Cancer treatment using PD-1 and PD-L1 antagonists in combination with radiotherapy

Abstract

To provide treatment methods combining radiation therapy with PD-1 and / or PD-L1 antagonists to realize maximal benefit to patients suffering from cancer.SOLUTION: A method of treating cancer in a patient comprises administering at least one dose of radiation therapy, and administering at least one PD-1 and / or PD-L1 antagonist, where the at least one PD-1 and / or PD-L1 antagonist is administered on the same day as or no later than 4 days after the administration of the radiation therapy.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Sequence List This application includes an electronically submitted sequence listing in ASCII format, the entirety of which is referenced by reference. The ASCII copy was created on June 16, 2015, named B7H1-275WO1_SL.txt, and has a size of 103,313 bytes.

[0002] field Cancer treatment methods. [Background technology]

[0003] Radiotherapy (RT) remains the most important non-surgical treatment in the management of solid malignancies, with approximately 50-60% of all cancer patients receiving this treatment. Inclusion of RT in treatment planning reduces disease recurrence and improves overall survival in the majority of common cancers (1-3). Although effective, many patients still experience problems with local recurrence and metastatic disease.

[0004] In addition to the direct cytoreductive effect of RT, emerging evidence suggests that the development of an antitumor immune response may play a significant role in the efficacy of this therapy (4,5). RT may lead to the expression of ectocalreticulin on tumor cells, as well as the release of several disorder-associated molecular patterns (DAMPs), including high-mobility box-1 (HMGB1) and ATP, which may result in the recruitment and activation of antigen-presenting cells (APCs) and priming of tumor antigen-specific T cell responses (6-10). Despite this, immune evasion is frequent, and tumor recurrence remains a leading cause of mortality in patients who have received RT (11). Identifying and inhibiting the main driving mechanisms of immunosuppression may enhance the antitumor immune response and potentially improve patient outcomes.

[0005] Therefore, new insights into treatment failure and more effective RT combination approaches are urgently needed.

[0006] The programmed death 1 (PD-1) / programmed death ligand 1 (PD-L1) system is involved in maintaining peripheral immune tolerance and regulating acute inflammatory responses through inhibiting T cell function and apoptosis of activated T cells (12, 13). In addition to binding to PD-1, PD-L1 can also suppress T cell function through interaction with CD80 (14). The expression of PD-L1 is inducible and is thought to respond to local inflammatory environments, particularly type I and II interferons (IFN) (12, 15, 16). Although mostly undetectable in most normal tissues, the expression of PD-L1 has been described in multiple malignancies (review (17)). Importantly, recent clinical trials using either a PD-1 or PD-L1 targeting monoclonal antibody (mAb) have demonstrated promising responses in patients with advanced diseases (18 - 21).

Summary of the Invention

Problems to be Solved by the Invention

[0007] There is a desire for an administration strategy that combines radiotherapy with PD-1 and / or PD-L1 antagonists to provide the greatest benefit to patients suffering from cancer.

Means for Solving the Problems

[0008] According to the description, a method of treating cancer in a patient comprises a. administering at least one dose of radiotherapy; and b. administering at least one PD-1 and / or PD-L1 antagonist wherein the at least one PD-1 and / or PD-L1 antagonist is administered on the same day as or up to 4 days after the administration of radiotherapy.

[0009] In another embodiment, the at least one PD-1 and / or PD-L1 antagonist is at least one PD-1 and / or PD-L1 antibody or a functional portion thereof.

[0010] In one aspect, the radiation therapy is fractionated radiation therapy. In one mode, the fractionated radiation therapy comprises from 2 to 7 fractions. In another mode, the fractionated radiation therapy comprises 5 fractions.

[0011] In one embodiment, the radiation therapy fractions are administered daily. In another embodiment, the radiation therapy fractions are administered on the 1st, 2nd, 3rd, 4th, and 5th days. In one embodiment, the radiation therapy comprises approximately 10 Gy in 5 fractions.

[0012] In one aspect, at least one PD-1 and / or PD-L1 antagonist is administered on at least the 1st day and / or the 5th day. In one mode, at least one PD-1 and / or PD-L1 antagonist is administered multiple times. For example, at least one PD-1 and / or PD-L1 antagonist may be administered 3 times a week.

[0013] In one embodiment, the anti-PD-1 and / or PD-L1 antibody or a functional portion thereof is MEDI4736.

[0014] In another embodiment, the anti-PD-1 and / or anti-PD-L1 antibody or a functional portion thereof is pembrolizumab, nivolumab, BMS-936558, AMP-224, or MPDL3280A.

[0015] In one aspect, the cancer is melanoma, colorectal cancer, or breast cancer. In another aspect, two or more treatment cycles are performed. In a further aspect, from 2 to 8 treatment cycles are performed. In one mode, the treatment cycles are weekly or biweekly.

[0016] Additional objectives and advantages are in part described in the following description, in part apparent from the description, or may be learned by practice. The objectives and advantages are realized and achieved by means of the elements and combinations particularly pointed out in the appended claims.

[0017] The general explanation above and the detailed explanation below are both specific examples and are for illustrative purposes only, and are not intended to limit the scope of the claims.

[0018] The accompanying drawings incorporated herein and forming part thereof illustrate one (or several) embodiments and, together with the descriptions, help illustrate the principles described herein. [Brief explanation of the drawing]

[0019] [Figure 1] Figures 1A–F illustrate the blockade of the PD-1 / PD-L1 system, which enhances the activity of fractionated radiotherapy. A and B are the median fluorescence intensity (A) and typical histogram (B) of PD-L1 expression on CT26 cells isolated from tumors 1, 3, or 7 days after treatment with approximately 10 Gy in five fractions of 2 Gy per day. C and D are mice with CT26 tumors that received approximately 10 Gy RT delivered in five fractions of 2 Gy per day, either alone or in combination with either αPD-1 (C) or αPD-L1 (D) mAb administered at 10 mg / kg 3 qw for up to 3 weeks. E is the tumor volume 10 days after the start of treatment in mice with 4T1 tumors that received 20 Gy RT delivered in five fractions of 4 Gy per day, either alone or in combination with αPD-L1 mAb. F consists of mice with 443 tumors that received approximately 10 Gy RT delivered in five divided daily doses of 2 Gy, either alone or in combination with an αPD-L1 mAb. The experimental group includes at least 7 mice and represents at least two independent experiments. A, E, and F show mean ± SEM. *, P<0.05**, P<0.01, Mann-Whitney test. C and D, *, significance compared to control mice. +, significance compared to monotherapy. *** / +++, P<0.001, log-rank (Mantel-Cox) test. [Figure 2]Figures 2A–C show that the therapeutic activity of the split RT and αPD-L1 mAb combination is dependent on the activity of CD8+ T lymphocytes. A is tumor volume at 7 and 11 days after five doses of 2 Gy in combination with αPD-L1 mAb. A subset of immune cells (either CD8, CD4, or NK cells) was depleted one day prior to treatment, and the depletion was maintained for two weeks. ***, P>0.001, *, P>0.01, *, P>0.05, Mann-Whitney test. B is survival curve. *** / +++, P<0.001, log-rank (Mantel-Cox) test. Data are representative of 10 mice per cohort. C is a typical density plot of peripheral blood supporting the depletion of the immune cell subset. [Figure 3] Figures 3A–C show that the combination of split RT and αPD-L1 mAb induces protective immunological memory. A is the survival curve of LTS mice following contralateral rechallenge of 5 × 10⁵ CT26 cells. *P<0.05 compared to control mice (log-rank; Mantel-Cox test). B is a typical dot blot of IFNγ production by CD8+ T cells isolated from either tumor-unsensitized or LTS mice initially treated with RT and αPD-L1 mAb. C is the frequency of IFNγ+CD8+ T cells isolated from either tumor-unsensitized or initially treated with RT and αPD-L1 mAb LTS mice, following co-culture with either an H2-Ld-restricted peptide (AH1(SPSYVYHQF)(SEQ ID NO: 91); a defined CT26 tumor-associated antigen or β-galactosidase (TPHPARIGL)(SEQ ID NO: 92); a prokaryotic control peptide) or CT26 cells irradiated with 50 Gy for 5 days, followed by priming with 50 Gy-irradiated CT26 cells. *P<0.05 (Mann-Whitney test). Representative data from two independent experimental sets. [Figure 4]Figures 4A–C illustrate that fractional RT increases tumor cell PD-L1 expression in vivo and is dependent on CD8+ T cells. A shows PD-L1 expression on CT26 cells after treatment with in vitro RT (2.5–10 Gy). B and C are typical contour plots (B) and median fluorescence intensity (C) of PD-L1 expression on CT26 cells (gated as CD45- cells) isolated from tumors 3 days after administration of approximately 10 Gy in five fractional doses of 2 Gy per day, in combination with either a CD8, CD4, or NK cell depletion antibody. *P<0.05 (Mann-Whitney test). The experimental group includes at least 5 mice and represents at least 2 independent experiments. [Figure 5] Figures 5A-E show that IFNγ production by CD8+ T cells, following cleavage RT, is involved in the upregulation of PD-L1 expression on tumor cells. A shows PD-L1 expression on wild-type CT26 tumor cells (WT) or transduced cells with either 20 ng / ml IFNγ, TNFα, or a combination of both cytokines, following 24 hours of co-culture. B is a typical density plot showing IFNγ and TNFα expression by CD8+ T cells following treatment with PBS or either phorbol 12-myristate 13-acetate (PMA) and ionomycin, and C shows the frequency of appearance of PD-L1-positive CT26 tumor cells (WT, NTC ShRNA, or IFNγR1 ShRNA) following 24 hours of co-culture with either PBS or PMA / ionomycin-activated splenocytes. n / s = P > 0.05, ***P < 0.001**P < 0.01 (two-sided Student's t-test). D and E are typical contour plots (D) and median fluorescence intensity (E) of PD-L1 expression on CT26 tumor cells following treatment with RT (approximately 10 Gy in 5 fractions) in the presence of an in vivo αIFNγ blocking mAb (or isotype control). **P < 0.01 (Mann-Whitney test). The experimental group included at least 5 mice. [Figure 6]Figures 6A-D show that the administration schedule affects prognosis, and RT synergy was observed only with concurrent αPD-L1 mAb therapy and not with sequential therapy. A is the schema for the administration schedule study. Mice were administered divided RT (approximately 10 Gy in 5 divided daily doses of 2 Gy) either alone or in combination with αPD-L1 mAb, initiated on either day 1 of the RT cycle (Schedule A), day 5 of the RT cycle (Schedule B), or 7 days after the last RT dose (Schedule C). B, Survival curves of the treatment. ++, P<0.01, compared to monotherapy (log-rank; Mantel-Cox test). C and D are the expression of PD-1onCD4+(C) and CD8+(D) T cells 24 hours and 7 days after the last RT dose. *P<0.05 (Mann-Whitney test). The experimental group included at least 5 mice and represented at least 2 independent experiments. [Figure 7] Figure 7 shows that blocking both PD-1 and PD-L1 does not further improve efficacy in combination with fractionated RT in mice with CT26 tumors. A) Survival curves following fractionated RT (approximately 10 Gy in 5 fractions of 2 Gy per day) administered alone or in combination with αPD-1, αPD-L1, or a combination of both mAbs administered 3 qw over 3 weeks. n / s, P>0.05, log-rank; Mantel-Cox test. The experimental group consisted of at least 5 mice and represented two (at 2) independent experiments. [Figure 8] Figures 8A-B show that combination therapy with fractional RT and either αPD-1 or αPD-L1 mAb was well tolerated in mice. Mice with CT26 tumors were administered 10 Gy RT delivered in five fractional daily doses of 2 Gy, either alone or in combination with αPD-L1 mAb administered at 10 mg / kg 3 qw for either 3 weeks or 1 week. The experimental group consisted of at least 7 mice and represented at least two independent experiments. n / s, P>0.05, Mann-Whitney test. [Figure 9]Figures 9A-C illustrate that in vitro treatment of tumor cells with αPD-1 or αPD-L1 mAbs does not sensitize them to irradiation-induced cell death. Clonal survival curves of CT26 cells (A), 4T1 cells (B), and 4434 cells (C) treated with RT (2.5-10 Gy) in the presence or absence of 2 μg / ml αPD1 or αPD-L1. [Figure 10] Figures 10A-C show how the administration schedule affects the prognosis. A) Tumor volume following fractional RT administered alone or in combination with αPD-L1 mAb (as 10 Gy in 5 fractional daily doses of 2 Gy), initiated on either day 1 of the RT cycle (Schedule A), day 5 of the RT cycle (Schedule B), or 7 days after the last RT dose (Schedule C). B and C) Tumor volume of RT-treated mice showing comparable tumor volume across different administration schedules. n / s, P>0.05, Mann-Whitney U test. The experimental group consisted of at least 5 mice and represented two (at 2) independent experiments. [Figure 11] Figure 11 shows that the comparison between acute and chronic administration of αPD-L1 mAb does not affect the efficacy of combination therapy. Mice with CT26 tumors were administered 10 Gy RT delivered in five divided daily doses of 2 Gy, either alone or in combination with αPD-L1 mAb administered at 10 mg / kg 3 qw for either 3 weeks or 1 week. The experimental group consisted of at least 7 mice and represented at least two independent experiments. n / s, P>0.05, log-rank; Mantel-Cox test. [Modes for carrying out the invention]

[0020] Sequence description Table 1 provides a list of specific sequences referenced in the embodiment under consideration. CDRs are shown in underlined bold.

[0021] [Table 1] TIFF0007879919000002.tif164125TIFF0007879919000003.tif158125TIFF0007879919000004.tif165125TIFF0007879919000005.tif16912 5TIFF0007879919000006.tif165125TIFF0007879919000007.tif163125TIFF0007879919000008.tif164125TIFF0007879919000009.tif60125

[0022] Description of the Embodiment I. Treatment This method encompasses cancer treatment using at least one dose of radiotherapy and at least one dose of at least one PD-1 and / or PD-L1 antagonist. For example, a method of treating cancer in a patient may include the steps of administering at least one dose of radiotherapy and administering at least one PD-1 and / or PD-L1 antagonist, wherein the at least one PD-1 and / or PD-L1 antagonist is administered on the same day as the radiotherapy or up to four days later.

[0023] A method of treating cancer may be performed once (as a single treatment cycle), or it may be performed more than once (i.e., in multiple treatment cycles) on a weekly, bi-weekly, quarterly, or monthly basis. If a method is performed more than once, it may be performed two, three, four, five, six, seven, or eight or more times.

[0024] While not bound by theory, the inventors discovered that, within the scope of established syngeneic tumor models, low-dose fractionated RT resulted in upregulation of PD-L1 expression in tumor cells in vivo. The inventors also found that fractionated RT delivered in combination with αPD-1 or αPD-L1 mAb was effective against tumor CD8 + This indicates that a response was generated, which improved local tumor control and long-term survival, and protected against tumor rechallenge by inducing a tumor antigen-specific memory immune response. IFNγ CD8+ T cell production has been found to be involved in the upregulation of PD-L1 on tumor cells following fractional RT. Furthermore, the scheduling of anti-PD-L1 mAbs in relation to the delivery of fractional RT appears to affect treatment outcomes; administration of the antagonist on the same day as RT or up to 4 days after the first fractional RT dose shows advantages over administration of the antagonist more than 7 days after the end of radiotherapy. And while not bound by theory, the upregulation of tumor cell PD-L1 expression in response to low doses of fractional RT, as conventionally used in clinics, appears to be an adaptive immunological resistance mechanism by tumor cells; therefore, the PD-L1 / PD-1 signaling pathway may potentially contribute to treatment failure. Combination therapy of RT with PD-1 / PD-L1 signaling pathway blockade has the potential to overcome this resistance, but our preclinical studies have shown that the timing of administration affects treatment outcomes and provide important new insights for bridging to clinics.

[0025] A. PD-1 and / or PD-L1 antagonists for use in therapeutic treatment In one embodiment, at least one PD-1 and / or PD-L1 antagonist for use in the present method is an anti-PD-1 and / or anti-PD-L1 antibody or a functional portion thereof.

[0026] At least one PD-1 and / or PD-L1 antagonist is administered on the same day as or up to four days after the administration of radiotherapy. For example, in one embodiment, if radiotherapy is provided on day 1 of the treatment cycle, at least one PD-1 and / or PD-L1 antagonist may also be administered on day 1 of the treatment cycle (i.e., on the same day as the radiotherapy administration). In another embodiment, if radiotherapy is provided on day 1 of the treatment cycle, at least one PD-1 and / or PD-L1 antagonist is administered on day 5 (i.e., four days later). In a further embodiment, the PD-1 and / or PD-L1 antagonist may be administered on days 1, 2, 3, 4, 5, 6, and / or 7 of the treatment cycle (including both single-session and multi-session treatment schedules).

[0027] In one embodiment, at least one PD-1 and / or PD-L1 antagonist is administered multiple times during a treatment cycle. In another embodiment, at least one PD-1 and / or PD-L1 antagonist is administered two, three, four, five or more times during a treatment cycle. For example, the antagonist may be administered three times per week over a one-week treatment cycle, or three times per week over a treatment cycle of two or more weeks as described above.

[0028] In one embodiment, the antibody or functional portion thereof is selected from those disclosed in U.S. Patent Publication No. 2010 / 0028330, which is incorporated by reference with respect to teaching these antibodies and functional portions thereof. In one embodiment, the antibody is MEDI4736.

[0029] In another embodiment, the anti-PD-1 and / or anti-PD-L1 antibody is pembrolizumab, nivolumab, BMS-936558, AMP-224, or MPDL3280A.

[0030] The antibody or its functional portion is administered in a therapeutically effective dose. Typically, the therapeutically effective dose may vary depending on the patient's age, condition, and sex, as well as the severity of the disease. The therapeutically effective dose of the antibody or its functional portion ranges from approximately 0.001 to 30 mg / kg body weight, 0.01 to 25 mg / kg body weight, 0.1 to 20 mg / kg body weight, or 1 to 10 mg / kg body weight. The dose may be adjusted as needed to suit the observed therapeutic effect. The appropriate dose is selected by the treating physician based on clinical indications.

[0031] The antibody may be administered as a bolus to maximize the circulating antibody level over the maximum time after administration. Continuous infusion may also be used after bolus administration.

[0032] In the use of this specification, the term antibody or its functional portion is used in its broadest sense. It may be artificial, such as monoclonal antibodies (mAbs) and / or functional fragments thereof, produced by conventional hybridoma technology, recombinant technology, etc. It may also include both unchanged immunoglobulin molecules such as polyclonal antibodies, monoclonal antibodies (mAbs), monospecific antibodies, bispecific antibodies, polyspecific antibodies, human antibodies, humanized antibodies, animal antibodies (e.g., camelid antibodies), chimeric antibodies, etc., as well as any portion, fragment, region, peptide, and derivative of such immunoglobulins lacking a light chain, such as Fab, Fab', F(ab')2, Fv, scFv, antibody fragments, bispecific antibodies, Fd, CDR regions, or any portion or peptide sequence of an antibody capable of binding to an antigen or epitope (provided by any known technology, such as, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant technology). In one embodiment, the functional portion is a single-chain antibody, a single-chain variable fragment (scFv), a Fab fragment, or an F(ab')2 fragment.

[0033] An antibody or functional moiety is said to be a "binding" molecule if it has the ability to react specifically with a molecule, and as a result the molecule binds to the antibody. The antibody fragment or moiety may lack the Fc fragment of the unchanged antibody, be removed from circulation more rapidly, and may have lower nonspecific tissue binding than the unchanged antibody. An example of an antibody that may be produced from an unchanged antibody using methods known in the art is proteolytic cleavage using enzymes such as papain (producing a Fab fragment) or pepsin (producing an F(ab')2 fragment). The antibody moiety may be produced by any of the above methods, or by the expression of a part of the recombinant molecule. For example, the CDR region of a recombinant antibody may be isolated and subcloned into a suitable expression vector.

[0034] In one embodiment, the antibody or functional moiety is a human antibody. The use of human antibodies for the treatment of humans may reduce the probability of side effects due to the immunological response in individual humans to non-human sequences. In another embodiment, the antibody or functional moiety is humanized. In yet another embodiment, the antibody or functional moiety is a chimeric antibody. In this way, sequences of interest, such as binding sites of interest, may be incorporated into the antibody or functional moiety.

[0035] In one embodiment, the antibody may have an IgG, IgA, IgM, or IgE isotype. In one embodiment, the antibody is IgG.

[0036] B. Radiation therapy used in treatment Radiotherapy, also known as high-dose ionization irradiation, is a component of the therapeutic approach under consideration.

[0037] In one embodiment, the radiotherapy is fractionated radiotherapy. In one embodiment, the fractionated radiotherapy comprises 2 to 7 fractions. In another embodiment, the fractionated radiotherapy comprises 3 to 6 fractions. In another embodiment, the fractionated radiotherapy comprises 4 to 5 fractions. In one embodiment, the fractionated radiotherapy comprises 2, 3, 4, 5, 6, or 7 fractions. In one embodiment, the fractionated radiotherapy comprises 5 fractions.

[0038] In one configuration, the radiotherapy fractions are administered daily. In another configuration, radiotherapy may include two or more doses daily and / or daily doses. In one configuration, the radiotherapy fractions are administered on days 1, 2, 3, 4, and 5. In yet another configuration, radiotherapy consists of five fractions totaling approximately 10 Gy (i.e., 2 Gy each day for 5 days).

[0039] Other fractionation schedules may be used, including accelerated fractionation (administering larger doses daily or weekly to shorten the number of treatment weeks), over-fractionation (administering smaller doses of radiation more than once a day), or under-fractionation (administering larger doses once a day or less, often shortening the number of treatments).

[0040] Radiotherapy may involve X-rays, gamma rays, or charged particles. Radiotherapy may be external beam radiotherapy or internal radiotherapy (also known as proximal radiotherapy). Whole-body radiotherapy using radioactive materials such as radioactive iodine may also be used.

[0041] External beam radiotherapy includes 3D structured radiotherapy, intensity-controlled radiotherapy, image-guided radiotherapy, tomotherapy, stereotactic radiotherapy, proton therapy, or other charged particle beam therapies.

[0042] C. Cancers that are treated This method may be used to treat various types of cancer. In one embodiment, the method may be used to treat melanoma, colorectal cancer, or breast cancer. In one embodiment, the breast cancer is triple-negative breast cancer.

[0043] In one embodiment, cancer is defined as adrenocortical tumor, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, central nervous system cancer, cervical cancer, thoracic cancer, colon cancer, colorectal cancer, endometrial cancer, epidermal carcinoid cancer, esophageal cancer, eye cancer, glioblastoma, glioma, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, head and neck cancer, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer (hepatocellular carcinoma, etc.), lung cancer (non-small cell, small cell, and pulmonary carcinoid). This includes tumors, lymph node cancer, lymphoma, cutaneous lymphoma, melanoma, mesothelioma, oral cancer, multiple myeloma, nasal cavity and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, pediatric malignancies, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0044] Herein, we refer in detail to exemplary embodiments under consideration, which are illustrated in the accompanying drawings. Where possible, the same reference numerals are used throughout the drawings to refer to identical or similar parts. Other embodiments will be apparent to those skilled in the art from the specification and consideration of implementation disclosed herein. Embodiments are further described in the following examples. These examples do not limit the scope of the claims and serve only to clarify specific embodiments. The specification and examples are for illustrative purposes only, and the true scope and spirit are intended to be shown by the following claims. [Examples]

[0045] Example 1. Method A) Mice and cell lines BALB / c and C57Bl / 6 mice were obtained from Harlan, UK. All animal experiments were approved by local ethics committees and conducted under UK Home Office licenses. CT26 mouse colon cancer cells (ATCC) and 4434 cells were bred using BRafV600E p16.- / - Cell lines were isolated from mice (Richard Marias, Cancer Research UK, Manchester Institute) and maintained in DMEM. 4T1 triple-negative breast cancer (ATCC) cells were maintained in RPMI-1640 supplemented with 10% FCS and 1% L-glutamine (Invitrogen, UK). All cell lines were routinely screened to confirm the absence of Mycoplasma contamination.

[0046] B) Oncology treatment The mouse has 5x10 5 1 x 10 CT26 5 4T1 or 5×10 6 One of the 4434 cells was subcutaneously inoculated (sc). Irradiation was performed 7-10 days after inoculation (when the tumor was at least 100 mm) using a Pantak HF-320 320 kV X-ray unit (Gulmay Medical, UK). 3This was performed at the time point of). The device was operated at 300 kV and 9.2 mA, and a filtration device was attached to the X-ray beam to give a radiation quality of 2.3 mm Cu half-value layer. The mice were placed at a distance of 350 mm from the X-ray focus, and the dose rate was 0.80 Gy / min. Administration of αPD-1 (clone RMPI-14), αPD-L1 (clone 10F.9G2) (both from Biolegend) or isotype control mAbs (IgG2a and IgG2b respectively) was started on day 1 of the fractionated RT cycle (unless otherwise specified), and administered intraperitoneally (i.p.) at a dose of 10 mg / kg in a volume of 100 μl / 10 g in PBS at 3qw for up to 3 weeks. For cell and cytokine depletion experiments, mice were administered either αCD8 mAb; clone YTS169 (gift from M. Glennie, Southampton University), αCD4 mAb; clone GK1.5 (Biolegend), αAsialo-GM1 (Wako Chemicals) or αIFNγ; clone XMG1.2 (BioXcell). Peripheral blood was collected during treatment to confirm cell depletion. In the tumor rechallenge experiment, tumor cells were transplanted contralaterally into long-term survival (LTS) mice at least 100 days after the previous tumor transplantation. Additional control mice also received transplants to confirm tumor growth. The experimental groups included at least 5 mice / group and represented at least 2 independent experiments.

[0047] C) CD8 isolated from long-term survival mice + Measurement of cytokine production by T cells In in vitro stimulation, 1 × 10 irradiated with 50 Gy 6 tumor cells or 1 μmol / ml of H2-Ld-restricted peptide SPSYVYHQF (SEQ ID NO: 91) (AH1) / TPHPARIGL (SEQ ID NO: 92) (β-galactosidase) (Anaspec, U.K.) in the presence of either LTS or control mice 6Splenocytes were cultured for 5 days in RPMI-1640 supplemented with 10% FCS, 100 U / ml penicillin, 100 μg / ml streptomycin, 1% L-glutamine, 50 μM 2-ME, and 10 IU / ml human recombinant IL-2. The experimental group consisted of 3-5 mice and represented two independent experiments. After 5 days of culture, the cells were re-stimulated with 50 Gy-irradiated tumor cells for 16 hours in a 1:1 ratio in the presence of 3 μg / ml Brefeldin A (BD Pharmingen, UK) and 100 IU / ml human recombinant IL-2 (Chiron, NL). For FACS analysis, the cells were washed and cultured with rat anti-CD16 / 32 (eBioscience, UK) to block nonspecific binding, and then stained with FITC-conjugated anti-CD8αmAb (eBioscience, UK). Next, the cells were fixed / permeabilized and stained for IFNγ expression using APC conjugate mAb (eBioscience, UK).

[0048] D) Determination of tumor and immune cell phenotypes by flow cytometry. To obtain single-cell suspensions, tumors were treated using the gentleMacs Dissociator and a mouse tumor isolation kit (Miltenyi Biotec, UK). For analysis, nonspecific binding was blocked as described above, and the expression of CD4, CD8 (BD Biosciences, UK), CD45, NKP46, PD-1, and PD-L1 was examined by multi-parameter flow cytometry (all from eBioscience unless otherwise noted).

[0049] E) Simultaneous in vitro culture Tumor cells were cultured for 24 hours in the presence of 20 ng / ml IFNγ and / or TNFα prior to the evaluation of PD-L1 expression by flow cytometry as described above. In co-culture assays, resting or activated splenocytes (treated with PBS or phorbol 12-myristate 13-acetate and ionomycin cell-stimulating mixture, (eBioscience, UK)) were co-cultured with tumor cells in a 1:1 ratio, and tumor cell expression of PD-L1 was evaluated as described above. Silencing of IFNγR1 expression was achieved by lentiviral transduction of cells with ShRNA (cells were also transduced with untargeted ShRNA as a control) (Thermo Scientific, UK). Measures of splenocyte cytokine production (IFNγ and TNFα) were measured by intracellular flow cytometry as described above.

[0050] Example 2. Blocking PD-1 or PD-L1 enhances the therapeutic effect of RT. While radiotherapy has been shown to modulate the immunogenicity of tumor cells, it rarely produces a durable therapeutic response that results only in systemic anti-tumor immunity. We show that low-dose topical fractionated radiotherapy (RT), delivered as approximately 10 Gy in five fractions, leads to increased PD-L1 expression in tumor cells, and this increase in expression is evident 1, 3, and 5 days after the final dose of RT compared to time-coordinated untreated (NT) mice (Figure 1A and B). This RT-mediated increase in tumor cell PD-L1 expression peaks 72 hours after the final dose of RT, and although it decreases significantly 7 days after RT (compared to expression on days 1 and 3; P<0.05 and P<0.01, Mann-Whitney test, respectively), it remains elevated compared to NT mice (P<0.05, Mann-Whitney test).

[0051] Given these observations, the inventors hypothesized that the immune response following RT may be restricted through the PD-1 / PD-L1 system. Local RT, delivered as approximately 10 Gy in five divided daily doses, was found to significantly improve survival in mice with established CT26 tumors compared to NT controls (Figure 1C and D; P<0.05 log-rank; Mantel-Cox test). The inventors' data show that this RT-mediated local tumor control can be substantially improved through combination therapy with either an αPD-L1 mAb or an αPD-1 mAb (Figure 1C and D; P<0.001 log-rank; Mantel-Cox test). The combination therapy resulted in a synergistic antitumor response, which was curative in 66% and 80% of mice administered RT in combination with either an αPD-L1 mAb or an αPD-1 mAb, respectively. In contrast to combination therapy with RT, monotherapy with either αPD-L1 mAb or αPD-1 mAb did not significantly improve survival (Figure 1C and D; P>0.05 log-rank; Mantel-Cox test). Furthermore, compared to combination therapy with either mAb alone, no significant benefit was observed when RT was administered to mice in combination with both αPD-1 and αPD-L1 mAbs (Figure 7).

[0052] PD-L1 blockade also improved the response to RT in mice with established 4T1 tumors, and combination therapy significantly reduced tumor volume by 38% compared to RT alone (Figure 1E; 10 days after initiation of treatment; 184.3 ± 13.5 mm, respectively). 2 Ratio 292.8 ± 14.3 mm 2(P<0.01 Mann-Whitney test) and significantly improved survival (P<0.001 log-rank; Mantel-Cox test; data not shown). Similar results were also observed in mice with established 4434 melanomas (Figure 1F). Combination therapy with topical RT targeting either PD-1 or PD-L1 and mAbs was well tolerated in both BALB / c and C57Bl / 6 mice (Figure 8A and B). These preclinical data clearly demonstrate the potential to improve prognosis following low-dose fractional RT in established solid tumors through blockade of the PD-1 / PD-L1 system.

[0053] Example 3. NK cells contribute to local tumor control following combination therapy, but long-term survival is limited to CD8 + T cell dependent Next, the inventors investigated the underlying mechanisms of long-term tumor control observed following combination RT and αPD-L1 mAb therapy. First, using a colony formation assay, they confirmed that αPD-1 and αPD-L1 mAbs did not act as radiosensitizers through direct interaction with tumor cells (Figure 9A-C). Using antibody depletion, the inventors then investigated the role of effector T cells and NK cells in mediating antitumor activity following RT / αPD-L1 mAb combination therapy. The inventors' data demonstrate a clear reduction in tumor burden in mice following combination therapy, compared to NT mice, as early as 7 days after the initiation of a 5-day fractional RT cycle (with αPD-L1 mAb therapy initiated on day 1 of the RT cycle) (207.5 ± 29.2 mm, respectively). 2 Ratio 409.4±86.88mm 2 (P=0.067, Mann-Whitney U test) (Figure 2A). However, this statistical trend of decreasing volume is CD8 +Following the depletion of either T cells or NK cells, tumor volume was lost, and there was no significant difference from the NT cohort (P=0.52 and P=0.70, respectively, Mann-Whitney U test), but it was significantly larger than in mice treated with combination therapy without immune cell depletion (P<0.01; CD8 depletion compared to combination therapy, and P<0.05; NK cell depletion compared to combination therapy, Mann-Whitney U test). By day 11 post-treatment, combination therapy significantly reduced tumor volume compared to NT controls (P<0.001, Mann-Whitney U test). CD8 at this point + Depletion of either T cells or NK cells reduces the effectiveness of combination therapy (P<0.001 and P<0.05, respectively, Mann-Whitney U test), while CD8 + The relative contributions of T cells and NK cells become more apparent, and tumor control is significantly reduced in CD8 vs. NK cell depleted mice (P<0.05, Mann-Whitney U test). Our data also show that CD4 + The study also shows that T cell depletion improved local tumor control following combination therapy (153.2 ± 27.0 mm, respectively). 2 Ratio 72.7±17.3mm 2 (P<0.05, Mann-Whitney U test).

[0054] In contrast to early tumor control following combination RT / αPD-L1 mAb therapy, long-term survival (LTS) was not affected by NK cell depletion (70% of LTS mice following RT / αPD-L1 treatment vs. 77.8% following combination therapy with NK cell depletion) (Figure 2B). CD4 + T lymphocyte depletion increased the incidence of LTS mice from 70% to 87.5% (combination vs. combination + CD4 depletion), but this difference was not statistically significant (P>0.05 log-rank; Mantel-Cox test). However, CD8 +T cell depletion completely suppressed the therapeutic effect of combination RT / αPD-L1 mAb therapy (P<0.001 log-rank; Mantel-Cox test). Depletion of the immune cell population was confirmed by flow cytometry on peripheral blood samples (Figure 2C). These data suggest that NK cells may express some local tumor control, but this did not affect overall survival and CD4 + T cells are unnecessary, or can even suppress the response, while CD8 + This suggests that T cells appear to mediate effective long-term tumor control following treatment with RT and αPD-L1 mAbs.

[0055] Example 4. Treatment with αPD-L1 mAb and RT induces a protective tumor antigen-specific memory T cell response. Next, the inventors investigated whether immunological memory developed following treatment with RT and αPD-L1 mAb. The inventors demonstrated that LTS mice initially treated with RT and αPD-L1 mAb were able to completely reject the tumor following contralateral rechallenge (Figure 3A). To quantify this memory response, splenocytes were harvested from LTS mice with disease-free survival periods exceeding 100 days and co-cultured with a peptide derived from CT26 tumor-associated antigen (AH1:SPSYVYHQF (SEQ ID NO: 91)), a control peptide (β-galactosidase:TPHARIGL (SEQ ID NO: 93)), or irradiated CT26 cells, followed by IFNγ-producing CD8 + The capabilities of T cells were evaluated (Figures 3B and C). Our data showed that LTS mice, following co-culture with AH1 peptide, produced significantly higher IFNγ-producing CD8 cells than unsensitized mice. + The frequency of T lymphocyte appearance was given (6.6% ± 0.8 vs. 2.3% ± 0.2, respectively; P < 0.05, Mann-Whitney test). A similar response was observed following co-culture of splenic cells with CT26 cells. Comparison of co-culture of peptides and tumor cells is shown in the memory CD8 +We revealed that the frequency of T cell appearance in LTS mice was approximately three times lower when co-cultured with tumor cells compared to co-cultured with AH1 peptide, which may reflect tumor cell-mediated suppression of T cell activation. Taken together, these data suggest that in a mouse model, RT, when combined with PD-1 / PD-L1 system blockade, can induce protective immunological memory in long-term survivors.

[0056] Example 5. Fractionated RT is used to improve PD-L1 expression in tumor cells. + This leads to T cell-dependent adaptive upregulation. First, the inventors confirmed that treatment of tumor cells with a series of RT administrations in vitro had no direct effect on PD-L1 expression (Figure 4A). To identify which cell populations within the tumor microenvironment are involved in regulating PD-L1 tumor cell expression, cleavage RT cycles were administered to mice in combination with CD8, CD4, or NK cell depletion antibodies. The inventors' data showed that CD8, not NK cells, was involved. + T cell depletion completely suppresses RT-mediated upregulation of PD-L1 on tumor cells (Figures 4B and C). Interestingly, CD4 + T cell depletion was found to further enhance RT-mediated upregulation of PD-L1 on tumor cells (approximately twice as much as treatment with RT alone).

[0057] Example 6. Adaptive upregulation of PD-L1 by tumor cells following fractional RT is IFNγ-dependent. Given the clinical correlation between IFNγ and PD-L1 expression in the tumor microenvironment (16) and the effect of TNFα on this response (22), we evaluated the effects of these cytokines on PD-L1 in our cell lines. Co-culture of tumor cells with recombinant IFNγ resulted in a remarkable 20-fold increase in PD-L1 cell surface expression in vitro (Figure 5A). Furthermore, while the addition of recombinant TNFα alone did not affect PD-L1 expression in tumor cells, a mixture of both IFNγ and TNFα could further enhance tumor cell PD-L1 expression (approximately 2-fold) compared to expression with IFNγ alone (Figure 5A). We demonstrate that silencing of IFN-γ receptor 1 (IFNγR1) on CT26 tumor cells using ShRNA completely suppresses the upregulation of PD-L1 following co-culture with recombinant IFNγ, or with both IFNγ and TNFα (Figure 5A).

[0058] To establish whether activated immune cells can induce adaptive changes in tumor cell PD-L1 expression through IFNγ and TNFα production, isolated resting (PBS-treated) and activated (phorbol 12-myristate 13-acetate (PMA) and ionomycin-treated) splenocytes (Figure 5B and C) were co-cultured with WT CT26 cells and cells transduced with either untargeted ShRNA (NTC ShRNA) or IFNγR1 ShRNA. Importantly, these experiments demonstrate that activated immune cells can elevate tumor cell PD-L1 expression to physiologically reasonable IFNγ and TNFα concentrations in an IFNγR1-dependent manner.

[0059] To confirm the role of IFNγ in tumor cell PD-L1 expression in response to in vivo RT, RT was administered to mice in combination with an αIFNγ blocking antibody (or isotype control). IFNγ blockade reduced tumor cell PD-L1 expression in NT mice by 2.6-fold compared to levels observed on in vitro cultured CT26 cells; suggesting adaptive upregulation of PD-L1 following tumor transplantation (Figures 5D and E). However, the significant upregulation of PD-L1 observed following RT (2.4-fold compared to NT controls) was completely suppressed by IFNγ blockade, confirming that this is the driving mechanism for adaptive tumor cell expression of PD-L1 following RT treatment.

[0060] Example 7. A combination therapy schedule of αPD-L1 mAb and RT produces an effective antitumor immune response. The inventors evaluated three different combination schedules in which mice with established CT26 tumors received approximately 10 Gy of fractional RT cycles in five fractional doses, along with administration of αPD-L1 mAb, initiated on either day 1 of the fractional RT cycle (Schedule A), day 5 of the cycle (Schedule B), or 7 days after RT completion (Schedule C) (Figure 6A). There was no significant difference in overall survival between Schedules A and B, with LTS being 60% and 57%, respectively (P>0.05 log-rank; Mantel-Cox test) (Figures 6B and 10A). In contrast, despite similar tumor loads across the entire test group, sequential treatment with RT followed by αPD-L1 mAb 7 days later (Schedule C) was completely ineffective in improving overall survival compared to RT alone (median survival times at 30 and 35 days, respectively; P>0.05 log-rank; Mantel-Cox test) (Figures 10B and C).

[0061] Tumor-infiltrating CD4 24 hours after the last dose of RT + and CD8 + T cell analysis showed increased PD-1 expression compared to time-matched NT controls (P<0.05, Mann-Whitney test) (Figure 6C). In contrast, 7 days after RT, CD4 +Changes in PD-1 expression on T cells were not clear, and compared to time-matched NT controls, CD8 + It was found to be significantly reduced on T cells (P<0.05, Mann-Whitney test) (Figure 6D). PD-1 expression was consistently reduced on CD4 + T cells are more tumor-infiltrating than CD8 + The results showed higher activity (Figures 6C and D). Next, we compared activity following acute versus long-term αPD-L1 mAb administration protocols, in which mice were administered either three doses of mAb concurrently with RT, or the same for an additional two weeks (3qw). No additional benefits were observed with long-term αPD-L1 mAb administration (Figure 11).

[0062] In summary, these data suggest that treatment with low-dose fractionated RT leads to an acute increase in PD-1 expression on T cells, and sequential therapy, in which the blockade of the PD-1 / PD-L1 signaling pathway is delayed until the completion of the RT cycle, is associated with tumor-responsive CD8 + This suggests that it may be potentially ineffective due to T cell deletion or anergy.

[0063] Example 8. Discussion of the above-described example Low-dose divided RT is CD8 + Secondarily to IFNγ production by T cells, this leads to upregulation of PD-L1 expression on tumor cells. In mouse models of melanoma, colorectal cancer, and breast cancer, we show that RT activity can be enhanced through combination with αPD-L1 mAb, leading to the development of immunological memory that protects against tumor recurrence in long-surviving mice. Furthermore, our data show that the administration sequence may affect prognosis, and that simultaneous but not sequential treatment is effective in improving local tumor control and survival.

[0064] This is because the split RT is CD8 +This is the first preclinical study to demonstrate that increased tumor cell expression is induced through IFNγ PD-L1 production by T cells. Here, tumor cell PD-L1 expression may function as a biomarker of the local antitumor response, and local RT may lead to CD8 + This suggests that it may be sufficient to stimulate a T cell response. However, while treatment with RT alone may not produce sustained anti-cancer immunity, activity was found to be enhanced through mAb-mediated blockade of either PD-1 or PD-L1, suggesting that a signaling pathway mediated by this system may limit the immune response to RT. The inventors did not observe any significant difference in overall survival between RT delivered in combination with either αPD-1 or αPD-L1, or in combination with both mAbs. Given this observation, it appears that when delivered in combination with RT, the activity of these mAbs occurs through blockade of PD-1 / PD-L1 signaling and not mediated through the PD-L1 / CD80 or PD-1 / PD-L2 systems, but further experiments are needed to confirm this.

[0065] Our preclinical data show that NK cell depletion reduces local tumor control at an early stage (up to 11 days after completion of RT), but does not affect overall survival. Furthermore, NK cell depletion does not affect PD-L1 expression in tumor cells following RT, suggesting their limited contribution to local IFNγ production. In contrast, CD4 + While T cell depletion did not affect survival time, a significant increase in PD-L1 expression in tumor cells was observed after RT. CD4 + T cell depletion affects both helper T cells and Treg counts. These CD4 + Further research is needed to describe the relative contribution of T cell subpopulations, but in our model, we found that CD8 cells are effective after RT / PD-L1 mAb therapy. + CD4 +Helper T cells are unnecessary, and it can be hypothesized that either type of helper T cell, possibly a Treg, may actively suppress IFNγ production within the local tumor environment. Therefore, the therapeutic ability to promote an antitumor response through Treg depletion may be at least partially attenuated through increased PD-L1 expression in tumor cells.

[0066] PD-L1 expression can be regulated by several cytokines, including type 1 and type 2 IFNs, TNFα, and TGFβ (16,22,30-32). We show that PD-L1 expression in tumor cells can be enhanced following co-culture with IFNγ, and that the addition of TNFα can further enhance this response. However, blockade of IFNγR1 or in vivo depletion of IFNγ demonstrates the dependence of PD-L1 upregulation on IFNγ-mediated signaling, and tumor cell PD-L1 expression cannot be regulated by TNFα alone. Interestingly, in vivo depletion of IFNγ reduces PD-L1 expression on syngeneic tumor cells, consistent with the in vitro tumor cell expression profile. This suggests that the local tumor microenvironment in the absence of therapeutic intervention may promote immunologically driven tumor adaptations that support tumorigenesis. Similarly, in human melanocyte lesions, PD-L1 expression is associated with CD8 + It has been shown to co-localize with T cell infiltration areas, which is thought to correspond to an adaptive mechanism of immune evasion (16). However, in our preclinical mouse model, monotherapy with PD-1 / PD-L1 targeted mAbs demonstrated only mild activity, suggesting that targeting only this system is unlikely to produce a sustained antitumor immune response in all situations, thus highlighting the need for a combinatorial approach.

[0067] In contrast to the use of a single 12 Gy dose (30), our results show that upregulation of PD-L1 occurs at a much lower bioeffective dose with fractional RT delivery using approximately 10 Gy in five divided daily doses. This is an important finding given that this fractional dosing is more commonly used in routine clinical practice. Several studies have evaluated the effects of fractional RT administration; comparing single resection doses, fractional doses, and fractional RT for antitumor immune response development and tumor microenvironment (4, 5, 25, 26, 33, 34). However, the results of these studies are ambiguous, and the effects of fractional RT administration on optimal RT delivery and PD-L1 expression in tumor cells require further investigation.

[0068] Previously, no studies had addressed the effect of the order of activity on RT and αPD-L1 mAb therapy. This is particularly relevant given the tendency in clinical settings to adopt combination of treatment sequences as a strategy to maximize tolerability. We demonstrate that PD-L1 blockade at the time of RT delivery alone can improve the therapeutic response in a mouse model, and that treatment 7 days after RT completion is no longer better than treatment with RT alone. Furthermore, our data show that PD-L1 expression increases up to 24 hours after completion of a fractional RT cycle and remains elevated at least 7 days after completion. In addition, we show that tumor-infiltrating CD4 at 24 hours after RT is higher compared to a control cohort. + and CD8 + We discovered increased PD-1 expression on T cells. PD-1 expression is associated with CD4 + CD8 in comparison to T cells + As shown above, it was consistently found to be higher. Taken together, these data suggest that locally fractionated RT is effective against CD8 +While T cell responses may be stimulated, these may be attenuated by signaling via the PD-1 / PD-L1 system, suggesting that initial inhibition of this system may lead to a sustained and effective antitumor response. Furthermore, the need for chronic blockade of the PD-1 / PD-L1 system, compared to acute blockade, when combined with RT, remains unclear. The inventors observed no difference in survival when αPD-L1 mAbs were administered concurrently with RT delivery for 3 quarter weeks compared to a long-term schedule of up to 3 weeks. These data suggest that the combination approach with RT may allow for a reduction in the duration of αPD-1 / PD-L1 mAb therapy required to achieve a therapeutic antitumor immune response. Collectively, these data have significant implications for clinical trial design.

[0069] In summary, this trial demonstrates that treatment with fractional RT leads to upregulation of PD-L1 expression in tumor cells, and that blocking the PD-1 / PD-L1 system can enhance the immune response to fractional RT in multiple syngeneic mouse models of cancer. Our data suggest that the RT and αPD-L1 mAb administration regimen may produce a therapeutic immune response by reducing tumor burden and improving survival. This therapeutic combination may be promising for addressing many solid malignancies for which RT is commonly used, and bridging to early clinical trials is clearly justified.

[0070] Example 9. Treatment Protocol Patient A has colorectal cancer. In week 1, Patient A receives an effective dose of radiotherapy in five fractions (fractopms). Also in week 1, Patient A receives a therapeutic dose of MEDI4736 on days 1, 3, and 5. Patient A repeats this schedule in weeks 2, 3, 4, and 5.

[0071] Patient B has breast cancer. In week 1, Patient B receives an effective dose of radiotherapy. Also in week 1, Patient B receives a therapeutic dose of MEDI4736 on day 5. The patient repeats this schedule in weeks 3, 5, and 7.

[0072] Example 10. Specific Embodiment The following items provide specific embodiments disclosed herein.

[0073] Item 1. a. The step of administering at least one dose of radiotherapy; b. The step of administering at least one PD-1 and / or PD-L1 antagonist. A method for treating cancer in a patient in whom at least one PD-1 and / or PD-L1 antagonist is administered on the same day as or up to four days after the administration of radiotherapy.

[0074] Item 2. The method of Item 1, wherein at least one PD-1 and / or PD-L1 antagonist is at least one PD-1 and / or PD-L1 antibody or a functional portion thereof.

[0075] Item 3. One of the methods from Items 1 or 2, wherein the dose of radiotherapy is approximately 11 Gy or less.

[0076] Item 4. The method of Item 3, wherein the dose of radiotherapy is approximately 10 Gy or less.

[0077] Item 5. The radiotherapy is fractionated radiotherapy, one of the methods from Items 1-4.

[0078] Item 6. Fractionated radiotherapy, the method described in Item 5, including 2 to 7 fractions.

[0079] Item 7. Fractionated radiotherapy, the method of Item 6, including five fractions.

[0080] Item 8. Radiotherapy fractions are administered daily, using one of the methods described in items 5-7.

[0081] Item 9. The fractional dose of radiotherapy is administered on days 1, 2, 3, 4, and 5, using one of the methods described in items 5-8.

[0082] Item 10. One of the methods from Items 7-9, consisting of radiotherapy with approximately 10 Gy in five fractional doses.

[0083] Item 11. One of the methods from items 1-10, in which at least one PD-1 and / or PD-L1 antagonist is administered on day 1.

[0084] Item 12. One of the methods described in items 1-11, in which at least one PD-1 and / or PD-L1 antagonist is administered on day 5.

[0085] Item 13. One of the methods described in items 1-12, in which at least one PD-1 and / or PD-L1 antagonist is administered multiple times.

[0086] Item 14. One of the methods described in items 1-13, in which at least one PD-1 and / or PD-L1 antagonist is administered three times weekly.

[0087] Item 15. One of the methods from items 1 to 14, wherein the anti-PD-1 and / or PD-L1 antibody or its functional portion is MEDI4736.

[0088] Item 16. One of the methods from Items 1 to 15, wherein the anti-PD-1 and / or anti-PD-L1 antibody or its functional portion is pembrolizumab, nivolumab, BMS-936558, AMP-224, or MPDL3280A.

[0089] Item 17. The cancer is melanoma, colorectal cancer, or breast cancer, according to one of the methods described in items 1-16.

[0090] Item 18. One of the methods from items 1-17, involving 2 or more treatment cycles.

[0091] Item 19.2 - The method described in Item 18, which involves 8 treatment cycles.

[0092] Item 20. One of the methods from items 18-19, with a treatment cycle of weekly.

[0093] Item 21. One of the methods from items 18-19, with a treatment cycle of every other week. The present invention also provides embodiments of the following items. [Item A1] a. The step of administering at least one dose of radiotherapy; b. A method for treating cancer in a patient, comprising the step of administering at least one PD-1 and / or PD-L1 antagonist, wherein at least one PD-1 and / or PD-L1 antagonist is administered on the same day as or up to four days after the administration of radiotherapy. [Item A2] The method according to [Item A1], wherein the at least one PD-1 and / or PD-L1 antagonist is at least one PD-1 and / or PD-L1 antibody or a functional portion thereof. [Item A3] The method according to [Item A1], wherein the dose of radiotherapy is approximately 70 Gy or less. [Item A4] The method according to [Item A3], wherein the dose of radiotherapy is approximately 50 Gy or less. [Item A5] The method described in [Item A4], wherein the radiotherapy is fractionated radiotherapy. [Item A6] The method described in [Item A5], wherein the fractionated radiotherapy comprises 2 to 7 fractional doses. [Item A7] The method according to [Item A6], wherein the fractionated radiotherapy comprises five fractional doses. [Item A8] The method described in [Item A7], wherein the fractionated dose of the aforementioned fractionated radiotherapy is administered on consecutive days. [Item A9] The method according to [Item A8], wherein the fractionated doses of the fractionated radiotherapy are administered on days 1, 2, 3, 4, and 5. [Item A10] The radiotherapy method described in [Item A9], wherein the radiotherapy comprises approximately 70 Gy in five fractional doses. [Item A11] The method according to [Item A1], wherein at least one PD-1 and / or PD-L1 antagonist is administered on day 1. [Item A12] The method according to [Item A1], wherein at least one PD-1 and / or PD-L1 antagonist is administered on day 5. [Item A13] The method according to [Item A1], wherein at least one PD-1 and / or PD-L1 antagonist is administered multiple times. [Item A14] The method according to [Item A13], wherein at least one PD-1 and / or PD-L1 antagonist is administered three times a week. [Item A15] The method according to [Item A14], wherein the anti-PD-1 and / or PD-L1 antibody or its functional portion is MEDI4736. [Item A16] The method according to [Item A15], wherein the anti-PD-1 and / or anti-PD-L1 antibody or its functional portion is pembrolizumab, nivolumab, BMS-936558, AMP-224, or MPDL3280A. [Item A17] The method according to [Item A1], wherein the cancer is melanoma, colorectal cancer, or breast cancer. [Item A18] The method described in [Item A1], wherein two or more treatment cycles are performed. [Item A19] The method described in [Item A18] involves 2 to 8 treatment cycles. [Item A20] The method according to [Item A19], wherein the treatment cycle is weekly or bi-weekly.

[0094] References 1.D. Verellen et al., Innovations in image-guided radiotherapy. Nat Rev Cancer 7, 949-960 (2007). 2.L. J. Lee et al., Innovations in radiation therapy (RT) for breast cancer. Breast 18 Suppl 3, S103-111 (2009). 3.E. Kapiteijn et al., Preoperative radiotherapy combined with total mesorectal excision for resectable rectal cancer. N Engl J Med 345, 638-646 (2001). 4.Y. Lee et al., Therapeutic effects of ablative radiation on local tumor require CD8+ T cells: changing strategies for cancer treatment. Blood 114, 589-595 (2009). 5.A. A. Lugade et al., Local radiation therapy of B16 melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor. J Immunol 174, 7516-7523 (2005). 6.L. Apetoh et al., Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 13, 1050-1059 (2007). 7.S. J. Gardai et al., Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 123, 321-334 (2005). 8. F. Ghiringhelli et al., Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors. Nat Med 15, 1170-1178 (2009). 9. Y. Ma et al., Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells. Immunity 38, 729-741 (2013). 10. J. Honeychurch et al., Immunogenic potential of irradiated lymphoma cells is enhanced by adjuvant immunotherapy and modulation of local macrophage populations. Leuk Lymphoma 54, 2008-2015 (2013). 11. B. Cummings et al., Five year results of a randomized trial comparing hyperfractionated to conventional radiotherapy over four weeks in locally advanced head and neck cancer. Radiother Oncol 85, 7-16 (2007). 12. H. 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[0095] equivalent The aforementioned written specification is deemed sufficient to enable those skilled in the art to carry out the embodiments. The foregoing description and examples detail specific embodiments and describe the best modes considered by the inventors. However, regardless of how the foregoing matters are detailed in the text, it will be understood that embodiments may be carried out in many forms and the claims include any equivalents thereof.

[0096] In the context of this specification, the term "approximately" refers to numerical values, including, for example, integers, fractions, and percentages, whether explicitly indicated or not. The term "approximately" usually refers to a range of numerical values ​​(e.g., + / - 5 to 10%) that a person skilled in the art would consider equivalent to the listed values ​​(e.g., having the same function or result). In some cases, the term "approximately" may include numerical values ​​rounded to the nearest significant figure.

Claims

1. A pharmaceutical product for use in the treatment of renal cancer, liver cancer, lung cancer, bladder cancer, head and neck cancer, pancreatic cancer, and / or ovarian cancer in a patient, comprising at least one PD-1 and / or PD-L1 antagonist, wherein in said use, the at least one PD-1 and / or PD-L1 antagonist is administered to the patient on the same day as or up to four days after the fractional dose of fractional radiotherapy, wherein the at least one PD-1 and / or PD-L1 antagonist is a functional portion thereof capable of binding to at least one anti-PD-1 antibody and / or anti-PD-L1 antibody, or an antigen or epitope, and the total dose of fractional radiotherapy is 11 Gy or less.

2. The pharmaceutical product according to claim 1, wherein the total dose of fractionated radiotherapy is approximately 10 Gy or less.

3. The pharmaceutical product according to claim 1 or 2, wherein the fractionated radiotherapy comprises two to seven fractional doses.

4. The pharmaceutical product according to any one of claims 1 to 3, wherein the fractionated radiotherapy comprises five fractional doses.

5. The pharmaceutical product according to any one of claims 1 to 4, wherein the fractionated dose of the fractionated radiotherapy is administered on consecutive days.

6. The pharmaceutical product according to claim 5, wherein the fractionated doses of the fractionated radiotherapy are administered on day 1, day 2, day 3, day 4, and day 5.

7. The pharmaceutical product according to claim 6, wherein the fractionated radiotherapy comprises approximately 10 Gy in five fractional doses.

8. The pharmaceutical product according to any one of claims 1 to 7, wherein at least one PD-1 and / or PD-L1 antagonist is administered on day 1 of the treatment cycle.

9. The pharmaceutical product according to any one of claims 1 to 7, wherein at least one PD-1 and / or PD-L1 antagonist is administered on the fifth day of the treatment cycle.

10. The pharmaceutical product according to any one of claims 1 to 9, wherein the at least one PD-1 and / or PD-L1 antagonist is administered multiple times.

11. The pharmacopoeia according to claim 10, wherein the at least one PD-1 and / or PD-L1 antagonist is administered three times a week.

12. The pharmaceutical product according to any one of claims 1 to 11, wherein the functional portion capable of binding to the anti-PD-L1 antibody, or to an antigen or epitope, is MEDI4736, or to an antigen or epitope.

13. The pharmaceutical product according to any one of claims 1 to 11, wherein the functional portion capable of binding to the anti-PD-1 antibody and / or anti-PD-L1 antibody, or to an antigen or epitope, is pembrolizumab, nivolumab, BMS-936558, AMP-224, or MPDL3280A.

14. The pharmaceutical product according to claim 13, wherein the anti-PD-1 antibody is pembrolizumab.

15. The pharmaceutical product according to claim 13, wherein the anti-PD-1 antibody is nivolumab.

16. The pharmaceutical product according to claim 13, wherein the anti-PD-L1 antibody is MPDL3280A.

17. The pharmaceutical product according to any one of claims 1 to 16, wherein two or more treatment cycles are performed in the treatment of the aforementioned cancer.

18. The pharmaceutical product according to claim 17, wherein two to eight treatment cycles are performed.

19. The pharmaceutical product according to claim 17, wherein the treatment cycle is weekly or bi-weekly.

20. The pharmaceutical product according to any one of claims 1 to 19, wherein the pharmaceutical product is for use in the treatment of kidney cancer, liver cancer, pancreatic cancer, and / or ovarian cancer, and the radiotherapy is external beam radiotherapy.

21. The pharmaceutical product according to claim 20, wherein the external beam radiotherapy includes stereotactic radiotherapy.

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