Methods of treating cancer with antagonists against PD-1 and PD-l1 in combination with radiation therapy

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 Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, the entire contents of which are incorporated by reference. Said ASCII copy was created on Jun. 16, 2015, is named B7H1-275WO1_SL.txt, and is 103,313 bytes in size.

[0002] Field Cancer treatment. [Background technology]

[0003] Radiation therapy (RT) remains the primary non-surgical treatment in the management of solid malignancies, with approximately 50-60% of all cancer patients receiving this treatment. Incorporation of RT into treatment plans reduces disease recurrence and improves overall survival in the majority of common cancers (1-3). Although effective, many patients continue to struggle with local recurrence and metastatic disease.

[0004] In addition to the direct cytoreductive effects of RT, emerging evidence suggests that the generation of antitumor immune responses may play a key role in the efficacy of this treatment (4,5). RT can result in the expression of ecto-calreticulin on tumor cells and the release of several damage-associated molecular patterns (DAMPs), including high-mobility group box 1 (HMGB1) and ATP, which can lead to the recruitment and activation of antigen-presenting cells (APCs) and the priming of tumor antigen-specific T cell responses (6-10). Despite this, immune escape is frequent and tumor recurrence remains the leading cause of mortality in patients undergoing RT (11). Identification and inhibition of the primary driving mechanisms of immune suppression may enhance antitumor immune responses and 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 the maintenance of peripheral immune tolerance and the regulation of acute inflammatory responses through inhibition of T cell function and apoptosis of activated T cells (12,13). In addition to binding PD-1, PD-L1 can also suppress T cell function through its interaction with CD80 (14). PD-L1 expression is inducible and appears to respond to the local inflammatory environment, particularly type I and II interferons (IFNs) (12,15,16). While nearly undetectable in most normal tissues, PD-L1 expression has been described in multiple malignancies (reviewed (17)). Importantly, recent clinical trials with either PD-1 or PD-L1-targeted monoclonal antibodies (mAbs) have demonstrated promising responses in patients with advanced disease (18-21). Summary of the Invention [Problem to be solved by the invention]

[0007] A combined administration strategy of radiation therapy and PD-1 and / or PD-L1 antagonists that provides the greatest benefit to patients suffering from cancer is desirable. [Means for solving the problem]

[0008] According to the description, the method of treating cancer in a patient includes: a. administering at least one dose of radiation therapy; 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 four days after the administration of radiation therapy.

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

[0010] In one embodiment, the radiation therapy is fractionated radiation therapy. In one mode, the fractionated radiation therapy comprises 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 days 1, 2, 3, 4, and 5. In one embodiment, the radiation therapy comprises about 10 Gy in 5 fractions.

[0012] In one embodiment, the at least one PD-1 and / or PD-L1 antagonist is administered at least on day 1 and / or day 5. In one mode, the at least one PD-1 and / or PD-L1 antagonist is administered multiple times. For example, the at least one PD-1 and / or PD-L1 antagonist may be administered three times per week.

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

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

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

[0016] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0017] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to be limiting of the scope of the appended claims.

[0018] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments and, together with the description, serve to explain the principles described herein. [Brief description of the drawings]

[0019] [Figure 1] Figure 1A-F illustrates blockade of the PD-1 / PD-L1 system to enhance the activity of fractionated radiation therapy. A and B, Median fluorescence intensity (A) and representative histogram (B) of PD-L1 expression on CT26 cells isolated from tumors 1, 3, or 7 days after treatment with ∼10 Gy in 5 daily fractions of 2 Gy. C and D, Mice bearing CT26 tumors administered ∼10 Gy RT delivered in 5 daily fractions of 2 Gy, either alone or in combination with either αPD-1 (C) or αPD-L1 (D) mAb administered at 10 mg / kg 3qw for up to 3 weeks. E, Tumor volume 10 days after initiation of treatment in mice bearing 4T1 tumors administered 20 Gy RT delivered in 5 daily fractions of 4 Gy, either alone or in combination with αPD-L1 mAb. F, Mice bearing 443 tumors administered ∼10 Gy RT delivered in five daily fractions of 2 Gy, either alone or in combination with αPD-L1 mAb. Experimental groups contained at least seven mice and are representative of at least two independent experiments. A, E and F show mean ± SEM. *, P<0.05**, P<0.01, Mann-Whitney test. C and D, *, indicates significance compared to control mice. +, indicates significance compared to monotherapy. *** / +++, P<0.001, log-rank (Mantel-Cox) test. [Diagram 2]Figure 2A-C shows that the therapeutic activity of the fractionated RT and αPD-L1 mAb combination depends on the activity of CD8+ T lymphocytes. A, Tumor volumes 7 and 11 days after five 2 Gy + αPD-L1 mAb combination treatments. Immune cell subsets (either CD8, CD4 or NK cells) were depleted 1 day before treatment and depletion was maintained for 2 weeks. ***, P>0.001; *, P>0.01; *, P>0.05, Mann-Whitney test. B, Survival curves. *** / +++, P<0.001, log-rank (Mantel-Cox) test. Data representative of 10 mice per cohort. C, Representative density plots of peripheral blood confirming depletion of immune cell subsets. [Diagram 3] Figure 3A-C show that the combination of split RT and αPD-L1 mAb results in protective immune memory. A, Survival curves of LTS mice following contralateral re-challenge with 5x105 CT26 cells. *P<0.05 compared to control mice (log-rank; Mantel-Cox test). B, Representative dot blots of IFNγ production by CD8+ T cells isolated from either tumor-naive or LTS mice initially treated with RT and αPD-L1 mAb. C, Frequency of IFNγ+CD8+ T cells isolated from either tumor-naive or LTS mice initially treated with RT and αPD-L1 mAb following co-culture with either H2-Ld-restricted peptide (AH1 (SPSYVYHQF) (SEQ ID NO: 91); defined CT26 tumor-associated antigen or β-galactosidase (TPHPARIGL) (SEQ ID NO: 92); control peptide of prokaryotic origin) or 50 Gy irradiated CT26 cells for 5 days followed by priming with 50 Gy irradiated CT26 cells. *P<0.05 (Mann-Whitney test). Data representative of two independent experiments. [Figure 4]Figure 4A-C illustrates that fractionated RT increases tumor cell PD-L1 expression in vivo and is dependent on CD8+ T cells. A, Expression of PD-L1 on CT26 cells after treatment with ex vivo RT (2.5-10 Gy). B and C, Representative 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 receiving approximately 10 Gy in five daily fractions of 2 Gy in combination with either CD8, CD4 or NK cell depleting antibodies. *P<0.05 (Mann-Whitney test). Experimental groups contained at least 5 mice and were representative of at least two independent experiments. [Diagram 5] Figure 5A-E shows that IFNγ production by CD8+ T cells is involved in upregulating PD-L1 expression on tumor cells following split RT. A, PD-L1 expression on wild-type CT26 tumor cells (WT), or cells transduced with either non-targeting control (NTC) or IFNγR1 ShRNA following 24 hours of co-culture with either 20 ng / ml IFNγ, TNFα, or a combination of both cytokines. B, Representative density plots showing expression of IFNγ and TNFαby CD8+ T cells following treatment with either PBS or phorbol 12-myristate 13-acetate (PMA) and ionomycin, and C, Frequency 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-tailed Student's t test). D and E, Representative contour plots (D) and median fluorescence intensity (E) of PD-L1 expression on CT26 tumor cells following treatment with RT (~10 Gy in five fractions) in the presence of αIFNγ blocking mAb (or isotype control) in vivo. **P<0.01 (Mann-Whitney test). Experimental groups contained at least 5 mice. [Figure 6]Figure 6A-D show that dosing schedule affects outcome, with RT synergy observed only with concurrent αPD-L1 mAb therapy but not with sequential therapy. A, Schema of dosing schedule study. Mice received fractionated RT (as approximately 10 Gy in five daily fractions of 2 Gy) alone or in combination with αPD-L1 mAb, starting either on RT cycle day 1 (schedule A), RT cycle day 5 (schedule B), or 7 days after the last dose of RT (schedule C). B, Survival curves of treatment regimens. ++, P<0.01 compared to monotherapy (log-rank; Mantel-Cox test). C and D, Expression of PD-1onCD4+ (C) and CD8+ (D) T cells at 24 hours and 7 days after the last dose of RT. *P<0.05 (Mann-Whitney test). Experimental groups included at least 5 mice and were representative of at least two independent experiments. [Figure 7] Figure 7 shows that blockade of both PD-1 and PD-L1 does not further enhance efficacy in combination with fractionated RT in CT26-tumor bearing mice. A) Survival curves following fractionated RT (~10 Gy in 5 daily fractions of 2 Gy) alone or in combination with αPD-1, αPD-L1 or a combination of both mAbs administered 3qw over 3 weeks. n / s, P>0.05, log-rank; Mantel-Cox test. Experimental groups contained at least 5 mice and are representative of 2 (at 2) independent experiments. [Figure 8] Figure 8A-B show that combination therapy with fractionated RT and either αPD-1 or αPD-L1 mAb was well tolerated in mice. Mice bearing CT26 tumors received 10 Gy RT delivered in five daily fractions of 2 Gy, either alone or in combination with αPD-L1 mAb administered at 10 mg / kg 3qw for either 3 weeks or 1 week. Experimental groups contained at least 7 mice and were representative of at least 2 independent experiments. n / s, P>0.05, Mann-Whitney test. [Figure 9]Figures 9A-C illustrate that treatment of tumor cells with αPD-1 or αPD-L1 mAb does not sensitize radiation-induced cell death in vitro. Clonogenic survival curves of CT26 cells (A), 4T1 cells (B), and 4434 cells (C) treated with RT (2.5-10 Gy) in the absence or presence of 2 μg / ml αPD1 or αPD-L1. [Figure 10] Figure 10A-C show that dosing schedule impacts outcome. A) Tumor volumes following fractionated RT administered (10 Gy in five daily fractions of 2 Gy) alone or in combination with αPD-L1 mAb starting either on RT cycle day 1 (schedule A), RT cycle day 5 (schedule B), or 7 days after the last dose of RT (schedule C). B and C) Tumor volumes of RT-treated mice showing comparable tumor volumes across different dosing schedules. n / s, P>0.05, Mann-Whitney test. Experimental groups contained at least 5 mice and are representative of two (at 2) independent experiments. [Figure 11] Figure 11 shows that acute versus chronic administration of αPD-L1 mAb does not affect efficacy of combination therapy. Mice bearing CT26 tumors received 10 Gy RT delivered in five daily fractions of 2 Gy, either alone or in combination with αPD-L1 mAb administered at 10 mg / kg 3qw for either 3 weeks or 1 week. Experimental groups contained at least 7 mice and were representative of at least two independent experiments. n / s, P>0.05, log-rank; Mantel-Cox test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Sequence Description Table 1 provides a list of specific sequences referenced in the embodiments under consideration. The CDRs are shown in bold and underlined.

[0021] [Table 1] TIFF2025028053000003.tif164125TIFF2025028053000004.tif158125TIFF2025028053000005.tif165125TIFF2025028053000006.tif16912 5TIFF2025028053000007.tif165125TIFF2025028053000008.tif163125TIFF2025028053000009.tif164125TIFF2025028053000010.tif60125

[0022] Description of the embodiments I. Treatment The methods encompass cancer treatment with administration of at least one dose of radiation therapy 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 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 up to four days after the administration of radiation therapy.

[0023] The 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), such as weekly, biweekly, every three weeks, or monthly cycles. When the method is performed more than once, it may be performed 2, 3, 4, 5, 6, 7, 8 or more times.

[0024] While not wishing to be bound by theory, the inventors discovered that low-dose fractionated RT resulted in upregulation of PD-L1 expression on tumor cells in vivo within established syngeneic tumor models. The inventors demonstrated that fractionated RT delivered in combination with αPD-1 or αPD-L1 mAb resulted in effective antitumor CD8 + We show that IFNγ produced a CD8 response that improved local tumor control and long-term survival, and protected against tumor rechallenge by inducing tumor antigen-specific memory immune responses.+ T cell production was found to be involved in the upregulation of PD-L1 on tumor cells following fractionated RT. Furthermore, the scheduling of anti-PD-L1 mAb relative to the delivery of fractionated RT appears to affect treatment outcome; administration of antagonist on the same day as RT or up to 4 days after the first dose of fractionated RT shows advantages over administration of antagonist more than 7 days after the end of radiotherapy. And while not wishing to be bound by theory, upregulation of tumor cell PD-L1 expression in response to low doses of fractionated RT, as routinely used in the clinic, appears to be an adaptive immunological resistance mechanism by tumor cells; thus, the PD-L1 / PD-1 signaling system potentially contributes to treatment failure. Combination therapy with RT and PD-1 / PD-L1 signaling system blockade has the potential to overcome this resistance, but our preclinical studies show that the timing of dosing affects treatment outcome, providing important new insights for translation to the clinic.

[0025] A. PD-1 and / or PD-L1 Antagonists for Use in Therapeutic Methods In one embodiment, the at least one PD-1 and / or PD-L1 antagonist for use in the methods is an anti-PD-1 and / or anti-PD-L1 antibody, or a functional portion thereof.

[0026] The at least one PD-1 and / or PD-L1 antagonist is administered on the same day or up to four days after the administration of radiation therapy. For example, in one embodiment, if radiation therapy is provided on day 1 of a treatment cycle, then the 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 radiation therapy administration). In another embodiment, if radiation therapy is provided on day 1 of a treatment cycle, then the 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 day 1, day 2, day 3, day 4, day 5, day 6, and / or day 7 of a treatment cycle (including both single and multiple 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 2, 3, 4, 5 or more times during a treatment cycle. For example, the antagonist may be administered 3 times per week for a one week treatment cycle, or 3 times per week for a two or more week treatment cycle as described above.

[0028] In one aspect, the antibody or functional portion thereof is selected from those disclosed in U.S. Patent Publication No. 2010 / 0028330, which is incorporated by reference for its teachings of these antibodies and functional portions thereof. In one embodiment, the antibody if 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 functional portion thereof is administered in a therapeutically effective amount. In general, the therapeutically effective amount may vary depending on the age, medical condition, and sex of the subject, as well as the severity of the subject's disease. The therapeutically effective amount of the antibody or functional portion thereof ranges from about 0.001 to about 30 mg / kg body weight, about 0.01 to about 25 mg / kg body weight, about 0.1 to about 20 mg / kg body weight, or about 1 to about 10 mg / kg. The dose may be adjusted as necessary to suit the observed therapeutic effect. The appropriate dose is selected by the treating physician based on the clinical indication.

[0031] The antibody may be administered as a bolus dose to maximize circulating levels of antibody for the maximum period of time following administration. Continuous infusion may also be used following the bolus dose.

[0032] As used herein, the term antibody or functional portion thereof is used in the broadest sense. It may be man-made, such as monoclonal antibodies (mAbs) and / or functional fragments thereof, produced by conventional hybridoma technology, recombinant technology. It may include both intact immunoglobulin molecules, such as polyclonal antibodies, monoclonal antibodies (mAbs), monospecific antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, animal antibodies (e.g. camelid antibodies), chimeric antibodies, as well as parts, fragments, regions, peptides, and derivatives thereof (provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant technology), such as, for example, immunoglobulins lacking light chains, 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. In one embodiment, the functional portion is a single chain antibody, a single chain variable fragment (scFv), a Fab fragment, or a F(ab')2 fragment.

[0033] An antibody or functional portion is said to be a "binding" molecule if it is capable of specifically reacting with a molecule such that the molecule binds to the antibody. Antibody fragments or portions may lack the Fc fragment of the intact antibody, be cleared from the circulation more quickly, and may have less non-specific tissue binding than the intact antibody. An example of an antibody that may be generated from an intact antibody using methods well known in the art is by proteolytic cleavage, for example, with enzymes such as papain (to generate Fab fragments) or pepsin (to generate F(ab')2 fragments). An antibody portion may be generated by any of the above methods, or may be generated by expression of a portion of a 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 portion is a human antibody. The use of human antibodies for human therapy may reduce the probability of side effects due to immunological reactions in human individuals to non-human sequences. In another embodiment, the antibody or functional portion is humanized. In another embodiment, the antibody or functional portion is a chimeric antibody. In this way, a sequence of interest, such as, for example, a binding site of interest, can be included in the antibody or functional portion.

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

[0036] B. Radiation therapy used in treatment Radiation therapy, also known as high-dose ionizing radiation, is a component of therapeutic approaches under investigation.

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

[0038] In one mode, the radiotherapy fractions are administered on consecutive days. In one mode, the radiotherapy may include more than one administration every day and / or daily administration. In one mode, the radiotherapy fractions are administered on the first, second, third, fourth and fifth days. In another mode, the radiotherapy includes about 10 Gy in five fractions (i.e., 2 Gy each day for five days).

[0039] Other fractionation schedules may be used, including accelerated fractionation (treatment given in larger daily or weekly doses, shortening the number of weeks of treatment), hyperfractionation (smaller radiation doses given more than once a day), or hypofractionation (larger doses given once a day or less, often shortening the number of treatments).

[0040] Radiation therapy may be X-rays, gamma rays, or charged particles.Radiation therapy may be external beam radiation therapy or internal radiation therapy (also called brachytherapy).Whole-body radiation therapy using radioactive materials such as radioactive iodine may also be used.

[0041] External beam radiation therapy includes 3D conformational radiation therapy, intensity-modulated radiation therapy, image-guided radiation therapy, tomotherapy, stereotactic radiation therapy, proton therapy, or other charged particle beams.

[0042] C. The cancer being treated The method may be used to treat a variety of cancer types. In one aspect, 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, the cancer is cancer is adrenal cortical tumor, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, epidermoid cancer, esophageal cancer, eye cancer, glioblastoma, glioma, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, head and neck, Hodgkin's disease, Kaposi's sarcoma, renal cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer (such as hepatocellular carcinoma), lung cancer (non-small cell, small cell, and lung carcinoid tumors, lymph node cancer, lymphoma, lymphoma of the skin, melanoma, mesothelioma, oral cavity cancer, multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, childhood malignancies, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland, sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0044] Reference will now be made in detail to the exemplary embodiments under consideration, examples of which are illustrated in the accompanying drawings. Whenever possible, the same reference numbers have been used throughout the drawings to refer to the same or like parts. Other embodiments will become apparent to those skilled in the art from consideration of the specification and practice disclosed herein. The embodiments are further described in the following examples. These examples do not limit the scope of the claims, but merely serve to clarify certain embodiments. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit being indicated 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 the local ethical committee and carried out under a UK Home Office license. CT26 mouse colon carcinoma cells (ATCC) and 4434 cells were transfected with the BRafV600E p16- / - Cell lines were isolated from mice (Richard Marias, Cancer Research UK, Manchester Institute) and maintained in DMEM, and 4T1 triple-negative breast cancer (ATCC) was maintained in RPMI-1640 supplemented with 10% FCS, 1% L-glutamine (Invitrogen, UK). All cell lines were routinely screened to confirm the absence of Mycoplasma contamination.

[0046] B) Tumor Treatment For mice, 5×10 5 CT26, 1×10 5 4T1 or 5x10 6 Each mouse was inoculated subcutaneously (sc) with either 4434 cells or 4434 cells. Irradiation was performed 7–10 days after inoculation (when tumors were at least 100 mm ) using a Pantak HF-320 320 kV X-ray unit (Gulmay Medical, UK). 3RT was performed at 100 s (times 10 min). The equipment was operated at 300 kV and 9.2 mA, and the X-ray beam was fitted with a filter to give a radiation quality of 2.3 mm Cu half-value layer. Mice were positioned at a distance of 350 mm from the X-ray focus, and the dose rate was 0.80 Gy / min. αPD-1 (clone RMPI-14), αPD-L1 (clone 10F.9G2) (both Biolegend) or isotype control mAb (IgG2a and IgG2b, respectively) treatment was administered intraperitoneally (ip) at a dose of 10 mg / kg in a volume of 100 μl / 10 g in PBS, starting on day 1 of the split RT cycle (unless otherwise noted), 3 qw for up to 3 weeks. Cell and cytokine depletion experiments Mice received 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. For tumor rechallenge experiments, long-term survivor (LTS) mice were implanted contralaterally with tumor cells a minimum of 100 days after the previous tumor implantation. Additional control mice were also implanted to confirm tumor growth. Experimental groups contained at least 5 mice / group and are representative of at least two independent experiments.

[0047] C) CD8 isolated from long-term surviving mice + Measurement of cytokine production by T cells In vitro stimulation was performed using 1 × 10 mice irradiated with 50 Gy. 6 3.5×10 6444 cells from either LTS or control mice in the presence of either 1 μmol / ml of tumor cells or the H2-Ld-restricting peptide SPSYVYHQF (SEQ ID NO: 91) (AH1) / TPHPARIGL (SEQ ID NO: 92) (β-galactosidase) (Anaspec, UK). 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. Experimental groups contained 3–5 mice and are representative of two independent experiments. After 5 days of culture, cells were restimulated with 50 Gy irradiated tumor cells at a 1:1 ratio for 16 h 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, cells were washed and cultured with rat anti-CD16 / 32 (eBioscience, UK) to block nonspecific binding, and then stained with FITC-conjugated anti-CD8α mAb (eBioscience, UK). Cells were then fixed / permeabilized and stained for IFNγ expression using an APC-conjugated mAb (eBioscience, UK).

[0048] D) Tumor and immune cell phenotype determination by flow cytometry Tumors were processed using a gentleMacs Dissociator and Mouse Tumor Dissociation Kit (Miltenyi Biotec, UK) to obtain single cell suspensions. For analysis, non-specific binding was blocked as described above and expression of CD4, CD8 (BD Biosciences, UK), CD45, NKP46, PD-1 and PD-L1 was examined by multiparameter flow cytometry (all eBioscience unless otherwise noted).

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

[0050] Example 2. Blockade of PD-1 or PD-L1 enhances the therapeutic effect of RT Although radiation therapy has been shown to modulate tumor cell immunogenicity, it rarely produces durable therapeutic responses that result only in systemic antitumor immunity. We show that low-dose, localized fractionated RT delivered as approximately 10 Gy in five fractions results in increased tumor cell PD-L1 expression, with elevated expression evident 1, 3, and 5 days after the final dose of RT compared to time-matched untreated (NT) mice (Figures 1A and B). This RT-mediated increase in tumor cell PD-L1 expression peaks 72 hours after the final dose of RT, which declines significantly 7 days after RT (compared to expression on days 1 and 3; P<0.05 and P<0.01, respectively, Mann-Whitney test), but remains elevated compared to NT mice (P<0.05, Mann-Whitney test).

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

[0052] Blockade of PD-L1 also improved the response to RT in mice bearing established 4T1 tumors, with the combination therapy significantly reducing tumor burden by 38% compared with RT alone ( Fig. 1E ; 10 days after the start of treatment; 184.3 ± 13.5 mm , respectively). 2 292.8±14.3mm 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 bearing established 4434 melanoma (Fig. 1F). Combination therapy with local RT and mAbs targeting either PD-1 or PD-L1 was well tolerated in both BALB / c and C57Bl / 6 mice (Fig. 8A and B). These preclinical data clearly demonstrate the potential for improving prognosis following low-dose fractionated RT of established solid tumors through blockade of the PD-1 / PD-L1 axis.

[0053] Example 3. NK cells contribute to local tumor control following combination therapy but long-term survival is not related to CD8 + Depends on T cells We next investigated the mechanisms underlying the long-term tumor control observed following combined RT and αPD-L1 mAb therapy. First using colony formation assays, we confirmed that αPD-1 and αPD-L1 mAb do not act as radiosensitizers through direct interactions with tumor cells (Figure 9A-C). Using antibody depletion, we then investigated the role of effector T and NK cells in mediating antitumor activity following RT / αPD-L1 mAb combination therapy. Our data demonstrate that as early as 7 days after initiation of a 5-day split RT cycle (with αPD-L1 mAb therapy beginning on day 1 of the RT cycle), tumor burden reduction is evident in mice following combination therapy compared to NT mice (207.5 ± 29.2 mm, respectively). 2 409.4±86.88mm 2 ; P = 0.067, Mann-Whitney U test) (Figure 2A). However, this statistical trend towards reduced volume was not observed in CD8 +Following depletion of either T or NK cells, tumor volumes were not significantly different from the NT cohort (P = 0.52 and P = 0.70, respectively, Mann-Whitney U test), but were significantly greater than mice receiving combination therapy without immune cell depletion (P < 0.01; CD8 depletion vs. combination therapy, and P < 0.05; NK cell depletion vs. combination therapy, Mann-Whitney U test). By day 11 post-treatment, combination therapy significantly reduced tumor burden compared to NT controls (P < 0.001, Mann-Whitney U test). CD8 + Depletion of either T cells or NK cells reduced the efficacy of the combination therapy (P<0.001 and P<0.05, respectively, Mann-Whitney U test), whereas CD8 + The relative contributions of T cells and NK cells become more evident, with CD8 vs. NK cell depletion resulting in significantly reduced tumor control (P<0.05, Mann-Whitney U test). Our data also support the conclusion that CD4 + We also show that T cell depletion improved local tumor control following combination therapy (153.2 ± 27.0 mm, respectively). 2 72.7±17.3mm 2 ; P < 0.05, Mann-Whitney U test).

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

[0055] Example 4. Treatment with αPD-L1 mAb and RT generates protective tumor antigen-specific memory T cell responses We next investigated whether immune memory arose following treatment with RT and αPD-L1 mAb. We show that LTS mice initially treated with RT and αPD-L1 mAb are 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 of over 100 days and expressed IFNγ-producing CD8+ cells following co-culture with a peptide derived from the CT26 tumor-associated antigen (AH1: SPSYVYHQF (SEQ ID NO: 91)), a control peptide (β-galactosidase: TPHARIGL (SEQ ID NO: 93)), or irradiated CT26 cells. + The ability of T cells to produce IFNγ was assessed (Figures 3B and C). Our data show that following co-culture with AH1 peptide, LTS mice harbored significantly higher IFNγ-producing CD8 T cells than naïve mice. + The frequency of T lymphocytes 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 splenocytes with CT26 cells. Comparison of peptide and tumor cell co-cultures showed that memory CD8 +We found that the frequency of T cells was approximately three-fold lower in LTS mice following coculture with tumor cells compared with coculture with the AH1 peptide, which may reflect tumor cell-mediated suppression of T cell activation. Taken together, these data indicate that when RT is combined with blockade of the PD-1 / PD-L1 axis in a mouse model, it can generate protective immunological memory in long-term survivors.

[0056] Example 5. Fractionated RT inhibits tumor cell PD-L1 expression and CD8 + Conferring T cell-dependent adaptive upregulation First, we confirmed that treatment of tumor cells with a series of RT doses in vitro did not have any direct effect on PD-L1 expression (Figure 4A). To identify which cell populations within the tumor microenvironment are involved in regulating tumor cell expression of PD-L1, split RT cycles were administered to mice in combination with CD8, CD4 or NK cell depleting antibodies. Our data suggest that CD8 but not NK cells are involved in regulating tumor cell expression of PD-L1. + We show that depletion of T cells completely abrogates RT-mediated upregulation of PD-L1 on tumor cells (Figures 4B and C). Interestingly, CD4 + We found that T cell depletion further enhanced RT-mediated upregulation of PD-L1 on tumor cells (approximately two-fold compared with treatment with RT alone).

[0057] Example 6. Adaptive upregulation of PD-L1 by tumor cells following fractionated RT is IFNγ dependent Given the clinical correlation between IFNγ and PD-L1 expression in the tumor microenvironment (16), and the influence of TNFα on this response (22), we assessed the impact of these cytokines on PD-L1 in our cell lines. Co-culture of tumor cells with recombinant IFNγ results in a striking 20-fold increase in cell surface expression of PD-L1 in vitro (Figure 5A). Furthermore, while the addition of recombinant TNFα alone does not affect tumor cell PD-L1 expression, a mixture of both IFNγ and TNFα can further enhance tumor cell PD-L1 expression (approximately 2-fold) compared to expression by IFNγ alone (Figure 5A). We show that silencing of IFN-γ-receptor 1 (IFNγR1) on CT26 tumor cells using ShRNA completely abrogates 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 the production of IFNγ and TNFα, we co-cultured isolated resting (PBS-treated) and activated (phorbol 12-myristate 13-acetate (PMA) and ionomycin-treated) splenocytes (Figure 5B and C) with WT CT26 cells and those transduced with either non-targeting ShRNA (NTC ShRNA) and IFNγR1 ShRNA. Importantly, these experiments show that activated immune cells can increase tumor cell PD-L1 expression to physiologically relevant concentrations of IFNγ and TNFα in an IFNγR1-dependent manner.

[0059] To confirm the role of IFNγ on 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 the levels observed on ex vivo cultured CT26 cells; suggesting adaptive upregulation of PD-L1 following tumor implantation (Figure 5D and E). However, the marked 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. Combination scheduling of αPD-L1 mAb and RT produces effective anti-tumor immune responses We evaluated three different combination schedules in which mice bearing established CT26 tumors were administered a fractionated RT cycle of approximately 10 Gy in five fractions with administration of αPD-L1 mAb starting either on day 1 of the fractionated RT cycle (schedule A), on day 5 of the cycle (schedule B) or 7 days after completion of RT (schedule C) (Figure 6A). No significant difference in overall survival was found between schedules A and B, with LTS of 60% and 57%, respectively (P>0.05 log-rank; Mantel-Cox test) (Figure 6B and Figure 10A). In contrast, despite similar tumor burden across groups, RT followed by sequential treatment with αPD-L1 mAb 7 days later (schedule C) was completely ineffective in improving overall survival compared to RT alone (median survival of 30 and 35 days, respectively; P>0.05 log-rank; Mantel-Cox test) (Figure 10B and C).

[0061] Tumor-infiltrating CD4 + and CD8 + Analysis of T cells shows increased expression of PD-1 compared to time-matched NT controls (P<0.05, Mann-Whitney test) (Figure 6C). In contrast, 7 days after RT, CD4 +No changes in PD-1 expression on T cells were evident, and CD8 + We found that PD-1 expression was significantly decreased on CD4 T cells (P<0.05, Mann-Whitney test) (Fig. 6D). + Tumor-infiltrating CD8 + was found to be higher than that of chronic αPD-L1 mAb (Figure 6C and D). We next compared activity following acute vs. chronic αPD-L1 mAb dosing protocols, in which mice were given either 3 doses of mAb concomitant with RT, or the same doses administered chronically for an additional 2 weeks (3qw). No additional benefit was observed with chronic αPD-L1 mAb dosing (Figure 11).

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

[0063] Example 8. Discussion of the Previous Examples Low-dose fractionated RT inhibits CD8 + Secondary to T cell IFNγ production, this leads to upregulation of PD-L1 expression on tumor cells. In mouse models of melanoma, colorectal, and breast cancer, we show that the activity of RT can be improved through combination with αPD-L1 mAb, leading to the development of immune memory that can protect against tumor recurrence in long-term surviving mice. Furthermore, our data show that dosing regimens may affect prognosis, and that concurrent, but not sequential, treatments are effective in improving local tumor control and survival.

[0064] This is because split RT is a CD8 +This is the first preclinical study to show that T cell production of IFNγ leads to increased tumor cell expression, where tumor cell PD-L1 expression may serve as a biomarker of local antitumor response, and that local RT is associated with increased expression of CD8 + It is suggested that RT alone may be sufficient to stimulate T cell responses. However, while treatment with RT alone cannot produce sustained anti-cancer immunity, activity was found to be enhanced through mAb-mediated blockade of either PD-1 or PD-L1, suggesting that signaling through this system may limit immune responses to RT. We did not observe a 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 is likely that the activity of these mAbs, when delivered in combination with RT, occurs through blockade of PD-1 / PD-L1 signaling and is not mediated through the PD-L1 / CD80 or PD-1 / PD-L2 systems, although further experiments are required to confirm this.

[0065] Our preclinical data show that NK cell depletion reduces local tumor control at early time points (up to 11 days after completion of RT) but does not affect overall survival. Furthermore, NK cell depletion does not affect PD-L1 expression on tumor cells following RT, suggesting their limited contribution to local IFNγ production. In contrast, following combination therapy, CD4 + While T cell depletion did not affect survival, a significant increase in PD-L1 expression on tumor cells after RT was observed. + T cell depletion affects both helper T cell and Treg numbers. These CD4 + Although further studies are required to delineate the relative contributions of T cell subpopulations, we found that in our model, effective CD8 T cell proliferation was observed following RT / PD-L1 mAb therapy. + To generate a T cell response, CD4 +One might speculate that helper T cells are dispensable and that either helper T cells, possibly Tregs, may actively suppress IFNγ production within the local tumor environment. Thus, the therapeutic ability to promote antitumor responses by Treg depletion may be attenuated, at least in part, through enhanced PD-L1 expression on tumor cells.

[0066] PD-L1 expression can be regulated by several cytokines, including type 1 and type 2 IFN, TNFα, and TGFβ (16,22,30-32). We show that tumor cell PD-L1 expression can be enhanced following coculture with IFNγ, and addition of TNFα can further enhance this response. However, blockade of IFNγR1 or in vivo depletion of IFNγ demonstrates the dependency of PD-L1 upregulation on IFNγ-mediated signaling, and TNFα alone cannot regulate tumor cell PD-L1 expression. Interestingly, in vivo depletion of IFNγ reduces PD-L1 expression on syngeneic tumor cells, consistent with the tumor cell expression profile in vitro. This suggests that the local tumor microenvironment in the absence of therapeutic intervention may foster immunologically driven tumor adaptation that may support tumor development. Similarly, in human melanocytic lesions, PD-L1 expression is upregulated by CD8 + It has been shown to colocalize with areas of T cell infiltration and is postulated to represent an adaptive mechanism of immune escape.(16) However, in our preclinical mouse models, monotherapy with PD-1 / PD-L1-targeted mAbs demonstrated only modest activity, suggesting that targeting this system alone is unlikely to result in a sustained antitumor immune response in all settings, highlighting the need for combinatorial approaches.

[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 biologically effective dose with fractionated RT delivery using approximately 10 Gy in five daily fractions. This is an important finding given that this fractionation is more commonly used in routine clinical practice. Several studies have evaluated the impact of RT fractionation; comparing a single ablative dose, hypofractionated, and fractionated RT on antitumor immune response development and the tumor microenvironment (4, 5, 25, 26, 33, 34). However, the results of these studies are equivocal, and the optimal RT delivery and the impact of RT fractionation on tumor cell PD-L1 expression require further investigation.

[0068] Previously, no study has addressed the effect of activity sequence on RT and αPD-L1 mAb therapy. This is particularly pertinent given the trend in the clinic to adopt sequence combination regimens as a strategy to maximize tolerability. We show that PD-L1 blockade at the time of RT delivery alone can improve therapeutic response in mouse models, with treatment 7 days after RT completion no longer being better than RT alone. Furthermore, our data show that PD-L1 expression is elevated by 24 hours after completion of a fractionated RT cycle and remains elevated at least 7 days after completion. Furthermore, we show that tumor-infiltrating CD4+ mAb expression is significantly higher in patients with RT than in those with αPD-L1 mAb 24 hours after RT compared to control cohorts. + and CD8 + We found elevated expression of PD-1 on T cells. PD-1 expression is associated with increased expression of CD4 + CD8 vs. T cells + Taken together, these data suggest that fractionated local RT increases the anti-tumor CD8 +Although αPD-L1 mAb may stimulate T cell responses, these may be attenuated by signaling through the PD-1 / PD-L1 system, suggesting that early inhibition of this system may result in a sustained and effective antitumor response. Furthermore, the need for chronic versus acute blockade of the PD-1 / PD-L1 system when combined with RT is unclear. We did not observe a difference in survival when αPD-L1 mAb was administered 3qw concomitantly with RT delivery for 1 week versus a chronic schedule of up to 3 weeks. These data suggest that a 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. Taken together, these data have important implications for clinical trial design.

[0069] In summary, this trial demonstrates that treatment with fractionated RT results in upregulation of PD-L1 expression on tumor cells and that blockade of the PD-1 / PD-L1 axis can enhance immune responses to fractionated RT in multiple syngeneic mouse models of cancer. Our data suggest that a regimen of RT and αPD-L1 mAb may generate a therapeutic immune response by virtue of its ability to reduce tumor burden and improve survival. This therapeutic combination may hold promise in addressing the treatment of many solid malignancies where RT is commonly used, and translation to early stage clinical trials is clearly justified.

[0070] Example 9. Treatment Protocol Patient A has colorectal cancer. In week 1, Patient A is administered an effective amount of radiation therapy in 5 fractopms. In week 1, Patient A is also administered 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 is administered an effective amount of radiation therapy. In week 1, Patient B is also administered a therapeutic dose of MEDI4736 on day 5. The patient repeats this schedule on weeks 3, 5, and 7.

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

[0073] Item 1. a. administering at least one dose of radiation therapy; b. 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 administration of radiation therapy.

[0074] Item 2. The method of item 1, 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.

[0075] Item 3. The method of any one of items 1-2, wherein said dose of radiation therapy is about 11 Gy or less.

[0076] Item 4. The method of item 3, wherein said dose of radiation therapy is about 10 Gy or less.

[0077] Item 5. The method of any one of items 1 to 4, wherein the radiation therapy is fractionated radiation therapy.

[0078] Item 6. The method of item 5, wherein the fractionated radiation therapy comprises 2 to 7 fractions.

[0079] Item 7. The method of item 6, wherein the fractionated radiation therapy comprises five fractions.

[0080] Item 8. The method of any one of items 5-7, wherein the radiation therapy fractions are administered on consecutive days.

[0081] Item 9. The method of any one of items 5-8, wherein the radiation therapy fractions are administered on days 1, 2, 3, 4, and 5.

[0082] Item 10. Any one of the methods of items 7 to 9, in which the radiation therapy comprises approximately 10 Gy in five fractions.

[0083] Item 11. The method of any one of items 1 to 10, wherein at least one PD-1 and / or PD-L1 antagonist is administered on day 1.

[0084] Item 12. The method of any one of items 1 to 11, wherein at least one PD-1 and / or PD-L1 antagonist is administered on day 5.

[0085] Item 13. The method of any one of items 1 to 12, wherein at least one PD-1 and / or PD-L1 antagonist is administered multiple times.

[0086] Item 14. The method of any one of items 1 to 13, wherein at least one PD-1 and / or PD-L1 antagonist is administered three times weekly.

[0087] Item 15. The method of any one of items 1 to 14, wherein the anti-PD-1 and / or PD-L1 antibody or functional portion thereof is MEDI4736.

[0088] Item 16. The method of any one of items 1 to 15, wherein the anti-PD-1 and / or anti-PD-L1 antibody, or functional portion thereof, is pembrolizumab, nivolumab, BMS-936558, AMP-224, or MPDL3280A.

[0089] Item 17. The method of any one of items 1 to 16, wherein the cancer is melanoma, colorectal cancer, or breast cancer.

[0090] Item 18. The method of any one of items 1 to 17, wherein two or more treatment cycles are performed.

[0091] Item 19. The method of item 18, wherein 2 to 8 treatment cycles are performed.

[0092] Item 20. Any one of the methods of items 18 to 19, wherein the treatment cycle is weekly.

[0093] Item 21. Any one of the methods of items 18 to 19, wherein the treatment cycle is every other week.

[0094] References 1.D. Verellen et al., Innovations in image-guided radiotherapy. Nat Rev Cancer 7, 949-960 (2007). 2.LJ 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. Dong et al., Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 8, 793-800 (2002). 13. G. J. Freeman et al., Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192, 1027-1034 (2000). 14. M. J. Butte et al., Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity 27, 111-122 (2007). 15. S. Spranger et al., Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells. Sci Transl Med 5, 200ra116 (2013). 16. J. M. Taube et al., Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med 4, 127ra137 (2012). 17. M. Sznol et al., Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer. Clin Cancer Res 19, 1021-1034 (2013). 18. J. R. Brahmer et al., Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J Clin Oncol 28, 3167-3175 (2010). 19. J. R. Brahmer et al., Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366, 2455-2465 (2012). 20. O. Hamid et al., Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med 369, 134-144 (2013). 21. E. J. Lipson et al., Durable cancer regression off-treatment and effective reinduction therapy with an anti-PD-1 antibody. Clin Cancer Res 19, 462-468 (2013). 22. A. Kondo et al., Interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes. Blood 116, 1124-1131 (2010). 23. S. L. Topalian et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443-2454 (2012). 24. S. L. Topalian et al., Durable Tumor Remission, and Long-Term Safety in Patients With Advanced Melanoma Receiving Nivolumab. J Clin Oncol, (2014). 25. M. Z. Dewan et al., Fractionated but not single-dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody. Clin Cancer Res 15, 5379-5388 (2009). 26. S. J. Dovedi et al., Systemic delivery of a TLR7 agonist in combination with radiation primes durable antitumor immune responses in mouse models of lymphoma. Blood 121, 251-259 (2013). 27. J. Honeychurch et al., Anti-CD40 monoclonal antibody therapy in combination with irradiation results in a CD8 T-cell-dependent immunity to B-cell lymphoma. Blood 102, 1449-1457 (2003). 28. B. C. Burnette et al., The efficacy of radiotherapy relies upon induction of type i interferon-dependent innate and adaptive immunity. Cancer Res 71, 2488-2496 (2011). 29. J. Y. Kim et al., Increase of NKG2D ligands and sensitivity to NK cell-mediated cytotoxicity of tumor cells by heat shock and ionizing radiation. Exp Mol Med 38, 474-484 (2006). 30. L. Deng et al., Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. J Clin Invest 124, 687-695 (2014). 31. JN Ou et al., TNF-alpha and TGF-beta counter-regulate PD-L1 expression on monocytes in systemic lupus erythematosus. Sci Rep 2, 295 (2012). 32. S. Terawaki et al., IFN-alpha directly promotes programmed cell death-1 transcription and limits the duration of T cell-mediated immunity. J Immunol 186, 2772-2779 (2011). 33. D. Schaue et al., Maximizing tumor immunity with fractionated radiation. Int J Radiat Oncol Biol Phys 83, 1306-1310 (2012).

[0095] equivalent The foregoing written specification is deemed sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and examples detail certain embodiments and set forth the best mode contemplated by the inventors. However, no matter how detailed the foregoing is in the text, it will be understood that the embodiments may be practiced in many ways, and the claims encompass any equivalents thereof.

[0096] As used herein, the term about refers to numerical values, including, for example, integers, fractions, and percentages, whether or not expressly indicated. The term about typically refers to a range of numerical values ​​(e.g., + / - 5 to 10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some cases, the term about may include numerical values ​​that are rounded to the nearest significant figure.

Claims

1. a. administering at least one dose of radiation therapy; 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 four days after the administration of radiation therapy.

2. The method of claim 1, 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.

3. 10. The method of claim 1, wherein the dose of radiation therapy is about 70 Gy or less.

4. 4. The method of claim 3, wherein the dose of radiation therapy is about 50 Gy or less.

5. 5. The method of claim 4, wherein the radiation therapy is fractionated radiation therapy.

6. 6. The method of claim 5, wherein the fractionated radiation therapy comprises 2 to 7 fractions.

7. 7. The method of claim 6, wherein the fractionated radiation therapy comprises five fractions.

8. 8. The method of claim 7, wherein the fractionated radiation therapy is administered on consecutive days.

9. 9. The method of claim 8, wherein the fractionated radiation therapy is administered on days 1, 2, 3, 4, and 5.

10. 10. The method of claim 9, wherein the radiation therapy comprises about 70 Gy in five fractions.

11. 2. The method of claim 1, wherein the at least one PD-1 and / or PD-L1 antagonist is administered on day 1.

12. 2. The method of claim 1, wherein the at least one PD-1 and / or PD-L1 antagonist is administered on day 5.

13. 2. The method of claim 1, wherein the at least one PD-1 and / or PD-L1 antagonist is administered multiple times.

14. 14. The method of claim 13, wherein the at least one PD-1 and / or PD-L1 antagonist is administered three times per week.

15. The method of claim 14, wherein the anti-PD-1 and / or PD-L1 antibody or functional portion thereof is MEDI4736.

16. 16. The method of claim 15, wherein the anti-PD-1 and / or anti-PD-L1 antibody or functional portion thereof is pembrolizumab, nivolumab, BMS-936558, AMP-224, or MPDL3280A.

17. 10. The method of claim 1, wherein the cancer is melanoma, colorectal cancer, or breast cancer.

18. 10. The method of claim 1, wherein two or more treatment cycles are performed.

19. 20. The method of claim 18, wherein 2 to 8 treatment cycles are administered.

20. 20. The method of claim 19, wherein the treatment cycle is weekly or biweekly.

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