Cancer treatment methods using a combination of antagonists to pd-1 and pd-l1 with radiotherapy

Abstract

The present invention relates to methods of cancer treatment using a combination of antagonists to PD-1 and PD-L1 with radiotherapy. The present application provides a method of treating cancer in a patient, the method comprising administering at least one dose of radiation therapy and 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 radiation therapy dose or up to and including 4 days thereafter.

Description

[0001] This application is a divisional application of Chinese patent application 201580032538.3, filed on June 17, 2015, entitled "Cancer treatment method using a combination of PD-1 and PD-L1 antagonists and radiotherapy".

[0002] sequence list This application contains a sequence list that has been submitted electronically in ASCII format and is hereby incorporated in its entirety by reference. The ASCII copy created on June 16, 2015, is named B7H1-275WO1_SL.txt and has a size of 103,313 bytes. Technical Field

[0003] Treatment methods for cancer. Background Technology

[0004] Radiation therapy (RT) remains the most important non-surgical treatment for the management of solid malignancies, accounting for approximately 50-60% of all cancer patients. RT is included in treatment regimens to reduce disease recurrence and improve outcomes in most common cancers. 1-3 Overall survival rate in ( ). Although effective, many patients suffer from local recurrence and metastatic disease.

[0005] In addition to the direct cytopenic effect of RT, evidence suggests that the generation of an antitumor immune response may play an important role in the effectiveness of this treatment. 4，5 RT can lead to the expression of outer calreticulin on tumor cells and the release of several damage-associated molecular patterns (DAMPs) (including high-mobility group 1 (HMGB1) and ATP), which can lead to the recruitment and activation of antigen-presenting cells (APCs) and the initiation of tumor antigen-specific T cell responses. 6-10 Despite the frequent occurrence of such immune escapes, tumor recurrence remains a leading cause of death in patients receiving RT. 11 Identification and inhibition of key drivers of immunosuppression can enhance antitumor immune responses and have the potential to improve patient outcomes.

[0006] Therefore, there is an urgent need for new insights into treatment failure and more effective combination therapies for RT.

[0007] The programmed death-1 (PD-1) / programmed death-ligand 1 (PD-L1) axis participates in maintaining peripheral tolerance and regulating acute inflammatory responses by inhibiting T cell function and through apoptosis of activated T cells. 12，13 In addition to binding to PD-1, PD-L1 can also suppress T cell function by interacting with CD80. 14PD-L1 expression is inducible and is thought to respond to local inflammatory environments, particularly type I and type II interferon (IFN). 12、15、16 Although almost undetectable in most normal tissues, PD-L1 expression has been described in a variety of malignant tumors (in ( 17 (Reviewed in )). Importantly, recent clinical studies using PD-1 or PD-L1 targeting monoclonal antibodies (mAbs) have demonstrated encouraging responses in patients with advanced disease. 18-21 ).

[0008] It is hoped that the dosing strategy used in combination with radiotherapy and PD-1 and / or PD-L1 antagonists will produce the greatest benefit for patients with cancer. Summary of the Invention

[0009] According to the description, a method for treating cancer in a patient includes... a. Administering at least one dose of radiation therapy; and b. Administer at least one PD-1 and / or PD-L1 antagonist. At least one of the PD-1 and / or PD-L1 antagonists is administered on the same day as the radiotherapy dose or up to and including 4 days later.

[0010] 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 a functional part thereof.

[0011] In one aspect, this radiotherapy is fractionated radiotherapy. In one mode, fractionated radiotherapy comprises 2 to 7 fractions. In another mode, fractionated radiotherapy comprises 5 fractions.

[0012] In one embodiment, these radiation therapy portions are administered over consecutive days. In another embodiment, these radiation therapy portions are administered on day 1, day 2, day 3, day 4, and day 5. In one embodiment, the radiation therapy comprises approximately 10 Gy in five portions.

[0013] In one aspect, 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 a week.

[0014] In one embodiment, the anti-PD-1 and / or PD-L1 antibody or its functional portion is MEDI4736.

[0015] In another embodiment, the anti-PD-1 and / or anti-PD-L1 antibody or its functional portion is pembrolizumab, nivolumab, BMS-936558, AMP-224, or MPDL3280A.

[0016] On one hand, the cancer is melanoma, colorectal cancer, or breast cancer. On the other hand, more than one treatment cycle is performed. On the other hand, 2-8 treatment cycles are performed. In one model, treatment cycles are performed weekly or every other week.

[0017] Further objects and advantages will be set forth in part in the description which follows, and some objects and advantages will be obvious from the description or will become apparent by practice. These objects and advantages will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims.

[0018] It should be understood that the general description above and the detailed description below are merely illustrative and explanatory, and not intended to limit the scope of the claims.

[0019] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments and, together with the specification, serve to explain the principles described herein. Attached Figure Description

[0020] Figure 1 A- Figure 1 F indicates the activity of blocking PD-1 / PD-L1 axis-enhanced fractionated radiotherapy. A and B The median fluorescence intensity of PD-L1 expression on CT26 cells isolated from the tumor on day 1, day 3, or day 7 after treatment with approximately 10 Gy (5 daily fractions of 2 Gy). A ) and representative histogram ( B ). C and D Mice carrying CT26 tumors received either αPD-1 alone or in combination with αPD-1 at 10 mg / kg three times a week for up to 3 weeks. C or αPD-L1 ( D The mAb combination delivers approximately 10 Gy RT in 5 daily portions of 2 Gy. E Tumor volume in mice carrying 4T1 tumors 10 days after the start of therapy was measured by administering 20 Gy RT in 5 daily fractions of 4 Gy alone or in combination with αPD-L1 mAb. FMice carrying 4434 tumors received approximately 10 Gy RT, delivered in five daily fractions of 2 Gy, alone or in combination with αPD-L1 mAb. The experimental group consisted of at least seven mice and represented at least two independent experiments. A, E and F The mean ± SEM is shown. * P <0.05, **, P <0.01, Mann-Whitney test. C and D , * Indicates significance when compared with control mice. + Indicates significance when compared to a single therapy. *** / +++ , P <0.001, log-rank (Mantel-Cox) test.

[0021] Figure 2 A- Figure 2 C demonstrates that the therapeutic activity of the combination of split RT and αPD-L1 mAb depends on the activity of CD8+ T-lymphocytes. A Tumor volume was measured on the day of treatment with a combination therapy of 2 Gy in 5 fractions and αPD-L1 mAb, and on days 7 and 11 thereafter. Immune cell subsets (CD8, CD4, or NK cells) were reduced one day before treatment, and the reduction was maintained for two weeks. *** , P >0.001, * , P >0.01, * , P >0.05, Mann-Whitney test 。B Survival curve. *** / +++ , P <0.001, log-rank (Mantel-Cox) test. Data represent 10 mice / group. C This represents a density map of peripheral blood that confirms the reduction of immune cell subsets.

[0022] Figure 3 A- Figure 3 C shows that the combination of split RT and αPD-L1 mAb generates protective immune memory. A Using 5×10 5 Survival curves of LTS mice after bilateral re-challenging of CT26 cells. *P<0.05 compared to control mice (log-rank; Mantel-Cox test). B This indicates CD8 isolates from tumor-naïve or LTS mice initially treated with RT and αPD-L1 mAb.+ Dot imprints produced by IFNγ on T cells. C IFNγ isolated from tumor-naïve or LTS mice initially treated with RT and αPD-L1 mAb was co-cultured for 5 days with H2-Ld restriction peptide (AH1 (SPSYVYHQF) (SEQ ID NO: 91); defined CT26 tumor-associated antigen or β-galactosidase (TPHPARIGL) (SEQ ID NO: 92); prokaryotic control peptide) or 50 Gy-irradiated CT26 cells, followed by initiation with 50 Gy-irradiated CT26 cells. + CD8 + Frequency of T cells. * P < 0.05 (Mann-Whitney test). Data represent two independent experiments.

[0023] Figure 4 A- Figure 4 C shows that segmentation RT increases PD-L1 expression in tumor cells in vivo and is CD8-dependent. + T cells. A The expression of PD-L1 on CT26 cells after in vitro treatment with RT (2.5-10 Gy). B and C CT26 cells isolated from the tumor (as CD45) were administered 3 days after receiving a combination of approximately 10 Gy of 2 Gy in 5 daily doses with CD8, CD4, or NK cell depletion antibodies. - Representative contour plot of PD-L1 expression on cell-gated (cell-gated) B ) and median fluorescence intensity ( C * P < 0.05 (Mann-Whitney test). The experimental group consisted of at least 5 mice and represented at least 2 independent experiments.

[0024] Figure 5 A- Figure 5 E shows the CD8 of IFNγ. + T cells are generated after segmentation of RT and are responsible for upregulating PD-L1 expression on tumor cells. A PD-L1 expression was observed in wild-type CT26 tumor cells (WT) or cells transduced with non-targeted control (NTC) or IFNγR1 ShRNA after co-culturing with 20 ng / ml IFNγ, TNFα or a combination of the two cytokines for 24 hours. B The results showed that after treatment with PBS or phorbol 12-myristate 13-acetate (PMA) and iomycin, CD8 + Density map of IFNγ and TNFα expression in T cells; CThe frequency of PD-L1-positive CT26 tumor cells (WT, NTC ShRNA, or IFNγR1 ShRNA) after co-culturing with spleen cells activated by PBS or PMA / iomycin for 24 hours. n / s = P> 0.05, *** P < 0.001 ** P< 0.01 (Double-tailed student) t test). D and E This represents the contour plot of PD-L1 expression on CT26 tumor cells after treatment with RT (approximately 10 Gy in 5 fractions) in vivo in the presence of αIFNγ blocking mAb (or isotype control). D ) and median fluorescence intensity ( E ). P <0.01 (Mann-Whitney test). The experimental group consisted of at least 5 mice.

[0025] Figure 6 A- Figure 6 D shows the effect of dosing regimen on the results, where RT enhancement was observed only in cases of simultaneous rather than sequential αPD-L1 mAb therapy. A A diagram illustrating the dose-time study. Mice received split doses of RT (approximately 10 Gy in 5 daily fractions of 2 Gy), either alone or in combination with αPD-L1 mAb, starting on day 1 of the RT cycle (Protocol A), day 5 of the RT cycle (Protocol B), or 7 days after the last dose of RT (Protocol C). B Survival curve of treatment. ++ Compared with monotherapy, P<0.01 (log-rank; Mantel-Cox test). C and D CD4 24 hours and 7 days after the last dose of RT + ( C ) and CD8 + ( D PD-1 expression on T cells. P <0.05 (Mann-Whitney test). The experimental group consisted of at least 5 mice and represented at least 2 independent experiments.

[0026] Figure 7 The study showed that blocking both PD-1 and PD-L1 did not further enhance the efficacy of the combination of PD-1 and PD-L1 with segmentation RT in mice carrying CT26 tumors. A Survival curves following a fractionated dose RT (approximately 10 Gy in 5 daily fractions of 2 Gy), administered alone or in combination with αPD-1, αPD-L1, or a combination of both mAbs, three times weekly for 3 weeks. n / sP >0.05, log-rank; Mantel-Cox test. The experimental group consisted of at least 5 mice and represented at least 2 independent experiments.

[0027] Figure 8 A- Figure 8 B demonstrates that the combination therapy using fractionated RT and αPD-1 or αPD-L1 mAb was well tolerated in mice. Mice carrying 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 three times weekly for 3 weeks or 1 week. The experimental group included at least 7 mice and represented at least 2 independent experiments. n / s, P >0.05, Mann-Whitney test.

[0028] Figure 9 A- Figure 9 C shows the insensitivity of tumor cells to in vitro radiation-induced cell death after treatment with αPD-1 or αPD-L1 mAb. 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 αPD-1 or αPD-L1.

[0029] Figure 10 A- Figure 10 C shows the effect of the dosing regimen on the results. A Tumor volume following fractionated dose RT (e.g., 10 Gy in 5 daily fractions of 2 Gy), alone or in combination with αPD-L1 mAb starting on day 1 of the RT cycle (Program A), day 5 of the RT cycle (Program B), or 7 days after the last dose of RT (Program C). B and C Tumor volume in RT-treated mice shows equal tumor volume across different dosing regimens. n / s P >0.05 (Mann-Whitney test). The experimental group consisted of at least 5 mice and represented at least 2 independent experiments.

[0030] Figure 11 The results showed that acute versus chronic administration of αPD-L1 mAb did not affect the efficacy of the combination therapy. A Mice carrying CT26 tumors received either αPD-L1 mAb alone or in combination with αPD-L1 mAb administered at 10 mg / kg three times weekly for 3 weeks or 1 week, delivered in five daily fractions at a dose of 2 Gy. The experimental group consisted of at least seven mice and represented at least two independent experiments. n / s P >0.05, log-rank; Mantel-Cox test.

[0031] Sequence Description Table 1 provides a list of certain sequences mentioned in the current embodiments. CDRs are indicated in bold and underlined. Detailed Implementation

[0032] I. Treatment methods The methods of the present invention cover the treatment of cancer with 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, wherein the at least one PD-1 and / or PD-L1 antagonist is administered on the same day as the radiation therapy dose or at least four days later.

[0033] This cancer treatment can be administered once (as a single treatment cycle) or it can be administered more than once (i.e., using multiple treatment cycles), such as weekly, every other week, every three weeks, or monthly cycles. If the treatment is administered more than once, it can be administered 2, 3, 4, 5, 6, 7, or 8 times or more.

[0034] Although not limited by theory, within the established syngeneic tumor models, low-dose fractionated RT was found to upregulate PD-L1 expression in tumor cells in vivo. This indicates that fractionated RT delivered in combination with αPD-1 or αPD-L1 mAb produced effective anti-tumor CD8+ expression. + This response improves local tumor control and long-term survival, and protects against tumor reactivation by inducing a tumor antigen-specific memory immune response. IFNγ's CD8... +T cell production has been found to be responsible for upregulating PD-L1 on tumor cells after fractionated radiotherapy (RT). Furthermore, the delivery schedule of anti-PD-L1 mAbs relative to fractionated RT appears to influence treatment outcomes; administration of the antagonist on the same day as RT or within 4 days after the first dose of fractionated RT shows superior benefit compared to administration more than 7 days after the end of radiotherapy. Moreover, while not theoretically confined, upregulation of PD-L1 expression on tumor cells in response to low-dose fractionated RT appears to be a mechanism of adaptive immune resistance in tumor cells, as is routinely used clinically; the PD-L1 / PD-1 signaling axis thus potentially contributes to RT treatment failure. Combination therapy using RT and blocking the PD-1 / PD-L1 signaling axis has the potential to overcome this resistance, but based on preclinical studies, dose-time affects treatment outcomes, providing important new insights for clinical translation.

[0035] A. PD-1 and / or PD-L1 antagonists used in treatment methods In one embodiment, at least one PD-1 and / or PD-L1 antagonist used in the method of the present invention is an anti-PD-1 and / or anti-PD-L1 antibody or a functional portion thereof.

[0036] In one aspect, the at least one PD-1 and / or PD-L1 antagonist is administered on the same day as the radiotherapy dose or up to and including four days later. For example, in one aspect, if radiotherapy is administered on day 1 of the treatment cycle, 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 the radiotherapy dose). In another aspect, if radiotherapy is administered on day 1 of the treatment cycle, the at least one PD-1 and / or PD-L1 antagonist is administered on day 5 (i.e., four days later). In yet another aspect, 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 single and multiple treatment regimens).

[0037] In one embodiment, the at least one PD-1 and / or PD-L1 antagonist is administered multiple times during a treatment cycle. In another embodiment, the 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 three times a week for one week of treatment, or three times a week for two weeks or more of treatment cycles, as described above.

[0038] In one respect, the antibody or its functional portion is selected from those disclosed in U.S. Publication 2010 / 0028330, which is incorporated by reference for the purpose of teaching those antibodies and their functional portions. In one embodiment, the antibody is MEDI4736.

[0039] In another embodiment, the anti-PD-1 and / or anti-PD-L1 antibody is pambrolizumab, nivorumab, BMS-936558, AMP-224, or MPDL3280A.

[0040] These antibodies or their functional portions are administered at a therapeutically effective dose. Generally, the therapeutically effective dose can vary depending on the subject's age, condition, sex, and the severity of their medical condition. The therapeutically effective dose of the antibody or its functional portion ranges from about 0.001 to about 30 mg / kg body weight, from about 0.01 to about 25 mg / kg body weight, from about 0.1 to about 20 mg / kg body weight, or from about 1 to about 10 mg / kg. This dose may be adjusted as needed to suit the observed therapeutic effect. The appropriate dose is selected by the treating physician based on the clinical indication.

[0041] These antibodies can be administered as a concentrated dose to maximize the duration of antibody circulation after that dose. Continuous infusion can also be used after the concentrated dose.

[0042] As used herein, the term antibody or its functional portion is used in the broadest sense. It can be artificial, such as monoclonal antibodies (mAbs) and / or functional fragments thereof produced by conventional hybridoma techniques, recombinant techniques. It can include complete immunoglobulin molecules, such as polyclonal antibodies, monoclonal antibodies (mAbs), monospecific antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, animal antibodies (e.g., camel antibodies), chimeric antibodies, and their portions, fragments, regions, peptides, and derivatives (provided by any known technique such as, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant techniques), such as immunoglobulins lacking a light chain, Fab, Fab', F(ab')2, Fv, scFv, antibody fragments, biantibodies, Fd, CDR regions, or any portion or peptide sequence of an antibody capable of binding an antigen or epitope. In one embodiment, the functional portion is a single-chain antibody, a single-chain variable region fragment (scFv), a Fab fragment, or an F(ab')2 fragment.

[0043] An antibody or functional moiety is said to be "capable of binding" to a molecule if it can specifically react with that molecule to bind to the antibody. Antibody fragments or moieties may lack the Fc fragment of the intact antibody, be cleared from circulation more rapidly, and may have less nonspecific tissue binding than the intact antibody. Examples of antibodies can be produced from intact antibodies using methods well known in the art, such as by proteolytic cleavage with enzymes such as papain (to produce the Fab fragment) or pepsin (to produce the F(ab')2 fragment). Antibody moieties can be produced by any of the methods described above, or by expressing a portion of a recombinant molecule. For example, one or more CDR regions of a recombinant antibody can be isolated and subcloned into a suitable expression vector.

[0044] In one embodiment, the antibody or functional portion is a human antibody. Using human antibodies in human therapies can reduce the likelihood of side effects due to immune responses against non-human sequences in an individual. In another embodiment, the antibody or functional portion is humanized. In yet another embodiment, the antibody or functional portion is a chimeric antibody. In this way, sequences of interest, such as binding sites of interest, can be included in the antibody or functional portion.

[0045] In one embodiment, the antibody may have an isotype of IgG, IgA, IgM, or IgE. In one embodiment, the antibody is IgG.

[0046] B. Radiation therapy used in treatment methods Radiotherapy, also known as high-dose ionizing radiation, is a component of the treatment method of this invention.

[0047] In one mode, 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 yet another embodiment, the fractionated radiotherapy comprises 4 to 5 fractions. In one mode, the fractionated radiotherapy comprises 2, 3, 4, 5, 6, or 7 fractions. In one embodiment, the fractionated radiotherapy comprises 5 fractions.

[0048] In one mode, these radiation therapy portions are administered over consecutive days. In another mode, the radiation therapy may include more than one dose per day and / or multiple doses over consecutive days. In one mode, these radiation therapy portions are administered on day 1, day 2, day 3, day 4, and day 5. In yet another mode, the radiation therapy comprises approximately 10 Gy in 5 portions (i.e., 2 Gy per day over 5 days).

[0049] Other fractionation schemes may be used, including accelerated fractionation (treatment administered at larger daily or weekly doses to reduce the number of treatment weeks), hyperfractionation (smaller doses of radiation administered more than once a day), or hypofractionation (larger doses administered once a day or at a lower frequency to reduce the number of treatments).

[0050] Radiation therapy can use X-rays, gamma rays, or charged particles. It can be external beam radiation therapy or internal radiation therapy (also known as short-range radiation therapy). Whole-body radiation therapy using radioactive materials such as radioactive iodine can also be employed.

[0051] External beam radiotherapy includes 3D conformational radiotherapy, intensity-modulated radiotherapy, image-guided radiotherapy, tomography, stereotactic radiosurgery, proton therapy, or other charged particle beams.

[0052] C. Cancer used for treatment The method of this invention can be used to treat various types of cancer. In one aspect, the method can be used to treat melanoma, colorectal cancer, or breast cancer. In one embodiment, the breast cancer is triple-negative breast cancer.

[0053] In one embodiment, the cancer is an adrenocortical tumor, adrenal carcinoma, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, thoracic cancer, colon cancer, colorectal cancer, endometrial cancer, epidermoid carcinoma, 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 (such as hepatocellular carcinoma), lung cancer (including non-small cell lung cancer). Cancer, small cell lung cancer and lung carcinoid tumors), lymph node cancer, lymphoma, cutaneous lymphoma, melanoma, mesothelioma, oral cancer, multiple myeloma, nasal cavity and sinus cancer, nasopharyngeal carcinoma, 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, thymic cancer, pharyngeal cancer, thyroid cancer, uterine cancer, vaginal cancer or vulvar cancer.

[0054] Reference will now be made in detail to the present exemplary embodiments, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numerals will be used throughout the drawings to refer to the same or similar parts. Other embodiments will also be apparent to those skilled in the art upon consideration of the description and practice disclosed herein. These embodiments are further explained in the following examples. These examples are not intended to limit the scope of the claims, but are merely illustrative of certain embodiments. It is intended that the specification and examples be considered exemplary only, wherein the true scope and spirit of the invention will be indicated by the following claims.

[0055] The present invention also relates to the following embodiments: 1. A method for treating a patient's cancer, the method comprising: a. Administration of at least one dose of radiation therapy; and b. Administer at least one PD-1 and / or PD-L1 antagonist. At least one of the PD-1 and / or PD-L1 antagonists is administered on the same day as the radiotherapy dose or up to and including 4 days later.

[0056] 2. The method as described in embodiment 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.

[0057] 3. The method as described in embodiment 1, wherein the radiation therapy dose is about 70 Gy or less.

[0058] 4. The method as described in embodiment 3, wherein the radiation therapy dose is about 50 Gy or less.

[0059] 5. The method as described in implementation scheme 4, wherein the radiotherapy is fractionated radiotherapy.

[0060] 6. The method as described in embodiment 5, wherein the fractionated radiotherapy comprises 2 to 7 segments.

[0061] 7. The method as described in embodiment 6, wherein the fractionated radiotherapy comprises 5 parts.

[0062] 8. The method as described in implementation scheme 7, wherein these radiotherapy portions are administered over consecutive days.

[0063] 9. The method as described in embodiment 8, wherein these radiotherapy portions are administered on day 1, day 2, day 3, day 4, and day 5.

[0064] 10. The method as described in embodiment 9, wherein the radiotherapy comprises approximately 70 Gy in 5 parts.

[0065] 11. The method of embodiment 1, wherein the at least one PD-1 and / or PD-L1 antagonist is administered on day 1.

[0066] 12. The method of embodiment 1, wherein the at least one PD-1 and / or PD-L1 antagonist is administered on day 5.

[0067] 13. The method of embodiment 1, wherein the at least one PD-1 and / or PD-L1 antagonist is administered multiple times.

[0068] 14. The method as described in embodiment 13, wherein the at least one PD-1 and / or PD-L1 antagonist is administered three times a week.

[0069] 15. The method as described in embodiment 14, wherein the anti-PD-1 and / or PD-L1 antibody or its functional portion is MEDI4736.

[0070] 16. The method of embodiment 15, wherein the anti-PD-1 and / or anti-PD-L1 antibody or its functional portion is pambrolizumab, nivorumab, BMS-936558, AMP-224 or MPDL3280A.

[0071] 17. The method as described in implementation scheme 1, wherein the cancer is melanoma, colorectal cancer, or breast cancer.

[0072] 18. The method as described in implementation scheme 1, wherein more than one treatment cycle is performed.

[0073] 19. The method as described in implementation scheme 18, wherein 2 to 8 treatment cycles are performed.

[0074] 20. The method as described in implementation scheme 19, wherein these treatment cycles are weekly or every other week.

[0075] Example Example 1. Method A) Mice and cell lines BALB / c and C57Bl / 6 mice were obtained from Harlan, United Kingdom. All animal experiments were approved by the local ethics committee and conducted with permission from the United Kingdom Home Office. They will be derived from BRafV600Ep16. - / -CT26 mouse colon cancer cells (ATCC) and 4434 cells isolated from mice (Richard Marias, Cancer Research UK, Manchester Institute) were maintained in DMEM, while 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 underwent routine screening to confirm the absence of mycoplasma contamination.

[0076] B) Tumor Therapy Mice were subcutaneously (sc) inoculated with 5 × 10 5 CT26 cells, 1×10 5 4 T1 cells, or 5 × 10 6 4434 cells. Using a Pantak HF-320 320 kV X-ray unit (Gulmay Medical, UK), 7-10 days after inoculation (when the tumor is at least 100 mm). 3 Irradiation was performed at 300 kV and 9.2 mA, with filters fitted into the X-ray beam to achieve a radiation mass of 2.3 mm Cu half-value layer. Mice were positioned 350 mm from the X-ray focal spot, with a dose rate of 0.80 Gy / min. αPD-1 (clone RMPI-14), αPD-L1 (clone 10F.9G2) (both from Biolegend), or isotype control mAbs (IgG2a and IgG2b, respectively) were administered intraperitoneally (ip) at a dose of 10 mg / kg in PBS at a dose volume of 100 μl / 10 g, starting on day 1 of the split RT cycle (unless otherwise stated), three times a week for three weeks. For cell and cytokine reduction assays, mice received αCD8 mAb; clone YTS169 (a gift from M. Glennie, University of Southampton); αCD4 mAb; clone GK1.5 (Biolegend); αAsialo-GM1 (Wako Chemicals); or αIFNγ; clone XMG1.2 (BioXcell). Peripheral blood was used for cytokine reduction during treatment. For tumor re-challenge assays, long-term survivor (LTS) mice were bilaterally engrafted with tumor cells at least 100 days after previous tumor implantation. Control mice were also engrafted to confirm tumor growth. Experimental groups consisted of at least 5 mice / groups and represented at least 2 independent experiments.

[0077] C) Measure CD8 isolated from long-lived mice + T cell cytokine production For in vitro stimulation, 3.5 × 10⁻⁶ ions from LTS or control mice will be used. 6 One splenocyte was irradiated with 50 Gy 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 at a dose of 50 Gy. 6 Tumor cells were cultured for 5 days in the presence of either 1 μmol / ml of the H2-Ld restriction peptide SPSYVYHQF (SEQ ID NO: 91) (AH1) / TPHPARIGL (SEQ ID NO: 92) (β-galactosidase) (Anaspec, UK). Experimental groups consisted of 3–5 mice and represented two independent experiments. After 5 days of culture, cells were restimulated for 16 hours at a 1:1 ratio with 50 Gy irradiated tumor cells in the presence of 3 μg / ml brevidin A (BD Pharmingen, UK) and 100 IU / ml human recombinant IL-2 (Chiron, NL). For FACS analysis, cells were washed and incubated with rat anti-CD16 / 32 (eBioscience, UK) to block non-specific binding and subsequently stained with FITC-conjugated anti-CD8α mAb (eBioscience, UK). Cells were then fixed / permeable and stained for IFNγ expression using APC-conjugated mAbs (eBioscience, UK).

[0078] D) Phenotypic analysis of tumor and immune cells by flow cytometry To obtain single-cell suspensions, tumors were processed using a mild Macs dissociator and a mouse tumor dissociation 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 (all from eBioscience unless otherwise stated) was examined by multiparameter flow cytometry.

[0079] E) In vitro co-culture Tumor cells were cultured for 24 hours in the presence of 20 ng / ml IFNγ and / or TNFα, and PD-L1 expression was evaluated by flow cytometry as described above. For co-culture assays, resting or activated spleen cells (treated with PBS or a mixture of phorbol 12-myristate 13-acetate and iomycin cell stimulation, respectively (eBioscience, UK)) were co-cultured with tumor cells at a 1:1 ratio, and PD-L1 expression in tumor cells was measured as described above. Silencing of IFNγR1 expression was achieved by transducing cells with lentiviruses containing shRNA (cells were also transduced with non-targeted shRNA as a control) (Thermo Scientific, UK). Intracellular flow cytometry was used to measure spleen cell cytokine production (IFNγ and TNFα) as described above.

[0080] Example 2. Blocking PD-1 or PD-L1 enhances the therapeutic efficacy of RT. Radiotherapy has been shown to modulate the immunogenicity of tumor cells, but rarely has it been able to generate a durable therapeutic response that brings about systemic antitumor immunity on its own. Low-dose locally fractionated RT, delivered as approximately 10 Gy in 5 fractions, was shown to produce increased PD-L1 expression in tumor cells compared to time-matched untreated (NT) mice, with the elevated expression being most pronounced on days 1, 3, and 5 following the last dose of RT. Figure 1 A and Figure 1 B This RT-mediated increase in PD-L1 expression in tumor cells peaked 72 hours after the last dose of RT, and although it significantly decreased 7 days after RT (when compared with expression on day 1 and day 3; respectively). P <0.05 and P <0.01, Mann-Whitney test), but still elevated when compared to NT mice ( P <0.05, Mann-Whitney test).

[0081] Given these observations, it is hypothesized that the immune response following RT can be limited via the PD-1 / PD-L1 axis. Local RT, delivered as approximately 10 Gy in 5 daily fractions, was found to significantly improve survival in mice carrying established CT26 tumors compared to the NT control. Figure 1 C and Figure 1 D ; P <0.05 log-rank; Mantel-Cox test). Data demonstrate that this RT-mediated local tumor control can be substantially improved by combination with αPD-L1 mAb or αPD-1 mAb. Figure 1 C and Figure 1 D ; P <0.001 log-rank; Mantel-Cox test). Combination therapy produced a synergistic antitumor response, effective in 66% and 80% of mice receiving RT in combination with αPD-L1 mAb or αPD-1 mAb, respectively. Monotherapy with αPD-L1 mAb or αPD-1 mAb did not significantly improve survival compared to combination with RT. Figure 1 C and Figure 1 D ; P >0.05 log-rank; Mantel-Cox test). Furthermore, no significant benefit was observed when mice received a combination of RT with both αPD-1 and αPD-L1 mAbs compared to any single mAb combination. Figure 7 ).

[0082] Blocking PD-L1 also improved the response to RT in mice carrying established 4T1 tumors, with the combination therapy significantly reducing tumor burden by 38% compared to RT alone. Figure 1 E ; 10 days after treatment began; distribution 184.3 ± 13.5 mm 2 Comparison: 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 carrying established 4434 melanomas ( Figure 1 F The combination therapy of local RT and mAbs targeting PD-1 or PD-L1 was well tolerated in both BALB / c and C57Bl / 6 mice. Figure 8 A and Figure 8 B These preclinical data clearly demonstrate the potential to improve outcomes following low-dose fractionation RT in established solid tumors by blocking the PD-1 / PD-L1 axis.

[0083] Example 3. NK cells contribute to local tumor control after combination therapy, but long-term survival depends on CD8. + T cells Next, the potential mechanisms of long-term tumor control observed after combination RT and αPD-L1 mAb therapy were investigated. Initially, colony formation assays were used to confirm that αPD-1 and αPD-L1 mAb do not act as radiosensitizers through direct interaction with tumor cells. Figure 9 A -9 C Using ablation antibodies, we next investigated the roles of effector T cells and NK cells in mediating antitumor activity following RT / αPD-L1 mAb combination therapy. Data demonstrated that, compared to NT mice, as early as 7 days after the start of the 5-day split RT cycle (with αPD-L1 mAb therapy starting on day 1 of that RT cycle), the reduced tumor burden was significant in mice following combination therapy (207.5 ± 29.2 mm). 2 Comparison: 409.4 ± 86.88 mm 2 ; P = 0.067, Mann-Whitney test) Figure 2 A However, this statistical trend of volume reduction is reflected in CD8. + After the reduction or loss of T cells or NK cells, the tumor volume was not significantly different from that in the NT group (respectively). P = 0.52 and P = 0.70, Mann-Whitney U test), but significantly larger than mice without immune cell reduction that received combination therapy ( P <0.01; combination therapy compared to CD8 reduction, and P <0.05; combination therapy versus NK cell reduction, Mann-Whitney U test). By day 11 post-treatment, the combination therapy significantly reduced tumor burden compared to the NT control ( P <0.001, Mann-Whitney U test). And at this time point, CD8 is reduced. + T-cell or NK-cell suppression reduces the efficacy of combination therapy (respectively). P <0.001 and P <0.05, Mann-Whitney U test), CD8 + The relative contributions of T cells and NK cells became more pronounced, with tumor control significantly reduced in mice with CD8-to-NK cell depletion. P <0.05, Mann-Whitney U test). The data also revealed CD4 + T-cell reduction improved local tumor control after combination therapy (153.2 ± 27.0 mm). 2 Comparison: 72.7 ± 17.3 mm 2 ; P <0.05, Mann-Whitney U test).

[0084] Compared to early tumor control, long-term survival (LTS) was not affected by NK cell depletion after combination therapy with RT / αPD-L1 mAb (70% LTS in mice treated with RT / αPD-L1 compared to 77.8% in mice with NK cell depletion after combination therapy). Figure 2 B CD4 + The reduction of T lymphocytes increased the frequency of LTS mice from 70% to 87.5% (combination vs. combination + CD4 reduction), but this was not statistically significant. P >0.05 log-rank; Mantel-Cox test). However, reducing CD8 + Therapeutic efficacy of T-cell complete elimination combined with RT / αPD-L1 mAb therapy ( P <0.001 log-rank; Mantel-Cox test). The reduction in immune cell populations was confirmed by flow cytometry in peripheral blood samples. Figure 2 C These data suggest that while NK cells can exert some local tumor control, this does not affect overall survival, and although CD4... + T cells are unnecessary or even capable of suppressing the response, but CD8 + T cells appear to mediate effective long-term tumor control following treatment with RT and αPD-L1 mAb.

[0085] Example 4. Treatment with αPD-L1 mAb and RT elicits a protective tumor antigen-specific memory T cell response. The next investigation focused on whether immune memory was generated after treatment with RT and αPD-L1 mAb. The results showed that LTS mice initially treated with RT and αPD-L1 mAb were able to completely reject the tumor after bilateral re-challenge. Figure 3 A To quantify this memory response, spleen cells harvested from LTS mice that exhibited greater than 100 days of disease-free survival and CD8+ after co-culturing 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 ( Figure 3 B and Figure 3 C Data revealed that LTS mice, after being co-cultured with the AH1 peptide, were conferred significantly greater CD8+ production of IFNγ compared to mice receiving the experiment for the first time. +The frequency of T lymphocytes was 6.6% ± 0.8 compared to 2.3% ± 0.2. P <0.05 (Mann-Whitney test). A similar response was observed after co-culturing spleen cells with CT26 cells. Comparison of peptide and tumor cell co-culture revealed memory CD8. + The frequency of T cells in LTS mice was approximately 3-fold lower after co-culturing with tumor cells than after co-culturing with AH1, reflecting tumor cell-mediated suppression of T cell activation. In summary, these data demonstrate that RT can generate protective immune memory in long-term survivors in mouse models when combined with blockade of the PD-1 / PD-L1 axis.

[0086] Example 5. Segmentation of CD8+ induced by RT to induce PD-L1 expression in tumor cells + T cell-dependent adaptive upregulation Initially, it was confirmed that treating tumor cells with a series of RT doses in vitro had no direct effect on PD-L1 expression. Figure 4 A To identify which cell populations within the tumor microenvironment are responsible for regulating PD-L1 expression in tumor cells, mice were subjected to a combination of split RT cycles and CD8, CD4, or NK cell depletion antibodies. Data revealed that CD8... + The reduction of T cells, rather than NK cells, completely eliminates RT-mediated upregulation of PD-L1 on tumor cells. Figure 4 B and Figure 4 C Interestingly, CD4 + The reduction of T cells was found to further enhance RT-mediated upregulation of PD-L1 on tumor cells (approximately twice that of RT alone).

[0087] Example 6. Adaptive upregulation of PD-L1 in tumor cells after RT segmentation is IFNγ dependent. Given the clinical relevance between IFNγ and PD-L1 expression in the tumor microenvironment ( 16 ) and the effect of TNFα on this response ( 22 The effects of these cytokines on PD-L1 were evaluated in cell lines. Co-culturing tumor cells with recombinant IFNγ resulted in a significant 20-fold increase in cell surface expression of PD-L1 in vitro. Figure 5 A Furthermore, while adding recombinant TNFα alone had no effect on PD-L1 expression in tumor cells, a mixture of IFNγ and TNFα could further enhance PD-L1 expression in tumor cells (approximately 2-fold) compared to IFNγ alone. Figure 5A The silencing of IFN-γ receptor 1 (IFNγR1) on CT26 tumor cells using ShRNA was completely eliminated after co-culture with recombinant IFNγ or both IFNγ and TNFα, followed by upregulation of PD-L1. Figure 5 A).

[0088] To determine whether activated immune cells can induce adaptive changes in PD-L1 expression in tumor cells through the production of IFNγ and TNFα, WT CT26 cells, along with those transduced with non-targeted ShRNA (NTC ShRNA) and IFNγR1 ShRNA, were co-cultured with isolated resting (PBS-treated) and activated (PFRA and iomycin-treated) spleen cells. Figure 5 B and Figure 5 C Importantly, these experiments demonstrate that activated immune cells can increase PD-L1 expression in tumor cells in an IFNγR1-dependent manner at physiologically relevant concentrations of IFNγ and TNFα.

[0089] To confirm the role of IFNγ in tumor cell expression of PD-L1 in response to RT in vivo, mice received a combination of RT and an αIFNγ blocking antibody (or an allotype control). IFNγ blockade reduced PD-L1 tumor cell expression in NT mice by 2.6-fold to levels observed in cultured CT26 cells in vitro; thus indicating that adaptive upregulation of PD-L1 occurs after tumor implantation. Figure 5 D and Figure 5 E However, the significant upregulation of PD-L1 observed after RT (2.4-fold compared to the NT control) was completely eliminated by IFNγ blockade, thus confirming that this is the driver of adaptive tumor cell expression of PD-L1 after RT treatment.

[0090] Example 7. The concomitant use of αPD-L1 mAb and RT to generate an effective antitumor immune response. Three different combination regimens were evaluated in which mice carrying established CT26 tumors received fractionated RT cycles of approximately 10 Gy in five fractions, with αPD-L1 mAb administered on day 1 of the fractionated RT cycle (Rule A), day 5 of the cycle (Rule B), or 7 days after completion of RT (Rule C). Figure 6 A No significant difference in overall survival was found between scheme A and scheme B, with LTS rates of 60% and 57%, respectively. P>0.05 log-rank; Mantel-Cox test) Figure 6 B and Figure 10 A In contrast, despite similar tumor burden across multiple groups, sequential therapy with RT followed by αPD-L1 mAb 7 days later (regimen C) was completely ineffective in enhancing overall survival compared to RT alone (median survival at 30 days vs. 35 days, respectively). P >0.05 log-rank; Mantel-Cox test) Figure 10 B and Figure 10 C ).

[0091] 24 hours after the last dose of RT, tumor-infiltrating CD4 + and CD8 + T cell analysis revealed increased PD-1 expression compared to time-matched NT controls. P <0.05, Mann-Whitney test) Figure 6 C In contrast, by 7 days post-RT, on CD4... + No significant changes were observed in PD-1 expression on T cells, and it was found that PD-1 expression was lower on CD8 cells compared to time-matched NT controls. + Significantly reduced on T cells ( P <0.05, Mann-Whitney test) Figure 6 D PD-1 expression was consistently found in tumor-infiltrating CD8+ cells. + T cells compared to CD4 + Higher on T cells ( Figure 6 C and Figure 6 D Next, the activity following acute-contrast prolonged αPD-L1 mAb dosing regimens was compared, with mice receiving three doses of mAb concurrently with RT, or the same mAb but with prolonged dosing (three times weekly) for an additional two weeks. No additional benefit was observed with prolonged αPD-L1 mAb administration. Figure 11 ).

[0092] In summary, these data demonstrate that low-dose fractionated RT treatment leads to an acute increase in PD-1 expression on T cells, and that sequential therapy (where blockade of the PD-1 / PD-L1 signaling axis is delayed until the completion of the RT cycle) may potentially benefit tumor-responsive CD8+. + The reduction or non-responsiveness of T cells is ineffective.

[0093] Example 8. Discussion of the above examples Low-dose fractionated RT leads to CD8 secondary to IFNγ. + T cell generation and upregulation of PD-L1 expression on tumor cells were observed. In mouse models of melanoma, colorectal cancer, and breast cancer, it was demonstrated that RT activity could be enhanced by combination with αPD-L1 mAb, leading to the development of immune memory in long-term surviving mice that protects against immunotumor recurrence. Furthermore, data revealed that dose-time can influence outcomes, with simultaneous rather than sequential therapy proving effective in improving local tumor control and survival.

[0094] This is used to prove that the segmentation RT is achieved through IFNγ's CD8. + This is the first preclinical study showing that T cell production leads to increased PD-L1 expression in tumor cells. Here, PD-L1 expression in tumor cells can serve as a biomarker for local antitumor responses, suggesting that local tumor response (RT) may be sufficient to elicit CD8+ responses. + T-cell response. However, while RT alone did not produce durable anti-cancer immunity, activity was found to be enhanced via mAb-mediated PD-1 or PD-L1 blockade, suggesting that signaling along this axis could limit the immune response to RT. No significant difference in overall survival was observed between RT delivered in combination with αPD-1 or αPD-L1, or in combination with both mAbs. Given these observations, it appears that the activity of these mAbs when delivered in combination with RT is mediated by blocking PD-1 / PD-L1 signaling, rather than through the PD-L1 / CD80 or PD-1 / PD-L2 axis, but further experiments are needed to confirm this.

[0095] Preclinical data revealed that NK cell reduction decreased local tumor control at an early time point (11 days after RT) but did not affect overall survival. Furthermore, NK cell reduction did not affect PD-L1 expression in tumor cells after RT, suggesting a limited contribution to local IFNγ production. In contrast, while CD4 reduction... + T cells did not affect survival after combination therapy, but a substantial increase in PD-L1 expression in tumor cells was observed after RT. CD4 + The reduction of T cells affects both helper T cells and Treg populations. Further research is needed to characterize CD4. + The relative contributions of these T cell subsets, but can be inferred from CD4 + Helper T cells were effective against CD8 after RT / PD-L1 mAb therapy in the model. +The generation of a T-cell response may be non-essential, and helper T cells, or more likely Tregs, can actively suppress IFNγ production in the local tumor environment. Therefore, the therapeutic potential to enhance anti-tumor responses through Treg attenuation may be at least partially attenuated by enhanced PD-L1 expression on tumor cells.

[0096] PD-L1 expression can be regulated by a variety of cytokines, including type I and type II IFN, TNFα, and TGFβ. 16、 22、30-32 Tumor cell responses to PD-L1 were enhanced upon co-culture with IFNγ, and the addition of TNFα further amplified this response. However, blocking IFNγR1 or in vivo ablation of IFNγ demonstrated the dependence of IFNγ-mediated signaling on PD-L1 upregulation, where TNFα alone could not regulate PD-L1 expression in tumor cells. Interestingly, in vivo ablation of IFNγ reduced PD-L1 expression on syngeneic tumor cells, thus matching the expression profile of tumor cells in vitro. This suggests that the local tumor microenvironment in the absence of therapeutic intervention can promote immune-driven tumor adaptation, which may support tumor development. Similarly, in human melanocytic lesions, PD-L1 expression has been shown to correlate with CD8+. + T cell infiltration is co-localized in certain areas and is considered to represent an adaptive mechanism of immune escape. 16 However, in preclinical mouse models, monotherapy using mAbs targeting PD-1 / PD-L1 showed only moderate activity, suggesting that targeting this axis alone may not elicit a durable anti-tumor immune response in all settings, and highlighting the need for combination approaches.

[0097] Compared to using a single 12 Gy dose ( 30 The results demonstrated that PD-L1 upregulation occurred at a lower biologically effective dose involving the delivery of fractionated RT at approximately 10 Gy across five daily fractions. This is an important finding given that this dosage per fraction is more commonly used in routine clinical practice. Several studies have evaluated the effects of RT dose fractionation on antitumor immune responses and the tumor microenvironment; comparing single ablation doses, macrofractionation, and fractionated RT (…). 4、5、25、26、33、34 However, the results of these studies are inconclusive, and the effects of optimal RT delivery and RT dose fractionation on PD-L1 expression in tumor cells require further investigation.

[0098] Previously, no studies addressed the impact of sequencing on the activity of RT and αPD-L1 mAb therapy. This is particularly relevant given the tendency to use combined sequencing regimens in clinical settings as a strategy to maximize tolerability. It was shown that blocking PD-L1 alone during RT delivery enhanced treatment response in mouse models, where treatment 7 days after completion of RT was no better than RT alone. Furthermore, data revealed that PD-L1 expression increased 24 hours after completion of the split RT cycle and remained elevated for at least 7 days after completion. Additionally, it was found that tumor-infiltrating CD4+ expression was higher 24 hours after RT compared to the control group. + T cells and CD8 + T cells show elevated PD-1 expression. PD-1 expression has been found in CD8 cells. + T cells versus CD4 + The levels were consistently higher on T cells. In summary, these data suggest that locally segmented RT can induce anti-tumor CD8... + T-cell responses are observed, but these may be attenuated via PD-1 / PD-L1 axis signaling, and early inhibition of this axis can produce durable and effective antitumor responses. Furthermore, the requirement for chronic versus acute blockade of the PD-1 / PD-L1 axis when combined with RT is unclear. No difference in survival was observed when αPD-L1 mAb was administered three times a week for one week concurrent with RT delivery, compared to an extended regimen up to three 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. These data together have significant implications for clinical trial design.

[0099] In summary, this study demonstrates that fractionated RT treatment leading to upregulation of PD-L1 expression in tumor cells and blocking the PD-1 / PD-L1 axis enhances the immune response to fractionated RT in multiple syngeneic mouse cancer models. Data indicate that the dose-time series of αPD-L1 mAb and RT can generate a therapeutic immune response with the ability to reduce tumor burden and improve survival. This treatment combination could be a promising approach for treating many solid malignancies, where RT is commonly used and has been clearly approved for transition to early-stage clinical trials.

[0100] Example 9. Treatment Plan Patient A has colorectal cancer. In week 1, Patient A received an effective dose of radiation therapy in five fractions. During week 1, Patient A also received treatment doses of MEDI 4736 on days 1, 3, and 5. Patient A repeated this regimen for weeks 2, 3, 4, and 5.

[0101] Patient B has breast cancer. In week 1, Patient B received an effective dose of radiation therapy. In week 1, Patient B also received a therapeutic dose of MEDI 4736 on day 5. Patient B repeated this regimen for weeks 3, 5, and 7.

[0102] Example 10. Some embodiments The following items provide some of the embodiments disclosed herein.

[0103] Project 1. A method for treating a patient's cancer, the method comprising: a. Administering at least one dose of radiation therapy; and b. Administer at least one PD-1 and / or PD-L1 antagonist. At least one of the PD-1 and / or PD-L1 antagonists is administered on the same day as the radiotherapy dose or up to and including 4 days later.

[0104] Project 2. The method of Project 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.

[0105] Item 3. The method as described in any one of Items 1-2, wherein the radiation therapy dose is about 11 Gy or less.

[0106] Item 4. The method described in Item 3, wherein the radiation therapy dose is about 10 Gy or less.

[0107] Item 5. The method of any one of Items 1-4, wherein the radiotherapy is fractionated radiotherapy.

[0108] Item 6. The method as described in Item 5, wherein the fractionated radiotherapy comprises 2 to 7 segments.

[0109] Item 7. As described in Item 6, wherein the fractionated radiotherapy comprises 5 parts.

[0110] Item 8. The method as described in any one of Items 5-7, wherein the radiotherapy portion is administered over consecutive days.

[0111] Item 9. The method as described in any one of Items 5-8, wherein the radiotherapy portions are administered on day 1, day 2, day 3, day 4, and day 5.

[0112] Item 10. The method as described in any one of Items 7-9, wherein the radiotherapy comprises approximately 10 Gy in 5 parts.

[0113] Item 11. The method of any one of items 1-10, wherein the at least one PD-1 and / or PD-L1 antagonist is administered on day 1.

[0114] Item 12. The method of any one of Items 1-11, wherein the at least one PD-1 and / or PD-L1 antagonist is administered on day 5.

[0115] Item 13. The method of any one of items 1-12, wherein the at least one PD-1 and / or PD-L1 antagonist is administered multiple times.

[0116] Item 14. The method of any one of Items 1-13, wherein the at least one PD-1 and / or PD-L1 antagonist is administered three times a week.

[0117] Item 15. The method of any one of Items 1-14, wherein the anti-PD-1 and / or PD-L1 antibody or its functional portion is MEDI4736.

[0118] Item 16. The method of any one of Items 1-15, wherein the anti-PD-1 and / or anti-PD-L1 antibody or its functional portion is pambrolizumab, nivorumab, BMS-936558, AMP-224 or MPDL3280A.

[0119] Item 17. The method of any one of items 1-16, wherein the cancer is melanoma, colorectal cancer, or breast cancer.

[0120] Item 18. The method of any one of items 1-17, wherein more than one treatment cycle is performed.

[0121] Project 19. As described in Project 18, wherein 2-8 treatment cycles are performed.

[0122] Item 20. The method as described in any one of Items 18-19, wherein the treatment cycles are weekly.

[0123] Item 21. The method as described in any one of Items 18-19, wherein the treatment cycles are every other week.

[0124] References 1. D. Verellen et al., Innovations in image-guided radiotherapy. Nature Review of Cancer ( Nat Rev Cancer )7,949-960 (2007).

[0125] 2. LJ Lee et al., Innovations in radiation therapy (RT) for breast cancer. Breast ( Breast Supplement 3, 18, S103-111 (2009).

[0126] 3. E. Kapiteijn et al., Preoperative radiotherapy combined with total mesorectal excision for resectable rectal cancer. The New England Journal of Medicine. N Engl J Med 345, 638-646 (2001).

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[0128] 5. AA Lugade et al., Local radiation therapy of B16 melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor. Journal of Immunology (Journal of Immunology) J Immunol 174, 7516-7523 (2005).

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[0131] 8. F. Ghiringhelli et al., Activation of the NLRP3 inflammasome indendritic cells induces IL-1β-dependent adaptive immunity against tumors. *Natural Medicine* Nat Med 15, 1170-1178 (2009).

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[0133] 10. J. Honeychurch et al., The immunogenic potential of irradiated lymphoma cells is enhanced by adjuvant immunotherapy and modulation of local macrophage populations. (Leukemia and Lymphoma) Leuk Lymphoma 54, 2008-2015 (2013).

[0134] 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. (Radiotherapy and Oncology) Radiother Oncol 85, 7-16 (2007).

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[0139] 16. JM Taube et al., Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. (Science Translational Medicine) Sci Transl Med 4,127ra137 (2012).

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[0145] 22. A. Kondo et al., in myelodysplastic syndromes, interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB activation in blasts. Blood ( Blood 116, 1124-1131 (2010).

[0146] 23. SL Topalian et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. The New England Journal of Medicine (NJM). N Engl J Med 366, 2443-2454 (2012).

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[0157] equivalent The foregoing written description is considered sufficient to enable those skilled in the art to practice these embodiments. The foregoing description and examples detail certain embodiments and describe the best mode expected by the inventors. However, it will be understood that these embodiments can be practiced in many ways, regardless of how detailed the foregoing is presented in textual form, and the claims include any equivalents thereof.

[0158] As used herein, the term approximation refers to numerical values, including, for example, integers, fractions, and percentages, whether explicitly indicated or not. The term approximation typically refers to a range of numerical values ​​(e.g., + / −5% to 10% of the listed values) that a person skilled in the art would consider equivalent (e.g., having the same function or result). In some cases, the term approximation may include numerical values ​​rounded to the nearest significant figure.

Claims

1. A method for treating a patient's cancer, the method comprising: a. Administration of at least one dose of radiation therapy; and b. Administer at least one PD-1 and / or PD-L1 antagonist. At least one of the PD-1 and / or PD-L1 antagonists is administered on the same day as the radiotherapy dose or up to and including 4 days later.

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

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

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

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

7. The method of claim 6, wherein the fractionated radiotherapy comprises 5 parts.

8. The method of claim 7, wherein the radiotherapy portion is administered over consecutive days.

9. The method of claim 8, wherein the radiotherapy portions are administered on day 1, day 2, day 3, day 4, and day 5.

10. The method of claim 9, wherein the radiotherapy comprises approximately 70 Gy in 5 portions.

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