, as described in A, using 5 - 6×10 5cells / well, normalized results (top) and representative responses (bottom). C. The ability to enhance TGFb-15 specific immune responses by repeated stimulation was analyzed in PBMCs from 15 healthy donors as described in A, using 2×10 5 cells / well, normalized results (top) and representative responses (bottom).

[0030] Figure 10 : No increase in the distribution-free resampling (DFR) response after repeated stimulation. A. Comparison of DFR responses and DFR2x responses in PC patients after one or repeated stimulations. B. As in A, but in healthy donors. Statistical analysis was performed using DFR and DFR2x methods.

[0031] Figure 11: Strong TGFb-33 specific responses before treatment onset predict better survival. A. Kaplan-Meier curves showing overall survival of patients with TGFb-33 responses above or below the median response. B. Kaplan-Meier curves showing progression-free survival of patients with TGFb-33 responses above or below the median response. Time-to-event analysis was performed using the log-rank test.

[0032] Figure 12 : T cell responses to TGFb-33 in pancreatic cancer patients receiving radiotherapy and anti-CTLA-4 and anti-PD-1 therapies. Peripheral blood mononuclear cells (PBMCs) from pancreatic cancer patients were stimulated once in vitro with TGFb-33 peptide and IL-2, incubated for 14 days, and then plated at a concentration of 2x10 5 cells / well in interferon-γ enzyme-linked immunosorbent spot assays (IFNγ ELISPOTs) and incubated overnight. The experiments were performed in duplicate or triplicate, with negative control wells not stimulated. Responses were characterized in 31 samples isolated at baseline (left) and 28 samples isolated after 4 cycles of treatment (right). The figure shows the normalized response magnitude to TGFb-33, which was calculated by subtracting the mean number of spots in the negative control wells from the mean number of spots in the peptide-stimulated wells.

[0033] Brief Description of Sequences

[0034] SEQ ID NO:1 is the amino acid sequence of the full-length precursor of human TGFb-1 (also known as TGFb-1 precursor 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 NO:5-64 are each the amino acid sequence of a polypeptide fragment from human TGFb-1.

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

[0040] SEQ ID NO:66 is the amino acid sequence of the minimal epitope sequence within the TGFb-1 5-peptide sequence (SEQ ID NO:28).

[0041] SEQ ID NO:66 is also referred to herein as "TGFb-15-15short".

[0042] SEQ ID NO:67 is the amino acid sequence of TGFb-A2-01. Detailed Description

[0043] It should be understood that different applications of the disclosed products and methods can be customized according to the specific needs in the art. It should also be understood that the terms used herein are for the purpose of describing particular embodiments of the present invention only and are not intended to be limiting.

[0044] Definitions

[0045] Unless otherwise defined herein, technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following term descriptions will apply, and where appropriate, terms used in the singular ("a", "an", and "the") will also include the plural and vice versa, unless the context clearly indicates otherwise. Thus, for example, reference to "a polypeptide" includes "polypeptides", etc. If any description of the terms set forth conflicts with any text incorporated herein by reference, the description of the terms set forth below shall prevail.

[0046] In cases where the terms "comprising" and "comprises" are used, "consisting essentially of" or "consisting of" the listed elements is also provided.

[0047] "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 peptide mimetics. Thus, the term "polypeptide" includes short peptide sequences as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including D or L optical isomers, as well as amino acid analogs and peptide mimetics.

[0048] The terms "patient" and "subject" are used interchangeably and generally refer to humans.

[0049] As used herein, "immunogenicity" means that a polypeptide is capable of eliciting an immune response against the TGFb protein, particularly the TGFb-1 protein, typically when the protein is present in or on cells expressing the TGFb-1 protein. In other words, the polypeptide can be described as being immunogenic for TGFb. Alternatively, the polypeptide can be described as an immunogenic fragment of TGFb. The immune response can refer to a T cell response, and thus the polypeptide can be described as an immunogenic fragment of TGFb that contains a T cell epitope. After administration of the polypeptide to the individual (or the sample), an immune response can be detected in at least one individual (or in a sample taken from an individual).

[0050] References herein to TGF-b, T-GF-beta, etc. correspond to references to TGF-β. However, the foregoing terms are used to avoid the use of Greek symbols and to aid in the reproducibility of the text.

[0051] TGFbeta1-specific T cell response

[0052] The present invention is based on the discovery that patients who exhibit a T cell response specific for TGFbeta represent a group of patients who can be particularly effectively treated with PD-1 / PD-L1 antibodies. In addition, it has also been found that the magnitude of the T cell response specific for TGFbeta indicates the possible degree of effectiveness of PD-1 / PD-L1 antibodies in the treatment of cancer. This means that it is possible to determine whether there is a specific T cell response against TGFbeta in a particular patient, or to quantify it, in order to determine how effective PD-1 / PD-L1 antibody treatment might be. Therefore, it can be used as a method for determining whether to treat a patient with PD-1 / PD-L1 antibodies. The discovery of a T cell response specific for TGFbeta indicates that, for those patients who do not have such a response or have only a weak response, the effectiveness of PD-1 / PD-L1 antibody treatment also means that the patient can be treated to trigger or increase such a response, thereby helping to improve the therapeutic effect of PD-1 / PD-L1 antibodies. A T cell response specific for TGFbeta can refer to a response against TGFbeta1. The present invention is particularly useful for cancers refractory to PD-1 / PD-L1 antibody treatment. In a particularly preferred embodiment, the cancer is pancreatic cancer and the antibody is nivolumab.

[0053] Specifically, whether there is a TGFbeta response or the magnitude of the response is measured at “baseline” prior to the start of treatment. It can also be measured during treatment. In a further embodiment, whether there is a TGFbeta response can be measured at baseline and then after treatment aimed at increasing the T cell response to TGFbeta to check whether the response has occurred or increased.

[0054] In some embodiments, it is measured whether a patient has a T cell response specific for TGFbeta. The results of a test sample from the patient can be compared with the results of a negative control, and if the results of the test sample do not increase significantly relative to the negative control, then no response can be considered to be present. Instead of simply determining the presence or absence of a response, the magnitude of the response can be measured because the inventors have found that patients with a higher response level show more positive results with PD-1 / PD-L1 antibodies.

[0055] Whether a patient has a TGFbeta-specific T cell response or the magnitude of the response can be measured by any suitable method. Whether such a response is present can be measured by determining whether 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 listed herein can be employed, and particularly preferred peptides of interest are the TGFbeta-15 peptide sequence (SEQ ID NO:28) or the TGFbeta short peptide sequence (SEQ ID NO:66). A further preferred peptide of interest is the TGFbeta-33 peptide sequence of SEQ ID NO:55.

[0056] A patient sample for evaluation can be a sample obtained from blood. Peripheral blood mononuclear cells (PBMCs) represent the preferred sample used herein. In another embodiment, the cells are purified T cells, such as T cells purified from the patient's blood. One method of measuring whether a subject shows a TGFbeta-specific T cell response is to use peripheral blood mononuclear cells PBMCs isolated from the subject and determine whether they show a response to TGFbeta-1 or a TGFbeta-1 peptide. A suitable assay is to incubate the PBMCs with TGFbeta-1 or a TGFbeta-1 peptide and then determine whether the cells present are activated by the TGFbeta-1 or a TGFbeta-1 peptide. A possible method of determining whether the activated T cells show a response to TGFbeta-1 is an enzyme-linked immunospot assay (ELISPOT) assay. The assays employed in the examples of the present application can be used to determine whether a response is present and, if so, to quantify it.

[0057] In one embodiment, the assay comprises: (a) incubating PBMCs isolated from a patient with TGFbeta-1 or a TGFbeta-1 peptide for 7 to 14 days; (b) plating a known number of cells onto a plate coated with a primary IFN-gamma specific antibody, incubating the plate overnight, and then removing the cells; (c) detecting the presence of bound IFN-gamma using a secondary antibody, streptavidin-ALP, and an enzyme substrate, wherein the wells are washed with PBS before and between each step, such that the location of activated cells secreting IFN-gamma can be identified as spots; (d) counting the number of spots, and thereby calculating the number of TGFbeta-1 specific cells. In step (a), IL-2 may also be included in the incubation step. The assay may include a control, such as a negative control sample that does not include TGFbeta-1 or a TGFbeta-1 peptide also being performed in the incubation step of (a). In one embodiment, if a subject gives a TGFbeta response that is the same or similar to the negative control, it is defined as the absence of a T cell response against TGFbeta. In one embodiment, a lack of response in an ELISPOT assay can be said to be no more than five-fold the number of spots compared to an equivalent negative control. Additionally or alternatively, the assay may also include a positive control, such as including a sample of T cells that exhibit a response against TGFbeta.

[0058] In another embodiment, the magnitude of any TGFbeta T cell response is measured. In a further embodiment, a method that allows for the counting of the number of T cells that produce a TGFbeta specific response can be used. One way to do this is to employ a spot-based assay. The ELISPOT assay represents the assay used herein. For example, the assay employed in the examples of the present application can be used.

[0059] The present invention may include generating the obtained value or comparing the obtained value with an expected value. A patient may be selected for treatment based on having a TGFbeta-1 specific baseline T cell response that is equal to or greater than a threshold. One way to set the threshold is according to the median value. In some embodiments, the median value may be the median baseline value observed in cancer patients, particularly cancer patients having the same type of cancer. The median may be calculated according to cancer patients of the same cancer type and gender. It may be calculated according to a representative sample of such cancer patients. The expected median value for a given value may have been calculated and may be used. In a further embodiment, another way to set the threshold is according to the 75th percentile value. The 75th percentile value may be the 75th percentile value of the baseline values observed in cancer patients, particularly cancer patients having the same type of cancer. The 75th percentile may be calculated according to cancer patients of the same cancer type and gender. It may be calculated according to a representative sample of such cancer patients. The expected 75th percentile value for a given value may have been calculated and may be used.

[0060] The present invention may relate to calculating which percentile within a population of patients having the same condition a patient's value falls within. The present invention can be used to give an indication of the likely efficacy of treatment with a PD-1 / PD-L1 antibody. In some embodiments, a "threshold" may be applied to decide whether to administer a PD-1 / PD-L1 antibody to a subject. In another embodiment, if the value is below the threshold, a patient may be selected for treatment to increase the TGFbeta specific T cell response to help improve the likely efficacy of the PD-1 / PD-L1 antibody. A TGFbeta-1 peptide or coding sequence may be administered to the subject to attempt to stimulate such a response.

[0061] Patient

[0062] The present invention may be applied to any suitable mammalian subject, although the patient is human. The patient may be male or female. In one embodiment, the patient is male.

[0063] The patient will develop 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 cancer, 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 head and neck squamous cell carcinoma. In another embodiment, the patient may have urothelial cancer. 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 fewer metastases.

[0064] In one embodiment, the cancer is a cancer refractory to PD-1 / PD-L1 antibody treatment. An example of such a refractory cancer is pancreatic cancer. In a particularly preferred embodiment, the subject may thus have pancreatic cancer. In one embodiment, the pancreatic cancer is metastatic cancer. In a further embodiment, the cancer is refractory metastatic pancreatic cancer (mPC). PD- 1 / PD-L1 antibody and additional cancer therapy

[0065] A PD-1 / PD-L1 antibody is generally an antibody that blocks the interaction between PD-1 and PD-L1. Thus, in one embodiment, the antibody binds to PD-1. In another embodiment, the antibody binds to PD-L1. Examples of PD-1 antibodies include nivolumab, pembrolizumab, cemiplimab, and dostarlimab. Examples of PD-L1 antibodies include atezolizumab, avelumab, and durvalumab.

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

[0067] A patient can be administered a PD-1 / PD-L1 antibody alone. PD-1 / PD-L1 antibodies are sometimes used in combination with other therapies, or as a treatment in other lines after another therapy. In one embodiment, which can be any of the embodiments listed herein, where a subject is given a PD-1 / PD-L1 antibody, they are also given a second cancer therapy. A patient can be given both a PD-1 / PD-L1 antibody and a CTLA-4 antibody. Preferably, the CTLA-4 antibody is ipilimumab. A patient can be given both a PD-1 / PD-L1 antibody and radiotherapy. A subject can be given a PD-1 / PD-L1 antibody, a CTLA-4 antibody, and radiotherapy. The preferred CTLA-4 antibody is ipilimumab. One form of radiotherapy is stereotactic body radiotherapy (SBRT). In such embodiments, nivolumab is the PD-1 antibody of interest. Thus, nivolumab can be administered alone. Nivolumab can be administered with a CTLA-4 antibody (such as ipilimumab). Radiotherapy can be performed in either case. Thus, in one embodiment, nivolumab, ipilimumab, and SBRT are administered. In one example, radiotherapy is provided in the form of stereotactic body radiotherapy (SBRT) at about 15 Gy at a single disease site. 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 then every 6 weeks (±3 days).

[0068] A CTLA-4 antibody can be given, or the patient has been treated with such an antibody. An example of a CTLA-4 antibody is ipilimumab, sold under the trade name Another further example of a preferred CTLA-4 antibody is tremelimumab.

[0069] In those patients in which it is determined to stimulate a TGFbeta-specific T cell response as a means of attempting to increase the likely efficacy of a PD-1 / PD-L1 antibody, the TGFbeta-1 peptides further described herein can be administered to the patient to stimulate such a response. In one embodiment, the TGFbeta-1 peptide is administered at a dose of about 200 μg. The TGFb-1 peptide can be administered in the form of an emulsion with an adjuvant. In one embodiment, the TGFb-1 peptide is administered in the form of an emulsion at a dose of about 200 μg with about 500 μl of Montanide ISA-51. The peptide-adjuvant emulsion can be administered on day 1 of the first 6 fourteen-day treatment cycles, followed by administration every 4 weeks (±3 days).

[0070] In cases where additional agents or therapies are to be administered to the patient along with a PD-1 / PD-L1 antibody, they can be administered simultaneously, separately, or sequentially. The two drugs can be administered in the same composition or in separate compositions. In embodiments where a TGFbeta-1 peptide is administered to attempt to stimulate a TGFbeta-specific T cell response, it can be administered before the PD-1 / PD-L1 antibody. In one embodiment, a TGFbeta peptide can be administered, the stimulation of the T cell response against TGFbeta can be confirmed, and then the PD-1 / PD-L1 antibody can be administered, or the PD-1 / PD-L1 antibody can be administered once the response value increases above a threshold. In another embodiment, the TGFbeta-1 peptide and the PD-1 / PD-L1 antibody can be administered simultaneously.

[0071] TGFbeta-1 epitopes and peptides

[0072] TGFbeta is generally TGFbeta-1. The TGFbeta1 sequence can be used depending on the presence of a T cell response against TGFbeta in the patient. Alternatively, the sequences discussed below can be used to stimulate such a response.

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

[0074]

[0075]

[0076] SAAPCCVPQA LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS

[0077] Table 1 below lists various TGFbeta-1 related sequences, including specific TGFbeta-1 peptides that can be used in the present invention. In Table 1 below, unless otherwise stated, "Start pos" and "End pos" represent positions within the full-length human TGFbeta-1 proprotein (SEQ ID NO:1).

[0078] Table 1

[0079]

[0080]

[0081]

[0082] The presence or magnitude of a TGFbeta-specific T cell response can be determined using TGFbeta-1 itself or using one of the TGFbeta-1 peptide sequences herein. The TGFb-1 peptide sequences discussed herein can also be used to generate or increase the magnitude of a TGFbeta-1 specific T cell response and thus improve the treatment of cancer. Thus, the sequences discussed below can be relevant to detecting / measuring a TGFbeta-specific T cell response and also to seeking to stimulate / increase such a response. In a particularly preferred embodiment, the peptide sequence is the sequence of the TGFb-15 peptide of SEQ ID NO:28. In a further particularly preferred embodiment, the peptide is the peptide sequence of SEQ ID NO:66. In a further particularly preferred embodiment, the peptide is the sequence of SEQ ID NO:55.

[0083] Preferred TGFbeta-1 peptide sequences for use in the present invention are immunogenic fragments of human TGFbeta-1 (SEQ ID NO:1) that comprise or consist of a sequence of at least 9 contiguous amino acids of SEQ ID NO:1. A sequence of at least 9 contiguous amino acids of SEQ ID NO:1 can, for example, correspond to a sequence of at least 9 contiguous amino acids of the signal peptide (SP) domain of TGFbeta-1, such as at least 9 contiguous amino acids of SEQ ID NO:2. It can correspond to a sequence of at least 9 amino acids of the latency-associated peptide (LAP) domain of TGFb-1, such as at least 9 contiguous amino acids of SEQ ID NO:3. It can correspond to a sequence of at least 9 contiguous amino acids within the LAP sub-region defined by amino acid positions 121 and 160 of SEQ ID NO:1, such as at least 9 contiguous amino acids of SEQ ID NO:65. It can correspond to a sequence of at least 9 contiguous amino acids of the mature TGFb1 polypeptide, such as at least 9 contiguous amino acids of SEQ ID NO:4.

[0084] 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 NO:2 and 5-67. The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NO: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, the polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NO:6, 42, 12, 23, 28, 49, 55, 63, 66, 67 or 5.

[0085] The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NO: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, the polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NO:66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63.

[0086] The maximum length of the polypeptide may be 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 by the corresponding amide. The polypeptide may be isolated.

[0087] In some embodiments, the polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NO:6, 42, 12, 23, 28, 49, 55 or 63. In further embodiments, the polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NO:66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63. Longer polypeptide fragments of SEQ ID NO:1 incorporating these sequences may also be used.

[0088] The polypeptide may comprise an HLA-A2 restricted epitope. In one embodiment, the HLA-A2 restricted epitope comprises or consists of the amino acid sequence of SEQ ID NO:66. In another embodiment, the peptide comprising the HLA-A2 restricted epitope consisting of the amino acid sequence of SEQ ID NO:66 is a peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs:28 - 31 or 65. Alternatively, the HLA-A2 restricted epitope comprises or consists of the amino acid sequence of SEQ ID NO:67. In some embodiments, the peptide comprising the HLA-A2 restricted epitope consisting of the amino acid sequence of SEQ ID NO:67 is a peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs:5, 8, 9 or 2.

[0089] In any polypeptide herein, the amino acid sequence may be modified by one, two, three, four or five (i.e., up to five) additions, deletions or substitutions, provided that the polypeptide with the modified sequence exhibits the same or increased immunogenicity against TGFb1 compared to the polypeptide with the unmodified sequence. "Same" should be understood to mean that the polypeptide with the modified sequence does not exhibit a significant decrease in immunogenicity against TGFb1 compared to the polypeptide with the unmodified sequence. Any comparison of immunogenicity between sequences should be made using the same assay. Unless otherwise stated, modifications to the polypeptide sequence are conservative amino acid substitutions. Conservative substitutions replace an amino acid with another amino acid having a similar chemical structure, similar chemical properties or similar side chain volume. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acid it replaces. Alternatively, a conservative substitution may introduce another aromatic or aliphatic amino acid in place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected based on the properties of the 20 major amino acids defined in Table 2 below. When amino acids have similar polarity, this can be determined by referring to the hydrophilicity scale of amino acid side chains in Table 3.

[0090] Table 2 - Chemical properties of amino acids

[0091] Ala(A) Aliphatic, hydrophobic, neutral Met(M) Hydrophobic, neutral Cys(C) Polar, hydrophobic, neutral Asn(N) Polar, hydrophilic, neutral Asp(D) Polar, hydrophilic, charged (-) Pro(P) Hydrophobic, neutral Glu(E) Polar, hydrophilic, charged (-) Gln(Q) Polar, hydrophilic, neutral Phe(F) Aromatic, hydrophobic, neutral Arg(R) Polar, hydrophilic, charged (+) Gly(G) Aliphatic, neutral Ser(S) Polar, hydrophilic, neutral His(H) Aromatic, polar, hydrophilic, charged (+) Thr(T) Polar, hydrophilic, neutral Ile(I) Aliphatic, hydrophobic, neutral Val(V) Aliphatic, hydrophobic, neutral Lys(K) Polar, hydrophilic, charged (+) Trp(W) Aromatic, hydrophobic, neutral Leu(L) Aliphatic, hydrophobic, neutral Tyr(Y) Aromatic, polar, hydrophobic

[0092] Table 3 Hydrophobicity scale

[0093]

[0094] In any polypeptide disclosed herein, any one or more of the following modifications can be made to improve physicochemical properties (e.g., stability), provided that the polypeptide exhibits the same or increased immunogenicity to TGFb1 compared to the polypeptide with the unmodified sequence: (i) replacing the C-terminal amino acid with the corresponding amide (which can increase resistance to carboxypeptidase); (ii) replacing the N-terminal amino acid with the corresponding acylated amino acid (which can increase resistance to aminopeptidase); (iii) replacing one or more amino acids with the corresponding methylated amino acids (which can improve proteolytic resistance); and / or (iv) replacing one or more amino acids with the corresponding amino acids in the D-configuration (which can improve proteolytic resistance).

[0095] Preferred peptides that can be employed are set forth in WO2020 / 245264A1 (PCT / EP2020 / 065472), which is incorporated herein by reference in its entirety, and the TGFb1 sequences disclosed therein are also incorporated specifically by reference, both in relation to potential epitopes against which a T cell response can be directed, but also as sequences for stimulating such a response.

[0096] In any embodiment of using a peptide to stimulate a response as set forth herein, alternatively, a sequence encoding such a peptide can be administered, such as can be a composition comprising the peptide or the coding sequence.

[0097] Compositions comprising polypeptides

[0098] The present invention provides the use of the TGFb1 peptides set forth herein for increasing or eliciting a TGFbeta-specific T cell response, wherein a PD-1 / PD-L1 antibody is also administered to a subject. The TGFbeta1 peptide can be provided in the form of a pharmaceutical composition for such a method. The composition can also comprise a PD-1 / PD-L1 antibody. The present invention also provides a PD-1 / PD-L1 antibody for use as described herein, and the PD-1 / PD-L1 antibody can be formulated in the manner currently sold, but any suitable formulation can be employed.

[0099] In some embodiments, the pharmaceutical composition comprises at least one adjuvant, a pharmaceutically acceptable carrier, a preservative, and / or an excipient. In some embodiments, the 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, a pharmaceutically acceptable carrier, a preservative, and / or an excipient. In some embodiments, the 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 coding polynucleotides of the present invention and at least one adjuvant, a pharmaceutically acceptable carrier, a preservative, and / or an excipient.

[0100] The carrier, preservative, and excipient must be "acceptable" in the sense of being compatible with the other components of the composition and harmless to the subject to whom the composition is administered. Generally, all components and the final composition are sterile and pyrogen-free. The composition can be a pharmaceutical composition. The composition can contain an adjuvant. An adjuvant is any substance whose mixing with the composition enhances or otherwise modifies the immune response elicited by the composition. An adjuvant, defined broadly, is a substance that promotes an immune response. Adjuvants can also have a depot effect in that they also cause slow and sustained release of the active agent from the site of administration. A general discussion of adjuvants can be found on pages 61 to 63 of Goding's *Monoclonal Antibodies: Principles & Practice* (Second Edition, 1986).

[0101] Adjuvants can be selected from: AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also known as des-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, also known as MTP-PE), RIBI (MPL+TDM+CWS), in 2% squalene / Tween-80.RTM. emulsion, lipopolysaccharide and its various derivatives, including lipid A, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, Merck adjuvant 65, polynucleotides (such as poly IC and poly AU acid), 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, cholera toxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrices or GMDP, interleukin 1, interleukin 2, Montanide ISA-51, and QS-21. Various saponin extracts have also been proposed to be used as adjuvants in immunogenic compositions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can also be used as an adjuvant.

[0102] Adjuvants for stimulating T cell responses used in conjunction with the present invention in embodiments include oil / surfactant-based adjuvants, such as Montanide adjuvants (available from Seppic, Belgium), such as Montanide ISA-51. Other adjuvants are bacterial DNA-based adjuvants, such as adjuvants comprising CpG oligonucleotide sequences. Still other adjuvants are viral dsRNA-based adjuvants, such as poly I:C. GM-CSF and imidazoquinoline are also examples of adjuvants.

[0103] In one embodiment, the adjuvant is a Montanide ISA adjuvant. In a further embodiment, the Montanide ISA adjuvant is Montanide ISA 51 or Montanide ISA 720.

[0104] It is also noted on pages 61 to 63 of Goding's "Monoclonal Antibodies: Principles and Practice" (Second Edition, 1986) that when the antigen of interest has a low molecular weight or poor immunogenicity, conjugation to an immunogenic carrier is recommended. Thus, the polypeptides of the present invention can be conjugated to a carrier. The carrier can exist independently of the adjuvant. The function of the carrier can be, for example, to increase the molecular weight of the polypeptide fragment to increase activity or immunogenicity, confer stability, increase biological activity, or increase serum half-life. In addition, the carrier can help present the polypeptide or its fragment to T cells. Thus, in the composition, the polypeptide can be combined with a carrier such as those listed below. The carrier can be any suitable carrier known to those skilled in the art, such as 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 can be tetanus toxoid or diphtheria toxoid. Alternatively, the carrier can be dextran, such as agarose gel. The carrier must be physiologically acceptable and safe for humans.

[0105] If a composition contains excipients, they must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to its recipients. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like can be present in the excipients. These excipients and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Also included therein can be pharmaceutically acceptable salts such as inorganic acid salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids, e.g., acetate, propionate, malonate, benzoate, etc. A comprehensive discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances is presented in Remington’s Pharmaceutical Sciences, Mack Pub. Co., N.J. 1991.

[0106] The formulation of suitable compositions can be carried out using standard pharmaceutical formulation chemistry and methods, all of which are readily available to those of ordinary skill in the art. Such compositions can be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable compositions can be prepared, packaged, or sold in unit dosage form, for example, in an ampoule or a multi-dose container. Some ampoules or multi-dose containers may contain preservatives. 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 the composition, the active ingredient is provided in a dry (e.g., powder or granule) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to administration of the reconstituted composition. The composition can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution can be formulated according to known techniques and, in addition to the active ingredient, can contain additional ingredients such as adjuvants, excipients, and auxiliary substances described herein. Such sterile injectable formulations can be prepared using non-toxic parenterally acceptable diluents or solvents, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic monoglycerides or diglycerides of glycerol. Other useful compositions include those comprising the active ingredient in microcrystalline form, in liposomal formulations, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation can contain pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts. Alternatively, the active ingredient of the composition can be encapsulated, adsorbed to, or associated with a particulate carrier. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, and PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolide). See, for example, Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers can also be used, such as polymers of polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules.

[0107] Compositions and methods for improving the use of PD-1 / PD-L1 antibodies

[0108] The present invention provides a PD-1 / PD-L1 antibody for use in a method of treating cancer in a patient, wherein the method comprises administering the PD-1 / PD-L1 antibody to the patient and wherein the patient has previously been identified as having a TGFbeta-1 T cell response. The present invention provides a PD-1 / PD-L1 antibody for use in a method of treating cancer in a patient, wherein the method comprises administering the PD-1 / PD-L1 antibody to the patient, and wherein any baseline TGFbeta-1 specific T cell response in the patient has been measured and determined to be equal to or greater than a threshold. Preferably, the threshold can be the median response value. In another embodiment, the threshold can be the 75th percentile of patients having the same cancer. Various thresholds are discussed elsewhere herein and can be employed. The same cancer means that if, for example, the present invention is applied to pancreatic cancer, the median value is the median of patients with pancreatic cancer in general.

[0109] Preferably, the patient may have been, will be, or also be being treated with a CTLA-4 antibody and / or radiotherapy. The CTLA-4 antibody is ipilimumab. Accordingly, the method of treatment can further comprise administering 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 a PD-1 / PD-L1 antibody and / or radiotherapy is administered to a patient, wherein the patient has previously been identified as having a TGFbeta specific T cell response or has been identified as having a TGFbeta specific T cell response equal to or greater than a threshold.

[0110] In any of the embodiments discussed above, a measurement of the TGFbeta specific T cell response may have been made. Alternatively, such measurement can form part of a method of its own.

[0111] The present invention also provides a method of stratifying cancer patients into at least two treatment groups, the method comprising: (i) assaying a sample previously obtained from a patient to detect the presence of a TGFbeta specific T cell response; (ii) if a TGFbeta specific response is present, assigning the patient to a first treatment group; or if a TGFbeta specific response is not present, assigning the patient to a second treatment group. In some embodiments, if a patient is assigned to the first treatment group, a PD-1 / PD-L1 antibody is administered to them. In some embodiments, if a patient is assigned to the second treatment group, a TGFbeta-1 peptide is administered to them to increase or generate a TGFbeta-1 specific T cell response. In some embodiments, patients assigned to the second treatment group will be administered a PD-1 / PD-L1 antibody concurrently with, separately from, or sequentially to the TGFbeta-1 peptide.

[0112] The present invention also provides a method of stratifying cancer patients into one of at least two treatment groups, the method comprising: (i) performing an assay on a sample previously obtained from the patient and detecting the level of a TGFbeta-specific T cell response if present; (ii) if the TGFbeta-specific T cell response is at least a threshold, assigning the patient to a first treatment group; or, if the TGFbeta-specific T cell response is below the threshold, assigning the patient to a second treatment group. In some embodiments, if the patient is assigned to the first treatment group, a PD-1 / PD-L1 antibody is administered to them. In some embodiments, if the patient is assigned to the second treatment group, a TGF-β peptide is administered to them to increase or generate a TGFbeta-specific T cell response. In some embodiments, patients assigned to the second treatment group will be administered a PD-1 / PD-L1 antibody concurrently, separately, or sequentially with a TGFbeta1 peptide.

[0113] For example, any threshold discussed herein can be used as a dividing line for stratification.

[0114] The value of the TGFbeta-specific T cell response observed for a given subject can be used to indicate to the patient the extent to which PD-1 / PD-L1 antibody treatment is likely to be successful. For example, the present invention provides a method of providing an indication to a patient of the extent to which PD-1 / PD-L1 antibody treatment is likely to be effective for that particular patient, comprising detecting the presence of a TGFbeta-specific T cell response and the intensity of the TGFbeta-specific T cell response (if present) in a sample from the patient, and then determining the likelihood of efficacy of the PD-1 / PD-L1 antibody for that subject based on that measurement. Such a method can include calculating where the response falls in percentiles. It can include determining the magnitude of the response (if present) compared to an expected median. For example, such a method can be used to determine a treatment plan. It can be used to decide whether to stimulate a TGFbeta-specific T cell response as described herein.

[0115] The present invention also provides immunogenic fragments of human TGFbeta for use in methods of treating cancer, wherein the method comprises administering to a cancer patient previously identified as having no TGFbeta-specific T cell response or having a TGFbeta-specific T cell response below a threshold an immunogenic fragment of human TGFbeta, and wherein a PD-1 / PD-L1 antibody is also administered to the patient. In some embodiments, the 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 the PD-1 / PD-L1 antibody. In one embodiment, the method may comprise checking that the TGFbeta-specific T cell response is elicited by the administration of the peptide or that the response is at least the threshold, and if so, administering the PD-1 / PD-L1 antibody. The present invention also provides PD-1 / PD-L1 antibodies for such methods. In some embodiments, CTLA-4 antibodies (such as ipilimumab) are provided for use in such methods. In some embodiments, radiotherapy is provided for such methods.

[0116] The present invention also provides the use of an immunogenic TGFbeta-1 peptide for improving the cancer treatment with PD-1 / PD-L1 antibodies. Any method described herein can be used to improve PD-1 / PD-L1 antibodies. The improvement can consist of increasing the overall survival time (OS). Additionally or alternatively, the improvement can consist of increasing the length of progression-free survival (PFS). In one embodiment, the treatment increases the chance that a patient shows a clinical benefit from the treatment for six months or longer from the start of the treatment.

[0117] In embodiments where the TGFbeta-1 peptide is administered to stimulate or enhance the response, the TGFbeta-1 peptide can be administered once. In embodiments, the TGFbeta-1 peptide can be administered repeatedly. For example, it can be administered one to five times, such as two, three, four, or five times. It can be administered repeatedly until a response is seen or the response rises to a desired level.

[0118] When a product for a treatment method is described herein, the present invention also provides the method itself. The present invention also provides the use of the substances described in the manufacture of a medicament for treating the said condition.

[0119] The features disclosed in the foregoing description and the following examples can be used alone or in any combination thereof as materials for implementing the present invention in different forms.

[0120] Examples

[0121] The following is a description of the various methods and materials used in the study. They are presented to provide a complete disclosure and description to one of ordinary skill in the art 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 following experiments have been conducted and are all the experiments that can be conducted. It should be understood that the exemplary descriptions written in the present tense are not necessarily performed, but the descriptions can be performed to generate data etc. related to the teachings of the present invention. Efforts have been made to ensure the accuracy of the numbers used (such as amounts, percentages, etc.), but some experimental errors and deviations should be considered.

[0122] Example 1

[0123] Introduction

[0124] We have investigated why not all patients show good improvement with antibodies against PD-1 / PD-L1, and as described herein, have determined the presence of TGFbeta-specific T cell responses in a given subject, particularly its magnitude, which indicates how well the treatment will work. This finding helps to predict the effectiveness of antibody treatment for a particular patient, and whether to boost TGFbeta-specific T cell responses prior to antibody treatment, where combination therapy increases the efficiency of antibody therapy.

[0125] Materials and methods

[0126] Patients and donors

[0127] Buffy coats from healthy donors were obtained anonymously from the blood bank at Rigshospitalet in Copenhagen, Denmark. According to Danish law, the use of anonymized biomaterials does not require approval from an ethics committee. The buffy coats from pancreatic cancer patients were provided by the Department of Oncology at Copenhagen University Hospital in Herlev, Denmark. Informed consent was obtained from all patients prior to the use of the buffy coats in accordance with the Helsinki Declaration.

[0128] Peptides

[0129] The sequence of the specific peptide TGFβ-15 is: REAVPEPVLLSRAELRLLRL (SEQ ID NO:28). The peptide was obtained at high purity (>90%) from Schafer (Copenhagen, Denmark) and dissolved in DMSO at a concentration of 10 mM. In vitro culture and enzyme-linked immunospot assay (ELISPOT)

[0130] As previously described ( M.O. & Andersen, M.H. Healthy, Cancers (Basel). 12, (2020)), peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved. PBMCs were pre-stimulated in vitro with TGFβ-15 epitopes. After 9 - 10 days of incubation, cells were counted using a Countess II automated cell counter (Thermo-Fisher), and the emergence of TGFβ-15-specific T cells was evaluated using an interferon-g (IFN-g) ELISPOT assay. Cells were plated in triplicate at a concentration of 200,000 cells per well and stimulated with peptides to a final concentration of 5 μM in the wells. PVDF membrane plates (Merck, Germany) coated with a primary IFN-γ-specific antibody (Mabtech, Sweden) were used for the ELISPOT assay. After overnight incubation, the cells were removed, and the wells were coated with a secondary antibody, streptavidin-ALP, and an enzyme substrate according to the manufacturer's protocol (Mabtech, Sweden), and the wells were washed with PBS before and between each step. The plates were counted using an ImmunoSpot S6 Ultimate analyzer (CTL Analyzers, Shaker Heights, Ohio, USA) when dry. The normalized mean spot was defined as the mean number of spots in the peptide-stimulated wells minus the mean number of spots in the negative control wells. In the repeated stimulation assay, the in vitro cultures received 2 μl of 10 mM TGFβ-15 on day 0 and then 120 U / mL of IL-2 (Novartis, Switzerland) on day 1. This was repeated one or two times every 7 days and incubation was continued for 9 - 10 days after the final stimulation, and then they were set up in the ELISPOT assay as described above.

[0131] Statistics

[0132] Statistical analysis of paired observations was performed using a two-tailed paired T-test, and the non-parametric Wilcoxon paired signed-rank test was used when the sample size was small. For unpaired observations, a two-tailed non-parametric Mann-Whitney test was used. These tests were completed using Graphpad Prism Version 9.

[0133] Survival analysis was performed using the survminer package in the statistical software R. All variables related to survival and progression-free survival found in the original trial (Chen, I.M. et al, 2022, ibid) were tested for their association with these variables in the patient cohort we used for the immune response test. This univariate analysis was performed using the log-rank analysis. All statistically significant parameters were included in the Cox proportional hazards model to analyze the independent association between immune response and overall survival and progression-free survival. Kaplan-Meier curves were made using Graphpad Prism Version 9. The analysis of ELISPOT data also used the distribution-free resampling (DFR) method and the more conservative DFR2x method.

[0134] Results

[0135] Patient characteristics

[0136] We carefully examined samples from metastatic pancreatic cancer (PC) patients who participated in the CheckPAC trial and showed spontaneous immune responses to the TGFbeta-15 epitope. CheckPAC is a phase II single-center trial in refractory metastatic PC patients conducted at the University Hospital of Copenhagen in Herlev, Denmark. The trial included 84 patients who received stereotactic body radiotherapy (SBRT) combined with immune checkpoint inhibitors (ICI). The trial included two arms: arm A received nivolumab alone, and arm B received nivolumab combined with ipilimumab. Among the 84 patients included, we analyzed the immune responses to the TGFb-15 epitope in 32 patients. Patient characteristics are shown in Table 4. Among the 32 patients analyzed, 7 (22%) achieved partial response (PR), 14 (44%) had stable disease (SD), and 11 (34%) showed progressive disease (PD). The achievement of PR or SD was defined as a clinical benefit of the treatment. PD was defined as no clinical benefit of the treatment. At a median follow-up time of 219 days (range 65 - 1511), two patients were still alive (6%), and one of them (3%) showed no signs of disease.

[0137] Table 4

[0138]

[0139]

[0140] Patients with clinical benefit contain TGFb-specific T cells

[0141] We analyzed the TGFb-15 specific responses in samples from patients and found that T cells isolated at baseline and after 4 cycles of treatment both showed responses to TGFb-15 ( Figure 1A -B). The magnitude of the baseline immune response in patients with clinical benefit was significantly higher than that in patients with progressive disease (PD), while we found no difference in the magnitude of the response after treatment (8 weeks) ( Figure 1C ). The data suggest that the TGFb specific immune response present before the start of treatment affects the clinical response to SRBT / ICI. Thus, our data suggest that TGFbeta specific T cells can be important in the response to clinical therapies. Notably, we found a significant decrease in the magnitude of the TGFb specific response at 8 weeks after the start of treatment in patients with clinical benefit (Figure 2A-B). Additionally, patients with durable clinical benefit (defined as PR or SD lasting more than 6 months) showed significantly lower TGFb specific responses at 8 weeks after treatment compared to patients without durable clinical benefit ( Figure 3 A-B).

[0142] Next, we analyzed serial PBMC samples from three patients with follow-up longer than 2 years. We analyzed the TGFb-15 specific immune response to assess the temporal changes in the response. Interestingly, we found that the magnitude of the response fluctuated over time ( Figure 2C ).

[0143] T cell responses specific for TGFb-15 were independently associated with improved survival

[0144] Our data strongly suggest that a complete TGFb specific immune response may affect the response to therapy. Thus, we investigated whether the baseline TGFb specific immune response was associated with survival in our patient cohort. We stratified patients according to whether they showed a response above or below the median of the standardized TGFb-15 specific immune response. Interestingly, we found that patients with a response magnitude above the median had significantly longer overall survival (OS) than those with a response below the median (univariate Cox regression, hazard ratio [HR]: 0.171, p = 2.54x10 -4 ; Figure 4A and Table 5 below).

[0145] Table 5

[0146]

[0147]

[0148]

[0149]

[0150] 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 might be related to survival, paying particular attention to the parameters included in the survival analysis in the original CheckPAC trial report. Univariate Cox regression analysis showed that <2 metastatic foci were associated with lower OS (HR = 2.64, p = 0.03), but no other parameters showed statistical significance (Table 5). Multivariate analysis including metastatic parameters and the TGFb-15 specific immune response showed that for patients with a response above the median magnitude, only the TGFb-15 specific immune response was independently associated with OS (HR: 0.18) (p = 8x10 -4 ; Table 5). Notably, univariate analysis showed borderline significance for reduced survival among patients receiving nivolumab monotherapy (HR: 2.04, p = 0.055). Therefore, we included this parameter along with metastases and the TGFb-15 specific immune response in another multivariate analysis. In the latter model, neither the metastatic variable nor the treatment variable was associated with OS; however, a TGFb-15 specific response above the median was still associated with improved survival (HR: 0.19, p = 0.0032; data not shown). The same analysis was performed for progression-free survival (PFS). In univariate analysis, the TGFb-15 specific response was associated with prolonged PFS (HR: 0.227, p = 0.0015; Figure 4B and Table 6 below). Other important parameters in the univariate analysis were: nivolumab treatment (HR: 2.86, p = 0.007), male gender (HR: 0.406, p = 0.029), and >36 g / L albumin in peripheral blood (HR: 4.68, p = 0.038). When these important parameters were included in a multivariate analysis together with the TGFb-15 specific immune response, only a TGFb-15 response above the median was still independently associated with PFS (HR: 0.322, p = 0.023; Table 6 below).

[0151] Interestingly, among the samples examined, patients with a TGFb-15 response above the 75th percentile had the highest OS and PFS( Figure 5A -B). These results further suggest that the level of TGFb-15 specific T cells in pancreatic cancer patients is important for achieving a clinical response to SBRT / ICI treatment.

[0152] Table 6

[0153]

[0154]

[0155]

[0156] The association between TGFb-15 specific responses and survival is not due to general immune dysfunction in non-responding patients

[0157] We investigated whether patients with low survival rates might have a dysfunctional immune system, and thus, the low response magnitude observed in these patients might be caused by general T cell dysfunction. We analyzed the magnitude of PBMC (peripheral blood mononuclear cell) responses 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 from influenza virus (Cl 8A2 Flu). The latter is a nonamer epitope restricted to HLA-A2; thus only samples with HLA-A2 + were analyzed for responses to the influenza epitope. We analyzed the tetanus responses of 23 patient samples and the influenza responses of 16 patient samples. Samples from patients with strong and weak TGFb-15 specific immune responses had similar response magnitudes to the tetanus and influenza epitopes (Figure 7A-D). In addition, we showed that the TGFb-15 response magnitude was not associated with the tetanus response (r 2 = 0.05) and the influenza response (r 2 = 0.15) (Figure 6A-B). Perhaps more importantly, the response magnitudes to the tetanus and influenza epitopes were not associated with OS or PFS ( Figure 8A -D).

[0158] Repeated antigen stimulation with TGFb-15 peptide led to T cell responses in PBMCs that did not show a response after a single in vitro stimulation

[0159] The above results strongly suggest that a measurable TGFb-15 specific immune response is important for the clinical response to SBRT / ICI treatment in pancreatic cancer patients. Therefore, we infer that in pancreatic cancer, when combined with SRBT / ICI, inducing a TGFb-15 response with a therapeutic peptide vaccine might be effective. We investigated whether the TGFb-15 specific immune response in PBMCs from healthy individuals and pancreatic cancer patients could be enhanced by repeated stimulation with the epitope. We chose to use PBMC samples from an earlier experiment that showed a weak or no response to TGFb-15 after a single in vitro stimulation. T cell responses were analyzed after a single in vitro stimulation and after one or two additional in vitro stimulations. We showed that repeated antigen stimulation increased the response in PBMCs from PC patients ( Figure 9A ) and healthy individuals ( Figure 9BThe TGFβ-15 specific immune response in PBMCs of -C). Among the pancreatic cancer patients tested, 27% had a DFR2x response after one stimulation, which increased to 66% after repeated stimulation ( Figure 10 A). Among the healthy donors tested, 9% had a DFR2x response after one stimulation, which increased to 71% after repeated stimulation ( Figure 10 B). These data indicate that repeated antigen stimulation with TGFβ-15 peptide increases the number of TGFβ-specific T cells in PBMCs.

[0160] Discussion

[0161] We investigated here the relationship between the TGFβ-15 specific immune response and clinical outcomes. Interestingly, compared with patients who did not show a clinical benefit from treatment, patients with a clinical benefit had a significantly stronger TGFβ-15 specific immune response before treatment initiation and a significantly stronger decline in TGFβ-15 specific T cell response after treatment initiation. The latter phenomenon may be explained by the migration of TGFβ-specific T cells to the tumor after the start of ICI treatment. These results are consistent with the striking observation that patients with a TGFβ-15 specific immune response above the median had significantly longer progression-free survival (PFS) and overall survival (OS) compared with patients with a response below the median.

[0162] We also investigated whether the difference in OS between patients with strong and weak TGFβ-15 specific immune responses could be attributed to differences in the overall immune status of the patients. We compared the spontaneous immune responses against two highly immunogenic epitopes derived from common pathogens (influenza virus and Clostridium tetani) in these two groups (Slinghuf et al., 2021, ibid.). We found no difference in the magnitude of the responses to influenza and tetanus between the groups. In addition, there was no correlation between the magnitude of the response to TGFβ-15 and the response to the tetanus or influenza epitopes. Interestingly, patients with a strong pathogen-specific immune response did not show an improvement in OS or PFS after ICI treatment. This means that the strength of the TGFβ-15 specific immune response is not dependent on the general immune constitution; rather, it reflects the number of anti-regulatory TGFβ-specific T cells.

[0163] These findings suggest that TGFbeta-specific regulatory T cells play a role in the response to ICI therapy in pancreatic cancer. Given the multiple roles of TGFbeta in pancreatic cancer, it is worth considering the potential immunomodulatory and tumor-suppressive effects that TGFbeta-specific T cells may have in patients. In pancreatic cancer, approximately 95% of patients have activating mutations in the KRAS gene (Prior, I.A., et al, Cancer Res. 72, 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)). Thus, TGFbeta-specific T cells act directly on transformed cells. However, several other common immunosuppressive cells will also be targeted.

[0164] Pancreatic cancer is characterized by a highly fibrotic stroma, and cancer-associated fibroblasts (CAFs) are responsible for 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 bone marrow-derived mesenchymal stem cells and pancreatic stellate cells (Moir, J.A.G., et al. Surg. Oncol. 24, 232-238 (2015)). By secreting TGFb, pancreatic cancer-derived cell lines can activate CAFs, which increase the deposition of extracellular matrix proteins and enhance fibrosis ( M. et al, Cancer Res. 61, 550 - 555 (2001) and Principe, D. R. et al. Cancer Res. 76, 2525 (2016)). In addition, activated CAFs also secrete TGFβ, which acts in an autocrine manner, leading to additional secretion of TGFβ. In particular, the expression of TGFβ within tumors has been shown to be associated with tissue fibrosis. Another study showed that pancreatic cancer patients with high levels of fibrosis and the CAF marker αSMA had poorer survival rates (Sadozai, H. et al., Front. Immunol. 12, 1 - 15 (2021)). This finding emphasizes the importance of CAFs in pancreatic cancer. The immunomodulatory role of CAFs has been widely studied, and the CAF populations are similar among different cancers (Kieffer, Y. et al., Cancer Discov. 10, 1330 - 1351 (2020)). Notably, a subset of CAFs is characterized by enhanced TGFβ signaling, the so-called myofibroblastic CAFs (myCAFs). Interestingly, a subset of myCAFs is associated with non-responsiveness to ICIs in multiple cancers ( D., JEM (2017) doi:10.1084 / jem.20162024)).

[0165] The composition of immune cells in pancreatic cancer is heterogeneous (Steele, N.G. et al., Nat. cancer 1, 1097 (2020)), but generally, the majority are myeloid-derived (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 TAM (tumor-associated macrophages), MDSC (myeloid-derived suppressor cells), and neutrophils, all of which express TGFβ, which regulates the TME (tumor microenvironment). Neutrophils have been shown to secrete large amounts of TGFb in the pancreatic cancer TME, which attracts and activates CAF (Aoyagi, Y. et al., Br. J. Cancer 2004917 91, 1316 - 1326 (2004)). In addition, local TGFb converts myeloid cells into M2 macrophages, TAM, and MDSC, which are elevated in pancreatic cancer (Clark, C.E. et al., Cancer Res. 67, 9518 - 9527 (2007)), and they have a negative impact on both PFS and OS in patients with pancreatic cancer (Ino, Y. et al., Br. J. Cancer 20131084 108, 914 - 923 (2013); Sadozai, H. et al (2021) ibid.; Tsujikawa, T. et al., Cell Rep. 19, 203 - 217 (2017)). These cells express TGFβ (Zhu, L., et al., CellBiol.Int. (2017) doi:10.1002 / cbin.10788), and in addition to their own immunosuppressive properties, they 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)). Thus, the presence of MDSC and Treg in the TME of pancreatic cancer depends on TGFb. In addition, CD8 in the TME +The levels of T cells were negatively correlated with the levels of myeloid cells and Tregs (Clark, C. E. et al., (2007) ibid.; Steele, N. G. et al., Nat. cancer 1, 1097 (2020) and Siret, C. et al., (2020) ibid.). Thus, TGFb is an attractive target for enhancing the efficacy of pancreatic cancer immunotherapy.

[0166] We have shown that the levels of TGFb-specific T cells are associated with disease outcomes in terms of PF and OS. We also believe that therapeutic cancer vaccines against TGFb-derived epitopes represent a potential future therapeutic modality in combination with ICI therapy in the future. In addition, repeated peptide vaccination can enhance the TGFbeta-specific immune response of patients. The rationale for repeated vaccination is to increase the number of TGFbeta-specific T cells migrating to the TME; these cells will attack cells expressing TGFbeta and release Thl cytokines; thus, TGFb signaling will be reduced and the TME will be transformed into an immune-permissive environment favorable for tumor-specific T cells to kill transformed cells.

[0167] In this study, we mimicked therapeutic vaccination by repeated antigen stimulation of PBMCs that showed weak / absent responses to TGFb-15. We found that repeated stimulation induced strong TGFb-15-specific immune responses in almost all cultures. This finding supports the view that repeated vaccination with TGFb-derived peptides can induce TGFb-specific immune responses. In addition, these findings suggest that TGFb-15-specific T cells are not ultimately exhausted or absent in non-responsive samples.

[0168] Conclusion

[0169] PBMC samples from pancreatic cancer patients receiving ICI and SBRT treatment showed immune responses to the TGFb-15 epitope. Patients with clinical benefit from the treatment had stronger TGFb-15-specific T cell responses than those with progressive disease. In addition, strong TGFb-15-specific T cell responses before the start of treatment were independently associated with prolonged progression-free survival and overall survival. We also showed that the low levels of TGFb-15-specific responses observed in some patients were not due to general dysfunction of the immune system, as these patients retained normal T cell responses to common pathogen-derived epitopes. In addition, we showed that repeated antigen stimulation can induce / enhance TGFb-15-specific immune responses. Thus, administration of therapeutic cancer vaccination to deliver repeated TGFb-15-antigen stimulation within patients can induce specific T cell responses that may lead to a clinical response.

[0170] Example 2

[0171] Materials and methods

[0172] All materials and methods were as in Example 1. The sequence of peptide TGFb-33 is: FCLGPCPYIWSLDTQYSKVL (SEQ ID NO: 55).

[0173] Results

[0174] T cell responses specific for TGFb-33 were independently associated with improved survival

[0175] To further investigate whether baseline TGFb-33-specific immune responses were associated with improved survival in a patient cohort similar to that of Example 1, patients were stratified based on whether they exhibited responses above or below or equal to the median of the standardized TGFb-33-specific immune response at baseline. We analyzed TGFb-33-specific responses in patient samples and found that T cells isolated at baseline and after four cycles of treatment all showed responses to TGFb-33( Figure 12 ).

[0176] In addition, we tested the association of clinical parameters that might affect patient survival with survival. Accordingly, the following parameters were tested for association with overall survival in a univariate Cox regression analysis (baseline values n = 33, follow-up values n = 28):

[0177] - Treatment group

[0178] - Gender

[0179] - Age

[0180] - Performance status

[0181] - Weight loss > 5%

[0182] - Number of metastatic sites > 1 or < 1

[0183] - Whipple procedure

[0184] - Biliary stent

[0185] - Tumor marker CA-119-9 above or below the median

[0186] - Neutrophil to lymphocyte ratio (NLR) > 5

[0187] - Bilirubin > 25 μmol / L

[0188] - Albumin < 36 g / L

[0189] - C-reactive protein > 10

[0190] - Modified Glasgow prognostic score (mGPS)

[0191] - Number of previous treatment lines

[0192] - Response to TGFb33 at baseline > median

[0193] - Response to TGFb33 at follow-up > median

[0194] Interestingly, it was found that patients with a TGFb-33 response amplitude higher than the median at baseline had significantly longer overall survival (OS) compared to patients with a response lower than or equal to the median (univariate Cox regression analysis, hazard ratio [HR]: 6.85, p = 3.00x10 -4 ; Figure 11A and Table 7 below). The only other parameter with a statistically significant correlation with overall survival rate was the number of metastatic sites, as patients with ≤1 metastatic lesion showed poorer OS (HR = 2.5, p = 0.0368) (Table 7). Only variables with a statistically significant correlation with overall survival in the univariate analysis are shown in Table 7.

[0195] Incorporating TGFb-33 response and number of metastatic sites into 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 = 1.1x10 -3 ; for patients with a response lower than or equal to the median amplitude) (Table 7).

[0196] Table 7

[0197]

[0198]

[0199] The same analysis was performed for progression-free survival (PFS). In the univariate analysis, the TGFb-33 specific response was associated with PFS (HR: 6.85, p = 3.00x10 -4 ; Figure 11Band Table 8 below). Other important parameters for univariate analysis were: male (HR: 0.38, p = 0.0188) and <1 metastasis (HR: 2.5, p = 0.0312). Multivariate analysis incorporating these statistically significant parameters 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 magnitude, Table 8 below). Only variables with statistically significant correlation with progression-free survival in the univariate analysis are shown in Table 8.

[0200] Table 8

[0201]

[0202] Example 3

[0203] The "non-immunogenicity" of pancreatic cancer (PC), which has a high proportion of immunosuppressive cells and usually lacks tumor-infiltrating effector lymphocytes, is considered one of the reasons for the lack of response to single-agent immunotherapy. Considering the emerging role of the tumor microenvironment, the combination of checkpoint-blocking antibodies with the immunomodulation of the tumor microenvironment can lead to better responses in tumors that have historically been resistant to radiation and checkpoint-blocking antibody approaches as a single modality. For example, data from the phase 2 study CheckPAC (NCT02866383) for patients with resistant metastatic PC showed durable clinical benefit in a small group of patients after adding 15 Gy of stereotactic body radiotherapy (SBRT) to the combination of nivolumab and ipilimumab (published in ASCO GI 2022, San Francisco, see the 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).

[0204] We now find that the TGFb-15 immune response is associated with clinical benefit, which supports the rationale for combining the TGFb-15 peptide vaccine with the CheckPAC strategy (15 Gy of SBRT in combination with nivolumab and ipilimumab). Accordingly, an interventional Phase I study, CheckVAC (NCT05721846), is underway to evaluate the safety and tolerability of nivolumab in combination with ipilimumab, the TGFβ-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.

[0205] The following inclusion criteria are used:

[0206] ● Signed informed consent form

[0207] ○ The subject must have signed and dated an IRB / IEC-approved written informed consent form in accordance with regulatory and institutional guidelines. This consent must be obtained prior to the execution of any protocol-related procedures that are not part of normal subject care.

[0208] ○ The subject must be willing and able to comply with the scheduled visits, treatment schedule, laboratory tests, and other requirements of the study.

[0209] ● Histological or cytological confirmation of advanced pancreatic cancer prior to entry into this study

[0210] ● Prior treatment requirements:

[0211] ○ There is no upper limit on the number of prior chemotherapy regimens received. Participants must have received at least 1 line of systemic chemotherapy (gemcitabine- or 5-FU-based regimens) in the metastatic setting and have progressed during or after chemotherapy.

[0212] ○ Notes

[0213] ■ If the participant received adjuvant / neoadjuvant systemic combination therapy and progressed within 6 months, the adjuvant / neoadjuvant treatment will be considered as 1 line of systemic treatment.

[0214] ■ Generally, discontinuation of one drug in a multi-drug treatment regimen and continuation of other drugs is considered part of the same line of treatment. A drug holiday or restarting the same treatment regimen after maintenance chemotherapy can also be considered part of the same line of treatment. Conversion from intravenous (5-FU) to an oral formulation (capecitabine) of the same drug is also considered part of the same line of treatment.

[0215] ■ The shortest time from the first systemic therapy to progression in recurrent / metastatic pancreatic cancer should be at least 3 months.

[0216] ● 18 years of age or older

[0217] ●ECOG performance status (PS) 0 - 1

[0218] ● All participants are required to undergo mandatory pre - treatment and

[0219] in - treatment biopsies at an acceptable clinical risk as judged by the investigator. Archived pre - treatment samples are not acceptable.

[0220] ● Participants must have normal organ and bone marrow function, defined as follows:

[0221] ○ Absolute neutrophil count (ANC) ≥ 1.5 x 10 9 / L

[0222] ○ Platelet count ≥ 75 x 10 9 / L

[0223] ○ Serum bilirubin ≤ 1.5 x upper limit of normal (ULN)

[0224] ○ AST / ALT ≤ 5 x ULN

[0225] ○ Serum creatinine ≤ 1.5 x ULN or CrCl ≥ 40 mL / min (using the Cockcroft - Gault formula)

[0226] ● Women of child - bearing potential (WOCBP) must use the contraceptive methods shown in Appendix 3. For teratogenic study drugs and / or when there is not enough information to assess teratogenicity (pre - clinical studies have not been conducted), highly effective contraceptive methods (failure rate less than 1% per year) are required. The individualized contraceptive method and duration should be determined in consultation with the investigator. When the half - life of the study drug is greater than 24 hours, WOCBP must follow the birth control instructions and should continue contraception for 30 days plus the time required for the study drug to undergo five half - lives. The half - lives of nivolumab and ipilimumab are up to 25 days and 18 days, respectively. Therefore, WOCBP should use appropriate methods to avoid pregnancy during treatment and for 23 weeks (30 days plus the time required for nivolumab to undergo five half - lives) after the last dose of the study drug.

[0227] ● Sexually active males with WOCBP must use any contraceptive method with an annual failure rate of less than 1%. The investigator should review the contraceptive method and the time period during which contraception must be followed. When the half-life of the study drug is greater than 24 hours, sexually active males with WOCBP must follow the birth control instructions and continue contraception for 90 days plus the time required for the study drug to undergo five half-lives. The half-life of nivolumab is up to 25 days. Sexually active males with WOCBP must continue contraception during treatment and for 31 weeks (90 days plus the time required for nivolumab to undergo five half-lives) after the last dose of the study drug. Women who are not fertile (i.e., postmenopausal or surgically sterilized women and azoospermic men) do not require contraception.

[0228] ● Subjects must sign and date a written informed consent form approved by BIOPAC in accordance with regulatory and institutional guidelines.

[0229] Use the following exclusion criteria:

[0230] ● Any severe or uncontrolled medical condition that the investigator believes may increase the risk associated with study participation or study drug administration, impair the subject's ability to receive protocol treatment, or interfere with the interpretation of study results

[0231] ● Previous treatment with anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CD137, or anti-CTLA-4 antibodies or any other antibody or drug specifically targeting the T cell costimulation or checkpoint pathway

[0232] ● Participants with active, known, or suspected autoimmune diseases. Participants may be allowed to enroll if they have vitiligo, type I diabetes, residual hypothyroidism due to autoimmune disease that requires only hormone replacement, psoriasis that does not require systemic treatment, or a disease that is not expected to recur without an external trigger.

[0233] ● Current or previous use of immunosuppressive drugs within 14 days before the first dose of nivolumab, ipilimumab, and radiotherapy in combination with TGFβ-15 peptide vaccine. The following are exceptions to this criterion:

[0234] o Intranasal, inhaled, or topical steroids; or local steroid injections (e.g., intra-articular injections)

[0235] o Systemic corticosteroids, with a physiological dose not exceeding 10 mg / day of prednisone or equivalent

[0236] o Steroids used as preoperative medication for hypersensitivity reactions (e.g., preoperative medication for CT scans)

[0237] ● Participants with a known history of positive human immunodeficiency virus (HIV) testing or known acquired immunodeficiency syndrome (AIDS) should be excluded.

[0238] ● Allergies and adverse drug reactions

[0239] o History of allergy to the study drug components

[0240] o History of severe allergic reactions to any monoclonal antibody

[0241] ● WOCBP who are pregnant or lactating

[0242] Use the following dosing regimen:

[0243]

[0244] It is expected that the administration of the TGFb-15 peptide vaccine will enhance the clinical benefits of SBRT, nivolumab, and ipilimumab in the treatment of refractory PC.

[0245] *****

[0246] Those skilled in the art will understand that the above-described embodiments can be changed without departing from their broad inventive concept. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention as defined by this specification.

[0247] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of the documents, acts, materials, devices, articles, etc. included in this specification is for the purpose of providing the background context of the present invention. Such discussion does not admit that any or all of these matters constitute a part of the prior art of any invention disclosed or claimed.

Claims

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

2. The PD-1 / PD-L1 antibody for use according to claim 1, wherein the TGFbeta-specific T cell response is directed against a peptide sequence having an amino acid sequence of SEQ ID NO: 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 directed against a peptide sequence having an 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 use according to claim 1 or 2, wherein the baseline TGFbeta-specific T cell response exhibited by the patient prior to treatment is at least the median baseline value of the TGFbeta-specific T cell response observed in cancer patients with the same type of cancer; preferably, wherein the baseline TGFβ-specific T cell response exhibited by the patient prior to the treatment is at least the 75th percentile of the baseline value of the TGFβ-specific T cell response observed in cancer patients with the same type of cancer.

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

5. The PD-1 / PD-L1 antibody for use according to any one of the preceding claims, wherein the PD-1 / PD-L1 antibody is nivolumab.

6. The PD-1 / PD-L1 antibody for use according to claim 5, wherein the patient is also administered a CTLA-4 antibody, preferably ipilimumab.

7. The PD-1 / PD-L1 antibody for use according to any one of the preceding claims, wherein the patient is also undergoing radiotherapy.

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

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

10. The method according to claim 9, wherein the TGFbeta-specific T cell response is directed against a peptide sequence having the amino acid sequence of SEQ ID NO: 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 directed 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 according to claim 9 or 10, wherein the baseline TGFbeta-specific T cell response exhibited by the patient prior to treatment is at least the median baseline value of the TGFbeta-specific T cell response observed in cancer patients with the same type of cancer; preferably, wherein the baseline TGFbeta-specific T cell response exhibited by the patient prior to the treatment is at least the 75th percentile of the baseline value of the TGFbeta-specific T cell response observed in cancer patients with the same type of cancer.

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

13. The method according to claim 12, wherein the patient is also administered a CTLA-4 antibody, preferably wherein the CTLA-4 antibody is ipilimumab.

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

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

16. A method of stratifying cancer patients into at least two treatment groups, the method comprising: i. assaying a sample previously obtained from the patient to detect the presence of a TGFb-specific T cell response; ii. if a TGFb-specific response is present, assigning the patient to a first treatment group, or, if no TGFb-specific response is present, assigning the patient to a second treatment group; wherein if the patient is assigned to the first treatment group, a PD-1 / PD-L1 antibody is administered to them.

17. A method of stratifying cancer patients into one of at least two treatment groups, the method comprising: i. assaying a sample previously obtained from the patient and detecting the level thereof if a TGFb-specific T cell response is present; ii. if the TGFb-specific response is at least a threshold of TGFb-specific T cells, assigning the patient to a first treatment group, and, if the TGFb-specific response is below the threshold, assigning the patient to a second treatment group; wherein if the patient is assigned to the first treatment group, a PD-1 / PD-L1 antibody is administered to them.

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

19. The method according to any one of claims 16 - 18, wherein: (a) The PD-1 / PD-L1 antibody is nivolumab, preferably, wherein the patient is also undergoing radiotherapy treatment and / or the CTLA4 antibody is ipilimumab, and preferably both; and / or (b) The cancer is pancreatic cancer, preferably, wherein the cancer is metastatic pancreatic cancer.

20. The method according to any one of claims 16 - 19, wherein the TGFbeta-specific T cell response is directed against a peptide sequence having the amino acid sequence of SEQ ID NO: 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 a peptide 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) comprising or consisting of a sequence of at least 9 contiguous 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 cancer in a patient, the method comprising: (i) administering to the patient an immunogenic fragment of human transforming growth factor b (TGFb) comprising or consisting of a peptide sequence of at least 9 contiguous 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 use according to claim 21 or the immunogenic fragment of human transforming growth factor (TGFb) for use according to claim 22, the method comprising first identifying the patient as a patient having no TGFbeta-specific T cell response or a patient having a TGFbeta-specific T cell response below a threshold.

24. The PD-1 / PD-L1 antibody for use according to claim 21 or 23 or the immunogenic fragment of human transforming growth factor (TGFb) for use according to 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 use according to any one of claims 22 - 24, wherein: (i) The immunogenic fragment of human TGFβ comprises the amino acid sequence of SEQ ID NO: 28, 55, 66, 29-31, 67, 5-9, 42-45, 12-15, 56-58, 23-26, 49-52, 63, 64, 65 or 2. Preferably, the immunogenic fragment of human TGFβ comprises the amino acid sequence of SEQ ID NO: 28 or 55, more preferably SEQ ID NO: 28; (ii) The immunogenic fragment of TGFβ is administered to the patient before or concurrently with other therapies; (iii) The immunogenic fragment of TGFβ is administered to the patient repeatedly; and / or (iv) The immunogenic fragment of TGFβ is administered to the patient repeatedly until the patient has a TGFβ-specific T cell immune response or a TGFβ-specific T cell immune response at least at a threshold level.

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