Combination therapy for the treatment of cancer comprising an anti-PD-L1 antibody and an anti-CD73 antibody

Abstract

The present disclosure relates to methods of treating a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of a T-cell checkpoint inhibitor in combination with chemotherapy and / or radiation therapy, wherein the subject has reduced levels of CD73 protein or CD73 activity compared to a normal subject. In some embodiments, the method further comprises administering a CD73 inhibitor prior to or concurrently with the combined T-cell checkpoint inhibitor and chemotherapy and / or radiation therapy.

Description

[Technical Field]

[0001] The invention disclosed herein relates to a method of treating a tumor in a subject comprising administering a CD73 inhibitor, a T-cell checkpoint inhibitor, and chemotherapy and / or radiation therapy. [Background technology]

[0002] Extracellular adenosine has emerged as a key regulator of immune processes within the tumor microenvironment and is thought to reduce the efficacy of T cell checkpoint inhibitors (Sidders B, et al., Clin Cancer Res 2020, 26:2176-87; Augustin RC, et al., J Immunother Cancer 2022, 10:e004089). Adenosine triphosphate (ATP) released from necrotic or damaged cells is hydrolyzed to adenosine by the sequential action of two ectonucleosidases acting in tandem: CD39 (ENTPD1) and CD73 (NT5E). The resulting adenosine acts as a readily diffusible immunosuppressive "smog," and cytotoxic drugs and radiation therapy likely exacerbate this process. In this regard, enhanced antitumor activity has been observed in preclinical models when radiation therapy was combined with anti-CD73 (aCD73) blocking antibody treatment (Wennerberg E, et al. Cancer Immunol Res 2020;8:465-78; and Wennerberg E, et al., Front Immunol 2017;8:229). These studies highlighted the important role of radiation-induced type 1 interferon (R1) in driving elevated levels of tumor cDC1 infiltration and the beneficial effect of CD73 inhibition on this biomarker when radiation-activated type 1 interferon levels are suboptimal. Despite emerging information on the role of CD73 in the context of radiation-based standard therapies, relatively little is known about the effects of adenosine pathway inhibitors within chemotherapy treatment regimens, despite growing evidence demonstrating the immunomodulatory axis of these agents (Coffelt SB, et al., Trends Immunol 2015;36:198-216). The inclusion of T cell checkpoint inhibitors within this paradigm provides further scope for enhancing cell-mediated activity by countering adaptive immune tolerance and unleashing antitumor immunity.

[0003] Olecurumab, a CD73-blocking human monoclonal IgG1-TM antigen (Hay CM, et al., Oncoimmunology 2016;5), is currently in Phase 2 / 3 clinical development in combination with durvalumab for the treatment of patients with various solid tumors. Data from a Phase 2 platform trial of durvalumab in combination with olecurumab in patients with unresectable stage III non-small cell lung cancer who had not progressed after prior chemoradiation therapy highlighted the significant benefit of the olecurumab component. A Phase 3 clinical trial is currently underway in the same patient population. Therefore, there remains a need for effective therapies. Summary of the Invention

[0004] The present disclosure relates to a method of inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a PD-L1 inhibitor in combination with chemotherapy and / or radiation therapy, wherein the subject has reduced levels of CD73 protein or CD73 activity compared to a normal subject.

[0005] The present disclosure also relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a PD-L1 inhibitor in combination with chemotherapy and / or radiation therapy, wherein the subject has reduced levels of CD73 protein or CD73 activity compared to a normal subject.

[0006] The present disclosure also relates to a method of generating a protective tumor memory response in a subject, the method comprising administering to the subject a therapeutically effective amount of a PD-L1 inhibitor in combination with chemotherapy and / or radiation therapy, wherein the subject has reduced levels of CD73 protein or CD73 activity compared to a normal subject.

[0007] The present disclosure also relates to a method of inhibiting tumor growth in a subject in need thereof, comprising administering to the subject therapeutically effective amounts of a CD73 inhibitor, a PD-L1 inhibitor, and chemotherapy and / or radiation therapy.

[0008] The present disclosure also relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject therapeutically effective amounts of a CD73 inhibitor, a PD-L1 inhibitor, and chemotherapy and / or radiation therapy.

[0009] The present disclosure also relates to a method of generating a protective tumor memory response in a subject, comprising administering to the subject therapeutically effective amounts of a CD73 inhibitor, a PD-L1 inhibitor, and chemotherapy and / or radiation therapy.

[0010] In one embodiment, the PD-L1 inhibitor and chemotherapy and / or radiation therapy are administered simultaneously. In another embodiment, the PD-L1 inhibitor and chemotherapy and / or radiation therapy are administered sequentially. In another embodiment, the CD73 inhibitor is administered prior to administration of the PD-L1 inhibitor and chemotherapy and / or radiation therapy.

[0011] In one aspect, the chemotherapy is docetaxel, 5-fluorouracil, and / or oxaliplatin.

[0012] In one aspect, the PD-L1 inhibitor is an anti-PD-L1 antibody or antigen-binding fragment thereof. In another aspect, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In another aspect, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a HC variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7, and a LC variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8. In another aspect, the anti-PD-L1 antibody is durvalumab.

[0013] In one aspect, the CD73 inhibitor is an anti-CD73 antibody or antigen-binding fragment thereof. In another aspect, the anti-CD73 antibody or antigen-binding fragment thereof comprises: (a) an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 14. In another aspect, the anti-CD73 antibody or antigen-binding fragment thereof comprises an HC variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 15 and an LC variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 16. In another aspect, the anti-CD73 antibody or antigen-binding fragment thereof comprises an HC comprising the amino acid sequence of SEQ ID NO: 17 and an LC comprising the amino acid sequence of SEQ ID NO: 18. In another aspect, the anti-CD73 antibody is oleclumab.

[0014] In one aspect, the administration results in upregulation of CXCR3 in the tumor microenvironment.

[0015] In one aspect, CD73 protein or CD73 activity levels are determined by immunohistochemistry (IHC), imaging mass cytometry (IMC), or mass spectrometry imaging (MSI).

[0016] In another aspect, the tumor or cancer is a solid tumor or a cancer arising from solid tumor growth. In another aspect, the solid tumor is a lung tumor, breast tumor, colon tumor, bladder tumor, prostate tumor, colorectal tumor, head and neck tumor, liver tumor, or pancreatic tumor. In another aspect, the lung tumor is non-small cell lung cancer.

[0017] In one aspect, the subject is a human.

[0018] The present disclosure also relates to the use of a CD73 inhibitor, a PD-L1 inhibitor, and chemotherapy and / or radiation therapy as described herein to treat cancer in a subject in need thereof. [Brief explanation of the drawings]

[0019] [Figure 1] Combination treatment with anti-CD73, anti-PD-L1, and 5FU+OHP results in enhanced complete responses in syngeneic mouse models. (A) Schematic of the experimental design. (B) BALB / c mice (CT26 cells in PBS) and (C) C57BL / 6J mice (MCA205 cells in 50% Matrigel + PBS) were implanted with 5 x 10 cells in the right flank and treated as indicated in the schematic. Growth curves are plotted from caliper measurements performed three times weekly. The addition of aCD73 and aPD-L1 to 5FU+OHP significantly increased the number of complete responders (CRs) in each model system: 50% in CT26 (p = 0.005 vs. 5FU+OHP treatment group) and 61.5% in MCA205 (p = 0.008 vs. 5FU+OHP treatment group) (Kruskal-Wallis test). [Figure 2] Figure 2 shows the response of aCD73 and aPD-L1, alone or in combination, in syngeneic mouse models. BALB / c mice (CT26 cells in PBS (Figure 2A)) and C57BL / 6J mice (MCA205 cells in 50% Matrigel + PBS (Figure 2B)) were implanted with 500,000 cells into the right flank and treated as outlined in Figure 1A. Growth curves were plotted from caliper measurements performed three times weekly. Anti-CD73 monotherapy did not show any effect compared to control-treated mice in both the CT26 and MCA205 models. Anti-PD-L1 monotherapy showed a very low response rate only in CT26 (1 / 13 CR). Combination treatment with aCD73 and aPD-L1 also did not show an enhanced response rate, with 1 / 13 CR mice observed in each of the CT26 and MCA205 tumor models. [Figure 3]Figure 3: Mass spectrometry imaging (MSI) confirmed adenosine pathway modulation by addition of anti-CD73 to 5FU+OHP. (3A) Schematic of the adenosine production pathway. (3B) MSI image showing the abundance of ATP and various metabolites of the adenosine pathway in CT26 tumors. 5FU+OHP resulted in a modest increase in ATP and AMP abundance compared to control-treated tumors, whereas addition of aCD73 to 5FU+OHP significantly reduced adenosine as well as inosine and xanthine. [Figure 4] Figure 4 shows that the addition of anti-CD73 to 5FU+OHP and docetaxel does not result in enhanced cytotoxicity in vitro. Ten thousand HT-29 (4A), HCT-116 (4B), CT26 (4C and 4E), and MCA-205 (4D) cells were seeded into 96-well plates and treated with serially diluted chemotherapeutic agents as indicated along with anti-CD73. Cytotoxicity was measured by CellTiter-Glo® luminescence assay after 72 hours of incubation with the drugs. As shown in the different result panels, none of the tested cell lines showed any additive effect of anti-CD73 to either 5FU+OHP or docetaxel. [Figure 5] Figure 5 shows that CD8 depletion leads to a loss of efficacy observed in aCD73, aPD-L1, and 5FU+OHP combination treatment in the syngeneic mouse model MCA205. (5A) Schematic of the experimental design. (5B) Growth curves in C57BL / 6J mice (5x10 cells in 50% Matrigel+PBS) of the MCA205 tumor model were plotted from caliper measurements taken three times per week. Selective depletion of CD8 T cells resulted in reduced efficacy of the combination treatment, resulting in significantly shorter survival times (Kaplan-Meier plot, log-rank test, p=0.02) compared to those without CD8 cell depletion, as shown in (5C). [Figure 6]IHC and MSI analyses confirm target (CD73) engagement and adenosine pathway modulation by the addition of aCD73 to 5FU+OHP. (6A) Schematic of the experimental design. (6B) Immunohistochemical analysis revealed low levels of surface-bound CD73 protein in the aCD73 / 5FU+OHP combination group. Results are expressed as the area ratio of specific staining to background staining (hematoxylin). (6C) Mass spectrometry imaging showed a significant trend toward adenosine, inosine, and xanthine suppression as early PD biomarkers in CT26 tumors from mice treated with the anti-CD73 triple combination. Results are reported as relative abundance in arbitrary units. (6D) Imaging mass cytometry highlighted that CT26 tumors from mice treated with the aCD73+5FU+OHP combination displayed low frequencies of cells expressing macrophage markers such as CD68, F4 / 80, and suppressive tumor-associated macrophage markers such as CD163 and CD206 [left panel], as well as markers known to be associated with cancer-associated fibroblasts, such as collagen-IV, alpha-smooth muscle actin, and vimentin, along with CD31. Results are expressed as the mean ± SEM of % positive cells. [Figure 7A] Figure 1 shows the pharmacodynamic effects of aCD73 + aPD-L1 + 5FU + OHP on the CT26 transcriptome. RNAseq analysis was used to confirm changes in the CT tumor transcriptome. The top panel shows a schematic of the experimental design. The bottom panel shows the contribution of individual components in the aCD73, aPD-L1, and 5FU + OHP combination treatment compared to the aCD73 + aPD-L1 group. As seen in the bottom left panel, the addition of aCD73 had the most significant effect, resulting in 589 differentially expressed (DE) genes, in contrast to aPD-L1 (bottom right panel), which resulted in only 35 DE genes. The addition of chemotherapy components to the antibody doublet (aCD73 + aPD-L1) resulted in 546 DE genes. The DE cutoff values ​​used were Abs(log2FC) ≥ 1 and Adj-pval < 0.05. [Figure 7B]Figure 1 shows transcriptome-based deconvolution of significant effects mediated by the combination. Deconvolution of RNA-seq analysis revealed that the key pathways affected by the addition of aCD73 and 5FU+OHP were immune response activation pathways. Enriched pathways for upregulated genes, including KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment (KEGG, green) and Gene Ontology > Biological Process (GO BP, red), are shown (DE cutoff values ​​used were log2FC≥1 and Adj-pval<0.05). [Figure 7C] This figure shows that the addition of aCD73 to aPD-L1 + 5FU + OHP promoted the increase of tumor-infiltrating lymphocytes (cytotoxic T cells, NK cells, B cells) and myeloid dendritic cells in CT26 tumors. The abundance of different cell populations was estimated using the MCP counter tool. As shown in the heatmap, the addition of aCD73 had the greatest effect on activating relevant immune cells, such as cytotoxic T cells, NK cells, B cells, and myeloid dendritic cells (DCs). The addition of aPD-L1 to 5FU + OHP did not result in an increase in DC infiltration in tumors, so the DC infiltration effect was mediated solely by the addition of aCD73 to 5FU + OHP. [Figure 8] Figure 8A shows that combined treatment with aCD73, aPD-L1, and docetaxel (DTX) results in enhanced complete responses in a CT26 syngeneic tumor model. (8A) Schematic of the experimental design. (8B) BALB / c mice were implanted with 5x105 CT26 cells in PBS and treated as shown in the schematic. Growth curves were plotted from caliper measurements taken three times weekly. The addition of aCD73 and aPD-L1 to docetaxel resulted in a significant (p=0.0001 vs. vehicle) increase in the number of complete responders (CRs) of 58%, compared to the 25% CRs (p=0.001) seen when aPD-L1 alone was added to docetaxel (Kruskal-Wallis test). [Figure 9]Figure 9. Combined treatment with aCD73, aPD-L1, and radiation therapy (RTx) results in enhanced complete responses in the MC38 syngeneic tumor model. (9A) Schematic of the experimental design. (9B) C57BL6 / J mice were implanted with 5x105 MC38 cells in PBS and treated as shown in the schematic. Growth curves were plotted from caliper measurements taken three times weekly. Concurrent treatment with aCD73, aPD-L1, and radiation therapy resulted in a significant (p=0.0001 vs. NT) complete responder (CR) rate of 58%, compared with no response in the RTx-only group (p=0.5 vs. NT) (Kruskal-Wallis test). [Figure 10] Figure 1 shows imaging mass cytometry (IMC) images of the pharmacodynamic changes observed upon addition of aCD73 to 5FU+OHP. IMC images of CT26 tumor-bearing mice treated with either 5FU+OHP or aCD73+5FU+OHP control. Tumors from aCD73+5FU+OHP combination-treated mice had a low frequency of cells expressing macrophage markers such as CD68, F4 / 80, and suppressive tumor-associated macrophage markers such as CD163 and CD206 [left panel], as well as markers known to be associated with cancer-associated fibroblasts, such as collagen-IV, α-smooth muscle actin, vimentin, and CD31. [Figure 11] Figure 1 shows that the top 50 differentially expressed genes for the triple combination vs. control were identified and the log2 fold change was shown for the different comparisons. The heatmap in the right panel shows the expression changes of selected immune-related genes across the different conditions. [Figure 12] Figure 1 shows a schematic diagram of treatment groups divided by aCD73, aPD-L1, and timing of radiation therapy determined using the MC38 syngeneic mouse model. Six groups of mice were implanted with 5x10 cells as indicated. [Figure 13] Figure 1 shows that mice co-treated with aCD73, aPD-L1 and RTx exhibited the highest levels of tumor inhibition and subsequent survival probability. [Figure 14]Figure 10 shows that mice co-treated with aCD73, aPD-L1 and RTx also showed induction of protective memory responses upon rechallenge using the B16F10 and MC38 mouse models. [Figure 15] Figure 1 shows a schematic representation of treatment groups divided by timing of individual aCD73, aPD-L1, and radiation therapy. MC38 cells were transplanted as before, and timing of individual treatments was mapped. [Figure 16] FIG. 1 shows that administering aCD73 therapy prior to aPD-L1 and RTx resulted in the greatest reduction in tumor volume, which correlated with the highest probability of survival. Detailed Description of the Invention

[0020] Using mouse models of cancer and a mouse surrogate of oleclumab (hereafter referred to as aCD73), we investigated the effects of CD73 inhibition in combination with chemotherapy or radiation therapy and PD-L1 blockade. As a corollary, we sought to determine whether the same approach could be applied to enhance the efficacy of radiation therapy. As described herein, these combinations were highly effective in improving tumor growth inhibition, inducing protective memory responses, and prolonging overall survival. Transcriptome-based pharmacodynamic assessment highlighted increased abundance of cytotoxic lymphocytes and immune-supportive myeloid populations in the tumor. Profiling treatment groups representing the various components of the combination allowed for deconvolution of the individual contributing treatment components and highlighted the effects conferred by CD73 inhibition in the context of chemotherapy and PD-L1 blockade.

[0021] 1.Definition In order that this disclosure may be more readily understood, certain terms are first defined. As used herein, unless expressly provided otherwise, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0022] Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular compositions or process steps, as such may vary. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as "one or more" and "at least one," may be used interchangeably herein.

[0023] Furthermore, "and / or," as used herein, should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0025] Units, prefixes, and symbols are shown in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects that may be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0026] Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the words "consisting of" and / or "consisting essentially of" are also provided.

[0027] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted single-letter codes.

[0028] An "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds that specifically binds to an antigen, or an antigen-binding portion thereof. Each H chain contains a heavy chain variable region (referred to herein as V H The heavy chain constant region comprises three constant domains: C H1 , C H2 and C H3 Each light chain comprises a light chain variable region (referred to herein as V L The light chain constant region comprises one constant domain, C L Includes V H and V LThe regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) and highly conserved, interspersed regions called framework regions (FRs). H and V L Each of the heavy and light chain variable regions contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise specified herein, amino acids in the variable region are numbered using the Kabat numbering system, and amino acids in the constant region are numbered using the EU system.

[0029] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the class or subclass of antibody (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. Terms such as "antibody" include, by way of example, monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless explicitly stated and unless the context indicates otherwise, terms such as "antibody" include monospecific, bispecific, or multispecific antibodies, as well as single-chain antibodies. In some embodiments, an antibody is a bispecific antibody. In other embodiments, an antibody is a monospecific antibody.

[0030] As used herein, an "IgG antibody" has the structure of a naturally occurring IgG antibody, i.e., it has the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, an anti-ICOS IgG1, IgG2, IgG3, or IgG4 antibody consists of two heavy chains (HC) and two light chains (LC), which are linked by the same number and positions of disulfide bridges as occur in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies, respectively (unless the antibody has been mutated to alter the disulfide bonds).

[0031] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds PD-L1 is substantially free of antibodies that specifically bind antigens other than PD-1). However, an isolated antibody that specifically binds PD-L1 may have cross-reactivity to other antigens, such as PD-L1 molecules from different species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0032] The antibody may be an altered antibody (e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). For example, the antibody may contain one or more variant amino acids (compared to a naturally occurring antibody) that alter the properties (e.g., functional properties) of the antibody. Many such alterations are known in the art that affect, for example, half-life, effector functions, and / or the immune response to the antibody in a patient. The term antibody also includes artificial polypeptide constructs that contain at least one antibody-derived antigen-binding site.

[0033] The term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., antibody molecules which are essentially identical in primary sequence and which display a single binding specificity and affinity for a particular epitope. A mAb is an example of an isolated antibody. MAbs may be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0034] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0035] A "humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not destroy the antibody's ability to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0036] A "chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, e.g., whose variable region is derived from a murine antibody and whose constant region is derived from a human antibody.

[0037] An "anti-antigen" antibody refers to an antibody that specifically binds to an antigen. For example, an anti-PD-L1 antibody specifically binds to PD-L1, and an anti-CD73 antibody specifically binds to CD73.

[0038] An "antigen-binding portion" (also called an "antigen-binding fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen to which the whole antibody binds. It has been shown that the antigen-binding function of an antibody can be performed by fragments or portions of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody, such as the anti-CD73 antibodies described herein, include: (1) Fab fragment (a fragment derived from papain cleavage) or a similar monovalent fragment consisting of the VL, VH, LC, and CH1 domains; (2) F(ab')2 fragment (pepsin cleavage-derived fragment) or a similar bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment consisting of a VH domain and a CH1 domain; (4) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (5) Single-domain antibody (dAb) fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-46), which consist of a VH domain; (6) bi-single domain antibodies consisting of two VH domains linked by a hinge (dual affinity retargeting antibodies (DARTs)); (7) dual variable domain immunoglobulins; (8) an isolated complementarity-determining region (CDR); and (9) Combinations of two or more isolated CDRs, optionally linked by a synthetic linker, are included. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker, allowing them to be produced using recombinant techniques as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0039] As used herein, the term "CD73 polypeptide" refers to the CD73 (cluster of differentiation 73) protein, also referred to in the literature as 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase, which is encoded by the NT5E gene. See, e.g., Misumi et al. Eur. J. Biochem. 191(3):563-9 (1990). The sequences of human and murine CD73 are available in the Uniprot database under accession numbers P21589 and Q61503, respectively. In defining any CD73 antibody epitope, the amino acid numbering used refers to the amino acid residues of the mature CD73 protein, not including the signal sequence residues. Thus, for example, an antibody that binds to amino acids Vall44, Lysl80, and Asnl85 refers to the amino acid positions after cleavage of the signal sequence, i.e., the amino acids in the mature protein.

[0040] A "T cell checkpoint inhibitor" or "immune checkpoint inhibitor" refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduction and complete blocking of function. In particular, the immune checkpoint protein is a human immune checkpoint protein. Thus, the immune checkpoint protein inhibitor is, in particular, an inhibitor of a human immune checkpoint protein.

[0041] "Programmed death-ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as five analogs that share at least one shared epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

[0042] As used herein, a "patient" includes any patient suffering from cancer (e.g., non-small cell lung cancer (NSCLC)). The terms "subject" and "patient" are used interchangeably herein.

[0043] "Administration" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration," as used herein, refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulations are administered via a parenteral route, in some embodiments, orally. Other parenteral routes include topical, epidermal, or mucosal administration routes, such as intranasal, intravaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0044] "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject, or administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing or preventing the onset, progression, occurrence, severity or recurrence of a symptom, complication or condition, or biochemical manifestations associated with a disease.

[0045] As used herein, "effective treatment" refers to treatment that results in a beneficial effect, e.g., an improvement in at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement over baseline, i.e., an improvement over measurements or observations made before initiation of treatment according to the method. A beneficial effect can also take the form of a halt, slowing, delay, or stabilization of adverse progression of markers of a solid tumor. Effective treatment can refer to the alleviation of at least one symptom of a solid tumor. Such effective treatment can, for example, relieve a patient's pain, reduce the size and / or number of lesions, reduce or prevent tumor metastasis, and / or slow tumor growth.

[0046] The term "effective amount" refers to the amount of an agent that provides a desired biological, therapeutic, and / or prophylactic result. The result can be reduction, amelioration, palliative, relief, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to shrink the tumor and / or reduce the rate of tumor growth (such as inhibiting tumor growth), or prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor onset. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition may (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent, and stop cancer cell invasion of peripheral organs; (iv) inhibit (i.e., slow to some extent, and stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor development and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. In one example, an "effective amount" is the amount of anti-CD73 antibody and the amount of anti-PD-L1 antibody, in combination, that has been clinically proven to affect a significant reduction in cancer or a slowing of the progression of cancer, such as an advanced solid tumor. As used herein, the term "progression-free survival," which may be abbreviated as PFS, refers to the length of time a patient lives with, but does not worsen, disease during and after treatment for a solid tumor (i.e., NSCLC).

[0047] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and proliferation leads to the formation of malignant tumors that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors.

[0048] As used herein, the term "tumor" refers to any mass of tissue resulting from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0049] An "immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced either by these cells or the liver, resulting in the selective targeting, binding, damaging, destroying, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0050] Various aspects of the disclosure are described in further detail in the following subsections.

[0051] 2. Methods of the Disclosure In one aspect, the present disclosure relates to a method for inhibiting tumor growth in a subject having reduced levels of CD73 protein expression or CD73 activity compared to a normal subject. Combination therapy of a T-cell checkpoint inhibitor with chemotherapy and / or radiation therapy results in better treatment outcomes (e.g., objective response rate and disease control rate). To improve the treatment of malignant tumors, in one aspect, the present disclosure provides for identifying patients with reduced CD73 protein expression or CD73 activity and providing combination therapy of a T-cell checkpoint inhibitor with chemotherapy and / or radiation therapy.

[0052] A wide variety of chemotherapeutic agents may be used in accordance with embodiments of the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to a compound or composition administered in the treatment of cancer. These agents or drugs are classified by their mode of activity within cells, for example, whether and at what stage they affect the cell cycle. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0053] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metoledopa, and uredopa; ethyleneimines and methylameramines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma II and calicheamicin omega II; dynemicin (including dynemicin A); bisphosphonates such as clodronate; esperamicin;Also included are neocarzinostatin chromophores and related enediyne antibiotic chromophores, aclacinomycin, actinomycin, ausrarnycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and detorubicin). oxidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, protease inhibitors, and steroids. teropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine, androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as mitotane and trilostane; folic acid supplements such as floric acid; aceglatone ;Aldophosphamide glycosides;Aminolevulinic acid;Eniluracil;Amsacrine;Vestravcil;Bisantrene;Edatraxate;Defofamine;Demecolcine;Diazicon;Elformitin;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidanine;Maytansinoids, such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidanol;Nitraerin;Pentostatin;Fenamet;Pirarubicin;Losoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2"-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids, e.g., Paclitaxel Clitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum complexes, e.g., cisplatin, oxaliplatin, carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0054] In another embodiment, the combination includes radiation therapy. Other widely used agents that cause DNA damage include what are commonly known as gamma rays, X-rays, and / or direct delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents, such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation, are also contemplated. All of these agents most likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50 to 200 roentgens over prolonged periods (3 to 4 weeks) to single doses of 2,000 to 6,000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0055] In one aspect, the disclosure relates to identifying patients with reduced levels of CD73 protein expression or CD73 activity and treating the subject by administering a T-cell checkpoint inhibitor (e.g., an anti-PD-L1 antibody) and chemotherapy and / or radiation therapy. In one aspect, the disclosure includes a method of identifying patients with reduced levels of CD73 protein expression or CD73 activity and treating the subject by administering an anti-PD-L1 antibody and chemotherapy and / or radiation therapy.

[0056] In another aspect, the disclosure relates to a method of inhibiting tumor growth in a subject comprising administering a combination therapy of a CD73 inhibitor, a T-cell checkpoint inhibitor, and chemotherapy and / or radiation therapy, hi one aspect, tumor growth in the subject is inhibited by administering an anti-CD73 antibody, an anti-PD-L1 antibody, and chemotherapy and / or radiation therapy.

[0057] CD73 is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein that catalyzes the hydrolysis of adenosine monophosphate (AMP) to adenosine, working in concert with CD39, which converts adenosine triphosphate (ATP) to AMP. The resulting adenosine functions as a signaling molecule that activates P1 receptors, which are expressed on the cell surface in many different tissues. Four G protein-coupled P1 receptors or adenosine receptors have been cloned and named A1, A2A, A2B, and A3. Adenosine influences a wide range of physiological processes, including neuronal function, vascular perfusion, and immune response. In doing so, this metabolite regulates CNS, cardiovascular, and immune system functions, to name a few.

[0058] Increasing evidence suggests that interactions between tumor cells and their microenvironment are essential for tumorigenesis. The purinergic signaling pathway, in which CD73 plays a key role, has emerged as a key player in cancer progression. In recent years, adenosine has been shown to be one of the most important immunosuppressive regulatory molecules in the tumor microenvironment, contributing to immune evasion and tumor progression.

[0059] CD73 is a key protein molecule in cancer development and has been found to be overexpressed in many cancer cell lines and tumor types, including breast cancer, colorectal cancer, ovarian cancer, gastric cancer, gallbladder cancer, and cancers associated with poor prognosis.

[0060] In addition to being a prognostic biomarker in cancer patients, overexpression of CD73 has also been shown to be functionally associated with resistance to treatment (e.g., cancer treatment): elevated levels of CD73 were initially associated with resistance to various chemotherapeutic agents, including vincristine and doxorubicin.

[0061] CD73 has also been shown to be involved in immunotherapy resistance. This ectonucleotidase participates in the process of tumor immune evasion by inhibiting the activation, clonal expansion, and homing of tumor-specific T cells (especially T helper and cytotoxic T cells), impairing tumor cell killing by cytolytic effector T lymphocytes, driving the suppressive capacity of Treg and Th17 cells via pericellular generation of adenosine, enhancing the conversion of type 1 macrophages to tumor-promoting type 2 macrophages, and promoting the accumulation of MDSCs.

[0062] In some aspects of the present disclosure, the subject being treated has decreased CD73 protein expression or CD73 activity. In some aspects, the decrease is caused by prior treatment with a CD73 inhibitor. In some aspects, the CD73 inhibitor is an anti-CD73 antibody or antigen-binding fragment thereof.

[0063] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof is an antibody described in WO 2016 / 075099. In some embodiments, the anti-CD73 antibody comprises HC CDRs 1-3 and LC CDRs 1-3 of SEQ ID NOs: 9-11 and 12-14, respectively. In some embodiments, the anti-CD73 antibody comprises VH and VL of SEQ ID NOs: 15 and 16, respectively. In some embodiments, the anti-CD73 antibody comprises heavy and light chains of SEQ ID NOs: 17 and 18, respectively. In some embodiments, the anti-CD73 antibody is olcurab.

[0064] In some embodiments, the method comprises administering an anti-CD73 antibody or antigen-binding fragment thereof prior to or concurrently with the T-cell checkpoint inhibitor and chemotherapy and / or radiation therapy. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof is an antibody described in WO 2016 / 075099. In some embodiments, the anti-CD73 antibody comprises HC CDRs 1-3 and LC CDRs 1-3 of SEQ ID NOS: 9-11 and 12-14, respectively. In some embodiments, the anti-CD73 antibody comprises VH and VL of SEQ ID NOS: 15 and 16, respectively. In some embodiments, the anti-CD73 antibody comprises heavy and light chains of SEQ ID NOS: 17 and 18, respectively. In some embodiments, the anti-CD73 antibody is olcurab.

[0065] In some embodiments, prior art antibodies may be used to reduce CD73 expression and / or activity. Exemplary anti-CD73 antibodies are described in WO 2018 / 137598, WO 2016 / 081748, WO 2017 / 064043, WO 2017 / 100670, and WO 2018 / 237157.

[0066] In one aspect, the disclosure includes a method of selecting a tumor in a human patient for immunotherapy, the method comprising: (a) determining the level of CD73 protein expression or CD73 activity in a tumor sample, and (b) selecting the tumor for immunotherapy if the tumor sample exhibits decreased CD73 protein expression or CD73 activity. In one aspect, the disclosure includes a method of identifying a tumor in a human patient that is likely to be responsive to immunotherapy, the method comprising: (a) determining the level of CD73 protein expression or CD73 activity in a tumor sample, and (b) identifying the tumor as likely to be responsive to treatment if the tumor exhibits decreased CD73 protein expression or CD73 activity. In some aspects, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor. In some aspects, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-PD-L1 antibody. In some aspects, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-PD-1 antibody. In some aspects, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-CTLA-4 antibody. In some embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor and chemotherapy and / or radiation therapy.

[0067] In another aspect, the disclosure includes a method for treating tumor growth in a human patient in need thereof, comprising administering to the patient a T-cell checkpoint inhibitor and chemotherapy and / or radiation therapy, wherein the patient has been identified as having reduced CD73 protein expression or CD73 activity prior to administration. In some aspects, the T-cell checkpoint therapy comprises administering a therapeutically effective amount of a PD-1 pathway inhibitor. In some aspects, the T-cell checkpoint therapy comprises administering a therapeutically effective amount of an anti-PD-L1 antibody.

[0068] In yet other aspects, the disclosure includes methods of reducing tumor size by at least 10% in a human patient afflicted with a tumor, comprising administering to the patient a combination therapy disclosed herein (e.g., an anti-CD73 antibody, an anti-PD-L1 antibody, and chemotherapy and / or radiation therapy; or an anti-PD-L1 antibody and chemotherapy and / or radiation therapy). In some aspects, the patient has been identified as having reduced CD73 protein expression or CD73 activity prior to administration, and the administration reduces the tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration.

[0069] The present disclosure may also include methods of preventing recurrence and / or inducing remission in a patient, the methods comprising administering to the patient a combination therapy disclosed herein (e.g., an anti-CD73 antibody, an anti-PD-L1 antibody, and chemotherapy and / or radiation therapy, or an anti-PD-L1 antibody and chemotherapy and / or radiation therapy). In some aspects, the methods of the disclosure include methods comprising: (i) identifying a patient with decreased CD73 protein expression or CD73 activity; and (ii) administering to the patient a combination therapy disclosed herein (e.g., an anti-CD73 antibody, an anti-PD-L1 antibody, and chemotherapy and / or radiation therapy, or an anti-PD-L1 antibody and chemotherapy and / or radiation therapy).

[0070] The methods of the present disclosure can treat malignant tumors, reduce tumor size, prevent tumor growth, eliminate tumors from patients, prevent tumor recurrence, induce remission in patients, or any combination thereof, as a result of administering a combination therapy disclosed herein. In certain aspects, administration of a combination therapy disclosed herein induces a complete response. In other aspects, administration of a combination therapy disclosed herein induces a partial response. In some aspects, the immunotherapy comprises administering a therapeutically effective amount of a PD-1 pathway inhibitor and chemotherapy and / or radiation therapy. In some aspects, the PD-1 pathway inhibitor is an anti-PD-L1 antibody. In some aspects, the combination therapy comprises administering therapeutically effective amounts of a CD73 inhibitor, a T-cell checkpoint inhibitor, and chemotherapy and / or radiation therapy. In some aspects, the combination therapy comprises administering therapeutically effective amounts of an anti-CD73 antibody, an anti-PD-L1 antibody, and chemotherapy and / or radiation therapy.

[0071] In some embodiments, CD73 expression or CD73 activity is determined by receiving the results of an assay in which CD73 expression / activity can be determined.

[0072] Measurement of CD73 activity / expression To assess CD73 expression / activity, in one embodiment, a test sample is obtained from a patient in need of treatment. In some embodiments, the test sample includes any clinically relevant sample, such as, but not limited to, a tumor biopsy, a core biopsy tissue sample, a fine needle aspirate, or a bodily fluid sample, such as blood, plasma, serum, lymph, ascites, cyst fluid, or urine. In some embodiments, the test tissue sample is from a primary tumor. In some embodiments, the test sample is from a metastasis. In some embodiments, test samples are collected from a subject at multiple time points, e.g., before, during, and / or after treatment. In some embodiments, test samples are collected from different locations in the subject, e.g., a sample from the primary tumor and a sample from a metastasis at a distant location.

[0073] In some embodiments, the tissue sample tested is a paraffin-embedded, fixed tissue sample. In some embodiments, the tissue sample tested is a formalin-fixed, paraffin-embedded (FFPE) tissue sample. In some embodiments, the tissue sample tested is a fresh tissue (e.g., tumor) sample. In some embodiments, the tissue sample tested is a frozen tissue sample. In some embodiments, the tissue sample tested is a fresh-frozen (FF) tissue (e.g., tumor) sample. In some embodiments, the tissue sample tested is cells isolated from a fluid. In some embodiments, the tissue sample tested comprises circulating tumor cells (CTCs). In some embodiments, the tissue sample tested comprises circulating lymphocytes. In some embodiments, the tissue sample tested is an archived tissue sample. In some embodiments, the tissue sample tested is an archived tissue sample with a known diagnostic, treatment, and / or outcome history. In some embodiments, the sample is a block of tissue. In some embodiments, the tissue sample tested is dispersed cells. In some embodiments, the sample size is between about 1 cell and about 1 x 10 6 In some embodiments, the sample size is from about 1 cell to about 1 x 10 5 In some embodiments, the sample size is from about 1 cell to about 10,000 cells. In some embodiments, the sample size is from about 1 cell to about 1,000 cells. In some embodiments, the sample size is from about 1 cell to about 100 cells. In some embodiments, the sample size is from about 1 cell to about 10 cells. In some embodiments, the sample size is a single cell.

[0074] In another embodiment, assessing CD73 activity / expression can be accomplished without obtaining a test tissue sample. In some embodiments, selecting an appropriate patient includes (i) providing a test tissue sample containing tumor cells, optionally obtained from a patient with cancer of the tissue, and (ii) assessing the percentage of cells in the test tissue sample that express CD73 on their cell surface based on assessing that the percentage of cells in the test tissue sample that express CD73 on their cell surface is below a predetermined threshold level.

[0075] However, it should be understood that in any of the methods involving measuring CD73 expression in a test sample, the step involving preparing a test sample obtained from a patient is an optional step. That is, in certain embodiments, the method includes this step, while in other embodiments, it does not. It should also be understood that in certain embodiments, the "measuring" or "assessing" step to identify or determine the number or percentage of cells in the test sample that express CD73 is performed by a transformation method that assays for CD73 expression, e.g., by performing a reverse transcriptase-polymerase chain reaction (RT-PCR) assay, an IHC, an imaging mass cytometry (IMC), or a mass spectrometry imaging (MSI) assay. In certain other embodiments, a transformation step is not included, and CD73 expression is assessed, e.g., by reviewing a test result report from a laboratory. In some embodiments, CD73 activity / expression is assessed, e.g., by reviewing the results of an immunohistochemistry assay from a laboratory. In certain embodiments, the step of preparing a test result is performed by a physician or someone acting under the direction of a physician. In other aspects, these steps are performed by an independent laboratory or by an independent individual, such as a laboratory technician.

[0076] In certain embodiments of any of the methods, the percentage of cells expressing CD73 is assessed by performing an assay to detect the presence of CD73 RNA. In further embodiments, the presence of CD73 RNA is detected by RT-PCR, in situ hybridization, or RNase protection. In some embodiments, the presence of CD73 RNA is detected by an RT-PCR-based assay. In some embodiments, scoring the RT-PCR-based assay comprises assessing the level of CD73 RNA expression in the test tissue sample relative to a predetermined level.

[0077] In other embodiments, the proportion of cells expressing CD73 is assessed by performing an assay to detect the presence of a CD73 polypeptide. In further embodiments, the presence of a CD73 polypeptide is detected by IHC, enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some embodiments, CD73 expression is assayed by IHC, imaging mass cytometry (IMC), or mass spectrometry imaging (MSI). In all other embodiments of these methods, cell surface expression of CD73 is assayed using, for example, IHC or in vivo imaging.

[0078] T cell checkpoint inhibitors In the tumor microenvironment, cancer cells can evade immune surveillance by altering their surface antigens, thus avoiding detection and destruction by host lymphocytes. A central mechanism of tumor-induced immune suppression is the increased expression of ligands capable of binding to inhibitory T cell receptors. These ligands, known as T cell or immune checkpoints, act under physiological conditions to prevent the development of autoimmunity at multiple stages during the immunological response. The primary mechanism involved in T cell regulation is the suppression of potentially autoreactive naive T cells (characterized by TCRs directed against self-antigens) at an early stage in lymph nodes or at a later stage of T cell inactivation in peripheral tissues. This process is called peripheral immune tolerance and is primarily exerted by immune checkpoints in the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) pathways. Tumor cells have developed a way to exploit peripheral immune tolerance by inducing unregulated immune checkpoint expression by T cells to evade immune recognition.

[0079] Other novel checkpoints have also been discovered. Next-generation immune checkpoints include, for example, lymphocyte-activation gene-3 (LAG-3), T-cell immunoglobulin and mucin domain-containing-3 (TIM-3), B- and T-cell lymphocyte attenuator (BTLA), T-cell immunoglobulin and ITIM domain (TIGIT), V-domain Ig suppressor of T-cell activation (VISTA), and B7 homolog 3 protein (B7-H3).

[0080] PD-1 pathway inhibitors In certain embodiments, the present application encompasses the use of anti-PD-L1 antibodies as T cell checkpoint inhibitors. In one embodiment, the anti-PD-L1 antibodies inhibit PD-L1 receptor binding, i.e., the binding of PD-1 to its ligand PD-L1.

[0081] Anti-human PD-L1 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of the disclosure can be generated using methods well known in the art. In certain embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is an antibody described in WO 2011 / 066389. In some embodiments, the anti-PD-L1 antibody comprises HC CDR1-3 and LC CDR1-3 of SEQ ID NOs: 1-3 and 4-6, respectively. In some embodiments, the anti-PD-L1 antibody comprises VH and VL of SEQ ID NOs: 7 and 8, respectively. In some embodiments, the anti-CD73 antibody is durvalumab.

[0082] Alternatively, art-recognized anti-PD-L1 antibodies can be used. For example, anti-PD-L1 antibodies useful in the claimed methods are disclosed in U.S. Patent No. 7,943,743. Such anti-PD-L1 antibodies include 12A4 (also known as BMS-936559). In some embodiments, the anti-PD-L1 antibody is atezolizumab (Tecentriq or RG7446) (see, e.g., Herbst et al. (2013) J Clin Oncol 31(suppl):3000). Abstract; U.S. Patent No. 8,217,149), or avelumab (Bavencio). Other art-recognized anti-PD-L1 antibodies that can be used include, for example, those described in U.S. Patent Nos. 7,635,757 and 8,217,149, U.S. Patent Application Publication No. 2009 / 0317368, and WO 2011 / 066389 and WO 2012 / 145493, which are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies or inhibitors for binding to PD-L1 can also be used.

[0083] In a specific embodiment, the antibody that cross-competes for binding to human PD-L1 with or binds to the same epitope region of human PD-L1 as the PD-L1 antibodies described above is a mAb. For administration to human subjects, these cross-competing antibodies can be chimeric, humanized, or human. Such chimeric, humanized, or human mAbs can be prepared and isolated by methods well known in the art.

[0084] In a specific embodiment, the PD-L1 antibody is durvalumab (IMFINZI™). Durvalumab is a human IgG1 kappa monoclonal anti-PD-L1 antibody.

[0085] In one particular embodiment, the PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0086] In one particular embodiment, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

[0087] Anti-PD-L1 antibodies that can be used in the disclosed methods also include isolated antibodies that specifically bind to human PD-L1 and cross-compete with any of the anti-PD-L1 antibodies disclosed herein, e.g., durvalumab, atezolizumab, and / or avelumab, for binding to human PD-L1. In some embodiments, the anti-PD-L1 antibody binds to the same epitope as any of the anti-PD-L1 antibodies described herein, e.g., durvalumab, atezolizumab, and / or avelumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the reference antibody due to their binding to the same epitope region of PD-L1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and / or avelumab in standard PD-L1 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO 2013 / 173223).

[0088] In certain embodiments, antibodies that cross-compete for binding to human PD-L1 with or bind to the same epitope region of the human PD-L1 antibody as durvalumab, atezolizumab, and / or avelumab are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric, engineered, humanized, or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0089] Anti-PD-L1 antibodies that can be used in the disclosed methods also include antigen-binding portions of such antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0090] Anti-PD-L1 antibodies suitable for use in the disclosed methods or compositions are those that bind to PD-L1 with high specificity and affinity, block PD-1 binding, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-L1 "antibody" includes an antigen-binding portion or fragment that binds to PD-L1 and exhibits functional properties similar to those of the whole antibody in inhibiting receptor binding and upregulating the immune system. In certain aspects, the anti-PD-L1 antibody or antigen-binding portion thereof cross-competes with durvalumab, atezolizumab, and / or avelumab for binding to human PD-L1.

[0091] 3.PD-1 inhibitors In some embodiments, the T cell checkpoint inhibitor is a PD-1 pathway inhibitor, e.g., an anti-PD-1 antibody. In some embodiments, the PD-1 pathway inhibitor is a PD-L2 binding agent, e.g., an anti-PD-L2 antibody. In further embodiments, the PD-L1 binding agent is a soluble PD-1 polypeptide, e.g., a PD-1-Fc fusion polypeptide capable of binding to PD-L1. In further embodiments, the PD-L2 binding agent is a soluble PD-1 polypeptide, e.g., a PD-1-Fc fusion polypeptide capable of binding to PD-L2.

[0092] Anti-human PD-1 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present disclosure can be generated using methods well known in the art, or art-recognized anti-PD-1 antibodies can be used.

[0093] In another embodiment, the anti-PD-1 antibody is nivolumab or BMS-936558, as described in WO 2006 / 121168. Other known PD-1 antibodies include lambrolizumab (MK-3475), as described in WO 2008 / 156712. Additional known PD-1 antibodies and other PD-1 inhibitors include those described in, for example, WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335, and WO 2011 / 161699, which are incorporated herein by reference. In one embodiment, the anti-PD-1 antibody is REGN2810. In one embodiment, the anti-PD-1 antibody is PDR001. Another known anti-PD-1 antibody is pidilizumab (CT-011).

[0094] In some embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with pembrolizumab. In some embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as pembrolizumab. In certain embodiments, the anti-PD-1 antibody has the same CDRs as pembrolizumab. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as "KEYTRUDA®," lambrolizumab, and MK-3475) is a humanized monoclonal IgG4 antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is discussed, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0095] In certain embodiments, the first antibody is an anti-PD-1 antagonist. One example of an anti-PD-1 antagonist is AMP-224, a B7-DC Fc fusion protein. AMP-224 is discussed in U.S. Patent Application Publication No. 2013 / 0017199.

[0096] In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with BGB-A317. In some embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as BGB-A317. In particular embodiments, the anti-PD-1 antibody has the same CDRs as BGB-A317. In particular embodiments, the anti-PD-1 antibody is the humanized monoclonal antibody BGB-A317. BGB-A317 is discussed in U.S. Patent Application Publication No. 2015 / 0079109.

[0097] In some embodiments, the antibody is pidilizumab (CT-011), an antibody previously reported to bind to PD-1 but believed to bind to a different target. Pidilizumab is described in U.S. Pat. No. 8,686,119 (B2) or WO 2013 / 014668 (A1).

[0098] In certain embodiments, antibodies that cross-compete with nivolumab for binding to human PD-1 or that bind to the same epitope region of human PD-1 as nivolumab are mAbs. For administration to human subjects, these cross-competing antibodies can be chimeric, humanized, or human antibodies. Such chimeric, humanized, or human mAbs can be prepared and isolated by methods well known in the art.

[0099] Other anti-PD-1 monoclonal antibodies are described, for example, in U.S. Pat. Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509; U.S. Patent Application Publication No. 2016 / 0272708; and International Publication No. WO WO 2012 / 145493, WO 2008 / 156712, WO 2015 / 112900, WO 2012 / 145493, WO 2015 / 112800, WO 2014 / 206107, WO 2015 / 35606, WO 2015 / 085847, WO 2014 / 179664, WO 2017 / 020291, WO 2017 / 020858, WO 2016 / 197367, WO 2017 / 024515, WO 2017 / 025051, WO 2017 / 123557 , WO 2016 / 106159, WO 2014 / 194302, WO 2017 / 040790, WO 2017 / 133540, WO 2017 / 132827, WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 106061, WO 2017 / 19846, WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540, each of which is incorporated by reference in its entirety.

[0100] Anti-PD-1 antibodies useful in the compositions of the present disclosure also include antigen-binding portions of such antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) Fab fragments, V fragments, and VI fragments. L , V H , C L and C H1 (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a V H and C H1(iv) a Fd fragment consisting of a V domain of a single arm of an antibody; L and V H and Fv fragments consisting of domains.

[0101] Anti-PD-1 antibodies that can be used in the disclosed methods also include isolated antibodies that specifically bind to human PD-1 and cross-compete with any of the anti-PD-1 antibodies disclosed herein for binding to human PD-1. In some embodiments, the anti-PD-1 antibody binds to the same epitope as any of the anti-PD-1 antibodies described herein. The ability of antibodies to cross-compete for binding to an antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties that are very similar to those of the reference antibody.

[0102] 4. LAG-3 inhibitors In some embodiments, the LAG-3 inhibitor is a LAG-3 binding agent, e.g., an anti-LAG-3 antibody. In some embodiments, the LAG-3 inhibitor is a soluble LAG-3 polypeptide, e.g., a LAG-3-Fc fusion polypeptide capable of binding to MHC class II.

[0103] Anti-human LAG-3 antibodies (or VH / VL domains derived therefrom) suitable for use in the present disclosure can be generated using methods well known in the art. Alternatively, art-recognized anti-LAG-3 antibodies can be used. In certain aspects, LAG-3 inhibitors include anti-LAG-3 bispecific antibodies.

[0104] In some embodiments, the anti-LAG-3 antibody is leratolimab or BMS-986016, which comprises the heavy and light chains described in US Patent Application No. 13 / 48999.

[0105] In another embodiment, the antibody competes for binding to and / or binds to the same epitope on LAG-3 as the above antibodies.

[0106] In some embodiments, art-recognized anti-LAG-3 antibodies can be used in the therapeutic methods of the present disclosure. For example, the anti-human LAG-3 antibody described in U.S. Patent Application Publication No. 2011 / 0150892(A1) and designated monoclonal antibody 25F7 ("25F7" and "LAG-3.1") can be used. Other art-recognized anti-LAG-3 antibodies that can be used include IMP731 (H5L7BW), described in U.S. Patent Application Publication No. 2011 / 007023, MK-4280 (28G-10), described in WO 2016028672, and the antibody described in Journal for ImmunoTherapy of Cancer, (2016) Vol. 4, Supplement 1 Abstract Examples thereof include REGN3767 described in International Publication No. P195, BAP050 described in International Publication No. WO 2017 / 019894, IMP-701 (LAG-525), Sym022, TSR-033, MGD013, BI754111, FS118, AVA-017, and GSK2831781.These and other anti-LAG-3 antibodies useful in the claimed disclosure are described in, for example, WO 2016 / 028672, WO 2017 / 106129, WO 2017 / 062888, WO 2009 / 044273, WO 2018 / 069500, WO 2016 / 126858, WO 2014 / 179664, WO 2016 / 200782, WO 2015 / 200119, WO 2017 / 019846, WO 2017 / 198741, WO 2017 / 220555, WO 201 Patent applications can be found in U.S. Patent Application Publication Nos. WO 7 / 220569, WO 2018 / 071500, WO 2017 / 015560, WO 2017 / 025498, WO 2017 / 087589, WO 2017 / 087901, WO 2018 / 083087, WO 2017 / 149143, WO 2017 / 219995, U.S. Patent Application Publication Nos. 2017 / 0260271, WO 2017 / 086367, WO 2017 / 086419, WO 2018 / 034227, and WO 2014 / 140180. In one embodiment, the LAG-3 inhibitor is IMP321 (efthiradimod alfa). The contents of each of these references are incorporated herein by reference in their entirety.

[0107] Antibodies that compete with any of the above art-recognized antibodies for binding to LAG-3 can also be used.

[0108] 5. CTLA-4 antagonists In certain embodiments, the present application encompasses the use of anti-CTLA-4 antibodies. In one embodiment, the anti-CTLA-4 antibody binds to and inhibits CTLA-4. In some embodiments, the anti-CTLA-4 antibody is ipilimumab (Yervoy), tremelimumab (ticilimumab; CP-675,206), AGEN-1884, or ATOR-1015.

[0109] In one embodiment, the CTLA-4 antagonist is a soluble CTLA-4 polypeptide. In one embodiment, the soluble CTLA-4 polypeptide is abatacept (Orencia), belatacept (Nulojix), RG2077, or RG-1046. In another embodiment, the CTLA-4 antagonist is a cell-based therapy. In some embodiments, the CTLA-4 antagonist is an anti-CTLA-4 mAb RNA / GITRL RNA-transfected autologous dendritic cell vaccine or an anti-CTLA-4 mAb RNA-transfected autologous dendritic cell vaccine.

[0110] 6. Additional immune checkpoint inhibitors In certain aspects, the immune checkpoint inhibitor is a CD80 antagonist, a CD86 antagonist, a TIM-3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, an IDO1 antagonist, a STING antagonist, a GARP antagonist, a CD40 antagonist, an A2aR antagonist, a CEACAM1 (CD66a) antagonist, a CEA antagonist, a CD47 antagonist, a PVRIG antagonist, a TDO antagonist, a VISTA antagonist, or a KIR antagonist.

[0111] In one aspect, the immune checkpoint inhibitor is a KIR antagonist. In certain aspects, the KIR antagonist is an anti-KIR antibody or antigen-binding fragment thereof. In some aspects, the anti-KIR antibody is lirilumab (1-7F9, BMS-986015, IPH 2101) or IPH4102.

[0112] In one embodiment, the immune checkpoint inhibitor is a TIGIT antagonist. In one embodiment, the TIGIT antagonist is an anti-TIGIT antibody or an antigen-binding fragment thereof. In a specific embodiment, the anti-TIGIT antibody is BMS-986207, AB 154, COM902 (CGEN-15137), or OMP-313M32.

[0113] In one embodiment, the immune checkpoint inhibitor is a TIM-3 antagonist. In certain embodiments, the TIM-3 antagonist is an anti-TIM-3 antibody or antigen-binding fragment thereof. In some embodiments, the anti-TIM-3 antibody is TSR-022 or LY3321367.

[0114] In one embodiment, the immune checkpoint inhibitor is an IDO1 antagonist. In another embodiment, the IDO1 antagonist is indoximod (NLG8189; 1-methyl- D -TRP), epacadostat (INCB-024360, INCB-24360), KHK2455, PF-06840003, navoximod (RG6078, GDC-0919, NLG919), BMS-986205 (F001287) or pyrrolidine-2,5-dione derivatives.

[0115] In one embodiment, the immune checkpoint inhibitor is a STING antagonist. In certain embodiments, the STING antagonist is a 2'- or 3'-monofluoro-substituted cyclic dinucleotide, a 2'3'-difluoro-substituted mixed linkage 2',5'-3',5' cyclic dinucleotide, a 2'-fluoro-substituted, bis-3',5' cyclic dinucleotide; a 2',2''-diF-Rp,Rp, bis-3',5' cyclic dinucleotide; or a fluorinated cyclic dinucleotide.

[0116] In one embodiment, the immune checkpoint inhibitor is a CD20 antagonist. In some embodiments, the CD20 antagonist is an anti-CD20 antibody or an antigen-binding fragment thereof. In one embodiment, the anti-CD20 antibody is rituximab (Rituxan; IDEC-102; IDEC-C2B8), ABP 798, ofatumumab, or obinutuzumab.

[0117] In one embodiment, the immune checkpoint inhibitor is a CD80 antagonist. In a particular embodiment, the CD80 antagonist is an anti-CD80 antibody or an antigen-binding fragment thereof. In one embodiment, the anti-CD80 antibody is galiximab or AV 1142742.

[0118] In one embodiment, the immune checkpoint inhibitor is a GARP antagonist. In some embodiments, the GARP antagonist is an anti-GARP antibody or an antigen-binding fragment thereof. In a particular embodiment, the anti-GARP antibody is ARGX-115.

[0119] In one embodiment, the immune checkpoint inhibitor is a CD40 antagonist. In certain embodiments, the CD40 antagonist is an anti-CD40 antibody directed against an antigen-binding fragment thereof. In some embodiments, the anti-CD40 antibody is BMS3h-56, lucatumumab (HCD122 and CHIR-12.12), CHIR-5.9, or dacetuzumab (huS2C6, PRO 64553, RG 3636, SGN 14, SGN-40). In another embodiment, the CD40 antagonist is a soluble CD40 ligand (CD40-L). In one embodiment, the soluble CD40 ligand is a fusion polypeptide. In one embodiment, the soluble CD40 ligand is CD40-L / FC2 or monomeric CD40-L.

[0120] In one embodiment, the immune checkpoint inhibitor is an A2aR antagonist. In some embodiments, the A2aR antagonist is a small molecule. In certain embodiments, the A2aR antagonist is CPI-444, PBF-509, istradefylline (KW-6002), preladenant (SCH420814), tozadenant (SYN115), bipadenant (BIIB014), HTL-1071, ST1535, SCH412348, SCH442416, SCH58261, ZM241385, or AZD4635.

[0121] In one embodiment, the immune checkpoint inhibitor is a CEACAM1 antagonist. In some embodiments, the CEACAM1 antagonist is an anti-CEACAM1 antibody or an antigen-binding fragment thereof. In one embodiment, the anti-CEACAM1 antibody is CM-24 (MK-6018).

[0122] In one embodiment, the immune checkpoint inhibitor is a CEA antagonist. In one embodiment, the CEA antagonist is an anti-CEA antibody or an antigen-binding fragment thereof. In a specific embodiment, the anti-CEA antibody is sergituzumab-amnaleukin (RG7813, RO-6895882) or RG7802 (RO6958688).

[0123] In one embodiment, the immune checkpoint inhibitor is a CD47 antagonist. In some embodiments, the CD47 antagonist is an anti-CD47 antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-CD47 antibody is HuF9-G4, CC-90002, TTI-621, ALX148, NI-1701, NI-1801, SRF231, or Effi-DEM.

[0124] In one embodiment, the immune checkpoint inhibitor is a PVRIG antagonist. In a particular embodiment, the PVRIG antagonist is an anti-PVRIG antibody or an antigen-binding fragment thereof. In one embodiment, the anti-PVRIG antibody is COM701 (CGEN-15029).

[0125] In one embodiment, the immune checkpoint inhibitor is a TDO antagonist. In one embodiment, the TDO antagonist is a 4-(indol-3-yl)-pyrazole derivative, a 3-indole substituted derivative, or a 3-(indol-3-yl)-pyridine derivative. In another embodiment, the immune checkpoint inhibitor is a dual IDO and TDO antagonist. In one embodiment, the dual IDO and TDO antagonist is a small molecule.

[0126] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of B7-H3, hi some embodiments, the inhibitor of B7-H3 is enoblituzumab, MGD009, or 8H9.

[0127] In some embodiments, the agonist of an immune checkpoint molecule is an agonist of OX40, CD27, CD28, GITR, ICOS, TLR7 / 8, and CD137 (also known as 4-1BB).

[0128] In some embodiments, the agonist of CD137 is urelumab. In some embodiments, the agonist of CD137 is utomilumab.

[0129] In some embodiments, the agonist of the immune checkpoint molecule is an inhibitor of GITR. In some embodiments, the agonist of GITR is TRX518, MK-4166, INCAGN1876, MK-1248, AMG228, BMS-986156, GWN323, MEDI1873, or MEDI6469. In some embodiments, the agonist of the immune checkpoint molecule is an agonist of OX40, e.g., an OX40 agonist antibody or an OX40L fusion protein. In some embodiments, the anti-OX40 antibody is INCAGN01949, MEDI0562 (tavolimab), MOXR-0916, PF-04518600, GSK3174998, BMS-986178, or 9B12. In some embodiments, the OX40L fusion protein is MEDI6383.

[0130] In some embodiments, the agonist of an immune checkpoint molecule is an agonist of ICOS, hi some embodiments, the agonist of ICOS is GSK-3359609, JTX-2011, or MEDI-570.

[0131] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of CD28, hi some embodiments, the agonist of CD28 is celalizumab.

[0132] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of CD27, hi some embodiments, the agonist of CD27 is valilumab.

[0133] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of TLR7 / 8. In some embodiments, the agonist of TLR7 / 8 is MEDI9197.

[0134] 7. Patient population Provided herein are clinical methods for treating tumors in a subject, e.g., a human patient, using the therapies disclosed herein, e.g., T-cell checkpoint inhibitors (e.g., anti-PD-L1 antibodies), CD73 inhibitors (e.g., anti-CD73 antibodies), and chemotherapy and / or radiation therapy.

[0135] Cancers and / or malignant tumors that may be treated using the methods of the present disclosure include liver cancer, hepatocellular carcinoma (HCC), bone cancer, pancreatic cancer, skin cancer, oral cancer, cancer of the head and neck, breast cancer, lung cancer, small cell lung cancer, NSCLC, cutaneous or intraocular melanoma, kidney cancer, uterine cancer, ovarian cancer, colon cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, cancer of the endometrium, cancer of the cervix, cancer of the vagina, cancer of the vulva, squamous cell carcinoma of the head and neck (SCCHN), non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, renal pelvis cancer, tumors of the central nervous system (CNS), Primary lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including asbestos-induced cancer, hematological malignancies such as multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, precursor T-lymphoblastic lymphoma, and any combination of such cancers. The present disclosure is also applicable to the treatment of metastatic cancers. In some embodiments, the cancer is renal cell carcinoma (RCC), gastric / gastroesophageal junction cancer, non-small cell lung cancer (NSCLC), melanoma, squamous cell carcinoma of the head and neck (SCCHN), hepatocellular carcinoma, or urothelial carcinoma.

[0136] In one embodiment, the human patient is afflicted with a malignancy that is resistant to treatment with an immune checkpoint inhibitor. In another embodiment, the patient is afflicted with a malignancy that is resistant to treatment with a PD-L1 inhibitor. In another embodiment, the patient is afflicted with a malignancy that is resistant to treatment with an anti-PD-L1 antibody. [Example]

[0137] Experimental Method Animal experiments In vivo studies were performed using 8-10 week-old BALB / cAnNCrl mice (Charles River, UK) or C57BL / 6. Mice were housed in AstraZeneca vivariums with free access to food and water and cared for daily by trained personnel. Mice were acclimated to the vivarium for one week and handled in accordance with the UK Home Office Animals Scientific Procedures Act 1986. All animal experiments were performed under Home Office approved project licenses (PPL P49077891) and PP3208003 (RTx Research) and in accordance with institutional guidelines. BALB / c mice were given 0.5e 6 CT26 (colorectal) tumor cells were implanted subcutaneously in C57BL / 6 mice at 0.5 e 6 MCA205 (fibrosarcoma) tumor cells were cultured in 50% Matrigel® (Corning, Cat. No. 356231) in PBS or in 0.5 e of PBS. 6MC38 cells were implanted subcutaneously. Tumors were measured with calipers three times weekly, starting on day -5. Tumor-bearing mice were treated with either oxaliplatin (Hospira, clinical grade, 5 mg / mL) at a dose level of 6 mg / kg and 5-fluorouracil (Hospira, clinical grade, 50 mg / mL) at a dose level of 50 mg / kg, or docetaxel (Accord, clinical grade, 20 mg / mL) administered at 10 mg / kg, plus mouse surrogate monoclonal antibodies to oleculab (clone 10.3, anti-CD73 with a murine IgG1 Fc sequence, 10 mg / kg or 20 mg / kg) and durvalumab (clone 80, chimeric rat anti-mouse PD-L1 antibody with an IgG1 Fc sequence, 10 mg / kg). The chemotherapy doses and schedules used were based on previously published information (Dosset Met al., Oncoimmunology 2018;7; Gao Q et al., J Immunother Cancer 2019;7:42). In an attempt to define the contribution of components, comparison groups were replicated with monotherapy and other combinations. Groups of mice were removed at appropriate time points for pharmacodynamic analysis (MSI and transcriptomics) and immunohistochemistry (IHC) analysis. For radiation therapy experiments, tumors were treated with five consecutive daily doses of 2 Gy fractions using an X-ray source self-contained cabinet irradiator (Xstrahl CIX3).

[0138] In vitro assays Cells (HCT-116, HT-29, CT-26, and MCA-205) were cultured in 96-well culture plates for 1 e 4Cells were seeded at 40 μl per well in complete culture medium and incubated for approximately 4 hours in a 37°C / 5% CO2 incubator to allow cell attachment. 10 μl per well of olecurab (5 nM) was added to the cells at a final concentration of 5x and incubated overnight in a 37°C / 5% CO2 incubator to allow for olecurab pretreatment. 50 μl per well of serially diluted chemotherapy drugs (5-fluorouracil, oxaliplatin, docetaxel) were added to the cells at a final concentration of 2x, depending on the treatment group, and incubated for 72 hours in a 37°C / 5% CO2 incubator. Cell viability was measured using the CellTiter-Glo® Luminescent Assay (Promega, Catalog No. G7573).

[0139] RNA preparation for bulk sequencing Tumor tissue was snap frozen in liquid nitrogen and stored at −80°C upon collection in the animal unit. ° The samples were stored at 4°C and shipped on dry ice to Novogene. Tissue processing and RNA extraction were performed by Novogene using the Qiagen RNeasy Plus Universal Kit (Qiagen, catalog number 73404) according to the manufacturer's protocol. Briefly, tissue samples were homogenized in QIAzol lysis reagent. After adding gDNA removal solution and chloroform, the homogenate was separated into aqueous and organic phases by centrifugation. The upper aqueous phase containing RNA was collected, and the RNA was purified using an RNeasy spin column. RNA quality check (QC) was performed by 1% agarose gel electrophoresis. Quantity and purity were determined using a Nanodrop™ system, and RNA integration numbers (RINs) were obtained using an Agilent Bioanalyzer 2100.

[0140] The NEBNext® Ultra RNA Library Prep Kit (Cat. No. E7530L) was used for library preparation for sequencing. Samples were sequenced using Illumina PE150 (50M reads / sample) bulk RNA-seq, and downstream bioinformatics analysis was performed.

[0141] Analysis of RNASeq data Reads were mapped to the Mus musculus genome (mm10) using Star (Dobin A et al., Bioinformatics 2013;29:15-21). Uniquely mapped reads were counted using htseq-count (Putri GH et al., Bioinformatics 2022;38:2943-5). Counts were normalized by size factor using DESeq2 (Love MI et al., Genome Biol 2014;15), and differentially expressed genes were identified across conditions using a threshold of abs(log2FC) > 1 and an adjusted p-value < 0.05.

[0142] Gene set enrichment analysis was performed using the R package "fGSEA" (Korotkevich G et al., bioRxiv 2021;060012) using hallmark gene sets from the Mouse MSigDB (Subramanian A. et al., Proc Natl Acad Sci USA 2005;102:15545-50). Enrichment p-values ​​were calculated as described in Korotkevich G et al., bioRxiv 2021;060012, and p-values ​​were adjusted using the Benjamini-Hochberg method. Gene Ontology biological process enrichment was identified using the R package topGO (Bioconductor-topGO) with an adj-pval < 0.05. KEGG pathway enrichment analysis was performed using the R package clusterProfiler (Wu T. et al., The Innovation 2021;2:100141). Using the MCPCounter tool (Becht E. et al., Genome Biol 2016;17:1-20), immune cell abundance was estimated for each sample and condition using immune gene signatures within MCPCounter.

[0143] Tissue preparation for mass spectrometry imaging (MSI) Tumors were flash-frozen in liquid nitrogen immediately after resection, and frozen tissue was embedded in HPMC / PVP hydrogel as previously described (Dannhorn A. et al., Anal Chem 2020;92:11080-8). Sectioning was performed on a CM3050 S cryostat (Leica Biosystems, Nussloch, Germany) at a section thickness of 10 μm. Tissue sections were immediately thaw-mounted, dried under a nitrogen stream, and sealed in a vacuum pouch to preserve the metabolic integrity of the sections. Tissue sections for DESI-MSI and IMC were thaw-mounted on Superfrost microscope slides (VWR, catalog number 630-2863), and sections prepared for MALDI-MSI were thaw-mounted on conductive indium tin oxide (ITO)-coated slides (Bruker Daltonik, catalog number 8237001). Polyvinylpyrrolidone (PVP) and (hydroxypropyl)methylcellulose (HPMC) were purchased from Merck (catalog no. PVP360; H8384). Methanol (catalog no. 15624680), iso-pentane (catalog no. 15692830), and isopropyl alcohol (catalog no. 10674732) were obtained from Fisher Scientific.

[0144] Mass Spectrometry Imaging (MSI) DESI-MSI analysis was performed on a Q-Exactive mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with an automated 2D-DESI ion source (Prosolia Inc., Indianapolis, IN, USA) operating in negative ion mode, covering an applicable mass range of up to 1000 m / z, with a nominal mass resolution of 70,000. The injection time was fixed at 150 ms, resulting in a scan rate of 3.8 pixels / s. The spatial resolution was 70 μm. A homemade Swagelok DESI nebulizer was operated to deliver a mixture of 95% methanol and 5% water at a flow rate of 2721.5 μL / min, nebulized with nitrogen at 6 bar backpressure. The resulting .raw files were converted to .mzML files using ProteoWizard msConvert (version 3.0.4043) and then compiled into .imzML files (imzML Converter version 1.3) (Race AM et al., J Proteomics 2012;75:5111-2). All subsequent data processing was performed in SCiLS Lab (version 2021b, Bruker Daltonik, Bremen, Germany).

[0145] MALDI-MSI analysis was performed on a RapifleX Tissuetyper instrument (Bruker Daltonik, Bremen, Germany) operated in negative detection mode. 9-Aminoacridine (9-AA) was prepared in 80:20 methanol:water and used as the MALDI matrix. It was spray-deposited using an automated spray system (M3-Sprayer, HTX technologies, Chapel Hill, NC, USA). MALDI experiments were performed with a spatial resolution of 50 μm. A total of 400 laser shots were summed pixel-by-pixel to obtain the final spectrum. For all experiments, the laser was operated at a repetition rate of 10 kHz. All raw data were directly uploaded and processed with the SCiLS Lab (version 2021b) software package. All DESI and MALDI data and images were normalized to the total ion current (TIC) to correct for signal fluctuations during the course of the experiment. Data analysis, performed with the SCiLS lab software package, involved classification of the dataset based on manual annotation of histology-guided MSI data to identify "tumor" and "necrosis," while removing background, where applicable. Tissue classification was performed using partial least squares discriminant analysis (PLS-DA). "Necrotic margins" were delineated and distinguished from "viable tumor" by subjecting the "tumor" tissue cluster to pixel-wise principal component analysis (PCA). Principal component (PC) loadings highlighting tissue compartments adjacent to necrotic regions were extracted and used to generate peak lists for unsupervised segmentation based on a bisection K-means classifier. All data shown were extracted from the "viable tumor" cluster.

[0146] Imaging Mass Cytometry (IMC) Imaging mass cytometry was performed on slides analyzed by DESI-MSI. The antibodies used for IMC staining are listed in Table 1.

[0147] [Table 1]

[0148] Antibodies were either purchased pre-conjugated with a heavy metal tag, or untagged antibodies were conjugated in-house using Fluidigm's Maxpar Antibody Labeling Kit according to the manufacturer's instructions. Slides were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes. Slides were washed 3 times for 5 minutes in PBS, permeabilized with a 1:1000 dilution of Triton X-100 in casein for 5 minutes, washed 3 times for 5 minutes in PBS, and blocked with casein for 30 minutes. Antibodies were diluted to the appropriate concentration, and slides were incubated with the antibody solution overnight at 4°C. Slides were washed 3 times for 5 minutes in PBS, and nuclei were stained for 30 minutes using DNA intercalator-iridium at a 1:400 dilution in PBS. Slides were washed 3 times for 5 minutes in PBS and 30 seconds in deionized water, then dried and stored at room temperature until analysis. Using the serial H&E-stained sections and MSI results, regions for IMC analysis were selected. Approximately 1.5 × 1.5–2.0 × 2.0 mm were selected for analysis to include necrotic, necrotic margin, and viable tumor areas. IMC analysis was performed using a Hyperion instrument (Fluidigm Corporation, San Francisco, CA, USA) with an ablation energy of 4 dB and an ablation frequency of 100 Hz.

[0149] IMC data were analyzed using Halo v.3.3.2541.366 (Indica Laboratories, Albuquerque, NM, USA). Tissue regions were classified into viable tumor, necrosis, necrotic margin, and outside tissue using Random Forest. Analysis was performed using Highplex FL v 4.0.4. Cell segmentation and thresholding were manually optimized. Nucleus segmentation was performed using the 193Ir DNA intercalator channel. IMC images were aligned and extracted using the built-in geometry tool.

[0150] immunohistochemistry A portion of each tumor was immersion-fixed in 10% neutral-buffered formalin and then processed into paraffin using standard techniques. Tissues were sectioned at 4 mm thickness and immunohistochemically stained using a rabbit monoclonal antibody against CD73 (D7F9A, Cell Signaling Technology) at a dilution of 0.5 μg / mL on an automated Leica Bond-RX immunostainer, using DAB as the chromogen and hematoxylin as the background stain. This antibody has previously been shown to have no binding inhibition in tissues previously treated with oleclumab (unpublished observations).

[0151] The resulting slides were digitally scanned at 20x magnification using an Aperio scanner (Leica Biosystems). For analysis, measurements of CD73- and hematoxylin-positive areas were performed using Halo image analysis software (Indica Labs). Briefly, tumor areas were manually annotated, and positive DAB and hematoxylin areas within the annotated areas were measured using a halo area quantification algorithm adapted to the tissue staining characteristics. The percentage of CD73- and hematoxylin-positive areas within the total tumor area was obtained. The final data output used was the ratio of CD73 to hematoxylin area. Hematoxylin area was considered a representative indicator of cellularity (because it primarily stains the nucleus), allowing for normalization of CD73-positive area to the overall cellularity of the tumor.

[0152] statistical analysis All in vivo data were collated in an Excel spreadsheet and transferred to GraphPad Prism 9.00 (GraphPad Software Inc.) for graphical display and statistical analysis. After outlier identification and a Shapiro-Wilk test for normal distribution, statistical significance was determined by one-way ANOVA, followed by Dunnett's multiple comparisons for comparisons of three or more groups if the data were normally distributed. If the data were not normally distributed, as detailed in the figure legends, a Kruskal-Wallis test with Dunn's multiple comparisons test was used. Survival studies were analyzed with the log-rank (Mantel-Cox) test, and only two survival curves were compared at a time. P values ​​were not adjusted for multiple testing.

[0153] Example 1: Combination treatment of anti-CD73, anti-PD-L1 antibody, and 5FU+OHP results in enhanced complete responses in a syngeneic mouse model Combination treatment with anti-CD73 (aCD73) and anti-PD-L1 (aPD-L1) antibodies in addition to 5-fluorouracil (5FU) plus oxaliplatin (OHP) resulted in enhanced efficacy and complete responses in two mouse syngeneic models of cancer—CT26 (p=0.005) and MCA205 (p=0.008) (Kruskal-Wallis test), as shown in Figures 1B and 1C. Treatment of CT26- or MCA205-tumor-bearing mice with aCD73 as monotherapy provided negligible benefit in terms of tumor growth inhibition. Anti-PD-L1 treatment slowed tumor growth rate in a proportion of mice bearing CT26 tumors but showed minimal activity in the MCA205 model (Figure 2). While 5FU+OHP chemotherapy inhibited tumor growth rate in both models, combining 5FU+OHP with either immuno-oncology (IO) agent (anti-CD73 or anti-PD-L1) provided minimal additional efficacy. Combination treatment utilizing anti-CD73 and anti-PD-L1 antibodies (aCD73+aPD-L1) resulted in similar levels of tumor growth control as anti-PD-L1 alone, highlighting the importance of the chemotherapy component within the chemotherapy+IO combination (Figures 1 and 2).

[0154] Example 2: Effect of aCD73 on chemotherapy-induced cytotoxicity in cell culture In parallel, we investigated whether the effects seen in vivo were tumor microenvironment (TME)-driven by determining whether anti-CD73 could enhance the direct cytotoxic effect of 5FU + OHP on the cell line itself. However, we found no evidence that this occurred in an in vitro setting (Figure 4). We also investigated whether the cytotoxic effect of docetaxel on cultured CT26 cells was affected by the presence of aCD73. No effect of the antibody was detected.

[0155] Example 3: CD8 cell depletion impairs the efficacy of the four-drug combination (aCD73+aPD-L1+5FU+OHP) in MCA205 tumor-bearing mice To test the hypothesis that CD8 T cells are involved in the enhanced efficacy of the combination, we selectively depleted CD8 T cells in the MCA205 model using IP injection of clone 53-6.7 starting 17 days after tumor implantation (schematic Figure 5A). This experiment confirmed that tumor control and long-term survival were significantly reduced (>50%) in the (5FU + OHP + aCD73 + aPD-L1) combination treatment group (log-rank test, p = 0.026, Figure 5C). These CD8 depletion data suggest that cell-mediated immunity plays an essential role in the observed activity of the chemotherapy-IO combination. This does not exclude contributions from other immune system components or from "traditional" cytotoxic or cytostatic effects mediated by 5FU + OHP.

[0156] Example 4: "Early" Pharmacodynamic Effects of aCD73+5FU+OHP on CD73 Expression, Adenosine Pathway Metabolites, and Stromal Cell Populations in a Murine CT26 Tumor Model The pharmacodynamic activity of the combination of aCD73 and aPD-L1 + 5FU + OHP chemotherapy was assessed using immunohistochemistry (IHC) and imaging mass cytometry (IMC) to understand the early changes mediated by CD73 blockade. As expected, the murine surrogate antibody for oleclumab reduced CD73 levels in CT26 tumors. IHC-based CD73 detection used an antigen that did not compete with the murine surrogate antigen for oleclumab; therefore, the reduction in CD73 protein levels was consistent with oleclumab's known ability to internalize CD73. IMC highlighted that CT26 tumors from αCD73-treated mice tended to contain lower frequencies of cells expressing markers known to be associated with cancer-associated fibroblasts and suppressive tumor-associated macrophages. Mass spectrometry imaging (MSI) revealed a significant trend toward adenosine, inosine, and xanthine suppression in CT26 tumors from mice treated with anti-CD73 (Figures 3 and 5). Conversely, adenosine monophosphate (AMP), the major substate of CD73, appeared elevated in tumor tissue from mice treated with the combination of chemotherapy and anti-CD73 compared with other treatment groups. These pharmacodynamic data are consistent with oleculab's proposed mechanism of action, with respect to CD73 targeting, enzyme inhibition, and adenosine pathway modulation.

[0157] Example 5: Pharmacodynamic effects of aCD73+aPD-L1+5FU+OHP on the CT26 transcriptome To further understand the mechanistic basis for the enhanced activity in the quadruple combination group, we used RNA sequencing to explore changes within the CT26 transcriptome. Using the DEseq2 and fGSEA packages, we identified minimal changes in the CT26 transcriptome after aCD73 or aPD-L1 treatment as monotherapy (see Table 2 below). In contrast, 5FU+OHP was highly perturbative, causing a significant upregulation of 277 genes and downregulation of an additional 158 compared to control tumors. Gene Ontology (GO BP) base pair enrichment and KEGG pathway analysis of these data highlighted significant effects on genes related to immune response, leukocytes, NK and T cell activation, T cell receptor signaling, and interferon (type 1 and type 2) production (adjusted pval < 0.05). Consistently, upregulated individual genes included CCL3, 4, 8, 17, CXCL10, GDF15, CD8α, IFNγ, perforin, granzymes, Lag-3, and PD-1. CXCL2 expression was decreased (Figure 11). These RNA-seq data highlight that 5FU+OHP treatment significantly perturbs genes associated with immune function in the CT26 mouse model of cancer.

[0158] In contrast to the minor transcriptome changes observed with constitutive monotherapy treatment, the combination of aCD73 and aPD-L1 resulted in 1236 differentially expressed genes. The most significant widespread transcriptome changes were achieved with the combination of 5FU+OHP+aCD73+aPD-L1, with 1490 genes upregulated and 128 downregulated compared to untreated tumors (Table 2).

[0159] [Table 2]

[0160] KEGG pathway and gene ontology enrichment analysis highlighted chemotactic recruitment, T cell activation, T cell receptor signaling, Th1 and Th2 cell differentiation, and natural killer cell-mediated cytotoxicity. The most affected immune-related genes are listed in Figure 11. There were several notable additions to the genes upregulated by 5FU+OHP treatment, including, but not limited to, CD38, CD39, CXCL1, CXCL3, CXCL5, CD163, CTLA4, CXCR3, granzyme A, ICAM1, Il6, P2RY1, TNF-α, IL2α, Il1a, ALOX15, SLC7a2, Ear 2, and Havc2.

[0161] The inclusion of treatment groups representing the various components of the combination allowed for a deconvolution analysis of the contributions of the individual treatment components (Figures 7A, 7B, and 11; Table 2). This analysis highlighted the important role of 5FU+OHP in driving activation of the interferon pathway (types 1 and 2), along with T cell and NK cell activation, cytotoxic activity, and IL2 / STAT5 pathway signaling. The effects of 5FU+OHP inclusion were significantly different from those mediated by adding either of the IO drug components to the treatment, as evidenced by the 360 ​​genes uniquely upregulated and 122 downregulated by the 5FU+OHP component. The inclusion of 5FU+OHP in the aCD73+aPD-L1 combination treatment approach helped to elevate the expression of key immune-related genes, such as interferon-gamma, TRIM6, CCL17, granzyme B, GDF15, perforin, and Lag3. Conversely, when 5FU+OHP was included, IL-10, IL-1b, CXCL2, and S100A8 were downregulated.

[0162] Adding CD73 blockade to the 5FU + OHP and αPD-L1 combination upregulated 510 genes and downregulated 8 genes, which were not modulated in other iterations of the combination, as shown in Figure 7A. Significantly affected genes included CXCR3, H2-AB1, Itgae, CXCL3, Mgl2, CXCR5, CD4, and Cybb. This also promoted higher expression of CCL17, a major tumor-infiltrating lymphocyte (TIL)-attracting chemokine, and CCL24, a chemokine known to preferentially chemoattract M1 macrophages (Xuan W, et al., J Leukoc Biol 2015;97:61-9). These data highlight a novel and impactful role for adenosine pathway inhibition in chemotherapy / checkpoint inhibitor combinations, particularly with regard to individual genes and signatures related to myeloid and B cell biology. CD38 and P2Y1, two genes known to be involved in the adenosine pathway itself, were also significantly upregulated when chemotherapy + anti-PD-L1 was augmented with anti-CD73. Withholding aPD-L1 from the 5FU + OHP + aCD73 combination treatment was detrimental in that it upregulated genes related to inflammation, immune response, and interferon-gamma pathway activation. In particular, ALOX15, a gene related to macrophage function and efferocytosis, and IL-1b were affected.

[0163] Next, we investigated whether genetic perturbations at this scale resulted in changes in the enumeration of cell populations. To this end, we used the MCP-Counter tool (Becht E, et al., Genome Biol 2016;17:1-20) to estimate the abundance of immune cell populations in treated CT26 tumors. This computational analysis highlighted the significant effect of oxaliplatin and 5-fluorouracil chemotherapy on increased lymphocyte expression, consistent with the significant increase in proinflammatory and chemotactic chemokine / cytokine gene expression levels after this treatment (Figure 7C). CT26 tumors from mice treated with monotherapy and the combination excluding the chemotherapy component showed significantly less lymphocyte infiltration. 5FU+OHP+aCD73+aPD-L1 treatment significantly enhanced tumor abundance of both lymphocytes (cytotoxic T cells, NK cells, and B cells) and myeloid cell populations (including monocytic dendritic cells), a profile conducive to improved tumor control and prolonged overall survival in cancer models (Petitprez F, et al., Nature 2020 577:7791; Voss MH, et al., JCO20203815_suppl5025 2020;38:5025-5025; Chambers AM, et al., Front Immunol 2018;9:2533; Masteric-Gavillet B, et al., J Immunother Cancer 2019;7:1-16).

[0164] Example 6: Combination treatment of aCD73, aPD-L1 and docetaxel results in enhanced complete responses in the CT26 syngeneic mouse model Importantly, the synergistic combination effect observed with 5FU + OHP was found to extend to a second chemotherapy class, namely, docetaxel (DTX). The combination approach was layered on the established tolerated dose scheme for this chemotherapy. In this case, as shown in Figure 8, the combination of DTX, aPD-L1, and aCD73 significantly improved tumor growth inhibition, resulting in 7 of 12 (58%) complete responses (p=0.0001), compared with a maximum of 3 of 12 (25%) complete responses (p=0.001) in the aPD-L1 + docetaxel combination group (Kruskal-Wallis test).

[0165] Example 7: Combination of aCD73 and aPD-L1 enhances fractionated radiotherapy in the MC38 syngeneic mouse model Previously published preclinical data highlight the role of CD73 in radiotherapy response in cancer (Wennerberg E et al., Cancer Immunol Res 2020 8:465-78; Tsukui H, et al., BMC Cancer 2020;20; and Nguyen AM et al., Molecular & Cellular Proteomics 2020;19:375-89). We investigated whether the addition of aCD73+aPD-L1 treatment also enhances radiotherapy response, consistent with chemotherapy data. Using the MC38 model of colorectal cancer, we validated the established efficacy of fractionated radiotherapy regimens and used a simultaneous approach, i.e., all treatments initiated on the same day (schematic Figure 9A). 70-120 mm CT scans were used for interventional treatment. 3 Tumors were enrolled between 0 and 1. Data shown in Figure 9B demonstrate a strong effect (p=0.0001) from RTx+aCD73+aPD-L1 treatment of MC38 tumor-bearing mice, confirmed by Kruskal-Wallis test.

[0166] Example 8: Optimal scheduling using aCD73 and aPD-L1 in the MC38 syngeneic mouse model The effects of aCD73, aPD-L1, and timing of radiation therapy were also determined using the MC38 syngeneic mouse model. As shown in Figure 12, six groups of mice were treated with 5 × 10 5 As shown in Figure 13, mice treated simultaneously with aCD73, aPD-L1, and RTx exhibited the highest levels of tumor inhibition and subsequent survival rates. This also correlated with the induction of protective memory responses upon rechallenge using the B16F10 and MC38 mouse models (Figure 14).

[0167] To determine whether the timing at which individual aCD73, aPD-L1, and radiation therapies were administered had an effect on tumor volume, MC38 cells were transplanted as before, and the timing of individual therapies was mapped as shown in Figure 15. Interestingly, administration of aCD73 therapy before aPD-L1 and RTx showed the greatest reduction in tumor volume, which correlated with the highest probability of survival (Figure 16).

[0168] Growing evidence supports the immunosuppressive role of extracellular adenosine within the tumor microenvironment, and adenosine-related gene signatures have been associated with poor outcomes and reduced response to T-cell checkpoint inhibitors in several indications (Sidders et al., and Allard D, et al., Immunol Lett 2019;205:31-9). Molecules targeting extracellular nodes within the adenosine production pathway are gaining attention in clinical development, and olecurumab, in combination with durvalumab, is currently in phase 3 clinical development in patients with stage III NSCLC (non-small cell lung cancer) previously treated with chemoradiotherapy (Global Study Evaluating the Effect of Durvalumab and Olecurumab or Durvalumab and Monalizumab Combinations Following Concurrent Chemoradiotherapy in Patients with Stage III Unresectable Non-Small Cell Lung Cancer - View Full Text - ClinicalTrials.gov).

[0169] Despite the widespread assumption that CD73 inhibition will be beneficially combined with cytotoxic therapies that promote extracellular ATP release via cell death, there is a paucity of published preclinical data to support this. The data presented herein explore the effects of CD73 inhibition in concert with chemotherapy and PD-L1 blockade, highlighting additive and novel biological effects mediated by the inclusion of CD73 blockade. To this end, a combination containing aCD73 + aPD-L1 + 5FU + OHP resulted in enhanced efficacy in two mouse models of cancer (colorectal and sarcoma). As judged by the effects of a CD8-depleting antibody in the MCA205 model, the activity of the combination therapy was dependent on CD8 T cells. These data suggest a significant contribution from the cell-mediated arm of the mouse immune system to the resulting antitumor effects. Consistent with this, RNA-seq analysis confirmed that αCD73 + αPD-L1 + 5FU + OHP drove increased tumor abundance of cytotoxic lymphocytes and other key immune cells, such as myeloid dendritic cells and B cells. Substitution of OHP+5FU with a taxane backbone, i.e., DTX, was also investigated and demonstrated a similarly enhanced efficacy profile in the CT26 model. These data support the complementarity of oleclumab and aPD-L1 antibodies with both platin- and taxane-based chemotherapy backbones. The data also highlight the additivity of radiation therapy in the MC38 model, extending the findings of Wennerberg et al. (Cancer Immunol Res 2020;J.8:465-78) to colon models and enhancing intervention strategies for PD-1 / PD-L1 axis inhibition.

[0170] Mechanistically, aCD73 antibodies rapidly reduced CD73 expression in CT26 tumors and modulated extracellular adenosine levels in a manner consistent with their proposed mechanism of action. However, imaging mass cytometry of the same samples revealed changes in CAF and TAM markers, which may reflect direct or downstream effects of aCD73 treatment. These observations are consistent with other publications linking CD73 inhibition to tumor macrophage and fibroblast proliferation (Magagna I, et al., Cancers (Basel) 2021;13; Yu M, et al., Nat Commun 2020;11).

[0171] Monotherapy 5FU+OHP treatment delayed tumor growth in some drug recipients with CT26 / MCA205 tumors. A notable finding from this study was the breadth of immune pathway gene regulation following 5FU+OHP treatment of CT26 tumors. These included gene signatures related to type 1 and type 2 interferons, cytotoxic lymphocytes, and effector molecules, as well as their associated receptors. These observations are consistent with many of the known immunomodulatory effects of these chemotherapies in in vitro model systems (Siew YY, et al., Int Immunol 2015;27:621-32) and now extend our knowledge of their in vivo effects (Dosset M, et al., Oncoimmunology 2018;7). In particular, the data presented herein confirm that 5FU+OHP drives the type I interferon pathway, which is known to exert a wide range of effects on immune cells and cancer cells within the tumor microenvironment (Zitvogel L, et al., Nat Rev Immunol 2015;15:405-14). Specific genes modulated by monotherapy 5FU+OHP treatment included IFN-γ, CCL3, CCL8, Lag-3, and granzyme B (upregulated), accompanied by downregulation of CXCL2, IL-1b, CD103, and XCR1. 5FU+OHP treatment elevated ARORA2 gene expression, but the upregulation of ARORA2 was counteracted when either aPD-L1 or aCD73 mAb was administered in combination with 5FU+OHP treatment. Gene signatures related to pro-inflammation, STAT5 pathway activation, and chemotaxis were significantly upregulated only after CD73 blockade was applied to OHP+5FU treatment (Table 2). GDF-15 is a pleiotropic cytokine of emerging interest in cancer (Wischhusen J, et al., Front Immunol 2020;11), and GDF15 transcript levels in CT26 tumors were significantly elevated by 5FU+OHP-containing treatment, mirroring findings from other mouse models and human cancer patients undergoing platinum-based therapy (Breen DM, et al., Cell Metab 2020;32:938-950.e6).

[0172] In contrast to tumor RNA-seq data obtained from mice treated with either IO agent alone (e.g., as monotherapy), the combination of aCD73 and aPD-L1 resulted in 1,236 differentially expressed genes, highlighting broader transcriptome changes associated with antibody-mediated targeting of multiple inhibitory checkpoints within the tumor microenvironment. The IO "doublet" was noted for activation of pathways and gene families related to inflammation, myeloid leukocyte migration, cell chemotaxis, cytokine-cytokine receptor interactions (including TNF), chemokine signaling pathways, and complement and coagulation cascades. The pathways and processes affected by the IO combination (aCD73 + aPD-L1) are largely distinct from those affected by 5FU + OHP treatment, and have the potential for complementarity when superimposed within a combination treatment paradigm. In particular, the "doublet" IO combination (aCD73+aPD-L1) is known to mediate a range of beneficial immunomodulatory effects within the tumor microenvironment, with less efficient activation of type 1 interferon pathway genes, correlating with favorable disease outcomes in patients with many forms of cancer (Zitvogel L, et al., Nat Rev Immunol 2015;15:405-14).

[0173] Despite the detected transcriptome changes, it is clear that a "pure" small molecule / large molecule approach (5FU + OHP chemotherapy or IO combination) failed to achieve the same level of efficacy as achieved by combining 5FU + OHP with aCD73 and aPD-L1. Therefore, it was instructive to delve into the specific transcriptome differences that might explain this, as these genes and their signatures may be useful beyond the context of adenosine pathway regulation. Surprisingly, the addition of aCD73 to 5FU + OHP + aPD-L1 significantly upregulated CXCR3 in the CT26 tumor microenvironment. CXCR3 is the cognate receptor on activated T cells for the IFN-inducible chemokine CXCL9-11, and its upregulation coincides with increased T cell abundance and increased expression of interferon-activated chemokines in CT26 tumors of mice receiving 5FU + OHP or its chemotherapy-containing combination. Recent publications have highlighted the importance of tumor chemotaxis of CXCR3-bearing T cells for preclinical and clinical responses to T cell checkpoint inhibitors (Marcovecchio PM, et al., J Immunother Cancer 2021;9; Qu Y, et al., Cell Rep 2020;32; Chow MT, et al., Immunity 2019;50:1498-1512.e5). Increased Pdcd1 (PD-1) expression suggests that tumor T cells in these CT26 tumor-bearing mice may be activated and / or exhausted, strongly supporting the inclusion of aPD-L1 to overcome adaptive immune resistance to antitumor effects. Notably, aPD-L1 and IO-containing combinations significantly upregulated 15-lipoxygenase (15-LOX), which is involved in various macrophage functions, including efferocytosis and ferroptosis. It is intriguing that Snodgrass et al. (Front Immunol 2018;9) identified a novel role for ALOX15 in CCL17 production in human macrophages, which is important to note in light of the apparent increase in CCL17 expression in most protective forms of treatment.

[0174] The modulation of genes related to dendritic cell biology and antigen presentation (MHC II, Itgae, Itgax, DCstamp, TARM1, CD301) is particularly interesting and is consistent with other studies exploring CD73 inhibition in general in conjunction with radiation therapy and adenosine pathway blockade. Wennerberg et al. (Cancer Immunol Res. 2020) have already highlighted the important role of radiotherapy-induced type 1 interferon (T1F) for reprofiling the tumor microenvironment, particularly with respect to cDC1. The same group highlighted the important therapeutic role of aCD73 in tumors due to its complementarity to CD73 blockade by radiation therapy, resulting in suboptimal induction of radiotherapy-induced type 1 interferon. The data presented herein appear to be largely consistent with those findings. Consistent with this, genes for MHC II molecules and macrophage galactose C-type lectin (MGL / CD301) expressed on DCs were upregulated within the CT26 TME. CD301 is thought to be involved in the recognition of both altered self and pathogen molecules due to its monosaccharide specificity for Gal and N-acetylgalactosamine. T-cell interaction-activating receptor-1 (TARM1; gene symbol Tarm1) on myeloid cells is a recently identified LILR family member encoded within the leukocyte receptor complex. TARM1 is expressed by DCs and is required for their activation. Tarm1 was highly expressed in inflammatory-type (IA / I-E+Ly6C+CD11b+CD11c+) DCs during draining LNs (dLNs) after induction of CIA in Tarm1+ / - mice. Another novel finding was the ability of aCD73-containing combination to independently drive high levels of Ear2 expression. Ear2 is an RNase and also forms part of a 14-gene signature expressed by non-classical monocytes (Ma RY, et al., Trends Immunol 2022;J.43:546-63). Emerging evidence indicates an immunomodulatory role for extracellular RNase molecules, thereby acting as alarmins (Lu L, et al., Front Immunol 2018;9:1012). Similarly, tumor RNase2a expression was significantly increased by combination therapy.Effects on NOX2 (gene Cybb) were also noted, potentially of note given its fundamental role in endowing macrophages with the ability to respond to extracellular ATP stimulation with robust changes in cellular oxidation (Moore SF, et al., Journal of Immunology 2009;183:3302-8) and its role in regulating ATM kinase activation in macrophages and efficacy against radiation therapy (Wu Q, et al., Cell Death Differ 2017;24:1632-44). Macrophage lectin-like oxidized LDL receptor-1 (LOX-1 / OLR1) was also upregulated in CT26 tumors from mice treated with 5FU, OHP, aCD73, and aPD-L1. This receptor is known to sense heat shock proteins and is significantly upregulated by TLR agonists and other pro-inflammatory stimuli.

[0175] The effect on B cell expression within CT26 tumors is also noteworthy (Figures 7A and 7C). B cell-related genes, such as JChain, CXCR5, and CXCL13, were significantly regulated when aCD73 was included in the 5FU+OHP+aPD-L1 combination (Figure 11). While the impact of B cell biology is less well understood than that of CD8 in cancer, there is growing interest in the role of B cells in the human tumor microenvironment (Fridman WH et al., Journal of Experimental Medicine 2021;218; Griss J, et al., Nature Communications 2019;10:1-14; Bruni D, et al., Nature Reviews Cancer 2020;20:662-80). The mouse B cell data are also consistent with recent publications on the direct effects of CD73-blocking antibodies on human B cells (Hair J, et al., Cancer Res 2021;81:1695-1695; Luke J, et al., J Immunother Cancer 2021;9:A729-A729). These mechanistic data are neatly aligned with the improved survival and tumor growth control profile in animals treated with 5FU+OHP+aCD73+aPD-L1.

[0176] array SEQ ID NO: 1: Heavy chain CDR1 amino acid sequence; anti-PD-L1 antibody durvalumab GFTFSRYWMS SEQ ID NO: 2: Heavy chain CDR2 amino acid sequence; anti-PD-L1 antibody durvalumab NIKQDGSEKYYVDSVKG SEQ ID NO: 3: Heavy chain CDR3 amino acid sequence; anti-PD-L1 antibody durvalumab EGGWFGELAFDY SEQ ID NO: 4 Light chain CDR1 amino acid sequence; anti-PD-L1 antibody durvalumab RASQRVSSSYLA SEQ ID NO: 5 Light chain CDR2 amino acid sequence; anti-PD-L1 antibody durvalumab DASSRAT SEQ ID NO: 6: Light chain CDR3 amino acid sequence; anti-PD-L1 antibody durvalumab QQYGSLPWT SEQ ID NO: 7: Heavy chain variable domain (VH) amino acid sequence; anti-PD-L1 antibody durvalumab EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS SEQ ID NO: 8. Light chain variable domain (VL) amino acid sequence; anti-PD-L1 antibody durvalumab EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK SEQ ID NO: 9 Heavy chain CDR1 amino acid sequence; anti-CD73 antibody olekumaab SYAYS SEQ ID NO: 10 Heavy chain CDR2 amino acid sequence; anti-CD73 antibody olekumaab AISGSGGRTYYADSVKG SEQ ID NO: 11 Heavy chain CDR3 amino acid sequence; anti-CD73 antibody olekumab LGYGRVDE SEQ ID NO: 12 Light chain CDR1 amino acid sequence; anti-CD73 antibody olekumab SGSLSNIGRNPVN SEQ ID NO: 13 Light chain CDR2 amino acid sequence; anti-CD73 antibody olekumab LDNLRLS SEQ ID NO: 14 Light chain CDR3 amino acid sequence; anti-CD73 antibody olekumab ATWDDSHPGWT SEQ ID NO: 15 Heavy chain variable domain (VH) amino acid sequence; anti-CD73 antibody olekumaab EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAYSWVRQAPGKGLEWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLGYGRVDEWGRGTLVTVSS SEQ ID NO: 16 Light chain variable domain (VL) amino acid sequence; anti-CD73 antibody olekumaab QSVLTQPPSASGTPGQRVTISCSGSLSNIGRNPVNWYQQLPGTAPKLLIYLDNLRLSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDSHPGWTFGGGTKLTVL SEQ ID NO: 17 Heavy chain amino acid sequence; anti-CD73 antibody olekumab EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAYSWVRQAPGKGLEWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLGYGRVDEWGRGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPCPPAFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 18 Light chain amino acid sequence; anti-CD73 antibody olekumab QSVLTQPPSASGTPGQRVTISCSGSLSNIGRNPVNWYQQLPGTAPKLLIYLDNLRLSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDSHPGWTFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Claims

1. 1. A method of inhibiting tumor growth in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a PD-L1 inhibitor in combination with chemotherapy and / or radiation therapy, wherein the subject has reduced levels of CD73 protein or CD73 activity compared to a normal subject.

2. 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a PD-L1 inhibitor in combination with chemotherapy and / or radiation therapy, wherein the subject has reduced levels of CD73 protein or CD73 activity compared to a normal subject.

3. 1. A method of generating a protective tumor memory response in a subject, comprising administering to the subject a therapeutically effective amount of a PD-L1 inhibitor in combination with chemotherapy and / or radiation therapy, wherein the subject has reduced levels of CD73 protein or CD73 activity compared to a normal subject.

4. A method of inhibiting tumor growth in a subject in need thereof, comprising administering to the subject therapeutically effective amounts of a CD73 inhibitor, a PD-L1 inhibitor, and chemotherapy and / or radiation therapy.

5. A method of treating cancer in a subject in need thereof, comprising administering to the subject therapeutically effective amounts of a CD73 inhibitor, a PD-L1 inhibitor, and chemotherapy and / or radiation therapy.

6. A method of inducing a protective tumor memory response in a subject, the method comprising administering to the subject therapeutically effective amounts of a CD73 inhibitor, a PD-L1 inhibitor, and chemotherapy and / or radiation therapy.

7. The method of any one of claims 1 to 6, wherein the PD-L1 inhibitor and the chemotherapy and / or radiation therapy are administered simultaneously.

8. The method of any one of claims 1 to 6, wherein the PD-L1 inhibitor and the chemotherapy and / or radiation therapy are administered sequentially.

9. The method of any one of claims 4 to 6, wherein the CD73 inhibitor is administered prior to administration of the PD-L1 inhibitor and the chemotherapy and / or radiation therapy.

10. The method of any one of claims 1 to 9, wherein the chemotherapy is docetaxel, 5-fluorouracil and / or oxaliplatin.

11. The method of any one of claims 1 to 10, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody or an antigen-binding fragment thereof.

12. 12. The method of claim 11 , wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

13. The method of claim 11 or 12, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a HC variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a LC variable domain (VL) comprising the amino acid sequence of SEQ ID NO:

8.

14. The method of any one of claims 11 to 13, wherein the anti-PD-L1 antibody is durvalumab.

15. The method of any one of claims 4 to 14, wherein the CD73 inhibitor is an anti-CD73 antibody or an antigen-binding fragment thereof.

16. 16. The method of claim 15, wherein the anti-CD73 antibody or antigen-binding fragment thereof comprises: (a) a HC CDR1 comprising the amino acid sequence of SEQ ID NO:9, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a HC CDR3 comprising the amino acid sequence of SEQ ID NO:11, and a LC CDR1 comprising the amino acid sequence of SEQ ID NO:12, a LC CDR2 comprising the amino acid sequence of SEQ ID NO:13, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:

14.

17. 17. The method of claim 15 or 16, wherein the anti-CD73 antibody or antigen-binding fragment thereof comprises a HC variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 15 and a LC variable domain (VL) comprising the amino acid sequence of SEQ ID NO:

16.

18. The method of any one of claims 15 to 17, wherein the anti-CD73 antibody or antigen-binding fragment thereof comprises a HC comprising the amino acid sequence of SEQ ID NO: 17 and a LC comprising the amino acid sequence of SEQ ID NO:

18.

19. The method of any one of claims 11 to 13, wherein the anti-CD73 antibody is oleclumab.

20. 20. The method of any one of claims 1 to 19, wherein the administration results in upregulation of CXCR3 in the tumor microenvironment.

21. 21. The method of any one of claims 1 to 3 and 7 to 20, wherein the CD73 protein or CD73 activity level is determined by immunohistochemistry (IHC), imaging mass cytometry (IMC) or mass spectrometry imaging (MSI).

22. 22. The method of any one of claims 1 to 21, wherein the tumor or cancer is a solid tumor or a cancer arising from solid tumor growth.

23. 23. The method of claim 22, wherein the solid tumor is a lung tumor, a breast tumor, a colon tumor, a bladder tumor, a prostate tumor, a colorectal tumor, a head and neck tumor, a liver tumor, or a pancreatic tumor.

24. 24. The method of claim 23, wherein the lung tumor is a non-small cell lung tumor.

25. The method of any one of claims 1 to 24, wherein the subject is a human.

26. 26. Use of a CD73 inhibitor according to any one of claims 1 to 25, a PD-L1 inhibitor, and chemotherapy and / or radiation therapy for treating cancer in a subject in need thereof.

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