Compositions and methods for treating lung cancer

Abstract

Disclosed is a method of treating locally advanced (stage III), unresectable non-small cell lung cancer (NSCLC) with an antibody that inhibits PD-1 / PD-L1 activity, concurrently with chemoradiotherapy (cCRT).

Description

[Background technology]

[0001] Lung cancer has been the most common cancer in the world for decades, with an estimated 1.8 million new cases by 2012, representing 12.9% of all new cancers. It is also the most common cause of cancer death, with 1.59 million deaths (19.4% of the total). Non-small cell lung cancer (NSCLC) accounts for approximately 80%-85% of all lung cancers, with 30% of patients presenting with stage III disease. The standard treatment for patients with good performance status (PS) and unresectable stage III NSCLC is concurrent platinum-based doublet chemotherapy and radiation therapy (cCRT) administered with curative intent. Meta-analyses of concurrent versus sequential CRT have demonstrated better outcomes with concurrent therapy, but even with cCRT, 5-year overall survival (OS) rates range from 15% to 32%. Thus, there is a great unmet need for novel therapeutic approaches to boost patient survival beyond cCRT.

[0002] Programmed cell death ligand 1 (PD-L1) on tumor and myeloid cells within the tumor microenvironment binds to and inhibits the activity of the immune checkpoint protein PD-1 on activated T cells. Durvalumab is a selective, high-affinity human IgG1 monoclonal antibody that blocks the binding of PD-L1 to PD-1 and CD80, allowing T cells to recognize and kill tumor cells. Durvalumab has shown encouraging antitumor activity in early clinical studies across multiple advanced solid tumors and is approved for post-platinum locally advanced or metastatic urothelial carcinoma.

[0003] In addressing the need for improved methods of clinical management of locally advanced cancer, the present disclosure provides methods comprising administration of durvalumab concomitantly with chemoradiotherapy (cCRT) to patients with late-stage, locally advanced, unresectable NSCLC. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure relates generally to methods of treating locally advanced (stage III), unresectable non-small cell lung cancer (NSCLC) with antibodies that inhibit PD-1 / PD-L1 activity, in conjunction with chemoradiotherapy (cCRT).

[0005] Provided herein is a method of extending progression-free survival (PFS) in patients with unresectable non-small cell lung cancer (NSCLC), comprising treating the patient concurrently with an anti-PD-L1 antibody and chemoradiation therapy.

[0006] Also provided herein is a method of increasing overall response rate (ORR) in patients with unresectable NSCLC, comprising treating the patient with an anti-PD-L1 antibody and concurrent chemoradiotherapy.Also provided herein is a combination comprising an anti-PD-L1 antibody and concurrent chemoradiotherapy for use in a method of extending progression-free survival (PFS) in patients with unresectable non-small cell lung cancer (NSCLC).

[0007] Also provided herein is a combination comprising an anti-PD-L1 antibody and concurrent chemoradiation therapy for use in a method of increasing the overall response rate (ORR) in patients with unresectable non-small cell lung cancer (NSCLC).

[0008] Also provided herein is a combination comprising an anti-PD-L1 antibody and concurrent chemoradiotherapy for the treatment of stage III unresectable non-small cell lung cancer (NSCLC).

[0009] Also provided is the use of a combination comprising a human anti-PD-L1 antibody and concurrent chemoradiation therapy in the manufacture of a medicament for use in a method of extending progression-free survival (PFS) in patients with unresectable non-small cell lung cancer (NSCLC).

[0010] Also provided is the use of a combination comprising a human anti-PD-L1 antibody and concurrent chemoradiation therapy in the manufacture of a medicament for use in a method of increasing the overall response rate (ORR) in patients with unresectable non-small cell lung cancer (NSCLC).

[0011] Also provided is the use of a combination comprising a human anti-PD-L1 antibody and concurrent chemoradiotherapy in the manufacture of a medicament for the treatment of stage III unresectable non-small cell lung cancer (NSCLC). [Brief description of the drawings]

[0012] [Figure 1]

[0021] FIG. 1 shows the general study design for the methods disclosed herein. [Diagram 2] [Figure 2A] Schema showing treatment schedule and defined endpoints. Each group contained 6 mice. Radiotherapy (RT) was administered when tumors were approximately 100-200 mm3. Assay time points were as indicated. [Figure 2B] Bubble diagram (from Table 4) of longitudinal pathway analysis. Pathways are listed on the Y axis and time points on the X. Bubble size indicates absolute fold change expression value for each pathway. [Figure 2C] Tumor volume at the time of resection is shown. Data are expressed as mean ± SEM. Each group contained 6 mice. *P<0.05, Mann-Whitney test. [Diagram 3][Figures 3A-3C] Network maps showing differentially regulated genes and upstream regulators between NT and RT tumors at each time point. Genes and upstream regulators are separated into their cellular compartments at 1 (Figure 3A), 3 (Figure 3B), and 7 (Figure 3C) days after treatment with RT. [Figure 3D] Bubble diagrams (derived from data in Table 3) of longitudinal pathway analysis. Pathways are listed on the Y axis and time points on the X axis. Bubble color indicates the direction of gene regulation. Bubble size indicates absolute fold change expression value of this pathway. Pathway abbreviations: Crosstalk between dendritic cells (DC) and natural killer (NK) cells; Bacterial and viral recognition - role of pattern recognition receptors in bacterial and viral recognition; Innate and adaptive immune system - communication between innate and adaptive immune cells. [Figure 4] [Figures 4A-4F] RT causes changes in tumor-infiltrating myeloid cell populations. Cells were isolated from untreated (NT) (black bars) or RT-treated (7 Gy) (gray bars) tumors 1, 3, or 7 days after RT (or time-matched NT controls). Figure 4A shows tumor samples analyzed by flow cytometry for the presence of F4 / 80+ cells. Figures 4B and 4C show the expression (MFI) of CD86 and CD206 on F4 / 80+ cells. Representational histograms are shown above the bar graphs corresponding to isotype control (black line), NT (solid black line), and RT (gray line). Figure 4D shows the percentage of CD86+ and CD206+ cells that were F4 / 80+, and box and whisker plots show the CD86+ / CD206+ ratio for NT (black) or RT-treated (gray) tumors. Figures 4E and 4F show the frequency of CD11b+Gr1lo and CD11b+Gr1hi cells in tumor tissues. Mean ± SEM is plotted. Each group contained 6 mice. *P<0.05 and **P<0.01 when comparing NT with the irradiated group. [Diagram 5][Figures 5A-5F] RT affected the frequency and phenotype of tumor-infiltrating lymphocytes. Cells were isolated from untreated (NT) (black bars) or RT-treated (7 Gy) (gray bars) tumors 1, 3, or 7 days after RT (or time-matched NT controls). Figures 5A and 5B show the frequency of CD4+ and CD8+ tumor-infiltrating T cells. Figures 5C and 5D show CD69 expression on CD4+ and CD8+ T cells. Figure 5E shows tumor-infiltrating CD4+CD25+FoxP3+ (Treg) cells expressed as a percentage of the CD4+ population. Figure 5F shows the ratio of CD8+ cells to Treg cells. Means ± SEM are plotted. Each group contained 6 mice. *P<0.05 and **P<0.01 when comparing NT with irradiated groups. [Figure 6] [Figures 6A-6G] RT resulted in high expression of both PD-1 and PD-L1 in tumors, which reduced the efficacy of treatment. Cells were isolated from untreated (NT) (black bars) or RT-treated (7 Gy) (gray bars) tumors 1, 3, or 7 days after RT (or time-matched NT controls). Figures 6A and 6B show PD-1 expression on CD4+ and CD8+ T cells. Figures 6C and 6D show PD-L1 expression on CD4+ and CD8+ T cells. Figure 6E shows PD-L1 expression on CD45- tumor cells. Mean ± SEM is plotted. *P<0.05 and **P<0.01 when comparing NT with the irradiated group. Figures 6F and 6G show tumor growth curves and Kaplan-Meier curves of mice with established tumors following treatment with 7 Gy RT alone or in combination with αPD-L1 mAb administered 3 qw per week at 10 mg / kg. Experimental groups contained at least 6 mice and are representative of two independent studies. ++P<0.01 compared to 7 Gy RT alone. **P<0.01 and ***P<0.001 compared to NT control. [Figure 7][Figures 7A-7B] Heatmaps showing fold changes of lineage (Figure 7A) and phenotypic (Figure 7B) markers from irradiated tumor tissues removed on days 1, 3, and 7 after 7 Gy RT as a percentage of time-matched untreated control tumors. Values ​​in bold boxes were statistically significant compared to time-matched untreated samples. Each group contained 6 mice. (Mann-Whitney U, P<0.05). [Figure 8] [Figures 8A-8D] Gating strategy used to analyze tumor cell populations. Figure 8A shows gating on live leukocytes isolated from spleen used to draw the leukocyte gate. Figure 8B shows CD4+CD25+ FoxP3+ cells in tumor tissue. Figure 8C shows CD11b+Gr1lo and hi populations in spleen and tumor tissue. Figure 8D shows CD45- tumor cells. [Figure 9] [Figure 9] Percentage of CD45+ cells in tumor tissues isolated on days 1, 3, or 7 after NT (black) or 7 Gy (gray) RT. P<0.01, Mann-Whitney U. Each group contained 6 mice. **P<0.01, Mann-Whitney test. [Figure 10] [Figures 10A-10B] Depictive histograms of CD69 (Figure 10A) and PD-1 (Figure 10B) expression in CD4+ and CD8+ cells from NT and RT-treated tumor tissues. Figure 10A shows that the blank histogram is the isotype control. Figure 10B shows that the patterned histogram is the isotype control, with the black line being NT and the grey line being 7Gy RT. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Unless otherwise defined, all scientific and technical terms used herein have the meanings commonly understood by those skilled in the art to which this disclosure belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). The following terms used herein have the meanings ascribed to them unless otherwise specified.

[0014] In this disclosure, "comprises," "comprising," "containing," "having," and the like, may have the meaning ascribed to them in U.S. patent law and may mean "includes," "including," and the like; "consisting essentially of" or "consisting essentially of" may likewise have the meaning ascribed to them in U.S. patent law and is open-ended, allowing for the presence of other things than are recited, but excluding prior art aspects, so long as the basic or novel characteristics of the recited items are not changed by the presence of other things than are recited.

[0015] Unless otherwise stated or clear from context, the term "or" as used herein is understood to be inclusive. Unless otherwise stated or clear from context, the terms "a," "an," and "the" as used herein are understood to be singular or plural.

[0016] Unless otherwise specified or clear from the context, the term "about" as used herein is understood to mean within normal tolerance in the art, for example, within 2 standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0017] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0018] Ranges provided herein are understood to be shorthand for all of the values ​​within that range. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0019] As used herein, an "anti-PD-L1 antibody" refers to an antibody or antigen-binding fragment thereof that specifically binds to a PD-L1 polypeptide. Exemplary anti-PD-L1 antibodies are described in, for example, U.S. Patent No. 8,779,108 and U.S. Patent No. 9,493,565, which are incorporated herein by reference.

[0020] The term "durvalumab" as used herein refers to an antibody that selectively binds to PD-L1 and blocks the binding of PD-L1 to the PD-1 and CD80 receptors, as disclosed in U.S. Patent No. 9,493,565 (wherein durvalumab is referred to as "2.14H9OPT"), which is incorporated herein by reference in its entirety. The crystallizable (Fc) domain fragment of durvalumab contains a triple mutation in the constant domain of the IgG1 heavy chain that reduces binding to complement components C1q and Fcγ receptors, which play a role in mediating antibody-dependent cell-mediated cytotoxicity ("ADCC"). Durvalumab can relieve PD-L1-mediated suppression of human T-cell activation in vitro and inhibits tumor growth in xenograft models through a T-cell-dependent mechanism.

[0021] "Complete response" (CR) refers to disappearance of all lesions, measurable or not, and no new lesions. Confirmation can be obtained using repeated consecutive evaluations for at least 4 weeks from the date of first description. New nonmeasurable lesions exclude CR.

[0022] "Partial response" (PR) refers to a reduction in tumor burden of ≥ 50% versus baseline. Confirmation can be achieved using consecutive repeat assessments for at least 4 weeks from the date of initial description.

[0023] "Progressive disease" (PD) refers to an increase in tumor burden of ≥ 25% relative to nadir. Confirmation can be achieved by continuous repeat evaluations for at least 4 weeks from the date of initial description. New non-measurable lesions do not define PD.

[0024] "Stable disease" (SD) refers to not meeting the criteria for CR, PR, or PD. SD indicates that a 50% reduction in tumor burden compared to baseline cannot be established, and a 25% increase compared to nadir cannot be established.

[0025] Non-small cell lung cancer (NSCLC) can refer to any of three major subtypes of NSCLC: squamous cell carcinoma, adenocarcinoma, and large cell (anaplastic) carcinoma. Other subtypes include adenosquamous carcinoma and sarcomatoid carcinoma.

[0026] As used herein, "PD-L1" may refer to a polypeptide or polynucleotide sequence having at least about 85%, 95%, or 100% sequence identity to a PD-L1 sequence, or a fragment thereof. PD-L1 is also referred to in the art as B7-H1. In some embodiments, the PD-L1 polypeptide, or a fragment thereof, has at least about 85%, 95%, or 100% sequence identity to NCBI Accession No. NP_001254635, and has PD-1 and CD80 binding activity. PD-L1 Polypeptide Sequence NCBI accession number NP_001254635 [ka]

[0027] In some embodiments, a "PD-L1 nucleic acid molecule" comprises a polynucleotide that encodes a PD-L1 polypeptide. An exemplary PD-L1 nucleic acid molecule sequence is provided in NCBI Accession No. NM_001267706. PD-L1 nucleic acid sequence NCBI accession number NM_001267706 mRNA [ka] [ka] [ka]

[0028] Programmed Death-1 ("PD-1") is an approximately 31 kD type I membrane protein member of the extended CD28 / CTLA4 family of T cell regulators (see Ishida, Y. et al. "Induced Expression Of PD-1, A Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death," EMBO J. 11:3887-95 (1992)).

[0029] PD-1 is expressed on activated T cells, B cells, and monocytes (Agata et al., “Expression of the PD-1 Antigen on the Surface of Stimulated Mouse T and B Lymphocytes,” Int. Immunol. 8(5):765-72 (1996); Yamazaki et al., “Expression Of Programmed Death 1 Ligands By Murine T Cells And APC,” J. Immunol. 169:5538-45 (2002)), and at low levels in natural killer (NK) T cells (Nishimura et al., “Facilitation of Beta Selection and Modification of Positive Selection in the Thymus of PD-1-Deficient Mice,” J. Exp. Med. 191:891-98 (2000); Martin-Orozco et al., “Inhibitory Costimulation and Anti-Tumor Immunity,” Semin. Cancer 199:111-112 (2002)). Biol. 17(4):288-98(2007)). PD-1 is a receptor responsible for downregulation of the immune system following activation by binding to PDL-1 or PDL-2 (Martin-Orozco, N. et al.(2007)), and functions as a cell death inducer (Ishida, Y. et al.(1992); Subudhi et al., "The Balance of Immune Responses: Costimulation Verse Coinhibition," J. Molec. Med. 83:193-202 (2005); Lazar-Molnar et al., "Crystal Structure of the Complex Between Programmed Death-1(PD-1)and Its Ligand PD-L2," Proc. Natl. Acad. Sci. USA 105(30):10483-88(2008)).This process is exploited in many tumours through overexpression of PD-L1, leading to suppression of the immune response.

[0030] PD-1 is a well-validated target for immune-mediated therapy in oncology, with positive clinical trials, especially in the treatment of melanoma and non-small cell lung cancer (NSCLC). Antagonistic inhibition of the PD-1 / PDL-1 interaction increases T cell activation and enhances the recognition and elimination of tumor cells by the host immune system. The use of anti-PD-1 antibodies to treat infections and tumors, and to upregulate adaptive immune responses has been proposed.

[0031] The term "antibody" as used herein refers to an immunoglobulin or a fragment or derivative thereof and includes any polypeptide containing an antigen-binding site, whether generated in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, human single chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. For purposes of this disclosure, except when modified by the term "intact", as in "intact antibody", the term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to specifically bind to PD-L1. Typically, such fragments contain the antigen-binding domain.

[0032] The term "human antibody", as used herein, includes antibodies having variable and constant regions substantially corresponding to human germline immunoglobulin sequences.

[0033] As used herein, the terms "antigen-binding domain", "antigen-binding fragment" and "binding fragment" refer to a portion of an antibody molecule that contains the amino acids responsible for the specific binding between the antibody and the antigen. In some cases, if the antigen is large, the antigen-binding domain may bind only a portion of the antigen. The portion of the antigen molecule responsible for the specific interaction with the antigen-binding domain is referred to as an "epitope" or "antigenic determinant". An antigen-binding domain typically comprises the antibody light chain variable region (V L ) and antibody heavy chain variable region (V H ), however, it is not necessary to have both. For example, so-called Fd antibody fragments include V H Although consisting of only a domain, it still retains some of the antigen-binding function of an intact antibody.

[0034] Binding fragments of antibodies are produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single chain antibodies. An antibody other than a "bispecific" or "bifunctional" antibody is understood to be an antibody in which each of its binding sites is identical. Digestion of an antibody with the enzyme papain produces two identical antigen-binding fragments, also known as "Fab" fragments, and an "Fc" fragment that has no antigen-binding activity but has the ability to crystallize. Digestion of an antibody with the enzyme pepsin produces an F(ab')2 fragment in which the two arms of the antibody molecule remain linked and provide two antigen-binding sites. The F(ab')2 fragment has the ability to crosslink antigens. As used herein, the term "Fv" refers to the minimum fragment of an antibody that retains both the antigen recognition and binding sites. As used herein, the term "Fab" refers to a fragment of an antibody that contains the constant domain of the light chain and the CHI domain of the heavy chain.

[0035] As used herein, the term "mAb" refers to a monoclonal antibody. Antibodies of the present disclosure include, but are not limited to, whole natural antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single chain V-region fragments (scFv), fusion polypeptides, and non-conventional antibodies.

[0036] As used herein, the terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components that normally accompany it as found in its native state. "Isolate" refers to some degree of separation from source or surroundings. "Purify" refers to a greater degree of separation than isolation. A "purified" or "biologically pure" protein is sufficiently free from other substances such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences.

[0037] As used herein, the term "specifically binds" is meant to refer to a compound (e.g., an antibody) that recognizes and binds to a molecule (e.g., a polypeptide) but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample. For example, two molecules that specifically bind form a complex that is relatively stable under physiological conditions. Specific binding is characterized by high affinity and low to moderate capacity, which is typically distinguished from nonspecific binding, which has low affinity and moderate to high capacity. Typically, binding is determined by an affinity constant, K A 10 6 M -1 or more preferably 10 8 M -1 A specific binding is considered to be specific when the binding affinity is higher than 0.01. If necessary, non-specific binding can be reduced by changing the binding conditions without substantially affecting the specific binding. The appropriate binding conditions to allow binding, such as the concentration of the antibody, the ionic strength of the solution, temperature, time, and the concentration of blocking agents (e.g., serum albumin, milk casein), can be optimized by those skilled in the art using routine techniques.

[0038] The terms "treat", "treating", "treatment" and the like as generally used herein refer to palliating, ameliorating, or slowing the progression of a disorder or disease and / or symptoms associated with the disorder or disease. Although not exclusive, it will be recognized that treating a disorder, disease, or condition does not require the complete elimination of the disorder, disease, or condition, or associated symptoms. In certain embodiments related to NSCLC, "treat", "treating", "treatment" may refer to achieving any one or combination of a primary or secondary clinical endpoint.

[0039] Provided herein is a method of extending progression-free survival (PFS) in patients with unresectable non-small cell lung cancer (NSCLC), comprising treating the patient concurrently with a human anti-PD-L1 antibody and chemoradiation therapy.

[0040] Also provided herein is a method of increasing the overall response rate (ORR) in a patient with unresectable NSCLC, comprising treating the patient concurrently with a human anti-PD-L1 antibody and chemoradiation therapy.

[0041] Also provided herein is a method of treating a patient with stage III unresectable NSCLC, comprising concurrently treating the patient with a human anti-PD-L1 antibody and chemoradiotherapy.

[0042] In some embodiments, the human anti-PD-L1 antibody is durvalumab, avelumab, atezolizumab, or sugemalimab. In some embodiments, the human anti-PD-L1 antibody is durvalumab, avelumab, or atezolizumab. In some embodiments, the human anti-PD-L1 antibody is durvalumab.

[0043] Durvalumab and antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain and a light chain, or a heavy chain variable region and a light chain variable region. The amino acid sequence of the durvalumab light chain variable region is set forth in SEQ ID NO: 1, and the amino acid sequence of the durvalumab heavy chain variable region is set forth in SEQ ID NO: 2. The amino acid sequences of the durvalumab heavy chain variable region complementarity determining regions (CDRs) are set forth in SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3), and the amino acid sequences of the durvalumab light chain variable region CDRs are set forth in SEQ ID NO: 6 (CDR1), SEQ ID NO: 7 (CDR2), and SEQ ID NO: 8 (CDR3).

[0044] In some embodiments, durvalumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, durvalumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 3-5, and the light chain variable region comprises Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6-8. One of skill in the art will readily identify Chothia-defined, Abm-defined, or other CDR definitions known to those of skill in the art. In some embodiments, durvalumab or an antigen-binding fragment thereof used in the methods provided herein comprises the variable heavy and variable light chain CDR sequences of the 2.14H9OPT antibody disclosed in U.S. Pat. Nos. 8,779,108 and 9,493,565, which are incorporated by reference in their entireties.

[0045] Durvalumab or an antigen-binding fragment thereof may be administered once every four weeks while providing benefit to the patient. In a further embodiment, the patient is administered additional subsequent doses. The subsequent doses may be administered at various time intervals depending on the patient's age, weight, clinical evaluation, tumor burden, and / or other factors, including the judgment of the attending physician.

[0046] In some embodiments, multiple doses of durvalumab or an antigen-binding fragment thereof are administered to the patient. In some embodiments, at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses, at least 10 doses, at least 15 doses, at least 26 doses, or more than at least 20 doses may be administered to the patient. In some embodiments, durvalumab or an antigen-binding fragment thereof is administered every 2 weeks, for 2 weeks, for a 4 week treatment period, for a 6 week treatment period, for an 8 week treatment period, for a 12 week treatment period, for a 24 week treatment period, for a 1 year treatment period, or for a treatment period of more than 1 year.

[0047] In some embodiments, the interval between doses may be every 3 weeks. In some embodiments, the interval between doses may be every 4 weeks (Q4W). In some embodiments, the interval between doses may be every 2 months (e.g., during the maintenance phase).

[0048] In some embodiments, the patient is administered one or more doses of anti-PD-L1 or an antigen-binding fragment thereof, which is a fixed dose of 1500 mg. In some embodiments, the patient is administered 1500 mg of human anti-PD-L1 every four weeks. In some embodiments, the patient is administered one or more doses of anti-PD-L1, which is about 20 mg / kg. In some embodiments, the patient is administered 1500 mg of human anti-PD-L1 antibody intravenously every four weeks (Q4W).

[0049] In some embodiments, the patient is administered one or more doses of durvalumab or an antigen-binding fragment thereof, the dose being a fixed dose of 1500 mg. In some embodiments, the patient is administered 1500 mg of durvalumab every four weeks. In some embodiments, the patient is administered one or more doses of durvalumab, the dose being about 20 mg / kg.

[0050] The amount of durvalumab or an antigen-binding fragment thereof to be administered to a patient may be adjusted according to and depend on various parameters, such as the patient's age, weight, clinical evaluation, tumor burden, and / or other factors, including the judgment of the attending physician. In some embodiments, the dose is a fixed dose.

[0051] In some embodiments, administration of durvalumab or an antigen-binding fragment thereof according to the methods provided herein is via parenteral administration. For example, durvalumab or an antigen-binding fragment thereof may be administered by intravenous infusion or subcutaneous injection. In some embodiments, the administration is by intravenous infusion.

[0052] In some embodiments, durvalumab or an antigen-binding fragment thereof is administered simultaneously with chemoradiotherapy. As used herein, the term "concurrently" refers to administration of durvalumab or an antigen-binding fragment thereof and administration of chemoradiotherapy within about 3 days of each other. In some embodiments, durvalumab or an antigen-binding fragment thereof is administered within about 2 days of chemoradiotherapy. In some embodiments, durvalumab or an antigen-binding fragment thereof is administered within about 1 day of chemoradiotherapy. In some embodiments, durvalumab or an antigen-binding fragment thereof is administered on day 1 of cycle 1 of chemoradiotherapy.

[0053] In some embodiments, the anti-PD-L1 antibody is administered on day 1 of chemoradiotherapy.

[0054] In some embodiments, the chemotherapy includes a platinum-based therapeutic agent.

[0055] In some embodiments, the concurrent chemoradiotherapy includes any accepted standard first-line treatment for patients with advanced NSCLC. In some embodiments, the standard first-line treatment may include chemotherapy, radiation therapy, or both (chemoradiotherapy). In some embodiments, the therapy may include one or more platinum-based chemotherapeutic agents. In some embodiments, the chemoradiotherapy is platinum-based. In some embodiments, the one or more platinum-based chemotherapeutic agents may be selected from carboplatin, cisplatin, oxaliplatin, or combinations thereof. The platinum-based therapy described herein may include, for example, a singlet or doublet regimen in which cisplatin or carboplatin is administered together with another anticancer agent, such as paclitaxel, docetaxel, etoposide, gemcitabine, vinorelbine, etc.

[0056] The present disclosure relates to a method of treating patients with unresectable locally advanced non-small cell lung cancer (NSCLC), comprising administering to the patient a human PD-L1 antibody and concurrent chemoradiotherapy. The disclosed method of treatment can achieve substantial improvements in patient progression-free survival (PFS), overall response rate (ORR), overall survival (OS), and proportion of patients alive 24 months from randomization (OS24).

[0057] In some embodiments, the method achieves an increase in PFS compared to placebo. In some embodiments, the method achieves an increase in ORR compared to placebo. In some embodiments, the method achieves an increase in OS versus placebo.

[0058] In some embodiments, a combination is provided comprising a human anti-PD-L1 antibody and concurrent chemoradiotherapy for use in a method of increasing progression-free survival (PFS) in patients with unresectable non-small cell lung cancer (NSCLC). In some embodiments, a combination is provided comprising a human anti-PD-L1 antibody and concurrent chemoradiotherapy for use in a method of increasing overall response rate (ORR) in patients with unresectable non-small cell lung cancer (NSCLC). In some embodiments, a combination is provided comprising a human anti-PD-L1 antibody and concurrent chemoradiotherapy for use in the treatment of stage III unresectable non-small cell lung cancer (NSCLC).

[0059] In some embodiments, there is provided the use of a combination comprising a human anti-PD-L1 antibody and concurrent chemoradiotherapy in the manufacture of a medicament for use in a method for extending progression-free survival (PFS) in patients with unresectable non-small cell lung cancer (NSCLC). In some embodiments, there is provided the use of a combination comprising a human anti-PD-L1 antibody and concurrent chemoradiotherapy in the manufacture of a medicament for use in a method for increasing overall response rate (ORR ... the treatment of stage III unresectable non-small cell lung cancer (NSCLC).

[0060] Overall survival (OS) refers to the time from the date of treatment to death from any cause. OS may refer to overall survival within a period of, for example, 12 months, 18 months, 24 months, etc. Such a period may be identified, for example, by Kaplan-Meier estimation of overall survival at 24 months, as "OS24," which refers to the number (%) of patients alive at 24 months after the start of treatment.

[0061] Progression-free survival (PFS) refers to the time period starting from the date of treatment to the date of objective disease progression (RECIST 1.1) or death (from any cause in the absence of progression). In some embodiments, the methods of the present disclosure achieve an increase in PFS. In some embodiments, the methods achieve a PFS of at least 9 months to at least about 24 months (e.g., at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, up to about 5 years).

[0062] Objective response rate (ORR) refers to the number (%) of patients with at least one visit response of complete response (CR) or partial response (PR) by RECIST 1.1.

[0063] The methods described herein and illustrated by the examples provide for the treatment of locally advanced unresectable NSCLC. In some embodiments, unresectable cancer includes cancer that cannot be completely removed by surgery due to at least one of several medical reasons. The reasons why a cancer may be unresectable include, for example, tumor size (e.g., too large to be safely removed and / or may require extensive removal of parts of essential organs), tumor location (e.g., tumors that are physically intertwined with vital structures such as blood vessels or nerves), tumor metastasis where removal of the tumor is not effective in controlling all cancers, or other medical conditions (e.g., heart disease, lung disease, diabetes) that increase the risk of surgery to an unacceptable level. Furthermore, unresectable NSCLC may not be permanently unresectable after aggressive treatments that may be effective in reducing the size of the tumor to a degree that allows possible surgical resection. Furthermore, unresectable NSCLC may also refer to NSCLC (or distant metastasis) that is not completely removed by surgery but may be partially removed by one or more surgical procedures. Examples include debulking surgery, and surgery to remove parts of lung cancer and parts of metastatic lesions.

[0064] In certain embodiments, the methods disclosed herein may be used for unresectable cancers.

[0065] The methods of the present disclosure described and illustrated herein can be used to treat patients with late stage (e.g., stage III) locally advanced, unresectable NSCLC. Cancer staging can be performed using any test commonly known and accepted in the art. In some embodiments, cancer staging can include the American Joint Committee on Cancer's (AJCC's) TNM system. In general, the TNM system provides the results of various tests and scans to determine the size and location of the primary tumor (tumor, T); whether the cancer has spread to lymph nodes, and if so, the location and number of involved lymph nodes (nodes, N); and whether the cancer has spread to other parts of the body, and if so, the extent and location of distant cancer (metastasis, M). Although each type of cancer may have its own specific system, the TNM staging system generally uses a scoring system that is scaled for each letter.

[0066] In some embodiments, the unresectable NSCLC is stage III. In some embodiments, the unresectable NSCLC is locally advanced. In some embodiments, the unresectable NSCLC is stage III and locally advanced.

[0067] For tumors, the "T" is associated with a number (e.g., 0-4) that describes the general tumor size, location, and whether it has invaded into nearby tissues. Larger or more invasive tumors are given a higher number, and depending on the cancer, further detail may be provided with the addition of a lower case letter, such as "a," "b," or "m" (if multifocal).

[0068] Similarly, for nodes, "N" can be associated with a number (e.g., 0-3) to describe whether the cancer was found in a lymph node, and can also indicate the number of lymph nodes that contain cancer. The more lymph nodes involved in the cancer, the higher the number is assigned.

[0069] For metastasis, "M" indicates whether the cancer has spread to other parts of the body; if not, it is labeled M0, or if not, M1.

[0070] The results of T, N, and M are combined to determine the stage of the cancer, typically one of four stages: stage I (1) to IV (4). Some cancers are also stage 0 (zero). Stage 0 describes intraepithelial carcinoma that is localized to the tissue of origin, without any spread to nearby tissues. Cancers at this stage are often highly likely to be cured, usually by removing the entire tumor through surgery. Stage I, or early stage cancer, is typically used to describe small cancers or tumors that have not grown deeper into nearby tissues and have not spread to lymph nodes or other parts of the body. Stages II and III describe larger cancers or tumors that have grown deeper into nearby tissues and may have spread to lymph nodes, but have not metastasized to other tissues. Stage IV describes cancer that has spread to other organs or parts of the body, and is often identified as advanced or metastatic cancer.

[0071] Staging may include the optional analysis of prognostic factors to provide chances of recovery and recommended treatments. Prognostic factors may include staging the cancer based on the appearance of the cancer cells, analysis of tumor marker expression, and analysis of tumor genetics.

[0072] The cancer may be staged again using the same initial system to determine the effectiveness of treatment or to obtain more information about recurrent cancer.

[0073] Staging of NSCLC: NSCLC has five stages: stage 0 (zero) and stages I-IV (1-4). Stage 0 NSCLC indicates that the cancer has not grown into nearby tissue and has not spread outside the lungs.

[0074] Stage I NSCLC indicates that the cancer is a small tumor that has not spread to the lymph nodes. Stage I is divided into two substages based on the size of the tumor: stage IA tumors are less than 3 centimeters (cm) wide, and stage IB tumors are more than 3 cm wide but less than 5 cm wide. Stage I NSCLC may allow for complete surgical removal of the cancer.

[0075] Stage II is divided into two substages (IIA and IIB). Stage IIA can be either a tumor larger than 5 cm wide but smaller than 7 cm wide that has not spread to nearby lymph nodes, or a small tumor smaller than 5 cm wide that has spread to nearby lymph nodes. Stage IIB can describe either a tumor larger than 5 cm wide but smaller than 7 cm wide that has spread to lymph nodes, or a tumor larger than 7 cm wide that may or may not have grown into nearby structures in the lungs, but has not spread to lymph nodes. While stage II NSCLC may be treatable surgically, other therapies are usually required to treat this stage of NSCLC.

[0076] Stage III includes substages IIIA or IIIB. For many stage IIIA cancers and almost all stage IIIB cancers, surgery is difficult or impossible due to spread of the cancer to lymph nodes or growth into nearby structures in the lungs. Surgery in either situation typically requires partial removal of the cancer.

[0077] Stage IV NSCLC is associated with spread to multiple regions in the other lung, the fluid surrounding the lung or heart, or distant metastasis in the body. NSCLC is more likely to spread to the brain, bone, liver, and adrenal gland. Stage IV NSCLC includes substage IVA (spread within the chest) and IVB (spread outside the chest). Surgery is rarely successful in most stage III or IV NSCLC, and removal may be impossible if it spreads to lymph nodes above the collarbone or to important structures in the chest (e.g., heart, large blood vessels, or major lung structures). In certain embodiments, the patient disclosed herein is a stage IV NSCLC patient.

[0078] After a course of treatment, recurrent NSCLC is detected.

[0079] The practice of the methods disclosed herein employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology"; "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). EXAMPLES

[0080] Example 1: Efficacy of durvalumab in combination with platinum-based chemoradiotherapy in patients with locally advanced, unresectable non-small cell lung cancer (stage III) This is a phase III, randomized, double-blind, placebo-controlled, multicenter, international study evaluating the efficacy and safety of durvalumab given concomitantly with platinum-based chemoradiotherapy (CRT) (durvalumab plus standard of care [SoC]CRT) in patients with locally advanced, unresectable NSCLC (stage III).

[0081] Approximately 390 patients with locally advanced, unresectable NSCLC (stage III) were recruited, and 300 patients were randomized in a 2:1 ratio to receive durvalumab + SoC CRT or placebo + SoC CRT. Patients were stratified by age (<65 vs. ≥65 years) and stage (IIIA vs. IIIB / C).

[0082] Subjects in this study included adult subjects ≥18 years of age with histologically or cytologically proven NSCLC presenting with locally advanced, unresectable (stage III) disease. All subjects were required to have adequate organ and marrow function.

[0083] Subjects were excluded from participation in the study if they had received previous or current treatment for NSCLC, including, but not limited to, radiation therapy, investigational drugs, chemotherapy, and mAbs.

[0084] All patients will receive one of the following platinum-based SoC chemotherapy options in addition to radiation therapy: cisplatin / etoposide, carboplatin / paclitaxel, pemetrexed / cisplatin, or pemetrexed / carboplatin. Chemotherapy treatment regimens are outlined in Table 1.

[0085] [Table 1]

[0086] [Table 2]

[0087] Patients will also receive durvalumab 1500 mg or placebo via intravenous infusion concurrently with SoC CRT (i.e., beginning on day 1 [± 3 days] of cycle 1) every 4 weeks. Patients with complete response (CR), partial response (PR), or stable disease (SD) at 16-week tumor assessment after completion of SoC CRT will continue to receive durvalumab / placebo as consolidation treatment (1500 mg q4w IV). Patients with RECIST 1.1-defined radiologically progressive disease at 16-week tumor assessment after completion of SoC CRT will proceed to follow-up. Based on a mean body weight of 75 kg, a fixed dose of 1500 mg durvalumab q4w is equivalent to 20 mg / kg q4w.

[0088] The primary objective of this study is to evaluate the efficacy of durvalumab plus SoC CRT compared with placebo plus SoC CRT in terms of progression-free survival by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) assessed by blinded independent central review (BICR). Key secondary endpoints (i.e., those included in the multiple study procedure) are objective response rate by RECIST 1.1, assessed by BICR, overall survival, and the proportion of patients alive at 24 months from randomization (OS24).

[0089] Example 2: Gene expression in a syngeneic mouse tumor model demonstrating the immunological consequences of radiation therapy Materials and Methods Mouse Tumor Model. Mice were housed under specific pathogen-free conditions in Tecniplast 1284IVC cages containing up to six animals with aspenchips-2 bedding, sizzlenest nesting material, and cardboard tunnels. Mice were housed on a 12 / 12 light / dark cycle, provided with filtered water, and fed Teklad Global 19% protein extruded rodent diet ad libitum.

[0090] CT26 colon adenocarcinoma cell line (purchased from ATCC in 2011) was cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) L-glutamine (Invivogen). Cells were not passaged for more than 3 months and were regularly screened for the absence of mycoplasmosis (PlasmoTest, Source BioScience LifeSciences, UK). 1 × 10 5 CT26 cells were injected subcutaneously (sc) into the back of Balb / c mice (Harlan Laboratories, UK), 1 cm from the base of the tail. Tumor volumes were measured using calipers as length x width x depth in mm 3 and body weight was monitored daily.

[0091] Tumor treatment. Tumors between 100 and 200 mm 3 Local radiation was delivered when tumor size reached 1000 mm. Mice were restrained in a lead shield to expose only the tumor and allowed local exposure to IR with a single dose of 7 Gy using 250 kV X-rays (MXR-320 / 36 x-ray tube, Comet AG, Switzerland) at 12 mA and a dose rate of 2 Gy / min. Mice were sacrificed 1, 3, and 7 days after irradiation along with time-matched untreated controls. Tumors were harvested and used fresh for analysis by flow cytometry. At least 20 mg of tissue from each tumor was snap frozen for gene microarray analysis. For combination studies, mice received 10 mg / kg αPD-L1 monoclonal antibody (mAb) (clone 10F.9G2, Biolegend, UK) following RT (administered 3 qw per week, starting on day 1 of RT). When tumors reached 1000 mm 3 Mice were sacrificed when the dose reached or for long-term surviving (LT) mice at day 100 after treatment. Flow cytometry phenotyping and combination studies are representative of two independent experiments.

[0092] Exon microarray analysis. A single study was performed with five different tumors sampled from each treatment group at each time point for microarray evaluation. Fresh frozen RNA extraction was performed using RNAStat 60 (Amsbio, UK) and quality control testing of total RNA was performed using 2100 Bioanalyzer (Agilent, UK). Samples were amplified using Ovation Pico WTA System v2 (NuGEN Technologies, Netherlands). After QC testing, cDNA was fragmented and labeled using Encore Biotin Module (NuGEN Technologies, Netherlands) before hybridization to mouse exon arrays according to NuGEN guidelines for Affymetrix GeneChip® arrays. Microarray analysis was performed using Mouse Exon 1.0 ST arrays (Affymetrix, UK). All microarray data have been deposited in GEO (accession number GSE74875).

[0093] Data analysis. Raw microarray data were preprocessed / normalized with respect to core transcript probes using the Robust Multichip Algorithm (RMA) (Bolstad et al., Bioinformatics 19(2):185-93 (2003)). Quality control was then performed after data integrity assessment by removing three outliers (one from each treatment group on day 1 and one from the radiation treatment group on day 3). Affy AFFX control transcripts were removed together with uninformative transcripts (log2 expression threshold < 3.6473 and variance threshold < 0.0088). 8500 reliably detected transcript IDs (Affymetrix transcript cluster IDs) were retained for analysis.

[0094] Comparisons were performed on treatment groups by time point (untreated vs. irradiated) to identify differentially expressed transcript IDs (up- or down-regulated) using a cutoff p-value of 0.05 (ANOVA). Median log2 intensity values ​​per transcript ID by time point were calculated for the untreated tumor group and then subtracted from treated samples at equivalent time points to obtain control-normalized transcript expression intensities by time point. Hierarchical cluster analysis (HCA); non-normalized, linkage=Ward and distance=uncentered correlation (Omicsoft ArrayStudio) was performed to cluster control-normalized transcript expression data by sample and transcript ID. Differentially regulated pathway categorized gene set data were plotted by time point as bubble diagrams (MatLab). Bubble color indicates the direction of gene regulation. Bubble size indicates absolute fold change expression value for each pathway. Mouse gene annotations were assigned to transcript IDs using BioMart (Mus Musculus Genes GRCm38.p2) and ID Converter (Alibes et al., BMC Bioinformatics 8:9. doi:10.1186 / 1471-2105-8-9 (2007)). Functional enrichment and network analysis was performed using Ingenuity Pathway Analysis (IPA, Ingenuity® Systems). Transcripts that were up- or down-regulated by at least 1.5-fold were mapped to pathways and to upstream regulators. Gene Ontology enrichment analysis was performed with the g:GOSt function within g:Profiler (Reimand et al., (2011 update), Nucleic Acids Res. 39(Web Server issue):W307-15. doi:10.1093 / nar / gkr378(2011)).

[0095] Flow cytometry. Tumors were divided into 1 mm 3The cells were cut into 100-μm pieces, incubated in 2 U / mL DNase (Sigma, UK), 300 CDU / mL collagenase I (Life Technologies, UK), and 0.9 mg / mL dispase II (Sigma, UK) in PBS for 40 min at 37°C, and then forced through a 100 μm cell strainer with FACS buffer (PBS with 10% FCS). After incubation with CD16 / CD32 Fc blocking antibody (Life Technologies, UK), the expression of CD4, CD8 (BD Biosciences, UK), CD11b, CD11c, CD45, CD69, CD86, CD206 (Biolegend, UK), MHC-II, F4 / 80, Gr1, NKp46, B220, PD-1, and CTLA-4 (all eBiosciences, UK unless otherwise stated) was analyzed by flow cytometry. A viability stain (Life Technologies, UK) was included to exclude dead cells. Regulatory T cells were analyzed using Mouse Regulatory T-cell Staining Kit #3 (eBioscience, UK).

[0096] Statistical analysis. Flow cytometry data and tumor volumes were compared between the two groups using the Mann-Whitney U test. Gene expression profiling data were evaluated as previously described. The log-rank Mantel-Cox test was performed on survival data. Data were considered statistically different if p<0.05.

[0097] RT causes activation of innate and adaptive immunity. Immunocompetent Balb / c mice bearing established CT26 tumors received a single 7 Gy dose of RT, and tumors were excised 1, 3, and 7 days after treatment for exon microarray analysis to identify early transcriptional changes (Figures 2A and 2C). Comparing the transcriptomes of irradiated tumors with those of non-treated (NT), time-matched controls, we identified 757 genes that were significantly differentially expressed (up- or down-regulated) (+ / -1.5 fold change and p ≤ 0.05) for at least one of the time points (Table 2). In addition to the expected p53 activation pathways (radiation-dependent DNA damage and cell death), IPA functional enrichment analysis of the data highlighted a strong bias for innate and adaptive immune functions. These included antigen presentation, T cell activation and cytotoxicity, and chemokine production (Figures 2B and 3, and Tables 3 and 4). It was noteworthy that the number of differentially regulated genes clearly increased from day 1 to day 7 (Table 2).

[0098] Table 2: Number of up- or down-regulated genes meeting the cut-off threshold (≧+ / -1.5 fold change and p≦0.05, ANOVA) at different time points.

[0099] [Table 3]

[0100] Table 3: Key dominant immunological pathways associated with differentially expressed genes (fold change = + / - 1.5 and p ≤ 0.05) at three time points (days 1, 3, and 7) in CT26 tumors after 7 Gy IR (analyzed by IPA software). *Pathways involved at multiple time points. Non-disease related immune pathways present in the top 10 most significant p-values ​​are highlighted.

[0101] [Table 4]

[0102] Table 4: Classification of key genes within each cluster (A-E) from the heatmap (Figure 2) into functional sets with immune or radioregulatory relevance. Gene expression fold change at each time point (radiation treated vs. untreated tumors) is shown, with significant p-values ​​indicated by asterisks (p≦0.05*; p≦0.01**; p≦0.005***). Functional classification was guided by the Nanostring nCounter Mouse PanCancer Immune Profiling Panel and the authors' knowledge.

[0103] [Table 5]

[0104] [Table 6]

[0105] Patterns of response are also evident in the network map augmented from the top immune-related upstream regulators that were significantly differentially regulated at each time point (Figure 3). Furthermore, pathway analysis highlighted that 30.8% of the up- and down-regulated genes were associated with immune system processes (GO:0002376), with the most significant enrichment p-value of 4.35E-72 seen with the gene set highlighting the predominance of immune processes within 7 days after irradiation. However, nearly more than 5% of the genes showed similar behavior across all three time points (38 up / 3 down), thus highlighting distinct stages of radiation response at the three time points evaluated.

[0106] Detailed hierarchical cluster analysis (HCA) of the differentially expressed genes identified five clusters, each containing genes that were co-regulated at different time points (Figure 2B). Genes within cluster A were significantly upregulated at day 7 when compared to expression in control tumors. Pathway analysis demonstrated that the majority of these genes are associated with adaptive immune cell responses, including T cell receptor signaling and CD28 signaling. Illustrative of this point is the increased expression of Cd3d / e / g, Cd8a, and Cd28, which are involved in the regulation of CD3 + / CD8 + This suggests increased infiltration or proliferation of T cells. A signature of an active antitumor immune response was also suggested by the upregulation of Ifnγ. Another upregulated gene of interest is Tnfsf10, which encodes the death receptor TRAIL. Radiation is known to induce tumor cell surface expression of death receptors and their ligands, and upregulation of TRAIL on T cells may help lead to tumor cell death via activation of the TRAIL receptor. Interestingly, the observed co-expression of inhibitory immune checkpoints (e.g., Pd-1, Lag3, and Ctla4) suggested that this adaptive immune response may be transient. The development of this immunosuppressive tumor microenvironment was also reinforced by the upregulation of Cd39 / Entpd1, which is known to contribute to adenosine-dependent immune cell suppression in conjunction with the enzymatic activity of Cd73.

[0107] The second cluster (B) is enriched by genes associated with innate immunity as well as genes encoding proteins involved in communication between the innate and adaptive arms of the immune system. These genes were continuously upregulated during the experiment. Although some genes were significantly upregulated on day 1, the expression of the majority of genes was significantly upregulated from day 3 onwards. This expression pattern suggests that the innate immune response is initiated early and remains relatively constant during the first week after a single dose of RT. Relevant genes within this cluster included Cd80 (a costimulatory receptor expressed on APCs) and IL15 (a cytokine expressed by monocytes and dendritic cells that serves as a potent inducer / activator of natural killer cells and T cells). In addition, cluster B contained Nos2 (an enzyme induced by IFNγ on activated macrophages), Cfb (complement factor B whose catalytic subunit Bb can activate C3 convertase and subsequently activate B cells), and toll-like receptor 3 (Tlr3). In addition, type I and type II interferon-regulated genes, including Irf7, Irf9, Mx1, Oas1a / g, and Oas2, as well as the chemokine genes Cxcl10, Ccl2 / 5 / 6 and 7, were enriched in this cluster.

[0108] The profile of cluster C was more consistent across time points and may be considered the "first gene cluster" with respect to chronological response. It contained a large set of genes that were significantly upregulated at earlier time points (days 1 and 3) with a smaller subset that was upregulated later (days 3-7). Innate immune response, radiation-dependent DNA damage repair and cell death, and chemokines were the dominant functional enrichments in this cluster, but several genes associated with T cells and cytotoxicity, as well as antigen presentation and B cells, were also present. Genes associated with innate immunity included those encoding complement, such as C3, C1ra, and C1rb, as well as interferon-regulating genes, including Stat1. Interestingly, Stat1 was highlighted as an upstream regulator at all three time points in the IPA functional enrichment analysis (Figure 3 and Table 4). Also, chemokines such as Ccl4 (a chemokine associated with macrophage and NK cell recruitment) were strongly upregulated at day 1; as were genes associated with antigen processing (Psmb8 / 9 / 10, Tap1, and Tapbp). Radiation-dependent DNA damage repair and cell death, as well as T cell and cytotoxicity, were the predominant functional enrichments in this cluster. A notable hallmark of the dataset was the expression of genes associated with p53 signaling, which correlates with the direct effects of IR. Cellular stress, such as DNA damage caused by IR, activates p53, inhibiting cell proliferation and prompting tumor cells to initiate apoptosis. For example, p53 activation induces the expression of an inhibitor of cell cycle progression gene Cdkn1a (1.7-fold upregulated at day 3, p<0.01). Similarly, the death receptor (1.8-fold upregulated at day 1, p<0.01) is known to be induced by activated p53, rendering cells hypersensitive to FAS-ligand expressing immune effector cells. A related upregulated gene associated with adaptive immunity is Cd40lg (normally activated CD4 +These included the immune checkpoint proteins Pd-l1 (Cd274) (significantly upregulated from day 3 onwards), and Gzmb (granzyme B) (which, in addition to its function in target cell killing, is involved in basement membrane remodelling and lymphocyte migration, and was significantly upregulated from day 1 onwards).

[0109] Genes within cluster D were transiently upregulated on day 1 before being significantly downregulated on day 3. Pathway analysis demonstrated enrichment of genes associated with antigen presentation (H2 genes, Cd74, Cd209d, and Cd209c) and B cell activation (Cd24a), along with chemokine expression (Cxcl9 and Cxcl12). None of the antigen presentation and B cell-related genes were significantly differentially regulated by day 7, suggesting that a cascade of effects proceeded further downstream in the immune function pathway.

[0110] The final cluster identified (cluster E) was the largest cluster and contained 180 genes. Radiation-dependent DNA damage repair was a key functional enrichment with significant downregulation evident for certain genes as early as day 1 (day 1 Lig4;1 and day 3 Gadd45a, or day 7 Brca1, Brca2, Ercc1, Pola1, and Parbp). This data supports previous studies revealing rapid repair of DNA within 24 hours after irradiation. Finally, a strong association of cholesterol biosynthesis downregulation with radiation was identified.

[0111] Overall, the transcriptome analysis confirms that a single dose of radiation to CT26 tumors triggers p53-dependent cell death. Triggering cell death is likely the rate-limiting step that initiates both innate and adaptive immune responses in irradiated tumors. Indeed, gene expression profiles suggest that RT-induced tumor cell death is responsible for the recruitment and activation of innate immunity (IFNα expression, antigen processing / presentation, macrophage recruitment, and dendritic cell maturation) followed by the activation of adaptive immune responses (IFNγ signaling, T cell cytotoxicity, T cell receptor signaling, and B cell activation). However, for any physiological system, this biological immune response is expected to be transient, as evidenced by the increased expression of several immunosuppressive molecules.

[0112] To confirm the gene microarray data and further explore the phenotypic changes of immune components following IR at the protein level, the same tissues were simultaneously analyzed by flow cytometry. Heat maps showing the fold changes of lineage and phenotypic markers from matched tumor tissues are summarized in Figure 7.

[0113] RT alters the phenotype of tumor-infiltrating myeloid cell populations. Macrophages exhibit a high degree of lineage plasticity. However, tumor-associated macrophages (TAMs) are predominantly skewed toward the M2 phenotype in several cancer types. M2 cells express CD206 (also known as mannose receptor or MRC1), are poor APCs, and may contribute to immune escape and disease progression through the release of proangiogenic and immunosuppressive factors. In contrast, M1-differentiated macrophages co-express costimulatory molecules such as CD86 that enable effective lymphocyte activation. Both the frequency and differentiation status of TAMs in RT-treated and time-matched NT control tumors were analyzed. F4 / 80 + The total number of TAMs did not change significantly after RT (Figure 4A), whereas the expression of CD86 and CD206 was modulated. Seven days after RT, F4 / 80 +Cells downregulated CD86 expression compared with NT time-matched controls (MFI of 695.0 ± 46.6 NT vs. 494.7 ± 13.9 irradiated, P < 0.05; Fig. 4B). Furthermore, CD206 expression was significantly increased in tumor-infiltrating F4 / 80 cells 3 days after RT. + CD86 expression was significantly downregulated on irradiated tumors (MFI of 263.7±23.8 in NT vs. 99.5±8.1 in irradiated tumors, P<0.001; Fig. 4C). This downregulation was also observed 7 days after RT. A similar pattern of response was also observed in the transcriptome analysis (Table 4). + , CD206 + positive F4 / 80 + The ratio of TAMs to TAM-positive cells was increased at day 3 compared to time-matched NT controls (1.8±0.05 NT vs. 2.7±0.12 irradiated, P<0.01) (Figure 4D). In addition to changes in CD86 and CD206 expression, a shift in macrophage phenotype is also suggested by the increased Nos2 and Stat1 expression observed in the gene profiling data after irradiation (Figure 3 and Table 4). Furthermore, a decrease in gene expression of resistin-like alpha (Retnla) and Cd163 (6.9- and 2-fold, respectively), which are associated with an M2-like phenotype, was also identified 3 days after RT (Figure 3 and Table 4). Given that no changes in the overall number of TAMs were found, these data suggest that RT causes a transient bias of macrophages toward an M1 phenotype.

[0114] Myeloid-derived suppressor cells (MDSCs) have the ability to suppress antitumor immune responses and may therefore affect the immunogenicity of RT. Tumor-infiltrating CD11b + Gr1 lo No change in the frequency of CD11b cells was observed at any time point after RT (Figure 4E and gating strategy in Figure 8). + Gr1 hi A 2.7-fold expansion of cells, phenotypically defined as MDSCs, was observed in RT-treated tumors at day 3 (Figure 4F). This expansion appeared to be transient, as by day 7, CD11b + Gr1 hiNo significant difference in cell frequency was observed between the NT and RT treatment groups.

[0115] RT causes T cell activation and alters the CD8:Treg ratio in tumors. RT inhibits tumor-infiltrating CD45 + This caused an overall increase in the proportion of CD4 + and CD8 + T cell numbers were found to be reduced by 52% and 63%, respectively, 3 days after RT when compared to time-matched controls (Figures 5A and 5B). + T cell numbers remained depleted 7 days after treatment, whereas CD8 + T cell numbers recovered and there was a strong trend towards proliferation in RT-treated tumors (15.9±3.0% to 25.9±3.8%; P=0.06). Interestingly, higher expression of the early activation marker CD69 was observed in the remaining tumor-infiltrating CD4 T cells compared to time-matched controls. + (Days 3 and 7 after RT) and CD8 + (1 and 3 days after RT) was observed on T cells (Figures 5C and 5D and Figure 10A), suggesting that RT induced T cell activation.

[0116] Even within NT tumors, CD4 + The percentage of Treg cells increased 3.3-fold from day 1 to day 7 (Figure 5E), demonstrating how the tumor microenvironment changes over time. However, comparison of time-matched tumors revealed that RT further increased the frequency of Tregs by an additional 32% when assessed 7 days after treatment. Despite this, the CD8:Treg cell ratio, which has often been reported to be associated with better prognosis in cancer, was 2.5-fold higher in RT-treated tumors when compared to NT controls at day 7 (Figure 5F).

[0117] RT causes expression of PD-1 and PD-L1 in the tumor microenvironment limiting antitumor efficacy. Gene profiling data revealed that RT causes increased expression of several co-inhibitory immune checkpoints in tumors. Given the consistent upregulation of Pd-l1 at the mRNA level observed after RT, and expression of Pdcd1 (PD-1) at day 7 (Figure 3 and Table 4), initial studies focused on the PD-1 / PD-L1 pathway. Flow cytometry allowed further contextualization of the mRNA data, revealing increased expression of CD4+ at 7 days after RT. + and CD8 + We demonstrated increased expression of both PD-1 and PD-L1 on T cells (Figures 6A-6D and Figure 10B). Furthermore, RT also caused increased expression of PD-L1 on tumor cells at all time points examined (Figure 6E). This data closely resembled the expression pattern observed at the mRNA level (Figure 3 and Table 4).

[0118] To determine whether blockade of the PD-1 / PD-L1 axis affects the antitumor efficacy of RT, a therapeutic study was performed. Mice received RT (7 Gy as a single dose) alone or in combination with αPD-L1 mAb. Median survival in the NT cohort was 15.5 days. This was not significantly improved by αPD-L1 mAb delivered 3qw as monotherapy (median survival = 18 days; Figures 6F and 6G). However, RT when administered in combination with αPD-L1 mAb caused a significant improvement in survival when compared to monotherapy, with >70% of mice experiencing a complete therapeutic response.

[0119] Sequence Listing SEQ ID NO:1 EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK SEQ ID NO:2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS SEQ ID NO:3 - VH CDR1 GFTFSRYWMS SEQ ID NO:4 - VH CDR2 NIKQDGSEKYYVDSVKG SEQ ID NO:5 - VH CDR3 EGGWFGELAFDY SEQ ID NO:6-VL CDR1 RASQRVSSSYLA SEQ ID NO:7-VL CDR2 DASSRAT SEQ ID NO:8 - VL CDR3 QQYGSLPWT

Claims

1. A pharmaceutical composition for extending progression-free survival (PFS) in patients suffering from inoperable non-small cell lung cancer (NSCLC), said pharmaceutical composition comprising an anti-PD-L1 antibody and being administered to said patient concurrently with chemoradiotherapy.

2. The pharmaceutical composition according to claim 1, wherein the anti-PD-L1 antibody is a human anti-PD-L1 antibody.

3. The chemoradiotherapy is platinum-based; or The chemoradiotherapy is platinum-based and the anti-PD-L1 antibody is a human anti-PD-L1 antibody, The pharmaceutical composition according to claim 1.

4. The human anti-PD-L1 antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2; or The human anti-PD-L1 antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, and the chemoradiotherapy is platinum-based, The pharmaceutical composition according to claim 2.

5. The human anti-PD-L1 antibody has VH CDR1 having the amino acid sequence of SEQ ID NO: 3; and has VH CDR2 having the amino acid sequence of SEQ ID NO: 4; and has VH CDR3 having the amino acid sequence of SEQ ID NO: 5; and has VL CDR1 having the amino acid sequence of SEQ ID NO: 6; and has VL CDR2 having the amino acid sequence of SEQ ID NO: 7; and has VL CDR3 having the amino acid sequence of SEQ ID NO: 8 ; or The human anti-PD-L1 antibody has VH CDR1 having the amino acid sequence of SEQ ID NO: 3; and has VH CDR2 having the amino acid sequence of SEQ ID NO: 4; and has VH CDR3 having the amino acid sequence of SEQ ID NO: 5; and has VL CDR1 having the amino acid sequence of SEQ ID NO: 6; and has VL CDR2 having the amino acid sequence of SEQ ID NO: 7; and has VL CDR3 having the amino acid sequence of SEQ ID NO: 8 and the chemoradiotherapy is platinum-based, The pharmaceutical composition according to claim 2.

6. The human anti-PD-L1 antibody is durvalumab, avelumab, atezolizumab, or sugemalimab; or The human anti-PD-L1 antibody is durvalumab, avelumab, atezolizumab, or sugemalimab and the chemoradiotherapy is platinum-based, The pharmaceutical composition according to claim 2.

7. The pharmaceutical composition is administered intravenously to the patient at a dose of 1500 mg of the anti-PD-L1 antibody every 4 weeks (Q4W), and / or the pharmaceutical composition is administered on the first day of radiotherapy. The pharmaceutical composition according to any one of claims 1 to 6.

8. The unresectable NSCLC is stage III and / or locally advanced; or The unresectable NSCLC is stage III and / or locally advanced, and the pharmaceutical composition is administered intravenously to the patient at a dose of 1500 mg of the anti-PD-L1 antibody every 4 weeks (Q4W), and / or the pharmaceutical composition is administered on the first day of chemoradiotherapy. The pharmaceutical composition according to any one of claims 1 to 6.

9. A pharmaceutical composition for increasing the overall response rate (ORR) in a patient suffering from unresectable non-small cell lung cancer (NSCLC), the pharmaceutical composition comprising an anti-PD-L1 antibody and being administered to the patient concurrently with chemoradiotherapy.

10. The anti-PD-L1 antibody is a human anti-PD-L1 antibody. The pharmaceutical composition according to claim 9.

11. The chemoradiotherapy is platinum-based; or The chemoradiotherapy is platinum-based and the anti-PD-L1 antibody is a human anti-PD-L1 antibody. The pharmaceutical composition according to claim 9.

12. The human anti-PD-L1 antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2; or The human anti-PD-L1 antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, and the chemoradiotherapy is platinum-based. The pharmaceutical composition according to claim 10.

13. The human anti-PD-L1 antibody VH CDR1 having the amino acid sequence of SEQ ID NO: 3; and VH CDR2 having the amino acid sequence of SEQ ID NO: 4; and VH CDR3 having the amino acid sequence of SEQ ID NO: 5; and VL CDR1 having the amino acid sequence of SEQ ID NO: 6; and VL CDR2 having the amino acid sequence of SEQ ID NO: 7; and VL CDR3 having the amino acid sequence of SEQ ID NO: 8 comprises; or The human anti-PD-L1 antibody VH CDR1 having the amino acid sequence of SEQ ID NO: 3; and VH CDR2 having the amino acid sequence of SEQ ID NO: 4; and VH CDR3 having the amino acid sequence of SEQ ID NO: 5; and VL CDR1 having the amino acid sequence of SEQ ID NO: 6; and VL CDR2 having the amino acid sequence of SEQ ID NO: 7; and VL CDR3 having the amino acid sequence of SEQ ID NO: 8 comprising, wherein said chemoradiotherapy is platinum-based, The pharmaceutical composition according to claim 10.

14. The human anti-PD-L1 antibody is durvalumab, avelumab, atezolizumab, or sugemalimab; or The human anti-PD-L1 antibody is durvalumab, avelumab, atezolizumab, or sugemalimab, and said chemoradiotherapy is platinum-based, The pharmaceutical composition according to claim 10.

15. The pharmaceutical composition is administered intravenously to the patient at a dose of 1500 mg of said anti-PD-L1 antibody every 4 weeks (Q4W); and / or the pharmaceutical composition is administered on the first day of chemoradiotherapy. The pharmaceutical composition according to any one of claims 9 to 14.

16. The unresectable NSCLC is stage III and / or locally advanced; or The unresectable NSCLC is stage III and / or locally advanced, and the pharmaceutical composition is administered intravenously to the patient at a dose of 1500 mg of said anti-PD-L1 antibody every 4 weeks (Q4W); and / or the pharmaceutical composition is administered on the first day of chemoradiotherapy. The pharmaceutical composition according to any one of claims 9 to 14.

17. A pharmaceutical composition for treating a patient suffering from unresectable non-small cell lung cancer (NSCLC) at stage III, wherein the pharmaceutical composition comprises an anti-PD-L1 antibody and is administered to the patient simultaneously with chemoradiotherapy.

18. The pharmaceutical composition according to claim 17, wherein the anti-PD-L1 antibody is a human anti-PD-L1 antibody.

19. The chemoradiotherapy is platinum-based; or The chemoradiotherapy is platinum-based and the anti-PD-L1 antibody is a human anti-PD-L1 antibody, The pharmaceutical composition according to claim 17.

20. The human anti-PD-L1 antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2; or The human anti-PD-L1 antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, and said chemoradiotherapy is platinum-based, The pharmaceutical composition according to claim 18.

21. The human anti-PD-L1 antibody is VH CDR1 having the amino acid sequence of SEQ ID NO: 3; and VH CDR2 having the amino acid sequence of SEQ ID NO: 4; and VH CDR3 having the amino acid sequence of SEQ ID NO: 5; and VL CDR1 having the amino acid sequence of SEQ ID NO: 6; and VL CDR2 having the amino acid sequence of SEQ ID NO: 7; and VL CDR3 having the amino acid sequence of SEQ ID NO: 8 comprising; or said human anti-PD-L1 antibody is VH CDR1 having the amino acid sequence of SEQ ID NO: 3; and VH CDR2 having the amino acid sequence of SEQ ID NO: 4; and VH CDR3 having the amino acid sequence of SEQ ID NO: 5; and VL CDR1 having the amino acid sequence of SEQ ID NO: 6; and VL CDR2 having the amino acid sequence of SEQ ID NO: 7; and VL CDR3 having the amino acid sequence of SEQ ID NO: 8 comprising, wherein said chemoradiotherapy is platinum-based, The pharmaceutical composition according to claim 18.

22. said anti-PD-L1 antibody is durvalumab, avelumab, atezolizumab, or sintilimab; or said anti-PD-L1 antibody is durvalumab, avelumab, atezolizumab, or sintilimab, and said chemoradiotherapy is platinum-based, The pharmaceutical composition according to claim 18.

23. said pharmaceutical composition is administered intravenously to said patient at a dose of 1500 mg of said anti-PD-L1 antibody every 4 weeks (Q4W), and / or said pharmaceutical composition is administered on the first day of chemoradiotherapy, The pharmaceutical composition according to any one of claims 17 to 22.

24. Use of an anti-PD-L1 antibody in the manufacture of a medicament used to extend progression-free survival (PFS) in a patient suffering from unresectable non-small cell lung cancer (NSCLC), wherein said medicament is administered concomitantly with chemoradiotherapy.

25. Use of an anti-PD-L1 antibody in the manufacture of a medicament used to increase overall response rate (ORR) in a patient suffering from unresectable non-small cell lung cancer (NSCLC), wherein said medicament is administered concomitantly with chemoradiotherapy.

26. Use of an anti-PD-L1 antibody in the manufacture of a medicament used for the treatment of unresectable non-small cell lung cancer (NSCLC) in stage III, wherein said medicament is administered concomitantly with chemoradiotherapy.

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