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

Abstract

This invention relates to improving cancer treatment methods using PD-1 / PD-L1 antibodies. In particular, this invention is based on the finding that patients with a TGFβ-specific T cell response are likely to show favorable outcomes in cancer treatment with PD-1 / PD-L1 antibodies. Therefore, this invention provides treatment for such patient groups with PD-1 / PD-L1 antibodies and actively promotes the TGFβ-specific T cell response in order to enhance the therapeutic effect of PD-1 / PD-L1 antibodies.

Description

[Technical Field]

[0001] Field of Invention This invention relates to the identification of cancer patients for whom treatment with PD-1 / PD-L1 antibodies is particularly effective. Furthermore, this invention relates to methods for enhancing the effectiveness of PD-1 / PD-L1 antibody therapy for a given group of cancer patients. This invention also relates to compositions and methods for treating cancer.

[0002] Background of the Invention The discovery of the immune checkpoints CTLA-4 and PD-1, and the subsequent emergence of immune checkpoint inhibitors (ICIs), has revolutionized the treatment and prognosis of several 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 investigating ICIs in patients with advanced pancreatic cancer have failed to demonstrate clinically relevant effects (Brahmer, JR et al., N. Engl. J. Med.366, 2455-2465 (2012); O'Reilly, EM et al., JAMA Oncol.5, 1431-1438 (2019); and Royal, RE et al., J. Immunother.33, 828-833 (2010)).

[0003] In recent years, a Phase II trial (CheckPAC) in patients with refractory metastatic pancreatic cancer investigated radiotherapy using the PD-1 antibody nivolumab (with and without ipilimumab) (Chen, IM et al. J. Clin. Oncol. 71, (2022)). A clinical response rate of 37.2% was observed in the combination group, but only 14% achieved a partial response. Therefore, an improved approach is still needed for treating cancers that are refractory to PD-1 / PD-L1 antibody therapy, especially pancreatic cancer. [Overview of the project]

[0004] This invention provides a method for enhancing the therapeutic effect of PD-1 / PD-L1 antibodies, particularly for enhancing the therapeutic effect of cancers that are refractory to such antibody therapy. This invention is based on the finding that the presence or absence of a TGF-beta-specific T cell response indicates the degree of effectiveness of PD-1 / PD-L1 antibody therapy, and that, if such a response exists, the magnitude of that response also indicates the degree of therapeutic effectiveness. Therefore, this invention identifies patient groups for whom PD-1 / PD-L1 antibody therapy is likely to be particularly effective. Furthermore, TGF-beta or TGF-beta-derived immunogenic peptides can be used to stimulate or boost the TGF-beta T cell response, thereby increasing the likely effect of PD-1 / PD-L1 antibody therapy against cancer.

[0005] The present invention is particularly effective for cancers that are refractory to treatment with PD-1 / PD-L1 antibodies, such as pancreatic cancer. The PD-1 / PD-L1 antibody that can be used in the present invention is nivolumab. Therefore, in one embodiment, the antibody is nivolumab and the cancer is pancreatic cancer. Particularly useful indicators are the presence of a T cell response to the TGF beta-15 peptide sequence represented by SEQ ID NO: 28, and, if present, its size.

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

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

[0008] The present invention also provides a method for stratifying cancer patients into at least one of two treatment groups, i) A step of performing an assay on a sample obtained in advance from a patient in order to detect the presence or absence of a TGFb-specific T cell response. ii) A step in which patients with a TGFb-specific response are assigned to the first treatment group, and patients without a TGFb-specific response are stratified to the second treatment group. The stratification method also provides the above, which includes the administration of PD-1 / PD-L1 antibodies to patients assigned to the first treatment group.

[0009] The present invention also provides a method for stratifying cancer patients into at least one of two treatment groups, i) If a TGFb-specific T cell response is present, the step of performing an assay on a sample previously obtained from the patient in order to detect its level. ii) If the TGFb-specific T cell response is at least above the threshold for TGFb-specific T cells, the patient is assigned to the first treatment group; if the TGFb-specific response is below the threshold, the patient is stratified to the second treatment group. The present invention provides a stratification method that includes the above, wherein if a patient is assigned to the first treatment group, they are administered a PD-1 / PD-L1 antibody.

[0010] The present invention also provides PD-1 / PD-L1 antibodies for use in a method of treating cancer, wherein the method is i) A step of administering to a patient an immunogenic fragment of human transforming growth factor (TGFb) comprising or consisting of a sequence of at least nine consecutive amino acids in Sequence ID No. 1, and ii) Step of administering PD-1 / PD-L1 antibody Includes.

[0011] The present invention further provides immunogenic fragments of human transforming growth factor b (TGFb) for use in a method of treating cancer in a patient, wherein the method is i) A step of administering to a patient an immunogenic fragment of human transforming growth factor b (TGFb) comprising a peptide sequence consisting of at least nine consecutive amino acids as in Sequence ID No. 1, and ii) The step of administering PD-1 / PD-L1 antibodies to the patient, Includes. [Brief explanation of the drawing]

[0012] [Figure 1A] TGFb-15 specific response in pancreatic cancer (PC) patients. A. Peripheral blood mononuclear cells (PBMCs) obtained from pancreatic cancer patients were stimulated once in vitro with TGFb-15 peptide and IL-2, incubated for 14 days, and then seeded at a concentration of 2 × 10⁵ cells / well in interferon-γ enzyme-coupled immunosorbent spots (IFNγ ELISPOTs), incubated overnight. The experiment was performed in triple replication, with the negative control well left unstimulated. Responses were characterized in 32 samples isolated at baseline (left) and 31 samples isolated after four treatments (right). Error bars represent the standard error of the mean, and statistics were performed using paired t-tests. Statistical analysis was performed using the Mann-Whitney U test. B. Representative photographs of the baseline response (top) and the response after four treatments. The amplitude of the response in clinically responsive patients and the amplitude of the response in patients with disease progression are compared using the normalized CA data. Normalization was performed by subtracting the average number of spots in the control well from the average number of spots in the peptide-stimulated well. Error bars represent the standard error of the mean, and statistics were performed using paired t-tests. Statistics were also performed using the Mann-Whitney test. [Figure 1B] This is a continuation of Figure 1A. [Figure 1C] This is a continuation of Figure 1B. [Figure 2-1]This shows that the amplitude of the TGFb-15 specific immune response fluctuates over time. A. The normalized number of spots at baseline was compared with the number of spots after four treatments (i.e., 8 weeks after the first treatment) for patients who achieved a clinical response (left) and patients with disease progression (right). Statistical analysis was performed using the Wilcoxon matched-pairs signed-rank test. B. This shows that the amplitude of the TGFb-15 specific immune response fluctuates over time. Representative photographs of the baseline response and the response after four treatments in patients who achieved a clinical response (top) and patients with disease progression (bottom). C. This shows that the amplitude of the TGFb-15 specific immune response fluctuates over time. Normalized TGFb-15 specific immune response analyzed over a long period (more than 2 years) in three patients who achieved a clinical response. Spot number normalization was performed using the method described above. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3] Differences in the decline of TGFb-15-specific immune response between patients with sustained clinical response and those without. Comparison of TGFb-15-specific immune responses at baseline and after four treatments in patients with sustained clinical response (A) and patients without sustained clinical response (B). Sustained clinical response was defined as partial response (PR) or stable disease (SD) lasting for 6 months or more. Statistical analysis was performed using Wilcoxon's matched-pair signed-rank test. [Figure 4A] A strong TGFb-15 specific response before the initiation of treatment is predicted to be associated with a high survival rate. A. Kaplan-Meier curves showing overall survival in patients with a response above and below the median response amplitude to TGFb-15. B. Kaplan-Meier curves showing progression-free survival in patients with a response above and below the median response amplitude to TGFb-15. Time-to-event analysis was performed using the log-rank test. [Figure 4B] This is a continuation of Figure 4A. [Figure 5A]Analysis of patient survival rate based on the amplitude of TGFb-15 specific response. A. Analysis of the overall survival period of patients based on the normalized TGFβ-15 response. Patients were divided into four groups based on the amplitude of the TGFb-15 specific response. B. Analysis of the progression-free survival period similar to A. Statistics were performed using the log-rank test. [Figure 5B] Continuation of Figure 5A. [Figure 6] Correlation between TGFb-15 specific response and response to tetanus epitope. A. The response to normalized TGFb-15 and tetanus response were plotted and the correlation was analyzed using simple regression. B. The response to normalized TGFb-15 and influenza response were plotted and the correlation was analyzed using simple regression. [Figure 7-1] The amplitude of the GFb-15 specific response is not associated with the amplitude of the response to the tetanus peptide. A. PBMC were tested for the response to the tetanus epitope "tetanus-long" using an in vitro IFNg ELISPOT assay. The amplitude of the normalized tetanus-long specific response was compared between patients showing a response above the median of the TGFb-15 specific immune response and those showing a response below it. The response to tetanus-long was not analyzed in baseline samples of 5 patients in the group showing a response above the median and 1 patient in the group showing a response below the median, but was analyzed in samples collected within 2 weeks from baseline. Error bars indicate the standard error of the mean. Statistics were performed using the Mann-Whitney test. B. Representative photographs of patients with no TGFb-specific immune response (upper) but with unmodified tetanus-specific immune response (lower). C. The same analysis as A using the short influenza virus-derived epitope "C18 A2 Flu" instead of the tetanus-derived epitope. Error bars indicate the standard error of the mean. Statistics were performed using the Mann-Whitney test. D. Representative photographs of patients with no TGFb-specific immune response (upper) but with unmodified influenza virus-specific immune response (lower). [Figure 7-2] Continuation of Figure 7-1. [Figure 8A]The specific response to Clostridium tetani indicates no association with survival duration. Kaplan-Meier curves showing the overall survival duration of patients with responses above and below the median response amplitude to A. Tetanus-Long. B. Kaplan-Meier curves showing the progression-free survival duration of patients with responses above and below the median response amplitude to Tetanus-Long. C. The influenza-specific response indicates no association with survival duration. Kaplan-Meier curve showing the overall survival duration of patients with responses above and below the median response amplitude to C18 A2 Flu. D. Kaplan-Meier curve showing the progression-free survival duration of patients with responses above and below the median response amplitude to C18 A2 Flu. Survival time analysis was performed using the log-rank test. [Figure 8B] Continuation of Figure 8A. [Figure 8C] Continuation of Figure 8B. [Figure 8D] Continuation of Figure 8C. [Figure 9A]Repeated in vitro stimulation of patient and healthy donor PBMCs with TGFb-15 peptide increases the amplitude of the TGFb-15 specific immune response. PBMCs obtained from 16 PC patients with a weak TGFb-15 response were cultured in vitro, and the response to TGFb-15 was tested after one in vitro stimulation with TGFb-15 peptide and after three in vitro stimulations with TGF beta-15 peptide. The normalized response is shown in the heatmap (top), and representative responses after one and three stimulations are shown (bottom). Some cultures received only two in vitro stimulations (indicated by black stars next to the number of spots). B. Regarding the ability of repeated stimulation to enhance the TGFb-15 specific immune response, PBMCs obtained from 7 healthy donors using 5-6 x 10⁵ cells / well were analyzed as described in A, and the normalized results (top) and representative responses (bottom) are shown. To assess the ability of PBMCs obtained from 15 healthy donors using 2x10⁵ cells / well to enhance the C.TGFb-15 specific immune response to repeated stimulation, the normalized results (top) and representative responses (bottom) are shown. [Figure 9B] This is a continuation of Figure 9A. [Figure 9C] This is a continuation of Figure 9B. [Figure 10] Enhancement of distribution-independent resampling (DFR) responses after repeated stimulation. A. Comparison of DFR responses and DFR2x responses after one or repeated stimulation in PC patients. B. Enhancement of distribution-independent resampling (DFR) responses after repeated stimulation. Similar comparison to A in healthy donors. Statistical analysis was performed using DFR and DFR2x methods. [Figure 11A] Figures 11A and 11B show that a strong TGFb-15 specific response before treatment initiation is predicted to be associated with a higher survival rate. A. Kaplan-Meier curves showing overall survival in patients with a response above or below the median response amplitude to TGFb-33. B. Kaplan-Meier curves showing progression-free survival in patients with a response above and below the median response amplitude to TGFb-33. Survival analysis was performed using the log-rank test. [Figure 11B] This is a continuation of Figure 11A. [Figure 12] T-cell response to TGFb-33 in pancreatic cancer patients treated with radiotherapy and anti-CTLA-4 and anti-PD-1. Peripheral blood mononuclear cells (PBMCs) from pancreatic cancer patients were stimulated once in vitro with TGFb-33 peptide and IL-2, incubated for 14 days, and then plated to interferon-γ enzyme-coupled immunosorbent spots (IFNγELISPOT) at a concentration of 2 x 10⁵ cells / well, and incubated overnight. Experiments were performed in double or triple replication, and negative control wells were left unstimulated. Responses were characterized in 31 samples isolated at baseline (left) and 28 samples isolated after four treatments (right). The figure shows the normalized response amplitude to TGFb-33, calculated by subtracting the average number of spots in the negative control well from the average number of spots in the peptide-stimulated well. [Modes for carrying out the invention]

[0013] [A brief explanation of arrays] Sequence ID 1 is the amino acid sequence of the full-length precursor of human TGFb-1 (also called TGFb-1 preprotein). Sequence ID 2 is the amino acid sequence of the signal peptide of human TGFb-1. Sequence ID 3 is the amino acid sequence of the LAP peptide of human TGFb-1. Sequence ID 4 is the amino acid sequence of mature human TGFb-1. Sequence IDs 5-64 are the amino acid sequences of polypeptide fragments derived from human TGFb-1, respectively. Sequence ID 65 is the amino acid sequence of the LAP subregion, which contains a high frequency of immunogenicity sequences. Sequence ID 66 is the amino acid sequence of the minimal epitope sequence within the TGFb-15 peptide sequence (Sequence ID 28). Sequence ID 66 is also referred to herein as "TGFb-15-15short". Sequence ID 67 is the amino acid sequence of TGFb-A2-01.

[0014] Detailed description of the invention It should be understood that the disclosed products and methods may be adapted to different applications to meet specific needs in the art. Furthermore, it should be understood that the terms used herein are solely for the purpose of describing specific embodiments of the invention and are not intended to limit them.

[0015] [Definition] The technical and scientific terms used herein have the meanings generally understood by those skilled in the art unless otherwise defined. The following definitions of terms apply to interpreting this specification, and the singular form of a term shall include the plural form, and the plural form of a term shall include the singular form, as appropriate (unless it is clearly evident from the context). For example, "one polypeptide" includes "multiple polypeptides." In the event of any conflict between the definitions of terms provided herein and any references incorporated herein by reference, the definitions provided below shall prevail.

[0016] When the terms "comprising" and "comprises" are used, things that "consist essentially of" or "consist of" what is described are also provided.

[0017] In this specification, “polypeptide” is used in its broadest sense to refer to a compound composed of two or more subunit amino acids, amino acid analogs, or other peptide mimics. Therefore, the term “polypeptide” includes not only short peptide sequences but also longer polypeptides and proteins. As used herein, the term “amino acid” refers to either natural and / or unnatural (i.e., synthetic) amino acids, including both D and L optical isomers, or to amino acid analogs and peptide mimics.

[0018] The terms "patient" and "subject" are used interchangeably and usually refer to human beings.

[0019] In this specification, "immunogenicity" typically means that, when TGFb protein, particularly TGFb-1 protein, is present in or on cells expressing TGFb-1 protein, the polypeptide can elicit an immune response to said protein. In other words, the polypeptide is immunogenic to TGFb. Alternatively, this polypeptide can be said to be an immunogenic fragment of TGFb. The immune response refers to a T cell response, and therefore the polypeptide can be said to be an immunogenic fragment of TGFb containing a T cell epitope. The immune response can be detected in at least one individual (or a sample taken from that individual) after administration of the polypeptide to said individual (or said sample).

[0020] In this specification, when TGF-b, T-GF-beta, etc., refer to TGF-β. However, to avoid the use of Greek symbols and to aid in the reproducibility of the text, the former nomenclature is used.

[0021] TGF-beta 1-specific T cell response This invention is based on the finding that patients exhibiting a T-cell response specific to TGF-beta are a group of patients for whom PD-1 / PD-L1 antibody therapy is considered particularly effective. Furthermore, it has been found that the magnitude of the TGF-beta-specific T-cell response indicates the degree to which PD-1 / PD-L1 antibody therapy is effective against cancer. In other words, by determining the presence or absence of a TGF-beta-specific T-cell response in a given patient, or by quantifying it, it is possible to determine the degree to which PD-1 / PD-L1 antibody therapy is effective. Therefore, this can be used as a method to decide whether or not to administer PD-1 / PD-L1 antibody therapy to a patient. The finding that a TGF-beta-specific T-cell response indicates the degree to which PD-1 / PD-L1 antibody therapy is effective also indicates that in patients who lack such a response or whose response is weak, measures can be taken to induce or enhance such a response, thereby improving the effectiveness of PD-1 / PD-L1 antibody therapy. A TGF-beta-specific T-cell response may refer to one directed at TGF-beta 1. The present invention is particularly useful for cancers that are refractory to treatment with PD-1 / PD-L1 antibodies. In a particularly preferred embodiment, the cancer is pancreatic cancer and the antibody is nivolumab.

[0022] Specifically, the presence or absence of a TGF beta response, and the magnitude of that response, are measured at the "baseline" stage, before the start of treatment. Measurements are also taken during the course of treatment. In a further embodiment, the presence or absence of a TGF beta response is measured at baseline, and then measured again after treatment to enhance the T cell response to the TGF beta response to confirm that a response has occurred or been enhanced.

[0023] In some embodiments, it is measured whether a patient has a T-cell response specific to TGF-beta. The results of the patient's test sample are compared to those of a negative control, and if the test sample results are not significantly increased compared to the negative control results, it can be said that there is no response. The inventors have found that patients with higher response levels yield better results with PD-1 / PD-L1 antibodies, so rather than simply determining the presence or absence of a response, the magnitude of the response is measured.

[0024] Whether a patient has a TGF beta-specific T cell response, or the magnitude of that response, can be measured by any suitable method. Whether such a response exists can be determined by determining whether T cells isolated from the patient show a response when exposed to TGF beta-1 or the TGF beta-1 peptide. Any of the TGF beta peptide sequences described herein may be used, with particularly preferred peptides of interest being the TGF beta-15 peptide sequence of (SEQ ID NO: 28) or the TGF beta short peptide sequence of (SEQ ID NO: 66). A further preferred peptide of interest is the TGF beta-33 peptide sequence of SEQ ID NO: 55.

[0025] Patient samples for evaluation may be blood-derived. Peripheral blood mononuclear cells (PBMCs) are a representative example of preferred samples used herein. In another embodiment, the cells are purified T cells, for example, T cells purified from the patient's blood. One way to measure whether a subject exhibits a TGF beta-specific T cell response is to assay whether peripheral blood mononuclear cell PBMCs isolated from the subject exhibit a response to TGF beta-1 or the TGF beta-1 peptide. One suitable assay involves incubating PBMCs with TGF beta-1 or the TGF beta-1 peptide and then determining whether the present cells are activated by TGF beta-1 or the TGF beta-1 peptide. A possible method for determining whether activated T cells exhibit a response to TGF beta-1 is the ELISPOT assay. The assays employed in the examples of this application can be used to determine whether a response is present and, if present, to quantify it.

[0026] In one embodiment, the assay comprises the following steps: (a) incubating PBMCs isolated from a patient with TGF-beta-1 or TGF-beta-1 peptide for 7 to 14 days; (b) plating a known number of cells on a plate coated with an IFN-γ-specific primary antibody, incubating the plate overnight, and then removing the cells; (c) detecting the presence of bound IFN-γ using a secondary antibody, streptavidin-ALP, and an enzyme substrate (washing the wells with PBS before and after each step, i.e., identifying the locations of activated cells secreting IFN-γ as spots); and (d) counting the number of spots, i.e., the number of cells specific to TGF-beta-1. Step (a) may also include IL-2 in the incubation step. The assay may include controls, such as performing the assay on a negative control sample in which TGF-beta-1 or TGF-beta-1 peptide is not included in the incubation step of (a). In one embodiment, subjects who have the same or similar results as the negative control are defined as lacking a T cell response to TGF-beta. In one embodiment, a no-response result in the ELISPOT assay indicates that the number of spots is 5 times or less compared to an equivalent negative control. Similarly, or instead, the assay may further include a sample containing a positive control, such as T cells that show a response to TGF-beta.

[0027] In another embodiment, the magnitude of any TGF-beta T cell response is measured. In a further embodiment, a method may be used that can enumerate the number of T cells that produce a TGF-beta-specific response. One such method is to use a spot-based assay. A typical assay used herein is the ELISpot assay. For example, the assay employed in the embodiments of this application may be used.

[0028] The present invention may include a step of generating or comparing an obtained value to an expected value. Patients may also be selected for treatment based on the criterion that the baseline T-cell response specific to TGF beta-1 is above a threshold. One way to set a threshold is the median. In some embodiments, the median may be the baseline median observed in cancer patients, particularly those with the same type of cancer. The median can be calculated from cancer patients of the same type and sex. It may also be calculated from a representative sample of such cancer patients. An expected median value for a given value can be calculated beforehand and used. In further embodiments, another way to set a threshold is in terms of the 75th percentile value. The 75th percentile value may be the 75th percentile value of the baseline observed in cancer patients, particularly those with the same type of cancer. The 75th percentile value can be calculated from cancer patients of the same type and sex. It may also be calculated from a representative sample of such cancer patients. An expected 75th percentile value for a given value can be calculated beforehand and used.

[0029] This invention allows us to determine which percentile a patient's value falls to within a patient population with the same condition. This invention can be used to indicate the effectiveness of PD-1 / PD-L1 antibody therapy. In some embodiments, a "threshold" can be applied to determine whether or not to administer PD-1 / PD-L1 antibodies to a subject. In another embodiment, if the value falls below the threshold, a treatment to enhance the TGF beta-specific T cell response is chosen for that patient to increase the effectiveness of PD-1 / PD-L1 antibodies. A TGF beta-1 peptide, or a sequence encoding it, may be administered to the subject to stimulate such a response.

[0030] patient Although the patient is human, the present invention can be applied to any suitable mammalian subject. The patient may be male or female. In one embodiment, the patient is male.

[0031] The patient is a cancer patient. In one embodiment, the cancer is selected from pancreatic cancer, melanoma, lung cancer, malignant pleural mesothelioma, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, urothelial carcinoma, colon cancer, esophageal squamous cell carcinoma, liver cancer, gastric cancer, and esophageal cancer 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 carcinoma. In a further embodiment, the patient may have high microsatellite instability or mismatch repair deficiency 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 metastatic sites.

[0032] In one embodiment, the cancer is refractory to treatment with PD-1 / PD-L1 antibodies. An example of such a refractory cancer is pancreatic cancer. Therefore, in a particularly preferred embodiment, the subject may have pancreatic cancer. In one embodiment, the pancreatic cancer is metastatic. In a further embodiment, the cancer is refractory metastatic pancreatic cancer (mPC).

[0033] PD-1 / PD-L1 antibodies and additional cancer therapies PD-1 / PD-L1 antibodies typically inhibit the interaction between PD-1 and PD-L1. Therefore, 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, semiprimab, and dostarimab. Examples of PD-L1 antibodies include atezolizumab, avelumab, and durvalumab.

[0034] Nivolumab is a particularly preferred PD-1 antibody for this purpose. Nivolumab is marketed under the brand name Opdivo (registered trademark). It may also be marketed under the names ONO-4538, BMS-936558, and MDX1106. Nivolumab is used to treat a variety of cancers.

[0035] In some cases, only the PD-1 / PD-L1 antibody may be administered to the patient. However, the PD-1 / PD-L1 antibody may also be administered in combination with other therapies or as a further line of treatment after treatment with other therapies. In one embodiment, a second cancer therapy may also be administered in any of the embodiments described herein in which the subject is administered the PD-1 / PD-L1 antibody. In some cases, both the PD-1 / PD-L1 antibody and the CTLA-4 antibody may be administered to the patient. Preferably, the CTLA-4 antibody is ipilimumab. In some cases, both the PD-1 / PD-L1 antibody and radiotherapy may be administered to the patient. In some cases, the PD-1 / PD-L1 antibody, the CTLA-4 antibody, and radiotherapy may be administered to the patient. The preferred CTLA-4 antibody is ipilimumab. One form of radiotherapy is stereotactic radiotherapy (SBRT). In such embodiments, nivolumab is the PD-1 antibody of interest. Therefore, nivolumab may also be administered alone. Nivolumab may also be administered together with a CTLA-4 antibody (e.g., ipilimumab). In either case, radiotherapy may be performed. Thus, in one embodiment, nivolumab, ipilimumab, and SBRT are administered. In one embodiment, radiotherapy is provided in the form of stereotactic radiotherapy (SBRT) of approximately 15 Gy to a single site of disease. In a further embodiment, SBRT is performed on day 1 of a 14-day treatment cycle. In one embodiment, nivolumab is administered at a dose of approximately 3 mg / kg (maximum approximately 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 approximately 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 once every 6 weeks (±3 days) thereafter.

[0036] A CTLA-4 antibody may be administered, or the patient may already be receiving treatment with such an antibody. An example of a CTLA-4 antibody is ipilimumab, marketed under the brand name Yervoy (registered trademark). A more preferred example of a CTLA-4 antibody is tremelimumab.

[0037] As a method to enhance the efficacy of PD-1 / PD-L1 antibodies, in patients for whom a TGF beta-specific T cell response has been determined, such a response can be stimulated by administering the TGF beta-1 peptide, as further described herein. In one embodiment, the TGF beta-1 peptide is administered in a dose of approximately 200 μg. The TGF beta-1 peptide may be administered as an emulsion with an adjuvant. In one embodiment, the TGF beta-1 peptide is administered in a dose of approximately 200 μg as an emulsion with approximately 500 μl of montanide ISA-51 added. The peptide-adjuvant emulsion is administered on day 1 of each of the first six 14-day treatment cycles, and thereafter once every four weeks (±3 days).

[0038] When additional drugs or therapies are administered to a patient in addition to PD-1 / PD-L1 antibodies, they may be administered simultaneously, separately, or sequentially. The two drugs may be administered together in the same composition or in separate compositions. In embodiments where TGF beta-1 peptide is administered to stimulate a TGF beta-specific T cell response, it may be administered before the PD-1 / PD-L1 antibody. In one embodiment, the TGF beta peptide may be administered, and the PD-1 / PD-L1 antibody may be administered after confirming stimulation of the T cell response to TGF beta, or after the response value has increased and exceeded a threshold. In another embodiment, the TGF beta-1 peptide and the PD-1 / PD-L1 antibody may be administered simultaneously.

[0039] TGF beta-1 epitope and peptide TGF-beta is typically TGF-beta-1. The TGF-beta-1 sequence may be used in terms of whether or not a T cell response to TGF-beta is present in the patient. Alternatively, sequences described later may be used to stimulate such a response.

[0040] The sequence of the full-length human TGF beta-1 preprotein (NP000651.3) (SEQ ID NO: 1) is shown below. TIFF2026512959000001.tif67160

[0041] Table 1 below shows various TGF-beta-1 related sequences, including specific TGF-beta-1 peptides, that can be used in the present invention. In Table 1 below, "start position" and "end position" refer to the position within the full-length human TGF-beta-1 preprotein (SEQ ID NO: 1) unless otherwise specified.

[0042] [Table 1] TIFF2026512959000003.tif214166

[0043] The presence or size of a TGF-beta-specific T cell response can be determined using TGF-beta-1 itself or using one of the TGF-beta-1 peptide sequences described herein. Furthermore, the TGF-beta-1 peptide sequences discussed herein can also be used to induce or increase the size of a TGF-beta-1-specific T cell response, thereby enhancing the therapeutic effect on cancer. Therefore, the sequences discussed below can be involved not only in the detection / measurement of a TGF-beta-specific T cell response, but also in attempts to stimulate / enhance such a response. In a particularly preferred embodiment, the peptide sequence is the sequence of the TGF-beta-15 peptide of SEQ ID NO: 28. In a more particularly preferred embodiment, the peptide sequence is the sequence of SEQ ID NO: 66. In a more particularly preferred embodiment, the peptide sequence is the sequence of SEQ ID NO: 55.

[0044] A preferred TGF-beta-1 peptide sequence for use in the present invention is an immunogenic fragment of human TGF-beta-1 (SEQ ID NO: 1), which includes or consists of a sequence of at least nine consecutive amino acids in SEQ ID NO: 1. The sequence of at least nine consecutive amino acids in SEQ ID NO: 1 may correspond to, for example, a sequence of at least nine consecutive amino acids in the signal peptide (SP) domain of TGF-beta-1, for example, a sequence of at least nine consecutive amino acids in SEQ ID NO: 2. It may also correspond to a sequence of at least nine amino acids in the latency-related peptide (LAP) domain of TGF-1, for example, a sequence of at least nine consecutive amino acids in SEQ ID NO: 3. It may also correspond to a sequence of at least nine consecutive amino acids located within the LAP subregion surrounded by amino acids at positions 121 and 160 of SEQ ID NO: 1, for example, a sequence of at least nine consecutive amino acids in SEQ ID NO: 65. It may also correspond to a sequence of at least nine consecutive amino acids in a mature TGF-b1 polypeptide, for example, a sequence of at least nine consecutive amino acids in SEQ ID NO: 4.

[0045] A polypeptide may contain, 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 in SEQ ID NO: 1. A polypeptide may contain, or consist of, an amino acid sequence represented by any one of SEQ ID NOs: 2 and 5-67. A polypeptide may contain, or consist of an amino acid sequence represented by any one of SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, 63, 5, 7-9, 43-45, 13-15, 24-26, 29-31, 50-52, 56-58, 64, 65, 2, 66, 67, or 5. In some embodiments, the polypeptide comprises or consists of an amino acid sequence represented by any one of SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, 63, 66, 67, or 5.

[0046] The polypeptide may contain, or consist of, an amino acid sequence represented by any one of SEQ ID NOs: 28, 66, 29-31, 67, 5-9, 42-45, 12-15, 55-58, 23-26, 49-52, 63, 64, 65, or 2. In some embodiments, the polypeptide contains, or consists of an amino acid sequence represented by SEQ ID NOs: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49, or 63.

[0047] The maximum length of a 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 long. The C-terminal amino acid of the polypeptide may be substituted with the corresponding amide. The polypeptide may be isolated.

[0048] In some embodiments, the polypeptide comprises or consists of an amino acid sequence represented by any one of SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, or 63. In further embodiments, the polypeptide comprises or consists of an amino acid sequence represented by any one of SEQ ID NOs: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49, or 63. Alternatively, a longer polypeptide fragment of SEQ ID NO: 1 incorporating these sequences may be used.

[0049] The polypeptide may contain an HLA-A2 restriction epitope. In one embodiment, the HLA-A2 restriction epitope contains or consists of the amino acid sequence represented by SEQ ID NO: 66. In another embodiment, the peptide containing the HLA-A2 restriction epitope consisting of the amino acid sequence represented by SEQ ID NO: 66 is a peptide containing or consisting of the amino acid sequence represented by any one of SEQ ID NOs: 28-31 or 65. Alternatively, the HLA-A2 restriction epitope contains or consists of the amino acid sequence represented by SEQ ID NO: 67. In some embodiments, the peptide containing the HLA-A2 restriction epitope consisting of the amino acid sequence represented by SEQ ID NO: 67 is a peptide containing or consisting of the amino acid sequence represented by any one of SEQ ID NOs: 5, 8, 9 or 2.

[0050] In any polypeptide described herein, the amino acid sequence may be modified by 1, 2, 3, 4, or 5 (i.e., up to 5) additions, deletions, or substitutions, provided that the sequence-modified polypeptide is the same as or higher in immunogenicity to TGFb1 than the unmodified polypeptide. "Same" means that the sequence-modified polypeptide is not significantly less immunogenic to TGFb1 than the unmodified polypeptide. The comparison of immunogenicity between sequences shall be performed using the same assay. Unless otherwise specified, polypeptide sequence modifications are conservative amino acid substitutions. A conservative substitution is the replacement of an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side chain length. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid being substituted. Alternatively, a conservative substitution may introduce another aromatic or aliphatic amino acid in place of an existing aromatic or aliphatic amino acid. Conservative amino acid substitutions are well known in this field and can be selected according to the properties of the 20 major amino acids defined in Table 2 below. If the amino acids have similar polarity, this can be determined by referring to the hydrophathy scale (a measure of hydrophobicity and hydrophilicity) of the amino acid side chains in Table 3.

[0051] [Table 2]

[0052] [Table 3]

[0053] Polypeptides disclosed herein may undergo one or more of the following modifications to improve their physicochemical properties (e.g., stability) (provided that the polypeptide has the same or higher immunogenicity to TGFb1 as the unmodified polypeptide): (i) substitution of the C-terminal amino acid with the corresponding amide (which may enhance resistance to carboxypeptidase); (ii) substitution of the N-terminal amino acid with the corresponding acylated amino acid (which may enhance resistance to aminopeptidase); (iii) substitution of one or more amino acids with the corresponding methylated amino acids (which may improve resistance to proteolysis); and / or (iv) substitution of one or more amino acids with the corresponding D-configured amino acids (which may improve resistance to proteolysis).

[0054] A preferred peptide that may be adopted is described in International Patent Publication No. WO2020 / 245264 (PCT / EP2020 / 065472), which is incorporated in its entirety by reference, and the TGFb1 sequence disclosed therein is also incorporated by reference specifically in relation to a potential epitope against which a T cell response may be countered, and as a sequence used to stimulate such a response.

[0055] In any of the embodiments described herein that use a peptide to stimulate a response, a sequence encoding such a peptide may be administered instead, or a composition containing the peptide or coding sequence may be administered instead.

[0056] composition containing polypeptide The present invention provides the use of the TGFb1 peptide described herein to enhance or induce a TGF beta-specific T cell response when a subject is also administered a PD-1 / PD-L1 antibody. The TGF beta1 peptide can be provided in the form of a pharmaceutical composition for use in such a manner. This composition may also include a PD-1 / PD-L1 antibody. The present invention also provides a PD-1 / PD-L1 antibody for use as described herein, which can be formulated as currently available on the market, but any suitable formulation means can also be employed.

[0057] In some embodiments, the pharmaceutical composition comprises at least one adjuvant, a pharmaceutically acceptable carrier, a preservative and / or 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, and at least eight different polypeptides and at least one adjuvant, a pharmaceutically acceptable carrier, a preservative and / or 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, and at least eight different polynucleotides coding the present invention and at least one adjuvant, a pharmaceutically acceptable carrier, a preservative and / or excipient.

[0058] Carriers, preservatives, and excipients must be compatible with the other components of the composition and “acceptable” in the sense that the composition is not harmful to the subject to whom it is administered. Typically, all components and the final composition are sterile and pyrogen-free. The composition may be a pharmaceutical composition. The composition may contain an adjuvant. An adjuvant is any substance that, when mixed with a composition, enhances or modifies the immune response induced by that composition. In a broad sense, an adjuvant is a substance that promotes an immune response. Adjuvants can also be said to have a depot effect in that they provide sustained and prolonged release of the active substance from the administration site. A general discussion of adjuvants is found on pages 61-63 of Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986).

[0059] The adjuvant can be selected from the following group: 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 called nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP RIBI (MPL+TDM+CWS) emulsion in 2% squalene / Tween-80 RTM (also known as 19835A, MTP-PE), lipopolysaccharides containing lipid A and their various derivatives, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, Merck adjuvant 65, polynucleotides (e.g., polyIC and polyAU acid), wax D derived 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 matrix or GMDP, interleukin 1, interleukin 2, montanide ISA-51 and QS-21. Various saponin extracts have also been suggested to be useful as adjuvants for immunogenic compositions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can also be used as an adjuvant.

[0060] Adjuvants used in embodiments of the present invention to stimulate T cell responses include oil / surfactant-based adjuvants such as montanido adjuvant (available from Seppic, Belgium), e.g., montanido ISA-51. Other adjuvants include bacterial DNA-based adjuvants such as adjuvants containing CpG oligonucleotide sequences. Still other adjuvants include viral dsRNA-based adjuvants such as poly(I:C). GM-CSF and imidazoquinoline are also examples of adjuvants.

[0061] In one embodiment, the adjuvant is montanide ISA adjuvant. In a further embodiment, the montanide ISA adjuvant is montanide ISA51 or montanide ISA720.

[0062] Pages 61-63 of Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986), also state that coupling with an immunogenic carrier is recommended when the target antigen is low molecular weight or poorly immunogenic. Therefore, the polypeptide of the present invention may be coupled to a carrier. The carrier may exist independently of the adjuvant. The function of the carrier may be, for example, to increase the molecular weight of the polypeptide fragment to enhance activity or immunogenicity, to confer stability, to enhance biological activity, or to extend the serum half-life. Furthermore, the carrier may be useful in presenting the polypeptide or its fragments to T cells. Therefore, in the present composition, the polypeptide may be conjugated to a carrier as described below. The carrier may be any suitable carrier known to those skilled in the art, for example, a protein or an antigen-presenting cell such as a dendritic cell (DC). Examples of carrier proteins include keyhole limpet hemocyanin, transferrin, bovine serum albumin, human serum albumin, serum proteins such as thyroglobulin or ovalbumin, immunoglobulins, hormones such as insulin, or palmitic acid. Alternatively, the carrier protein may be tetanus toxoid or diphtheria toxoid. Alternatively, the carrier may be dextran such as cephalos. The carrier must be physiologically acceptable and safe for humans.

[0063] If a composition contains excipients, they must be "pharmaceutically acceptable" in the sense that they are compatible with the other components of the composition and are not harmful to the recipient. Auxiliary substances such as wetting agents, emulsifiers, and pH buffers may be present in the excipients. These excipients and auxiliary substances are generally pharmaceutical agents that can be administered without inducing an immune response in the individual receiving the composition and without excessive toxicity. Examples of pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate, and salts of organic acids such as acetate, propionate, malonate, and benzoate, may also be included. A thorough discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances is found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0064] The formulation of suitable compositions can be carried out using standard pharmaceutical chemistry and methodologies, all of which are readily available to those skilled in the art. Such compositions can be prepared, packaged, or sold in forms suitable for bolus or serial administration. Injectable compositions can be prepared, packaged, or sold in unit dose forms, such as ampoules or multi-dose containers. 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 a 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. Compositions can be prepared, packaged, or sold in the form of sterile, injectable aqueous or oily suspensions or solutions. These suspensions or solutions can be formulated according to known art and may contain additional components, in addition to the active ingredient, such as adjuvants, excipients, and auxiliary substances described herein. Such sterile injectable formulations can be prepared using, for example, water or a non-toxic, parenterally administered diluent or solvent such as 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixative oils such as synthetic mono- or di-glycerides. Other useful compositions include those containing the active ingredient in microcrystalline form in liposome formulations or as a component of biodegradable polymer systems. Compositions for sustained-release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, poorly soluble polymers, and poorly soluble salts. Alternatively, the active ingredient of the composition may be encapsulated or adsorbed or bound to 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). For example, see Jeffery et al., (1993) Pharm.Res.10:362-368.Additionally, other particulate systems and polymers can be used, such as polymers like polylysine, polyarginine, polyornithine, spermine, and spermidine, or conjugates of these molecules.

[0065] Compositions and methods for improving the use of PD-1 / PD-L1 antibodies 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 the step of administering the PD-1 / PD-L1 antibody to the patient, and the patient has been previously identified as having a TGF beta-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 the step of administering the PD-1 / PD-L1 antibody to the patient, and the baseline value of the patient's TGF beta-1 specific T cell response has been measured and identified as being equal to or greater than a threshold. Preferably, the threshold is the median value of the response. In another embodiment, the threshold may be the 75th percentile of patients with the same cancer. Various thresholds are described elsewhere in this specification and may be adopted. The same cancer means, for example, if the present invention is applied to pancreatic cancer, the median value means the value for all patients with pancreatic cancer.

[0066] Preferably, the patient has already received, will receive, or is currently receiving treatment with a CTLA-4 antibody and / or radiotherapy. The CTLA-4 antibody is ipilimumab. Therefore, the treatment method may further comprise the administration of CTLA-4 (e.g., ipilimumab) and / or radiotherapy. The present invention also provides a CTLA-4 antibody (e.g., ipilimumab) for use in a method of treating cancer, wherein the patient has been administered a PD-1 / PD-L1 antibody and / or radiotherapy, and the patient has been previously identified as having a TGF beta-specific T cell response, or has been identified as having a TGF beta-specific T cell response equal to or greater than the threshold.

[0067] In any of the embodiments described above, the measurement of the TGF beta-specific T cell response may have already been performed, or such measurement may constitute part of the method referred to in itself.

[0068] The present invention further provides a method for stratifying cancer patients into at least one of two treatment groups, the method comprising the steps of: (i) performing an assay on a sample pre-obtained from the patient to detect the presence or absence of a TGF beta-specific T cell response; (ii) assigning the patient to a first treatment group if a TGF beta-specific response is present, or to a second treatment group if a TGF beta-specific response is absent. In some embodiments, if the patient is assigned to the first treatment group, a PD-1 / PD-L1 antibody is administered. In some embodiments, if the patient is assigned to the second treatment group, a TGF beta-1 peptide is administered to enhance or induce a TGF beta-1 specific T cell response. In some embodiments, patients assigned to the second treatment group are administered the PD-1 / PD-L1 antibody and the TGF beta-1 peptide simultaneously, separately, or sequentially.

[0069] The present invention further provides a method for stratifying patients into one of at least two treatment groups, the method comprising the steps of: (i) performing an assay on a pre-obtained sample from the patient to detect the level of a TGF beta-specific T cell response if one is present; (ii) assigning the patient to a first treatment group if the TGF beta-specific T cell response is at least a threshold, or assigning the patient to a second treatment group if the TGF beta-specific response is below a threshold. In some embodiments, if the patient is assigned to the first treatment group, a PD-1 / PD-L1 antibody is administered. In some embodiments, if the patient is assigned to the second treatment group, a TGF-beta-1 peptide is administered to enhance or induce a TGF beta-specific T cell response. In some embodiments, patients assigned to the second treatment group are administered the PD-1 / PD-L1 antibody and the TGF beta-1 peptide simultaneously, separately, or sequentially.

[0070] Any of the thresholds discussed herein can be used, for example, as a dividing line for stratification.

[0071] The value of the TGF beta-specific T cell response observed in a particular subject can be used to provide the patient with an indicator of how likely treatment with a PD-1 / PD-L1 antibody is to be successful. For example, the present invention provides a method for providing a patient with an indicator of how likely treatment with a PD-1 / PD-L1 antibody is to be effective for that particular patient, comprising the steps of detecting the presence or absence of a TGF beta-specific T cell response in a sample obtained from the patient, and, if present, the intensity of the TGF beta-specific T cell response, and then determining from the measured value the likelihood of the PD-1 / PD-L1 antibody being effective for the subject. Such a method may include the step of determining where the response is located in percentile units. It may also include the step of determining the magnitude of the response compared to the expected median. Such a method can be used, for example, to determine a treatment plan. Such a method can also be used to determine whether to stimulate the TGF beta-specific T cell response as described herein.

[0072] The present invention also provides an immunogenic fragment of human TGF-beta for use in a method for treating cancer, the method comprising administering the immunogenic fragment of human TGF-beta to a cancer patient who has been previously identified as lacking or having a TGF-beta-specific T cell response below a threshold, the patient also receiving a PD-1 / PD-L1 antibody. In some embodiments, the immunogenic fragment of human TGF-beta is a TGF-beta-1 peptide. In some embodiments, the method may include administering the TGF-beta-1 peptide first, before administering the PD-1 / PD-L1 antibody. In one embodiment, the method may include confirming that the administration of the peptide has resulted in a TGF-beta-specific T cell response, or that the response is at least at a threshold, and administering the PD-1 / PD-L1 antibody if a response has occurred. The present invention further provides a PD-1 / PD-L1 antibody for use in such a method. In some embodiments, a CTLA-4 antibody (e.g., ipilimumab) is provided for use in such a method. In some embodiments, radiotherapy for use in such a method is provided.

[0073] The present invention also provides the use of immunogenic TGF beta-1 peptide to enhance the efficacy of cancer treatment with PD-1 / PD-L1 antibodies. The potency of PD-1 / PD-L1 antibodies can be improved using any of the methods described herein. This improvement may be in terms of extending overall survival (OS). In addition, or instead, it may be in terms of extending progression-free survival (PFS). In one embodiment, the likelihood of a patient showing a clinical response to treatment increases for more than six months from the start of the given treatment.

[0074] In embodiments where the TGF beta-1 peptide is administered to stimulate or enhance a response, the peptide may be administered once. In embodiments, the TGF beta-1 peptide may be administered repeatedly. It may be administered 1 to 5 times (e.g., 2, 3, 4, 5 times, etc.). It can be administered repeatedly until a response is observed or rises to a desired level.

[0075] Where a product for use in a treatment method is described herein, the present invention also provides the method itself. The present invention also provides the use of the described products in the manufacture of pharmaceuticals for treating the above condition.

[0076] The features disclosed in the above description and the following embodiments, individually or in any combination thereof, can serve as materials for realizing the present invention in its various forms. [Examples]

[0077] The following is a description of the various methods and materials used in the research. These are provided to give a complete disclosure and explanation of the manufacturing and use methods of the present invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be the present invention, nor are the following experiments intended to represent all experiments that have been performed or may be performed. Illustrative descriptions written in the present tense should be understood as not necessarily performed, but rather as experiments that may be performed to generate data, etc., related to the teachings of the present invention. While efforts have been made to ensure accuracy with respect to numerical values ​​(e.g., quantities, percentages, etc.) used, some experimental error and deviation should be expected.

[0078] [Example 1] Introduction The inventors investigated why not all patients show good improvement with antibodies against PD-1 / PD-L1 and identified the presence, and in particular its size, of a TGF beta-specific T cell response in a given subject as an indicator of how effective the treatment is, as described herein. This finding helps predict how effective antibody therapy will be for a particular patient, and also whether a TGF beta-specific T cell response should be promoted before antibody therapy, as combination therapy can enhance the effectiveness of antibody therapy.

[0079] material and method [Patient and Donor] Buffy coat from healthy donors was obtained anonymously from the blood bank of the National Hospital of Denmark (Rigshospitalet, Copenhagen, Denmark). Under Danish law, approval from an ethics committee is not required for the anonymous use of biological material. Access to buffy coat from pancreatic cancer patients was obtained from the oncology department of the University Hospital of Copenhagen (Hellev, Denmark). Informed consent was obtained from all patients before using the buffy coat, in accordance with the Declaration of Helsinki.

[0080] [peptide] The specific peptide sequence for TGFβ-15 is as follows: REAVPEPVLLSRAELRLLRL (SEQ ID NO: 28). This peptide was obtained in high purity (>90%) from Schafer (Copenhagen, Denmark) and dissolved in DMSO at a concentration of 10 mM.

[0081] [In vitro culture and enzyme-linked immunosorbent spot method (ELISPOT)] Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved (as described in Holmstrom, MO & Andersen, MH Healthy, Cancers (Basel). 12, (2020)). PBMCs were pre-stimulated in vitro with a TGFβ-15 epitope. After incubation for 9-10 days, cell counts were performed using a Countess II automated cell counter (Thermo-Fisher), and the development of TGFβ-15-specific T cells was evaluated using the interferon-g (IFN-g) ELISPOT assay. Three wells were prepared, each plated with a concentration of 200,000 cells, and stimulated with a peptide at a final concentration of 5 μM in the wells. PVDF membrane plates coated with primary IFN-γ specific antibody (Mabtech, Sweden) (Merck, Germany) were used for the ELISPOT assay. After incubation overnight, the cells were flushed out, and the wells were coated with the secondary antibody streptavidin-ALP and enzyme substrate according to the manufacturer's protocol (Mabtec, Sweden). The wells were washed with PBS before and between each step. Once the plates were dry, the spots were counted using an ImmunoSpot S6 Ultimate analyzer (CTL analyzer, Shaker Heights, Ohio, USA). The normalized mean spot count 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, in vitro cultures were stimulated with 2 μl of 10 mM TGFβ-15 on day 0, followed by stimulation with 120 U / mL of IL-2 (Novartis, Switzerland) on day 1. This was repeated 1-2 times every 7 days, and culture was continued for 9-10 days after the final stimulation before setting up in the ELISPOT assay as described above.

[0082] [statistics] Statistical analysis of paired observations was performed using a two-tailed paired t-test, or a nonparametric Wilcoxon matched pair signed-rank test for small sample sizes. For unpaired observations, a nonparametric two-tailed Mann-Whitney test was used. These tests were performed using Graphpad Prism Version 9.

[0083] Survival analysis was performed using the statistical software R with the survminer package. All variables found to be associated with overall survival and progression-free survival in the initial study (Chen, IM et al, 2022, cited above) were examined for their association with immune responses in the patient cohort. This univariate analysis was performed using log-rank analysis. To analyze the independent association of immune responses with overall survival and progression-free survival, all statistically significant parameters were included in the Cox proportional hazards model. Kaplan-Meier curves were created using Graphpad Prism Version 9. Distribution-free resampling (DFR) and the more conservative DFR2x method were also used for the analysis of ELISPOT data.

[0084] result [Patient characteristics] A sample of patients with metastatic pancreatic cancer (PC) enrolled in the CheckPAC trial was examined to show innate immune responses to the TGF-beta-15 epitope. CheckPAC was a phase II single-center trial conducted at Copenhagen University Hospital (Hellef, Denmark) for patients with refractory metastatic PC. The trial included 84 patients receiving stereotactic body radiotherapy (SBRT) in combination with immune checkpoint inhibitors (ICIs). The trial had two groups: Group A received nivolumab monotherapy, and Group B received nivolumab and ipilimumab in combination. Of the 84 participating patients, the immune response to the TGF-beta-15 epitope was analyzed in 32 patients. Patient characteristics are shown in Table 4. Of the 32 patients analyzed, 7 (22%) achieved a partial response (PR), 14 (44%) achieved stable disease (SD), and 11 (34%) experienced disease progression (PD). A clinical response to treatment was defined as achieving either a partial response (PR) or stable disease (SD). Progressive disease (PD) was defined as no clinical response to treatment. During a median follow-up period of 219 days (range 65–1511 days), two patients (6%) maintained survival, and one of them (3%) showed no signs of disease.

[0085] [Table 4] TIFF2026512959000007.tif85164

[0086] [Patients who achieved a clinical response possessed T cells specific to TGFb.] Analysis of TGFb-15 specific responses in patient samples revealed that T cells isolated at baseline and after four treatment cycles all showed a response to TGFb-15 (Figure 1A-B). Patients who achieved a clinical response had significantly higher baseline immune response amplitudes than patients with disease progression (PD), but there was no difference in response amplitude after treatment (8 weeks) (Figure 1C). This data indicates that the TGFb-specific immune response present before treatment initiation influences the clinical response to SRBT / ICI. Therefore, our data suggest that TGFbeta-specific T cells may be important in the clinical response to treatment. Notably, patients who achieved a clinical response showed a significant decrease in TGFβ-specific response amplitude 8 weeks after treatment initiation (Figure 2A-B). Furthermore, patients with sustained clinical response (defined as PR or SD lasting more than 6 months) showed a significant decrease in TGFβ-specific response 8 weeks after treatment initiation compared to patients with unsustained clinical response (Figure 3A-B).

[0087] Next, we analyzed consecutive PBMC samples from three patients with follow-up periods exceeding two years. We analyzed the TGFb-15 specific immune response and evaluated the temporal variation of the response. Interestingly, we found that the amplitude of the response varied over time (Figure 2C).

[0088] [TGFb-15-specific T cell responses were independently associated with improved survival.] The data strongly suggested that the unmodified TGFb-specific immune response may influence the response to treatment. Therefore, we investigated whether the baseline TGFb-specific immune response was associated with survival in the patient cohort. Patients were stratified based on whether their response was above or below the median normalized TGFb-15-specific immune response. Interestingly, patients with a response amplitude 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 (See Figure 4A and Table 5 below).

[0089] [Table 5] TIFF2026512959000009.tif239164

[0090] Next, we investigated whether the TGFb-15 specific immune response was independently associated with overall survival (OS). Univariate survival analyses were performed on clinical parameters potentially associated with survival, with particular attention paid to parameters included in the survival analysis of the initial CheckPAC trial report. Univariate Cox regression analysis showed that <2 metastatic sites were associated with a shortened OS (HR=2.64, p=0.03), but other parameters did not show statistical significance (Table 5). In multivariate analysis including metastatic parameters and the TGFb-15 specific immune response, only the TGFb-15 specific immune response was independently associated with OS in patients showing a response above the median (HR:0.18) (p=8x10). -4(Table 5). Notably, the univariate analysis showed a borderline significance of reduced survival in patients treated with nivolumab monotherapy (HR: 2.04, p=0.055). Therefore, this parameter was included in another multivariate analysis along with metastasis and TGFb-15-specific immune response. In the latter model, neither the metastasis variable nor the treatment variable was associated with OS, but the TGFb-15-specific response above the median was still associated with improved survival (HR: 0.19, p=0.0032; data not shown). A similar analysis was performed for progression-free survival (PFS). In the univariate analysis, the TGFb-15-specific response was associated with extended PFS (HR: 0.227, p=0.0015; Figure 4B and Table 6 below). Other significant parameters identified in univariate analysis were nivolumab treatment (HR: 2.86, p=0.007), male gender (HR: 0.406, p=0.029), and peripheral blood albumin >36 g / L (HR: 4.68, p=0.038). When these significant parameters were included in multivariate analysis along with the TGFb-15 specific immune response, only the TGFb-15 response above the median was independently associated with PFS (HR: 0.322, p=0.023; Table 6 below). Interestingly, patients with a TGFb-15 response above the 75th percentile had the highest OS and PFS among the samples examined (Figure 5A-B). These results further demonstrate that TGFb-15 specific T cell levels are important for obtaining a clinical response to SBRT / ICI therapy in pancreatic cancer patients.

[0091] [Table 6] TIFF2026512959000011.tif251165TIFF2026512959000012.tif45164

[0092] [The association between TGFb-15 specific response and survival was not due to general immunodeficiency in non-responding patients.] We investigated whether patients with short survival times may have immune system dysfunction, and therefore whether the low response amplitude observed in these patients may be caused by general T cell dysfunction. We analyzed the response amplitude of PBMCs (peripheral blood mononuclear cells) when stimulated with two common immunogenic epitopes (one derived from *Bacillus tetanus* (Slingluff, CL et al. J. Immunother. Cancer 9, (2021)) (tetanus-long) and the other from influenza virus (C18 A2 Flu)). Since the latter was a nonameric epitope limited to HLA-A2, we analyzed the response to the influenza epitope for HLA-A2. + Only samples were analyzed. 23 samples were analyzed for the response to tetanus, and 16 samples for the response to influenza. Samples from patients with strong and weak TGFb-15 specific immune responses showed similar response amplitudes to both tetanus and influenza epitopes (Figure 7A-D). Furthermore, the TGFb-15 response amplitude was similar to that of the tetanus response (r 2 It was shown that there was no correlation with either the tetanus epitope (r = 0.05) or the influenza response (r² = 0.15) (Figure 6A-B). Perhaps more importantly, the response amplitudes to the tetanus epitope and the influenza epitope were not associated with either OS or PFS (Figure 8A-D).

[0093] [Repeated antigen stimulation with TGFb-15 peptide resulted in a T cell response in PBMCs that did not show a response after only one stimulation in vitro.] 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, it was inferred that inducing a TGFb-15 response with a therapeutic peptide vaccine may be effective when used in combination with SBRT / ICI in pancreatic cancer. We investigated whether repeated stimulation with an epitope enhances the TGFb-15-specific immune response in PBMCs of healthy individuals and pancreatic cancer patients. We used PBMC samples from the above experiment in which the response to TGFb-15 was weak or not observed after a single in vitro stimulation. T cell responses were analyzed after one in vitro stimulation and after one or two further in vitro stimulations. Repeated antigen stimulation was shown to enhance the TGFb-15-specific immune response in PBMCs of both pancreatic cancer patients (Figure 9A) and healthy individuals (Figure 9B-C). Among the pancreatic cancer patients examined, 27% showed a DFR2x response after a single stimulation, and this increased to 66% with repeated stimulation (Figure 10A). In healthy donors examined, 9% showed a DFR2x response after a single stimulation, and this increased to 71% with repeated stimulation (Figure 10B). These data indicate that repeated antigen stimulation with TGFβ-15 peptide increases the number of TGF beta-specific T cells in PBMCs.

[0094] [Consideration] Here, we investigated the relationship between the TGFb-15 specific immune response and clinical outcomes. Interestingly, compared to patients who did not respond to treatment, patients who did respond had a significantly stronger TGFb-15 specific immune response before treatment initiation, and a significantly greater decrease in the TGFb-15 specific T cell response after treatment initiation. The latter phenomenon can be explained by the migration of TGF beta-specific T cells to the tumor after the initiation of ICI treatment. These results were consistent with the remarkable observation that patients with a TGFb-15 specific immune response above the median had significantly longer progression-free survival (PFS) and overall survival (OS) compared to patients with a response below the median.

[0095] We also investigated whether the difference in overall survival (OS) observed between patients with a strong and weak TGFb-15 specific immune response was due to differences in the patients' overall immune status. We compared the innate immune responses of these two groups to two highly pathogenic epitopes derived from common pathogens: influenza virus and tetanus (Slinghuf et al., cited above). No difference in response amplitude was observed between these groups for either influenza or tetanus. Furthermore, there was no correlation between the response amplitude to TGFb-15 and the response to the tetanus or influenza epitopes. Interestingly, patients with a strong pathogen-specific immune response did not show improvement in either OS or PFS after ICI treatment. This suggests that the strength of the TGFb-15 specific immune response is not due to overall immune constitution, but rather reflects the number of antiregulatory TGF beta-specific T cells.

[0096] These findings indicate that TGF-beta-specific antiregulatory T cells play a role in the response to ICI therapy in pancreatic cancer. Given the many effects of TGF-beta in pancreatic cancer, it is worth considering the immunomodulatory and tumor suppressor effects that TGF-beta-specific T cells may have in patients. In pancreatic cancer, approximately 95% of patients have activating mutations in the KRAS gene (Prior, IA, et al, Cancer Res. 72, 2457-2467 (2012)). These mutations induce TGF-beta production in transformed cells (Zdanov, S. et al. Res. 4, 354-365 (2016) and Cheng, H. et al., Cancer Lett. 446, 103-111 (2019)). Therefore, TGF-beta-specific T cells would act directly on transformed cells. However, several other immunosuppressive cells would also be targeted.

[0097] Pancreatic cancer is characterized by high levels of stromal deplasia, and cancer-associated fibroblasts (CAFs) are the main culprits behind this characteristic (Kalluri, R., Nat. Rev. Cancer 16, 582-598 (2016) and Kobayashi, H. et al., Nat. Rev. Gastroenterol. Hepatol. 16, 282-295 (2019)). CAFs are thought to originate from both bone marrow-derived mesenchymal stem cells and pancreatic astrocytic cells (Moir, JAG, et al. Surg. Oncol. 24, 232-238 (2015)). Through TGFb secretion, pancreatic cancer-derived cell lines can activate CAFs, which increase the deposition of extracellular matrix proteins and promote fibrosis (Lohr, M. et al., Cancer Res. 61, 550-555 (2001) and Principe, DR et al. Cancer Res. 76, 2525 (2016)). Furthermore, activated CAFs also secrete TGFb, and since this TGFb acts in an autocrine manner, it leads to further TGFb secretion. In particular, TGFb expression within tumors has been shown to correlate with tissue fibrosis. Another study showed that pancreatic cancer patients with high levels of fibrosis had poorer survival rates, coupled with the CAF marker aSMA (Sadozai, H. et al., Front.Immunol.12, 1-15 (2021)). This finding highlights the importance of CAFs in pancreatic cancer. The immunomodulatory effects of CAFs have been widely studied, and CAF populations are similar across different cancers (Kieffer, Y. et al. Cancer Discov. 10, 1330-1351 (2020)). Notably, a group of CAFs are characterized by increased TGFb signaling (so-called myofibroblastic CAFs (myCAFs)). Interestingly, a subset of myCAFs was associated with a lack of responsiveness to ICIs in several cancers (Ohlund D., JEM (2017) doi:10.1084 / jem.20162024).

[0098] The composition of immune cells in pancreatic cancer is heterogeneous (Steele, NG et al. Nat. cancer 1, 1097 (2020)), but generally most are of myeloid origin (Steele, NG et al. Nat. cancer 1, 1097 (2020); Vayrynen, SA et al., Clin. Cancer Res. 27, 1069-1081 (2021); and Elyada, E. et al., Cancer Discov. 9, 1102-1123 (2019)). These myeloid cells include TAMs (tumor-associated macrophages), MDSCs (myeloid-derived suppressor cells), and neutrophils, all of which express TGFβ, which regulates the TME (tumor microenvironment). Neutrophils have been shown to secrete large amounts of TGFb in pancreatic cancer TMEs, attracting and activating CAFs (Aoyagi, Y. et al. Br. J. Cancer 2004 917 91, 1316-1326 (2004)). Furthermore, local TGFb converts myeloid cells into M2 macrophages, TAMs, and MDSCs, which are elevated in pancreatic cancer (Clark, CE et al. Cancer Res. 67, 9518-9527 (2007)), and negatively impacts both PFS and OS in pancreatic cancer patients (Ino, Y. et al., Br. J. Cancer 2013 1084 108, 914-923 (2013); Sadozai, H. et al (2021) cited above; Tsujikawa, T. et al., Cell Rep. 19, 203-217 (2017)). These cells express TGFβ (Zhu, L., et al, Cell Biol. Int. (2017)). (doi:10.1002 / cbin.10788), in addition to its own immunosuppressive properties, it also mediates the conversion of naive T cells to Tregs (Huang, B. et al., Cancer Res. 66, 1123-1131 (2006); Siret, C. et al., Front.Immunol. 10, 3070 (2020)). Therefore, the appearance of MDSCs and Tregs in TMEs of pancreatic cancer is both TGFb-dependent. Furthermore, CD8 in TMEs+ T cell levels are inversely correlated with myeloid cell and Treg levels (Clark, CE et al., (2007) op. cit.; Steele, NG et al., Nat. cancer 1, 1097 (2020); and Siret, C. et al., (2020) op. cit.). Thus, TGFb is an attractive target for enhancing the efficacy of cancer immunotherapy in pancreatic cancer.

[0099] The inventors have shown that the level of TGFb-specific T cells correlates with disease outcomes in terms of both PFS and OS. Furthermore, they believe that therapeutic cancer vaccines using TGFβ-derived epitopes, when used in combination with ICI therapy, could become a future therapeutic modality. In addition, repeated vaccination with peptide vaccines may enhance the TGFbeta-specific immune response in the patient's body. The rationale for repeated vaccination is to increase the number of TGFbeta-specific T cells migrating to the TME. These cells attack cells expressing TGFbeta and release Th1 cytokines. Consequently, TGFb signaling decreases, and the TME is transformed into an immune-opermissive environment favorable to the killing of transformed cells by tumor-specific T cells.

[0100] In this study, we mimicked therapeutic vaccination by repeatedly stimulating PBMCs with a weak or no response to TGFb-15 with antigen stimulation. The results showed that repeated antigen stimulation induced a strong TGFb-15-specific immune response in almost all cultured cells. This finding supports the idea that repeated vaccination with TGFb-derived peptides can induce a TGFb-specific immune response. Furthermore, these findings indicate that in samples that did not respond, TGFb-15-specific T cells were not excessively exhausted or absent.

[0101] conclusion PBMC samples from pancreatic cancer patients treated with ICI and SBRT showed an immune response to the TGFb-15 epitope. Patients who achieved a clinical response to treatment had a stronger TGFb-15 specific T cell response than patients whose disease progressed. Furthermore, a strong TGFb-15 specific T cell response before the initiation of treatment was independently associated with extended progression-free survival and overall survival. In addition, the low levels of TGFb-15 specific T cell response observed in some patients were not due to general immune system dysfunction, as these patients retained a normal T cell response to epitopes derived from common pathogens. Furthermore, it was shown that the TGFb-15 specific immune response can be induced / enhanced by repeated antigen stimulation. As a result, administering a therapeutic cancer vaccine and repeatedly stimulating patients with the TGFb-15 antigen may induce a specific T cell response that is more likely to lead to a clinical response.

[0102] [Example 2] material and method All materials and methods are the same as in Example 1. The sequence of peptide TGFb-33 is as follows: FCLGPCPY IWSLDTQYSKVL (SEQ ID NO: 55).

[0103] result [T-cell responses specific to TGFb-33 were independently associated with prolonged survival.] In a patient cohort similar to that of Example 1, patients were stratified based on whether their response was above, below, or equal to the median normalized TGFb-33-specific immune response at baseline, in order to further investigate whether baseline TGFb-33-specific immune response was associated with extended survival. Analysis of TGFb-33-specific responses in patient samples revealed that both T cells isolated at baseline and T cells isolated after four treatment cycles showed a response to TGFb-33 (Figure 12).

[0104] Furthermore, we examined the relationship between survival and clinical parameters that may affect patient survival. Therefore, we tested the association of the following parameters with overall survival using univariate Cox regression analysis (n=33 for baseline values, n=28 for follow-up values): -Treatment group -sex -age - Performance status -Weight loss >5 - Whether the number of metastatic sites is >1 or <1 - Whipple surgery - Biliary stent - Is the tumor marker CA-119-9 above or below the median value? - Neutrophil-to-leukocyte ratio (NLR) > 5 - Bilirubin > 25 μmol / L - Albumin <36 g / L -C-responsive protein > 10 - Modified Glasgow Prognosis Score (mGPS) - Number of pretreatment lines -Baseline response to TGFb33 > median - Response to TGFb33 at follow-up > median

[0105] Interestingly, patients with baseline TGFb-33 response amplitudes above the median were found to have significantly longer overall survival (OS) compared to patients with responses below the median (univariate Cox regression, hazard ratio [HR]: 6.85, p=3.00x10). -4 (Figure 11A and Table 7 below). The only other parameter statistically significant in association with overall survival was the number of metastatic sites; patients with one or fewer metastatic sites had a poorer OS (HR=2.5, p=0.0368) (Table 7). Only variables statistically significant in association with overall survival in univariate analysis are shown in Table 7.

[0106] When TGFb-33 response and the number of metastatic sites were incorporated into a multivariate Cox regression model, the amplitude of the baseline TGFb-33-specific immune response was independently associated with overall survival (HR: 6.1, p=1.1x10). -3 (Patients showing a response below the median amplitude) (Table 7).

[0107] [Table 7]

[0108] Similar analyses were performed for progression-free survival (PFS). In the univariate analysis, the TGFb-33 specific response was associated with PFS (HR: 6.85, p=3.00x10). -4 Patients showing a response below the median amplitude (Figure 11B, Table 8 below). Other significant parameters obtained from univariate analysis were male (HR: 0.38, p=0.0188) and metastatic site <1 (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, patients showing a response below the median amplitude, Table 8 below). Only variables that showed a statistically significant association with progression-free survival in univariate analysis are shown in Table 8.

[0109] [Table 8]

[0110] [Example 3] The "non-immunogenicity" of pancreatic cancer, characterized by a high number of immunosuppressive cells and typically low levels of tumor-infiltrating effector lymphocytes, is considered one reason why it does not respond to monotherapy. Considering the new role of the tumor microenvironment, combining checkpoint inhibitor antibodies with immunomodulation of the tumor microenvironment may yield better responses in tumors that were previously refractory to radiotherapy or checkpoint inhibitor antibody therapy alone. For example, data from the Phase II trial CheckPAC (NCT02866383) in patients with refractory metastatic pancreatic cancer showed that adding 15 Gy of stereotactic body radiotherapy (SBRT) to combination therapy with nivolumab and ipilimumab resulted in sustained clinical response in a small subgroup of patients (see abstract by Chen et al. presented at ASCO GI 2022 in San Francisco, "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).

[0111] The TGFβ-15 immune response has been shown to correlate with clinical response, which supports the rationale for combining the TGFβ-15 peptide vaccine with the CheckPAC strategy (15 Gy of SBRT with nivolumab and ipilimumab). Therefore, the Phase I intervention trial CheckVAC (NCT05721846) is being conducted to evaluate the safety and tolerability of the combination of nivolumab and ipilimumab with the TGF beta-15 peptide vaccine and SBRT for refractory PC. This trial will measure adverse events, overall response rate, overall survival, progression-free survival, duration of response, best overall outcome, and disease control rate.

[0112] The following inclusion criteria are used: ● Signed informed consent • Subjects must sign and date a written informed consent form approved by the IRB / IEC, in accordance with regulatory authority and facility guidelines. This consent form must be obtained before any protocol-related procedures not included in routine subject care are performed. • Participants must be willing and able to comply with scheduled visits, treatment schedules, clinical examinations, and other requirements of this study. ● Prior to participating in this study, the progression of pancreatic cancer must be confirmed histologically or cytologically. ● Prior therapy requirements • There is no limit to the number of chemotherapy regimens previously received. Participants must be currently receiving or have recently received at least one line of systemic chemotherapy (gemcitabine or 5-FU-based regimen) for metastatic disease. · Notes - If a participant has been receiving adjuvant / neoadjuvant systemic combination therapy for less than 6 months, the adjuvant / neoadjuvant treatment will be considered one line of systemic treatment. Generally, discontinuing one drug in a multi-drug regimen and continuing the others is considered part of the same line of treatment. Restarting the same regimen after a drug break or maintenance chemotherapy is also considered part of the same line of treatment. Switching from intravenous administration of the same drug (5-FU) to oral administration (capecitabine) is also considered part of the same line of treatment. - The shortest time from initial systemic therapy to disease progression for recurrent / metastatic pancreatic adenocarcinoma should be at least 3 months. ● Must be 18 years of age or older ● ECOG Performance Status (PS) must be 0-1 ● All participants will be required to undergo biopsies before and during treatment if the principal investigator determines they are at acceptable clinical risk. Pre-treatment storage samples will not be accepted. Participants must have normal organ and bone marrow functions as defined below · Absolute neutrophil count (ANC) ≥ 1.5 × 10 9 / L · Platelet count ≥ 75 x 10 9 / L · Serum bilirubin ≤ 1.5 × upper limit of normal (ULN) · AST / ALT ≤ 5 × ULN · Serum creatinine ≤ 1.5 × ULN or CrCl ≥ 40 mL / min (by Cockcroft - Gault formula) ● Women of childbearing potential (WOCBP) must use contraception methods described in Attachment 3. In cases where teratogenic drugs and / or information for evaluating teratogenicity are insufficient (no pre - clinical trials have been conducted), highly effective contraception methods (failure rate less than 1% per year) are required. The individual contraception method and period shall be determined by consulting with the principal investigator of the clinical trial. WOCBP must follow contraception instructions when the half - life of the investigational drug exceeds 24 hours and continue contraception for 30 days + the period required for the investigational drug to pass through its half - life 5 times. The half - lives of nivolumab and ipilimumab are up to 25 days and 18 days respectively. Therefore, WOCBP must avoid pregnancy appropriately during treatment and for 23 weeks after the last dose of the investigational drug (30 days + the period required for nivolumab to pass through its half - life 5 times). ● Men who have sexual intercourse with WOCBP must use contraception methods with a failure rate less than 1% per year. The principal investigator of the clinical trial shall consider the contraception method and the period during which contraception must be practiced. Men who have sexual intercourse with WOCBP must follow contraception instructions when the half - life of the investigational drug exceeds 24 hours and continue contraception for 90 days + the period required for the investigational drug to pass through its half - life 5 times. The half - life of nivolumab is up to 25 days. Men who have sexual intercourse with WOCBP must continue contraception during treatment and for 31 weeks after the last dose of the investigational drug (90 days + the period required for nivolumab to pass through its half - life 5 times). Women with no possibility of pregnancy (post - menopausal or surgically sterile women and men with azoospermia do not require contraception). ● Participants must sign and date a written informed consent form approved by BIOPAC, in accordance with regulatory authority and facility guidelines.

[0113] The following exclusion criteria are used. ●In the opinion of the principal investigator, individuals with serious or uncontrolled medical conditions that may increase the risk associated with participation in the trial or administration of the investigational drug, impair the ability of subjects to receive protocol treatment, or interfere with the interpretation of trial results. ● Individuals with a history of treatment with anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CD137 antibodies, anti-CTLA-4 antibodies, or other antibodies or drugs that specifically target the T-cell costimulation pathway or checkpoint pathway. ● Participants with known or suspected active autoimmune disease. Enrollment is permitted for participants with vitiligo, type 1 diabetes, residual hypothyroidism due to autoimmune disease requiring hormone replacement only, psoriasis that does not require systemic treatment, or conditions where relapse is not expected unless there is an external trigger. ●Patients who have currently or previously used immunosuppressants within 14 days prior to the first dose of nivolumab, ipilimumab, or combination therapy with radiation and TGFβ-15 peptide vaccine. The following are exceptions to this criterion: • Nasal, inhaled, or topical steroids; or topical steroid injections (such as intra-articular injections). • Systemic corticosteroids or equivalent medications in physiological doses not exceeding 10 mg / day of prednisone. • Steroids as premedication for hypersensitivity reactions (e.g., premedication for CT scans) ● Participants who are HIV-positive or have a history of acquired immunodeficiency syndrome (AIDS) will be excluded. ● Allergies and side effects • Individuals with a history of allergies to the test drug components. • Individuals with a history of severe hypersensitivity reactions to any monoclonal antibody. ● WOCBP during pregnancy or breastfeeding

[0114] The following administration regimens are used:

[0115] [Table 9]

[0116] Administration of the TGFb-15 peptide vaccine is expected to enhance the clinical response rates of SBRT, nivolumab, and ipilimumab in the treatment of refractory PC.

[0117] Those skilled in the art will understand that modifications can be made to the above-described embodiments without departing from the broader concept of the invention. Therefore, it will be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to encompass modifications within the spirit and scope of the invention as defined herein.

[0118] Various publications, articles, and patents are cited or referenced throughout the background and specification, and each of these documents is incorporated herein by reference in its entirety. Discussions of documents, acts, materials, apparatus, articles, etc., included herein are for the purpose of providing context for the invention. Such discussions do not constitute an admission that any or all of these matters form part of the prior art with respect to the disclosed or claimed invention.

Claims

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

2. The PD-1 / PD-L1 antibody for use according to claim 1, wherein the TGF beta-specific T cell response is to a peptide sequence having an amino acid sequence represented by SEQ ID NOs: 28, 55, 66, 29-31, 67, 5-9, 42-45, 12-15, 56-58, 23-26, 49-52, 63, 64, 65, or 2, preferably the TGF beta-specific T cell response is to a peptide sequence having an amino acid sequence represented by SEQ ID NOs: 28, 55, or 66, more preferably SEQ ID NOs:

28.

3. A PD-1 / PD-L1 antibody for use according to claim 1 or 2, wherein the patient exhibits a pretreatment baseline TGF-beta-specific T cell response that is at least the median baseline TGF-beta-specific T cell response seen in cancer patients with the same type of cancer, preferably the patient exhibits a pretreatment baseline TGF-beta-specific T cell response that is at least the 75th percentile of the median baseline TGF-beta-specific T cell response seen in cancer patients with the same type of cancer.

4. A PD-1 / PD-L1 antibody for use according to any one of claims 1 to 3, wherein the PD-1 / PD-L1 antibody is a PD-1 antibody.

5. A PD-1 / PD-L1 antibody for use according to any one of claims 1 to 4, wherein the PD-1 / PD-L1 antibody is nivolumab.

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

7. A PD-1 / PD-L1 antibody for use according to any one of claims 1 to 6, wherein the patient is also receiving radiation therapy.

8. A PD-1 / PD-L1 antibody for use according to any one of claims 1 to 7, wherein the cancer is pancreatic cancer, preferably metastatic pancreatic cancer.

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

10. The method according to claim 9, wherein the TGF beta-specific T cell response is to a peptide sequence having an amino acid sequence represented by SEQ ID NOs: 28, 55, 66, 29-31, 67, 5-9, 42-45, 12-15, 56-58, 23-26, 49-52, 63, 64, 65, or 2, preferably to a peptide sequence having an amino acid sequence represented by SEQ ID NOs: 28, 55, or 66, more preferably to SEQ ID NOs:

28.

11. The method according to claim 9 or 10, wherein the patient exhibits a pre-treatment baseline TGF beta-specific T cell response that is at least the same as the baseline median TGF beta-specific T cell response seen in cancer patients with the same type of cancer, preferably the patient exhibits a pre-treatment baseline TGF beta-specific T cell response that is at least the 75th percentile of the baseline TGF beta-specific T cell response seen in cancer patients with the same type of cancer.

12. The method according to any one of claims 9 to 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 ipilimumab.

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

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

16. A method for stratifying cancer patients into at least one of two treatment groups, i) A step of performing an assay on a sample obtained in advance from the patient to detect the presence or absence of a TGFb-specific T cell response; ii) The process of assigning patients to a first treatment group if a TGFb-specific response is present, and stratifying patients to a second treatment group if a TGFb-specific response is not present; A method comprising, wherein, if the patient is assigned to the first treatment group, the PD-1 / PD-L1 antibody is administered.

17. A method for stratifying cancer patients into at least one of two treatment groups, i) If a TGFb-specific T cell response is present, the step of performing an assay on a sample previously obtained from the patient to detect its level; ii) If the TGFb-specific T cell response is at least above the threshold for TGFb-specific T cells, the patient is assigned to the first treatment group; if the TGFb-specific response is below the threshold, the patient is stratified to the second treatment group; A method comprising, wherein, if the patient is assigned to the first treatment group, the PD-1 / PD-L1 antibody is administered.

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

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

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

28.

21. PD-1 / PD-L1 antibody, (i) The step of administering to a patient an immunogenic fragment of human transforming growth factor (TGFb) which contains or consists of a sequence of at least nine consecutive amino acids in Sequence ID No. 1, and (ii) The step of administering PD-1 / PD-L1 antibodies, The above-mentioned PD-1 / PD-L1 antibody for use in cancer treatment methods including [specific cancer treatment method].

22. An immunogenic fragment of human transforming growth factor b (TGFb), (i) administering to a patient an immunogenic fragment of human transforming growth factor b (TGFb) comprising or consisting of a peptide sequence comprising at least nine consecutive amino acids as in Sequence ID No. 1; and (ii) The process of administering PD-1 / PD-L1 antibodies to the patient. An immunogenic fragment for use in cancer treatment methods for patients including [specific condition].

23. The method comprising the step of first identifying a patient as a patient lacking a TGF beta-specific T cell response or a patient whose TGF beta-specific T cell response is below a threshold, wherein the method is a PD-1 / PD-L1 antibody for use according to claim 21 or an immunogenic fragment of human transforming growth factor (TGFb) for use according to claim 22.

24. (i) The cancer is pancreatic cancer, preferably metastatic pancreatic cancer; (ii) The method further comprises the step of administering radiotherapy; and / or (iii) The method further includes the step of administering ipilimumab, A PD-1 / PD-L1 antibody for use according to claim 21 or 23, or an immunogenic fragment of a human transforming growth factor (TGFb) for use according to claim 22 or 23.

25. (i) The immunogenicity fragment of human TGFβ comprises an amino acid sequence represented by SEQ ID NOs: 28, 55, 66, 29-31, 67, 5-9, 42-45, 12-15, 56-58, 23-26, 49-52, 63, 64, 65 or 2, preferably the immunogenicity fragment of human TGFβ comprises an amino acid sequence represented by SEQ ID NOs: 28 or 55, more preferably SEQ ID NOs: 28; (ii) The immunogenic fragment of TGFβ is administered to the patient prior to or concurrently with other treatments; (iii) The immunogenic fragment of TGFβ is repeatedly administered to the patient; and / or (iv) The patient is repeatedly administered an immunogenic fragment of TGFβ until the patient develops a TGFβ-specific T cell response, or at least a TGFβ-specific T cell response above a threshold. An immunogenic fragment of a human transforming growth factor (TGFb) for use according to any one of claims 22 to 24.

Images