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

Abstract

The present disclosure provides a method 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 a reduced level of CD73 protein or CD73 activity compared to a normal subject. In some aspects, the methods comprise further administration of a CD73 inhibitor prior to or contemporaneously with a combination of a T cell checkpoint inhibitor and chemotherapy and / or radiation therapy.

Description

Technical Field

[0001] The invention disclosed herein relates to methods of treating tumors in a subject, the methods comprising administering a CD73 inhibitor, a T cell checkpoint inhibitor, and chemotherapy and / or radiotherapy. Background Art

[0002] Extracellular adenosine has emerged as an important regulator of immune processes within the tumor microenvironment and is thought to reduce the effectiveness of T cell checkpoint inhibitory drugs (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 ectonucleotidases, CD39 (ENTPD1) and CD73 (NT5E), working in tandem. The resulting adenosine acts as an immunosuppressive "smog" that readily diffuses, and cytotoxic agents and radiotherapy may exacerbate this process. In this regard, enhanced antitumor activity has been observed in preclinical models when radiotherapy is combined with anti - CD73 (aCD73) inhibitory antibody therapy (Wennerberg E et al., Cancer Immunol Res 2020; 8:465 - 78; and Wennerberg E et al., Front Immunol 2017; 8:229.). Those studies highlighted the important role of radiation - induced type I interferon in driving elevated levels of tumor cDC1 infiltration and the beneficial effect of CD73 inhibition on this biomarker when radiation - activated type I interferon levels are suboptimal. Despite new information on the role of CD73 in radiation - based standards of care, the role of adenosine pathway inhibitory drugs in the context of chemotherapy treatment regimens remains poorly understood, despite growing evidence of the immunomodulatory axis of these agents (Coffelt SB et al., Trends Immunol 2015; 36:198 - 216.). The inclusion of T cell checkpoint inhibitors in this paradigm provides further room for enhancing cell - mediated activity; by counteracting adaptive immune resistance and unleashing antitumor immunity.

[0003] Oleclumab is a CD73-inhibiting human monoclonal IgG1-TM antibody (Hay CM et al., Oncoimmunology 2016;5) and 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 study of the combination of durvalumab and oleclumab in patients with unresectable stage III non-small cell lung cancer (patients who had not progressed after prior chemoradiation) highlighted the significant benefit of the oleclumab component. A phase 3 clinical trial is now underway in the same patient population. Thus, 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 combination of a PD-L1 inhibitor with chemotherapy and / or radiotherapy; wherein the subject has a reduced level 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, the method comprising administering to the subject a therapeutically effective amount of a combination of a PD-L1 inhibitor with chemotherapy and / or radiotherapy; wherein the subject has a reduced level 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 combination of a PD-L1 inhibitor with chemotherapy and / or radiotherapy; wherein the subject has a reduced level 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, the method comprising administering to the subject a therapeutically effective amount of a CD73 inhibitor, a PD-L1 inhibitor, and chemotherapy and / or radiotherapy.

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

[0009] 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 CD73 inhibitor, a PD-L1 inhibitor, and chemotherapy and / or radiotherapy.

[0010] In one aspect, the PD-L1 inhibitor is administered concurrently with chemotherapy and / or radiotherapy. In another aspect, the PD-L1 inhibitor and chemotherapy and / or radiotherapy are administered sequentially. In another aspect, a CD73 inhibitor is administered prior to the administration of the PD-L1 inhibitor and chemotherapy and / or radiotherapy.

[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 an antigen-binding fragment thereof. In another aspect, the anti-PD-L1 antibody or an antigen-binding fragment thereof comprises: (a) a heavy chain (HC) CDR1 having the amino acid sequence SEQ ID NO: 1, an HC CDR2 having the amino acid sequence SEQ ID NO: 2, and an HC CDR3 having the amino acid sequence SEQ ID NO: 3; and a light chain (LC) CDR1 having the amino acid sequence SEQ ID NO: 4, an LC CDR2 having the amino acid sequence SEQ ID NO: 5, and an LC CDR3 having the amino acid sequence SEQ ID NO: 6. In another aspect, the anti-PD-L1 antibody or an antigen-binding fragment thereof comprises an HC variable domain (VH) having the amino acid sequence SEQ ID NO: 7 and an LC variable domain (VL) having the amino acid sequence 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 an antigen-binding fragment thereof. In another aspect, the anti-CD73 antibody or an antigen-binding fragment thereof comprises: (a) an HC CDR1 having the amino acid sequence SEQ ID NO: 9, an HC CDR2 having the amino acid sequence SEQ ID NO: 10, and an HC CDR3 having the amino acid sequence SEQ ID NO: 11; and an LC CDR1 having the amino acid sequence SEQ ID NO: 12, an LC CDR2 having the amino acid sequence SEQ ID NO: 13, and an LC CDR3 having the amino acid sequence SEQ ID NO: 14. In another aspect, the anti-CD73 antibody or an antigen-binding fragment thereof comprises an HC variable domain (VH) having the amino acid sequence SEQ ID NO: 15 and an LC variable domain (VL) having the amino acid sequence SEQ ID NO: 16. In another aspect, the anti-CD73 antibody or an antigen-binding fragment thereof comprises an HC having the amino acid sequence SEQ ID NO: 17 and an LC having the amino acid sequence SEQ ID NO: 18. In another aspect, the anti-CD73 antibody is olamkicept.

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

[0015] In one aspect, the level of CD73 protein or CD73 activity is 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 resulting from the growth of a solid tumor. 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 a non-small cell lung tumor.

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

[0018] The present disclosure also relates to the use of the CD73 inhibitor, PD-L1 inhibitor, and chemotherapy and / or radiotherapy described herein for treating cancer in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A through Figure 1 C show that combined anti-CD73, anti-PD-L1, and 5FU+OHP treatment results in enhanced complete responses in syngeneic mouse models. (A) Schematic of the experimental design. 5×10 5 cells were implanted into the right flanks of (B) BALB / c (CT26 cells in PBS) and (C) C57BL / 6J mice (MCA205 cells in 50% Matrigel+PBS) and treated as shown in the schematic. Growth curves were plotted based on caliper measurements performed three times per week. Addition of aCD73 and aPD-L1 to 5FU+OHP resulted in a significant increase in the number of complete responders (CR) in each model system - 50% in CT26 (p = 0.005, relative to the 5FU+OHP treatment group) and 61.5% in MCA205 (p = 0.008, relative to the 5FU+OHP treatment group); KruskalWallis test.

[0020] Figure 2 A through Figure 2 B show the responses to aCD73 and aPD-L1 treatment alone or in combination in syngeneic mouse models. 500,000 cells were implanted into the right flanks of BALB / c (CT26 cells in PBS ( Figure 2 A)) and C57BL / 6J mice (MCA205 cells in 50% Matrigel+PBS ( Figure 2 B)) and treated as Figure 1Treatment was carried out as shown in the schematic diagram of A. Growth curves were plotted based on caliper measurements performed three times a week. In both the CT26 and MCA205 models, anti-CD73 monotherapy did not show any effect compared to mice treated as controls. Anti-PD-L1 monotherapy had only a very slight response (1 / 13 CR) only in CT26. The combination treatment of aCD73 and aPD-L1 also did not show any enhanced response rate, and 1 / 13 CR mice were observed in each of the CT26 and MCA205 tumor models.

[0021] Figure 3 A to Figure 3 B shows that mass spectrometry imaging (MSI) confirmed the regulation of the adenosine pathway by adding anti-CD73 to 5FU + OHP. (3A) Schematic diagram of the adenosine production pathway. (3B) The MSI image shows the abundance of ATP and different metabolites of the adenosine pathway in CT26 tumors. Compared to tumors treated as controls, 5FU + OHP led to a slight increase in the abundance of ATP and AMP. However, adding aCD73 to 5FU + OHP led to a significant decrease in adenosine, as well as inosine and xanthine.

[0022] Figure 4 A to Figure 4 E shows that adding anti-CD73 to 5FU + OHP and docetaxel did not result in enhanced in vitro cytotoxicity. 10,000 cells of each of HT-29 (4A), HCT-116 (4B), CT26 (4C and 4E), and MCA-205 (4D) cells were seeded in 96-well plates and treated with serial dilutions of the indicated chemotherapeutic agents together with anti-CD73. Cytotoxicity was measured by luminescence assay after incubation with the drugs for 72 hours. As shown in the different result graphs, none of the tested cell lines showed any additive effect of anti-CD73 on 5FU + OHP and docetaxel.

[0023] Figure 5 A to Figure 5 C shows that CD8 depletion in the syngeneic mouse model MCA205 led to a loss of efficacy seen in the combined treatment of aCD73, aPD-L1, and 5FU + OHP. (5A) Schematic diagram of the experimental design. (5B) Growth curves of C57BL / 6J mice in the MCA205 tumor model (5 × 10 5 cells in 50% Matrigel + PBS) were plotted based on caliper measurements performed three times a week. Selective depletion of CD8 T cells led to a decrease in the efficacy of the combined treatment, resulting in a significantly shorter survival time (Kaplan Meier plot, log-rank test, p = 0.02) compared to those without CD8 cell depletion, as shown in (5C).

[0024] Figure 6 A to Figure 6 D shows that IHC and MSI analysis confirmed the regulation of target (CD73) engagement and adenosine pathway by adding aCD73 to 5FU+OHP. (6A) Schematic diagram of the experimental design. (6B) Immunohistochemical analysis revealed lower levels of surface-bound CD73 protein in the aCD73 combined with 5FU+OHP group. Results are expressed as the area ratio of specific staining to background staining (hematoxylin). (6C) Mass spectrometry imaging showed a significant inhibitory trend of adenosine, inosine, and xanthine, as early PD biomarkers, in CT26 tumors from mice treated with the triple combination group containing anti-CD73. 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 showed a lower frequency of cells expressing the following markers: macrophage markers (such as CD68, F4 / 80) and inhibitory tumor-associated macrophage markers (such as CD163 and CD206) [left panel] and markers known to be associated with cancer-associated fibroblasts (such as type IV collagen, α-smooth muscle actin, vimentin, and CD31). Results are expressed as the mean ± SEM of positive cells%.

[0025] Figure 7A Shows the pharmacodynamic effect of aCD73+aPD-L1+5FU+OHP on the CT26 transcriptome. RNAseq analysis was used to observe changes in the CT tumor transcriptome. The upper panel shows a schematic diagram of the experimental design. The lower panel shows the contribution of individual components in the combined treatment of aCD73, aPD-L1, and 5FU+OHP relative to the aCD73+aPD-L1 group. As seen in the lower left panel, adding aCD73 produced the most significant effect, resulting in 589 differentially expressed (DE) genes, while aPD-L1 only resulted in 35 DE genes (lower right panel). Adding the chemotherapy component to the antibody doublet (aCD73+aPD-L1) resulted in 546 DE genes. The DE cut-off used was absolute (log2FC) >= 1 and adjusted p-value < 0.05.

[0026] Figure 7B Shows transcriptome-based deconvolution of the key roles mediated by the combination. Deconvolution of RNAseq analysis revealed that the key pathways affected by adding aCD73 and 5FU+OHP were immune response activation pathways. Enriched pathways showing upregulated genes, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment KEGG, green) and Gene Ontology > Biological Process (GOBP, red) (the DE cut-off used was log2FC >= 1 and adjusted p-value < 0.05).

[0027] Figure 7C It shows that adding aCD73 to aPD-L1+5FU+OHP drives an increase in 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, adding aCD73 has the most significant effect on driving related immune cells such as cytotoxic T cells, NK cells, B cells, and myeloid dendritic cells (DCs). The DC infiltration effect is mediated only by adding aCD73 to 5FU+OHP, as adding aPD-L1 to 5FU+OHP does not result in an increase in DC infiltration in tumors.

[0028] Figure 8 A to Figure 8 B shows that combined treatment with aCD73, aPD-L1, and docetaxel (DTX) results in enhanced complete responses in the CT26 syngeneic tumor model. (8A) Schematic of the experimental design. (8B) 5×10 5 CT26 cells in PBS were implanted into BALB / c and treated as shown in the schematic. Growth curves were plotted based on caliper measurements taken three times a week. Adding aCD73 and aPD-L1 to docetaxel led to a significant (p = 0.0001, relative to vehicle) increase in the number of complete responders (CR), 58%, compared to 25% CR (p = 0.001) seen when aPD-L1 was added alone to docetaxel: KruskalWallis test.

[0029] Figure 9 A to Figure 9 B shows that simultaneous treatment with aCD73, aPD-L1, and radiotherapy (RTx) results in enhanced complete responses in the MC38 syngeneic tumor model. (9A) Schematic of the experimental design. (9B) 5×10 5 MC38 cells in PBS were implanted into C57BL6 / J and treated as shown in the schematic. Growth curves were plotted based on caliper measurements taken three times a week. Simultaneous treatment with aCD73, aPD-L1, and radiotherapy led to a significant (p = 0.0001, relative to NT) 58% of complete responders (CR) compared to no complete responders seen in the RTx alone group (p = 0.5, relative to NT): Kruskal Wallis test.

[0030] Figure 10Imaging mass cytometry (IMC) showing the pharmacodynamic changes observed by adding aCD73 to 5FU+OHP. IMC images of CT26 tumor-bearing mice treated with control, 5FU+OHP, and aCD73+5FU+OHP. Tumors from mice treated with the aCD73+5FU+OHP combination showed a lower frequency of cells expressing the following markers: macrophage markers (such as CD68, F4 / 80) and inhibitory tumor-associated macrophage markers (such as CD163 and CD206) [left panel] and markers known to be associated with cancer-associated fibroblasts (such as collagen iv, α-smooth muscle actin, vimentin, and CD31).

[0031] Figure 11 Shows the top 50 differentially expressed genes of the triple compared to the control and the log2 fold change for different comparisons. The heatmap on the right panel shows the expression changes of selected immune-related genes under different conditions.

[0032] Figure 12 Shows a schematic of the treatment groups divided according to the timing of aCD73, aPD-L1, and radiotherapy using the MC38 syngeneic mouse model. As shown, 5×10 5 cells were implanted into six groups of mice.

[0033] Figure 13 Shows that mice treated simultaneously with aCD73, aPD-L1, and RTx showed the highest level of tumor suppression and subsequent survival probability.

[0034] Figure 14 Shows that mice treated simultaneously with aCD73, aPD-L1, and RTx also showed induction of a protective memory response after re-challenge using the B16F10 and MC38 mouse models.

[0035] Figure 15 Shows a schematic of the treatment groups divided according to the timing of aCD73, aPD-L1, and radiotherapy alone. As before, MC38 cells were implanted as before, and the timing of individual therapies was plotted.

[0036] Figure 16 Shows that administration of aCD73 therapy before aPD-L1 and RTx led to the greatest reduction in tumor volume, which was associated with the highest survival probability. Detailed Description

[0037] Using a murine cancer model and a murine surrogate of oleclumab (hereinafter referred to as aCD73), the effects of CD73 inhibition in combination with chemotherapy or radiotherapy and PD-L1 blockade were explored. As a corollary, the same approach was explored to determine whether it could be applied to enhance the effects of radiotherapy. As described herein, these combinations are highly effective in terms of improved tumor growth inhibition, induction of protective memory responses, and overall survival benefits. Transcriptomics-based pharmacodynamic assessments highlighted increased abundances of cytotoxic lymphocyte and immune-supportive myeloid cell populations in tumors. Analysis of treatment groups representing the various components of the combination allowed for deconvolution of the individual treatment components contributing; highlighting the role conferred by CD73 inhibition in the context of combined chemotherapy and PD-L1 blockade.

[0038] 1. Definitions

[0039] To enable easier understanding of the present disclosure, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0040] Before describing the present disclosure in detail, it is to be understood that the present disclosure is not limited to particular compositions or method steps, and may thus vary. Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" as used in this specification and the appended claims include plural referents. The terms "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein.

[0041] Furthermore, as used herein, "and / or" is considered to be a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase 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" as used in a phrase such as "A, B, and / or C" is intended to cover 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).

[0042] 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, ConciseDictionary OfBiomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide a general dictionary of many terms used in the present disclosure for those skilled in the art.

[0043] Units, prefixes, and symbols are expressed in their recognized SI (International System of Units) form. Numerical ranges include the values defining the range. Unless otherwise indicated, amino acid sequences are written left to right in an amino to carboxyl orientation. The headings provided herein are not limitations on the various aspects, which can be obtained by reference to the entire specification. Accordingly, the terms defined immediately below are more fully defined by reference to the entire content of the specification.

[0044] It should be understood that wherever aspects are described herein in the language "comprising", other similar aspects are also provided described in terms of "consisting of" and / or "consisting essentially of".

[0045] Amino acids are represented herein by the commonly known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides are represented by the commonly accepted single-letter codes.

[0046] "Antibody" (Ab) shall include, but not be limited to, glycoprotein immunoglobulins that specifically bind an antigen and contain at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain contains a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region includes three constant domains C H1 , C H2 and C H3 . Each L chain contains a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region contains one constant domain C L . The V H and V L regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)), interspersed with more conserved regions (called framework regions (FRs)). Each V H and V LIt contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including 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, the amino acids in the variable regions are numbered using the Kabat numbering system, and those in the constant regions are numbered using the EU system.

[0047] The immunoglobulin 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 encoded by the heavy chain constant region gene (e.g., IgM or IgG1). By way of example, the term "antibody" includes 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 otherwise specified, the term "antibody" includes monospecific, bispecific, or multispecific antibodies, as well as single-chain antibodies, unless the context clearly indicates otherwise. In some aspects, the antibody is a bispecific antibody. In other aspects, the antibody is a monospecific antibody.

[0048] 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), wherein the two heavy chains and light chains are linked by the same number and positions of disulfide bridges that are present in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bonds).

[0049] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to PD-L1 is substantially free of antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds to PD-L1 may have cross-reactivity with other antigens, such as PD-L1 molecules from different species. In addition, an isolated antibody can be substantially free of other cellular materials and / or chemicals.

[0050] The antibody can be an antibody that has been altered (e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). For example, the antibody can include one or more variant amino acids (compared to a naturally occurring antibody) that alter the properties of the antibody (e.g., functional properties). For example, many such alterations are known in the art and affect, for example, the half-life, effector function, 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.

[0051] The term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of antibody molecules consisting of a single molecule, i.e., antibody molecules that have substantially the same primary sequence and exhibit a single binding specificity and affinity for a particular epitope. mAbs are an example of isolated antibodies. MAbs can be produced by hybridoma technology, recombinant technology, transgenic technology, or other techniques known to those skilled in the art.

[0052] A "human" antibody (HuMAb) is an antibody that has variable regions in which both the framework regions and the CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-specific mutagenesis or in vivo by somatic mutation). 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 transplanted into human framework sequences. The terms "human" antibody and "fully human" antibody are used as synonyms.

[0053] A "humanized antibody" is an antibody in which some, most, or all of the amino acids outside the CDR structures of a non-human antibody have been replaced with the corresponding amino acids derived from a human immunoglobulin. In one aspect of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR structures 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 have not changed. Small additions, deletions, insertions, substitutions, or modifications of the amino acids are permitted, provided that they do not eliminate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to the original antibody.

[0054] A "chimeric antibody" is an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0055] An "anti-antigen" antibody is 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.

[0056] The "antigen-binding portion" (also referred to as "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 bound by the whole antibody. It has been demonstrated that the antigen-binding function of an antibody can be carried out by fragments or portions of the full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (e.g., the anti-CD73 antibody described herein) include:

[0057] (1) Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of the VL, VH, LC, and CH1 domains;

[0058] (2) F(ab’)2 fragment (fragment from pepsin cleavage) or a similar divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region;

[0059] (3) Fd fragment consisting of the VH and CH1 domains;

[0060] (4) Fv fragment consisting of the VL and VH domains of a single arm of the antibody;

[0061] (5) Single-domain antibody (dAb) fragment consisting of the VH domain (Ward et al.,

[0062] (1989) Nature 341:544-46);

[0063] (6) Bispecific single-domain antibody (Dual Affinity ReTargeting antibody (DART)) consisting of two VH domains linked by a hinge;

[0064] (7) Bispecific variable domain immunoglobulin;

[0065] (8) Isolated complementarity-determining region (CDR); and

[0066] (9) A combination of two or more isolated CDRs optionally linked by a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that enables them to form a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science

[0067] 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA

[0068] 85: 5879-5883). Such single-chain antibodies are also intended to be encompassed within the terms "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 screening these fragments in the same manner as intact antibodies is of no utility. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0069] As used herein, the term "CD73 polypeptide" refers to CD73 (cluster of differentiation 73) protein, also known in the literature as 5'-nucleotidase (5'-NT) or extracellular 5'-nucleotidase, which is encoded by the NT5E gene. See, for example, Misumi et al. Eur. J. Biochem. 191(3): 563-9 (1990). The corresponding sequences of human and murine forms of 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 without the signal sequence residues. Thus, for example, an antibody binding to amino acids Val144, Lys180, and Asn185 refers to the amino acid positions after signal sequence cleavage, i.e., the amino acids in the mature protein.

[0070] A "T cell checkpoint inhibitor" or "immune checkpoint inhibitor" refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduced function and complete blockade. In particular, the immune checkpoint protein is a human immune checkpoint protein. Thus, an immune checkpoint protein inhibitor is particularly an inhibitor of a human immune checkpoint protein.

[0071] "Programmed death ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other cell surface glycoprotein ligand is PD-L2), which 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, isotypes, and species homologs of hPD-L1, as well as five analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

[0072] As used herein, "patient" includes any patient suffering from cancer (e.g., non-small cell lung cancer (NSCLC)). The terms "subject" and "patient" are used interchangeably herein.

[0073] "Administration" means physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode 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, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some aspects, the formulation is administered via a non-parenteral route, and in some aspects, orally. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical. Administration may also be carried out, for example, once, multiple times and / or over one or more extended periods of time.

[0074] "Treatment" or "therapy" of a subject means any type of intervention or process performed on a subject, or the administration of an active agent to a subject, with the aim of reversing, alleviating, ameliorating, inhibiting, slowing or preventing the onset, progression, development, severity or recurrence of symptoms, complications or disorders, or biochemical markers associated with a disease.

[0075] As used herein, "effective treatment" means a treatment that produces a beneficial effect, e.g., improvement of at least one symptom of a disease or disorder. The beneficial effect may take the form of an improvement relative to a baseline, i.e., relative to a measurement or observation made prior to the start of therapy according to the method. The beneficial effect may also take the form of preventing, slowing, delaying or stabilizing the deleterious progression of a solid tumor marker. Effective treatment may mean alleviating at least one symptom of a solid tumor. Such effective treatment may, for example, relieve patient pain, reduce the size and / or number of lesions, may reduce or prevent tumor metastasis, and / or may slow tumor growth.

[0076] 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 a reduction, amelioration, mitigation, alleviation, delay, and / or remission of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. With reference to solid tumors, an effective amount includes an amount sufficient to cause tumor shrinkage and / or reduce the tumor growth rate (such as inhibiting tumor growth) or prevent or delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. The effective amount can be administered in one or more administrations. An effective amount of a drug or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) to some extent inhibit, delay, slow down, and can prevent cancer cells from infiltrating into peripheral organs; (iv) inhibit (i.e., to some extent slow down and can prevent tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) to some extent alleviate one or more symptoms associated with cancer. In one example, an "effective amount" is a combination of an amount of an anti-CD73 antibody and an amount of an anti-PD-L1 antibody that is clinically proven to affect a significant reduction in cancer or slow the progression of cancer (such as advanced solid tumors). As used herein, the term "progression-free survival" may be abbreviated as PFS and refers to the length of time during and after treatment of a solid tumor (i.e., NSCLC) that a patient lives with the disease without the disease getting worse.

[0077] "Cancer" refers to a large group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors.

[0078] As used herein, the term "tumor" refers to any mass of tissue caused by excessive cell growth or proliferation, whether benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0079] "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 produced by any of these cells or the liver (including antibodies, cytokines, and complement), which results in the selective targeting, binding, injury, destruction, and / or elimination of invading pathogens, cells or tissues infected with pathogens, cancer cells, or other abnormal cells in the vertebrate body, or normal cells or tissues in the case of autoimmunity or pathological inflammation.

[0080] The various aspects of the present disclosure are described in more detail in the following subsections.

[0081] 2. Methods of the Present Disclosure

[0082] In one aspect, the present disclosure relates to a method of inhibiting tumor growth in a subject having a reduced level of CD73 protein expression or CD73 activity as compared to a normal subject. A combination therapy of a T cell checkpoint inhibitor and chemotherapy and / or radiotherapy results in better treatment outcomes (e.g., objective response rate and disease control rate). To improve the treatment of malignancies, in one aspect, the present disclosure provides for identifying a patient having reduced CD73 protein expression or CD73 activity, and providing a combination therapy of a T cell checkpoint inhibitor and chemotherapy and / or radiotherapy.

[0083] According to this aspect, a variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell, e.g., whether and at what stage these agents or drugs affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, insert into DNA, or induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[0084] Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; podophyllotoxins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including the synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide and uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I); dynemicin, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycins, actinomycin C, carabicin, carminomycin, carcinomycin, chromomycinis, dactinomycin, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, puromycin, quinamycin, rodorubicin, streptozocin, streptothricin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thioguanine, tioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didanosine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal, such as mitotane and trilostane; folic acid supplements, such as folinic acid; glucuronolactone; aldophosphamide glycoside; aminolevulinic acid; enuracil; aclarubicin; bestrabucil; bisantrene; edatrexate; defofamine; colchicine; diaziquone; ifosfamide; elfornithine; epothilones; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-acetylhydrazine; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2”-trichloroethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman;Metoprine; arabinoside ("Ara-C"); cyclophosphamide; taxane-like agents such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; navelbine; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitor, transplatinum, and pharmaceutically acceptable salts, acids or derivatives of any of the above chemotherapeutic agents.

[0085] In another aspect, the combination includes radiotherapy. Other factors that cause DNA damage and have been widely used include what are commonly referred to as gamma rays, X-rays, and / or direct delivery of radioactive isotopes to tumor cells. Other forms of DNA-damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and ultraviolet irradiation. Most likely, all of these factors will cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range of X-rays is from 50 roentgens to 200 roentgens per day for a long period (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dose range of radioactive isotopes varies widely, depending on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by tumor cells.

[0086] In one aspect, the present disclosure relates to identifying a patient with reduced CD73 protein expression or CD73 activity level, and treating a subject by administering a T cell checkpoint inhibitor (e.g., an anti-PD-L1 antibody) and chemotherapy and / or radiotherapy. In one aspect, the present disclosure includes a method of identifying a patient with reduced CD73 protein expression or CD73 activity level and treating a subject by administering an anti-PD-L1 antibody and chemotherapy and / or radiotherapy.

[0087] In another aspect, the present disclosure relates to a method of inhibiting tumor growth in a subject, the method comprising administering a combination therapy of a CD73 inhibitor, a T cell checkpoint inhibitor, and chemotherapy and / or radiotherapy. In one aspect, tumor growth in a subject is inhibited by administering an anti-CD73 antibody, an anti-PD-L1 antibody, and chemotherapy and / or radiotherapy.

[0088] CD73 is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein that catalyzes the hydrolysis of adenosine monophosphate (AMP) to adenosine and acts in concert with CD39, which converts adenosine triphosphate (ATP) to AMP. The resulting adenosine serves as a signaling molecule that activates P1 receptors expressed on the cell surfaces of many different tissues. Four G protein-coupled P1 or adenosine receptors have been cloned and designated A1, A2A, A2B, and A3. Adenosine affects a wide range of physiological processes, including neural function, vascular perfusion, and immune responses. In doing so, the metabolite regulates CNS, cardiovascular, and immune system functions, to name just a few.

[0089] Increasing evidence suggests that the interaction between tumor cells and their microenvironment is crucial for tumorigenesis. The purinergic signaling pathway, in which CD73 plays a key role, has emerged as an important player in cancer progression. In recent years, it has become clear that adenosine is one of the most important immunosuppressive regulatory molecules in the tumor microenvironment and contributes to immune escape and tumor progression.

[0090] CD73 is a key protein molecule in cancer development. CD73 has been found to be overexpressed in many cancer cell lines and tumor types, including, for example, breast cancer, colorectal cancer, ovarian cancer, gastric cancer, gallbladder cancer, and cancers associated with poor prognosis.

[0091] In addition to serving as a prognostic biomarker in cancer patients, overexpression of CD73 has also been found to be functionally associated with resistance to therapies, such as cancer therapies. Elevated levels of CD73 were initially associated with resistance to multiple chemotherapeutic agents, including vincristine and doxorubicin.

[0092] CD73 has also been shown to be involved in immune therapy resistance. This ectonucleotidase is involved in the process of tumor immune escape by inhibiting the activation, clonal expansion, and homing of tumor-specific T cells, particularly T helper cells and cytotoxic T cells; impairing the tumor cell killing by cytotoxic effector T lymphocytes; driving the inhibitory capacity of Tregs and Th17 cells via adenosine production in the cell periphery; enhancing the conversion of type 1 macrophages to tumor-promoting type 2 macrophages; and promoting the accumulation of MDSCs.

[0093] In some aspects of the present disclosure, the CD73 protein expression or CD73 activity of a subject to be treated is reduced. In some aspects, the reduction is caused by prior treatment with a CD73 inhibitor. In some aspects, the CD73 inhibitor is an anti-CD73 antibody or an antigen-binding fragment thereof.

[0094] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof is an antibody described in PCT Publication No. WO2016 / 075099. In some aspects, the anti-CD73 antibody comprises HC CDR1-3 and LC CDR1-3 of SEQ ID NOs: 9-11 and 12-14, respectively. In some aspects, the anti-CD73 antibody comprises VH and VL of SEQ ID NOs: 15 and 16, respectively. In some aspects, the anti-CD73 antibody comprises a heavy chain and a light chain of SEQ ID NOs: 17 and 18, respectively. In some aspects, the anti-CD73 antibody is oleclumab.

[0095] In some aspects, the method comprises administering the anti-CD73 antibody or antigen-binding fragment thereof before or concurrently with a T cell checkpoint inhibitor and chemotherapy and / or radiotherapy. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof is an antibody described in PCT Publication No. WO2016 / 075099. In some aspects, the anti-CD73 antibody comprises HC CDR1-3 and LC CDR1-3 of SEQ ID NOs: 9-11 and 12-14, respectively. In some aspects, the anti-CD73 antibody comprises VH and VL of SEQ ID NOs: 15 and 16, respectively. In some aspects, the anti-CD73 antibody comprises a heavy chain and a light chain of SEQ ID NOs: 17 and 18, respectively. In some aspects, the anti-CD73 antibody is oleclumab.

[0096] In some aspects, existing antibodies can be used to reduce CD73 expression and / or activity. Exemplary anti-CD73 antibodies are described in PCT Publications Nos. WO2018 / 137598; WO2016 / 081748; WO2017 / 064043; WO2017 / 100670; WO2018 / 237157.

[0097] In one aspect, the present disclosure includes a method of selecting a tumor of 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 reduced CD73 protein expression or CD73 activity. In one aspect, the present disclosure includes a method of identifying a tumor in a human patient that is likely to respond 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 respond to treatment if the tumor exhibits reduced 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 aspects, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor and chemotherapy and / or radiotherapy.

[0098] In another aspect, the present disclosure includes a method of treating tumor growth in a human patient in need thereof, the method comprising administering to the patient a T cell checkpoint inhibitor and chemotherapy and / or radiotherapy, wherein prior to administration, the patient has been identified as having reduced CD73 protein expression or CD73 activity. 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.

[0099] In other aspects, the present disclosure includes a method of reducing the size of a tumor in a human patient having a tumor by at least 10%, the method 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 radiotherapy; or an anti-PD-L1 antibody and chemotherapy and / or radiotherapy). In some aspects, prior to administration, the patient has been identified as having reduced CD73 protein expression or CD73 activity, wherein the administration reduces the size of the tumor 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 size of the tumor prior to administration.

[0100] The present disclosure may also include a method of preventing recurrence and / or inducing remission in a patient, the method comprising administering to the patient the combination therapy disclosed herein (e.g., an anti-CD73 antibody, an anti-PD-L1 antibody, and chemotherapy and / or radiotherapy; or an anti-PD-L1 antibody and chemotherapy and / or radiotherapy). In some aspects, the method of the present disclosure comprises (i) identifying a patient having reduced CD73 protein expression or CD73 activity; (ii) administering to the patient the combination therapy disclosed herein (e.g., an anti-CD73 antibody, an anti-PD-L1 antibody, and chemotherapy and / or radiotherapy; or an anti-PD-L1 antibody and chemotherapy and / or radiotherapy).

[0101] Due to the administration of the combination therapy disclosed herein, the methods of the present disclosure can treat a malignancy, reduce tumor size, prevent tumor growth, eliminate a tumor from a patient, prevent tumor recurrence, induce remission in a patient, or any combination thereof. In certain aspects, administration of the combination therapy disclosed herein induces a complete response. In other aspects, administration of the 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 radiotherapy. In some aspects, the PD-1 pathway inhibitor is an anti-PD-L1 antibody. In some aspects, the combination therapy comprises administering a therapeutically effective amount of a CD73 inhibitor, a T cell checkpoint inhibitor, and chemotherapy and / or radiotherapy. In some aspects, the combination therapy comprises administering a therapeutically effective amount of an anti-CD73 antibody, an anti-PD-L1 antibody, and chemotherapy and / or radiotherapy.

[0102] In some aspects, CD73 expression or CD73 activity is determined by receiving the results of an assay capable of determining CD73 expression / activity.

[0103] Measurement of CD73 Activity / Expression

[0104] To assess CD73 expression / activity, in one aspect, a test sample is obtained from a patient in need of treatment. In some aspects, the test sample includes, but is not limited to, any clinically relevant sample, such as a tumor biopsy, a core biopsy tissue sample, a fine needle aspirate, or a body fluid sample (such as blood, plasma, serum, lymph fluid, ascites, cyst fluid, or urine). In some aspects, the test tissue sample is from a primary tumor. In some aspects, the test sample is from a metastasis. In some aspects, the test sample is collected from a subject at multiple time points, e.g., before treatment, during treatment, and / or after treatment. In some aspects, the test sample is collected from different locations of a subject, e.g., a sample from a primary tumor and a sample from a metastasis at a distal location.

[0105] In some aspects, the test tissue sample is a paraffin-embedded fixed tissue sample. In some aspects, the test tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample. In some aspects, the test tissue sample is a fresh tissue (e.g., tumor) sample. In some aspects, the test tissue sample is a frozen tissue sample. In some aspects, the test tissue sample is a fresh frozen (FF) tissue (e.g., tumor) sample. In some aspects, the test tissue sample is cells isolated from a liquid. In some aspects, the test tissue sample includes circulating tumor cells (CTCs). In some aspects, the test tissue sample includes circulating lymphocytes. In some aspects, the test tissue sample is an archived tissue sample. In some aspects, the test tissue sample is an archived tissue sample with a known diagnosis, treatment, and / or outcome history. In some aspects, the sample is a tissue block. In some aspects, the test tissue sample is dispersed cells. In some aspects, the sample amount is from about 1 cell to about 1×1 0 6 cells or more. In some aspects, the sample amount is from about 1 cell to about 1×10 5 cells. In some aspects, the sample amount is from about 1 cell to about 10,000 cells. In some aspects, the sample amount is from about 1 cell to about 1,000 cells. In some aspects, the sample amount is from about 1 cell to about 100 cells. In some aspects, the sample amount is from about 1 cell to about 10 cells. In some aspects, the sample amount is a single cell.

[0106] In another aspect, the assessment of CD73 activity / expression can be achieved without obtaining a test tissue sample. In some aspects, selecting a suitable patient includes (i) optionally providing a test tissue sample obtained from the tissue of a patient with cancer, the test tissue sample containing tumor cells; and (ii) evaluating the proportion of cells expressing CD73 on the cell surface in the test tissue sample based on the assessment that the proportion of cells expressing CD73 on the cell surface in the test tissue sample is lower than a predetermined threshold level.

[0107] However, in any method that includes measuring CD73 expression in a test sample, it should be understood that the step of providing a test sample obtained from a patient is an optional step. That is, in some aspects, the method includes this step, while in other aspects, this step is not included in the method. It should also be understood that in some aspects, the "measurement" or "assessment" step of identifying or determining the number or proportion of cells expressing CD73 in a test sample is carried out by a method of measuring CD73 expression transformation, such as by performing a reverse transcription polymerase chain reaction (RT-PCR) assay, IHC, imaging mass cytometry (IMC), or mass spectrometry imaging (MSI) assay. In some other aspects, no transformation step is involved, and CD73 expression is evaluated by, for example, reviewing the test result report of a laboratory. In some aspects, CD73 activity / expression is evaluated by reviewing, for example, the results of immunohistochemical assays of a laboratory. In some aspects, the step of providing test results is carried out by a medical practitioner or a person acting under the guidance of a medical practitioner. In other aspects, these steps are carried out by an independent laboratory or by independent personnel (such as laboratory technicians).

[0108] In some aspects of any method of the present invention, the proportion of cells expressing CD73 is evaluated by performing an assay to detect the presence of CD73 RNA. In additional aspects, the presence of CD73 RNA is detected by RT-PCR, in situ hybridization, or ribonuclease protection. In some aspects, the presence of CD73 RNA is detected by an RT-PCR-based assay. In some aspects, scoring an RT-PCR-based assay includes evaluating the level of CD73 RNA expression in a test tissue sample relative to a predetermined level.

[0109] In other aspects, the proportion of cells expressing CD73 is evaluated by performing an assay to detect the presence of CD73 polypeptide. In additional aspects, the presence of CD73 polypeptide is detected by IHC, enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some aspects, CD73 expression is assayed by IHC, imaging mass cytometry (IMC), or mass spectrometry imaging (MSI). In other aspects of all these methods, cell surface expression of CD73 is assayed using, for example, IHC or in vivo imaging.

[0110] T Cell Checkpoint Inhibitors

[0111] In the tumor microenvironment, cancer cells can evade immune surveillance by altering their surface antigens, thus avoiding detection and destruction by host lymphocytes. The core mechanism of tumor-induced immunosuppression is the increased expression of ligands that can bind to inhibitory T cell receptors. These ligands are called T cell or immune checkpoints and function under physiological conditions to prevent the development of autoimmunity at multiple steps during the immune response. The main mechanisms involved in T cell regulation are the inhibition of potential autoreactive naive T cells (characterized by TCRs against self-antigens) in lymph nodes at the initial stage, or the inactivation of T cells in peripheral tissues at a later stage. This process is called peripheral tolerance and is mainly mediated by the immune checkpoint cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) pathways. Tumor cells have developed ways to exploit peripheral tolerance by inducing the expression of immune checkpoints that disrupt T cell function to avoid immune recognition.

[0112] Other new 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 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).

[0113] PD-1 Pathway Inhibitors

[0114] In certain aspects, the present application encompasses the use of anti-PD-L1 antibodies as T cell checkpoint inhibitors. In one aspect, the anti-PD-L1 antibody inhibits the binding of the PD-L1 receptor, namely PD-1, to its ligand PD-L1.

[0115] Anti-human PD-L1 antibodies (or VH and / or VL domains derived therefrom) suitable for the methods of the present disclosure can be generated using methods well known in the art. In certain aspects, the anti-PD-L1 antibody or its antigen-binding fragment is the antibody described in PCT Publication No. WO2011 / 066389. In some aspects, the anti-PD-L1 antibody comprises the HC CDR1-3 and LC CDR1-3 of SEQ ID NOs: 1-3 and 4-6, respectively. In some aspects, the anti-PD-L1 antibody comprises the VH and VL of SEQ ID NOs: 7 and 8, respectively. In some aspects, the anti-CD73 antibody is durvalumab.

[0116] Alternatively, anti-PD-L1 antibodies recognized in the art 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 aspects, the anti-PD-L1 antibody is atezolizumab (Tcentriq 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 anti-PD-L1 antibodies recognized in the art that can be used include, for example, those described in U.S. Patent Nos. 7,635,757 and 8,217,149, U.S. Publication No. 2009 / 0317368, and PCT Publications WO 2011 / 066389 and WO 2012 / 145493, which are incorporated herein by reference. Antibodies that competitively bind to PD-L1 with any of these art-recognized antibodies or inhibitors can also be used.

[0117] In certain aspects, antibodies that cross-compete with the above-referenced PD-L1 antibodies for binding to human PD-L1 or that bind to the same epitope region of human PD-L1 are mAbs. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, or can be humanized or human antibodies. Such chimeric, humanized, or human mAbs can be prepared and isolated by methods well known in the art.

[0118] In certain aspects, the PD-L1 antibody is durvalumab (IMFINZI TM ). Durvalumab is a human IgG1K monoclonal anti-PD-L1 antibody.

[0119] In certain aspects, the PD-L1 antibody is atezolizumab Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0120] In certain aspects, the PD-L1 antibody is avelumab Avelumab is a human IgG1λ monoclonal anti-PD-L1 antibody.

[0121] Anti-PD-L1 antibodies useful in the disclosed methods also include isolated antibodies that specifically bind to human PD-L1 and cross-compete for binding to human PD-L1 with any of the anti-PD-L1 antibodies disclosed herein (e.g., durvalumab, atezolizumab, and / or avelumab). In some aspects, 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 specific epitope region. Since these cross-competing antibodies bind to the same epitope region of PD-L1, it is expected that these cross-competing antibodies will have functional properties very similar to those of the reference antibodies. Cross-competing antibodies can be readily identified in standard PD-L1 binding assays, such as Biacore assays, ELISA assays, or flow cytometry, based on their ability to cross-compete with atezolizumab and / or avelumab (see, e.g., WO 2013 / 173223).

[0122] In certain aspects, the antibodies that cross-compete for binding to human PD-L1 or bind to the same epitope region of the anti-human PD-L1 antibody as durvalumab, atezolizumab, and / or avelumab are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0123] Anti-PD-L1 antibodies useful in the methods of the present disclosure also include antigen-binding portions of the above-described antibodies. It has been well demonstrated that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody.

[0124] Anti-PD-L1 antibodies suitable for the disclosed methods or compositions are antibodies 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 full antibody in inhibiting receptor binding and upregulating the immune system. In certain aspects, the anti-PD-L1 antibody or its antigen-binding portion cross-competes for binding to human PD-L1 with durvalumab, atezolizumab, and / or avelumab.

[0125] 3. PD-1 Inhibitors

[0126] In some aspects, the T cell checkpoint inhibitor is a PD-1 pathway inhibitor, such as an anti-PD-1 antibody. In some aspects, the PD-1 pathway inhibitor is a PD-L2 binder, such as an anti-PD-L2 antibody. In other aspects, the PD-L1 binder is a soluble PD-1 polypeptide, such as a PD-1-Fc fusion polypeptide capable of binding to PD-L1. In other aspects, the PD-L2 binder is a soluble PD-1 polypeptide, such as a PD-1-Fc fusion polypeptide capable of binding to PD-L2.

[0127] The anti-human PD-1 antibody (or VH and / or VL domains derived therefrom) applicable to the present disclosure can be produced using methods well known in the art. Alternatively, anti-PD-1 antibodies recognized in the art can be used.

[0128] In other aspects, the anti-PD-1 antibody is nivolumab or BMS-936558 described in WO 2006 / 121168. Other known PD-1 antibodies include lambrolizumab (MK-3475) described in WO 2008 / 156712. Additional known PD-1 antibodies and other PD-1 inhibitors include, for example, those antibodies described in WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335, and WO 2011 / 161699, which are incorporated herein by reference. In one aspect, the anti-PD-1 antibody is REGN2810. In one aspect, the anti-PD-1 antibody is PDR001. Another known anti-PD-1 antibody is pidilizumab (CT-011).

[0129] In some aspects, the anti-PD-1 antibody or its fragment cross-competes with pembrolizumab. In some aspects, the anti-PD-1 antibody or its fragment binds to the same epitope as pembrolizumab. In certain aspects, the anti-PD-1 antibody has the same CDRs as pembrolizumab. In another aspect, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as 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 described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0130] In certain aspects, the first antibody is an anti-PD-1 antagonist. An example of an anti-PD-1 antagonist is AMP-224, a B7-DC Fc fusion protein. AMP-224 is discussed in U.S. Publication No. 2013 / 0017199.

[0131] In other aspects, the anti-PD-1 antibody or fragment thereof cross-competes with BGB-A317. In some aspects, the anti-PD-1 antibody or fragment thereof binds to the same epitope as BGB-A317. In certain aspects, the anti-PD-1 antibody has the same CDRs as BGB-A317. In certain aspects, the anti-PD-1 antibody is BGB-A317, a humanized monoclonal antibody. BGB-A317 is described in U.S. Publication No. 2015 / 0079109.

[0132] In some aspects, the antibody is pidilizumab (CT-011), an antibody that has been previously reported to bind to PD-1 but is thought to bind to a different target. Pidilizumab is described in U.S. Patent No. 8,686,119 B2 or WO2013 / 014668 A1.

[0133] In certain aspects, the antibody that cross-competes with nivolumab for binding to human PD-1 or binds to the same epitope region of human PD-1 as nivolumab is an mAb. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies or humanized or human antibodies. Such chimeric, humanized or human mAbs can be prepared and isolated by methods well known in the art.

[0134] Other anti-PD-1 monoclonal antibodies have been described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, U.S. Publication No. 2016 / 0272708, and PCT Publications WO2012 / 145493, WO2008 / 156712, WO2015 / 112900, WO2012 / 145493, WO2015 / 112800, WO2014 / 206107, WO2015 / 35606, WO2015 / 085847, WO2014 / 179664, WO2017 / 020291, WO2017 / 020858, WO2016 / 197367, WO2017 / 024515, WO2017 / 025051, WO2017 / 123557, WO2016 / 106159, WO2014 / 194302, WO2017 / 040790, WO2017 / 133540, WO2017 / 132827, WO2017 / 024465, WO2017 / 025016, WO2017 / 106061, WO2017 / 19846, WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540, the entire contents of each of which are incorporated herein by reference.

[0135] Anti-PD-1 antibodies useful in the compositions of the present disclosure also include antigen-binding portions of the above antibodies. It has been 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 within the term "antigen-binding portion" of an antibody include (i) Fab fragments or monovalent fragments consisting of V L 、V H 、C L and C H1 domains; (ii) F(ab′)2 fragments, divalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) Fd fragments consisting of V H and C H1 domains; and (iv) Fv fragments consisting of the V L and V H domains of a single arm of an antibody.

[0136] Anti-PD-1 antibodies useful 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 aspects, 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 antigen binding 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 specific epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the reference antibody.

[0137] 4. LAG-3 Inhibitors

[0138] In some aspects, the LAG-3 inhibitor is a LAG-3 binder, such as an anti-LAG-3 antibody. In some aspects, the LAG-3 binder is a soluble LAG-3 polypeptide, such as a LAG-3-Fc fusion polypeptide capable of binding to MHC class II.

[0139] Anti-human LAG-3 antibodies (or VH / VL domains derived therefrom) suitable for the present disclosure can be generated using methods well known in the art. Alternatively, anti-LAG-3 antibodies recognized in the art can be used. In certain aspects, the LAG-3 inhibitor includes an anti-LAG-3 bispecific antibody.

[0140] In some aspects, the anti-LAG-3 antibody is relatlimab or BMS-986016 comprising a heavy chain and a light chain as described in PCT / US13 / 48999.

[0141] In another aspect, the antibody competes with the above antibodies for binding to the same epitope on LAG-3.

[0142] In some aspects, anti-LAG-3 antibodies recognized in the art can be used in the therapeutic methods of the present disclosure. For example, the anti-human LAG-3 antibody called monoclonal antibody 25F7 (also known as "25F7" and "LAG-3.1") described in US2011 / 0150892A1 can be used. Other anti-LAG-3 antibodies recognized in the art that can be used include IMP731 (H5L7BW) described in US2011 / 007023, MK-4280 (28G-10) described in WO2016028672, REGN3767 described in Journal for ImmunoTherapy of Cancer, (2016) Volume 4, Supplement Issue 1 Abstract Number: P195, BAP050, IMP-701 (LAG-525), Sym022, TSR-033, MGD013, BI754111, FS118, AVA-017, and GSK2831781 described in WO2017 / 019894. These and other anti-LAG-3 antibodies that can be used in the claimed disclosure can be found, for example, in: WO2016 / 028672, WO2017 / 106129, WO2017 / 062888, WO2009 / 044273, WO2018 / 069500, WO2016 / 126858, WO2014 / 179664, WO2016 / 200782, WO2015 / 200119, WO2017 / 019846, WO2017 / 198741, WO2017 / 220555, WO2017 / 220569, WO2018 / 071500, WO2017 / 015560, WO2017 / 025498, WO2017 / 087589, WO2017 / 087901, WO2018 / 083087, WO2017 / 149143, WO2017 / 219995, US2017 / 0260271, WO2017 / 086367, WO2017 / 086419, WO2018 / 034227, and WO2014 / 140180. The content of each of these references is incorporated herein by reference in its entirety.

[0143] Antibodies that compete with any of the above-mentioned antibodies recognized in the art for binding to LAG-3 can also be used.

[0144] 5. CTLA-4 Antagonists

[0145] In certain aspects, the present application encompasses the use of anti-CTLA-4 antibodies. In one aspect, the anti-CTLA-4 antibody binds to and inhibits CTLA-4. In some aspects, the anti-CTLA-4 antibody is ipilimumab (YERVOY), tremelimumab (ticilimumab; CP-675,206), AGEN-1884 or ATOR-1015.

[0146] In one aspect, the CTLA-4 antagonist is a soluble CTLA-4 polypeptide. In one aspect, the soluble CTLA-4 polypeptide is abatacept (Orencia), belatacept (Nulojix), RG2077 or RG-1046. In another aspect, the CTLA-4 antagonist is a cell-based therapy. In some aspects, the CTLA-4 antagonist is an anti-CTLA-4mAb RNA / GITRL RNA-transfected autologous dendritic cell vaccine or an anti-CTLA-4mAb RNA-transfected autologous dendritic cell vaccine.

[0147] 6. Additional Immune Checkpoint Inhibitors

[0148] 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.

[0149] In one aspect, the immune checkpoint inhibitor is a KIR antagonist. In certain aspects, the KIR antagonist is an anti-KIR antibody or an antigen-binding fragment thereof. In some aspects, the anti-KIR antibody is lirilumab (1-7F9, BMS-986015, IPH 2101) or IPH4102.

[0150] In one aspect, the immune checkpoint inhibitor is a TIGIT antagonist. In one aspect, the TIGIT antagonist is an anti-TIGIT antibody or an antigen-binding fragment thereof. In certain aspects, the anti-TIGIT antibody is BMS-986207, AB 154, COM902 (CGEN-15137) or OMP-313M32.

[0151] In one aspect, the immune checkpoint inhibitor is a TIM-3 antagonist. In certain aspects, the TIM-3 antagonist is an anti-TIM-3 antibody or an antigen-binding fragment thereof. In some aspects, the anti-TIM-3 antibody is TSR-022 or LY3321367.

[0152] In one aspect, the immune checkpoint inhibitor is an IDO1 antagonist. In another aspect, 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 a pyrrolidine-2,5-dione derivative.

[0153] In one aspect, the immune checkpoint inhibitor is a STING antagonist. In certain aspects, 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; 2',2”-diF-Rp,Rp, a bis-3',5' cyclic dinucleotide; or a fluorinated cyclic dinucleotide.

[0154] In one aspect, the immune checkpoint inhibitor is a CD20 antagonist. In some aspects, the CD20 antagonist is an anti-CD20 antibody or an antigen-binding fragment thereof. In one aspect, the anti-CD20 antibody is rituximab (RITUXAN; IDEC-102; IDEC-C2B8), ABP 798, ofatumumab or obinutuzumab.

[0155] In one aspect, the immune checkpoint inhibitor is a CD80 antagonist. In certain aspects, the CD80 antagonist is an anti-CD80 antibody or an antigen-binding fragment thereof. In one aspect, the anti-CD80 antibody is galiximab or AV1142742.

[0156] In one aspect, the immune checkpoint inhibitor is a GARP antagonist. In some aspects, the GARP antagonist is an anti-GARP antibody or an antigen-binding fragment thereof. In certain aspects, the anti-GARP antibody is ARGX-115.

[0157] In one aspect, immune checkpoint inhibitors are CD40 antagonists. In certain aspects, CD40 antagonists are anti-CD40 antibodies or their antigen-binding fragments. In some aspects, anti-CD40 antibodies are BMS3h-56, lucatumumab (lucatumumab) (HCD122 and CHIR-12.12), CHIR-5.9 or dacetuzumab (dacetuzumab) (huS2C6, PRO64553, RG3636, SGN 14, SGN-40). In another aspect, CD40 antagonists are soluble CD40 ligands (CD40-L). In one aspect, soluble CD40 ligands are fusion polypeptides. In one aspect, soluble CD40 ligands are CD40-L / FC2 or monomeric CD40-L.

[0158] In one aspect, the immune checkpoint inhibitor is an A2aR antagonist. In some aspects, the A2aR antagonist is a small molecule. In certain aspects, the A2aR antagonist is CPI-444, PBF-509, istradefylline (KW-6002), preladenant (SCH420814), tozadenant (SYN115), vipadenant (BIIB014), HTL-1071, ST1535, SCH412348, SCH442416, SCH58261, ZM241385 or AZD4635.

[0159] In one aspect, the immune checkpoint inhibitor is a CEACAM1 antagonist. In some aspects, the CEACAM1 antagonist is an anti-CEACAM1 antibody or an antigen-binding fragment thereof. In one aspect, the anti-CEACAM1 antibody is CM-24 (MK-6018).

[0160] In one aspect, immune checkpoint inhibitors are CEA antagonists. In one aspect, CEA antagonists are anti-CEA antibodies or their antigen-binding fragments. In some aspects, anti-CEA antibodies are cergutuzumab amu leukin (RG7813, RO-6895882) or RG7802 (RO6958688).

[0161] In one aspect, the immune checkpoint inhibitor is a CD47 antagonist. In some aspects, the CD47 antagonist is an anti-CD47 antibody or an antigen-binding fragment thereof. In certain aspects, the anti-CD47 antibody is HuF9-G4, CC-90002, TTI-621, ALX148, NI-1701, NI-1801, SRF231, or Effi-DEM.

[0162] In one aspect, the immune checkpoint inhibitor is a PVRIG antagonist. In certain aspects, the PVRIG antagonist is an anti-PVRIG antibody or an antigen-binding fragment thereof. In one aspect, the anti-PVRIG antibody is COM701 (CGEN-15029).

[0163] In one aspect, the immune checkpoint inhibitor is a TDO antagonist. In one aspect, 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 aspect, the immune checkpoint inhibitor is an IDO / TDO dual antagonist. In one aspect, the IDO / TDO dual antagonist is a small molecule.

[0164] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of B7-H3. In some embodiments, the inhibitor of B7-H3 is enoblituzumab, MGD009, or 8H9.

[0165] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of OX40, CD27, CD28, GITR, ICOS, TLR7 / 8, and CD137 (also known as 4-1BB).

[0166] In some embodiments, the agonist of CD137 is urelumab. In some embodiments, the agonist of CD137 is utomilumab.

[0167] 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, such as 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.

[0168] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of ICOS. In some embodiments, the agonist of ICOS is GSK-3359609, JTX-2011, or MEDI-570.

[0169] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of CD28. In some embodiments, the agonist of CD28 is theralizumab.

[0170] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of CD27. In some embodiments, the agonist of CD27 is varlilumab.

[0171] 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.

[0172] 7. Patient Populations

[0173] Provided herein are clinical methods for treating tumors in a subject (e.g., a human patient) using the therapies disclosed herein, such as T cell checkpoint inhibitors (e.g., anti-PD-L1 antibodies), CD73 inhibitors (e.g., anti-CD73 antibodies), and chemotherapy and / or radiotherapy.

[0174] Examples of cancers and / or malignancies treatable using the methods of the present disclosure include liver cancer, hepatocellular carcinoma (HCC), bone cancer, pancreatic cancer, skin cancer, oral cancer, head or neck cancer, breast cancer, lung cancer, small cell lung cancer, NSCLC, cutaneous or uveal malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, head and neck squamous cell carcinoma (SCCHN), non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including asbestos-induced cancers), hematological malignancies (including, for example, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T lymphoblastic lymphoma), and any combination of the foregoing cancers. The present disclosure may also be applied to the treatment of metastatic cancers. In some aspects, the cancer is renal cell carcinoma (RCC), gastric / gastroesophageal junction cancer, non-small cell lung cancer (NSCLC), melanoma, head and neck squamous cell carcinoma (SCCHN), hepatocellular carcinoma, or urothelial carcinoma.

[0175] In one aspect, a human patient has a malignancy that is refractory to treatment with immune checkpoint inhibitors. In another aspect, the patient has a malignancy that is refractory to treatment with a PD-L1 inhibitor. In another aspect, the patient has a malignancy that is refractory to treatment with an anti-PD-L1 antibody.

[0176] Examples

[0177] Experimental Methods

[0178] Animal Studies

[0179] In vivo studies were performed using 8 - 10 - week - old BALB / cAnNCrl mice (Charles River UK) or C57BL / 6. The mice were housed at the AstraZeneca animal facility, had free access to food and water, and were cared for daily by trained personnel. The mice were acclimatized to the animal facility conditions for one week and were handled in accordance with the Home Office Animals Scientific Procedures Act, 1986, UK. All animal work was carried out under a project license (PPL P49077891) and PP3208003 (RTx study) approved by the Home Office and in accordance with institutional guidelines. 0.5e 6 CT26 (colorectal) tumor cells were implanted subcutaneously into BALB / c mice. 0.5e (Corning, catalog number 356231) and 0.5e 6 MCA205 (fibrosarcoma) tumor cells in PBS or 0.5e 6 MC38 cells in PBS were implanted subcutaneously into C57BL / 6 mice. Tumors were measured three times a week with calipers starting from day 5. Tumor - bearing mice were treated with oxaliplatin (Hospira, clinical grade, 5mg / mL) at a dose level of 6mg / Kg and 5 - fluorouracil (Hospira, clinical grade, 50mg / mL) at a dose level of 50mg / Kg or docetaxel (Accord, clinical grade, 20mg / mL) administered at 10mg / Kg, as well as a combination of olelutamab (clone 10.3, an anti - CD73 with murine IgG1 Fc sequence, 10mg / Kg or 20mg / Kg) and durvalumab (clone 80, a chimeric rat anti - mouse PD - L1 antibody with IgG1 Fc sequence, 10mg / Kg), a murine surrogate monoclonal antibody. The chemotherapy doses and regimens used were based on previously published information (Dosset M et al., Oncoimmunology 2018;7; Gao Q et al., JImmunotherCancer 2019;7:42). To clarify the contribution of components, comparator groups received single therapies and other combination iteration regimens. At appropriate time points, mice from these groups were sacrificed for pharmacodynamic analysis (MSI and transcriptomics) and immunohistochemistry (IHC) analysis. For radiotherapy experiments, a tumor was treated continuously daily with five fractions of 2Gy using an X - ray source standalone cabinet irradiator (Xstrahl CIX3).

[0180] In Vitro Assays

[0181] Cells (HCT-116, HT-29, CT-26, and MCA-205) were seeded at 1e 4 cells / well in a 96-well culture plate with 40 μl of complete medium and cultured in a 37°C / 5% CO2 incubator for approximately 4 hours to allow the cells to adhere. 10 μl / well of 5-fold the final concentration of olalizumab (5 nM) was added to the cells, and the cells were cultured overnight in a 37°C / 5% CO2 incubator for olalizumab pretreatment. According to the treatment groups, 50 μl / well of 2-fold the final concentration of serially diluted chemotherapeutic drugs (5-fluorouracil, oxaliplatin, docetaxel) was added to the cells, and the cells were cultured for 72 hours in a 37°C / 5% CO2 incubator. Cell viability was measured using luminescence assays (Promega, catalog number G7573).

[0182] RNA Preparation for Bulk Sequencing

[0183] Tumor tissues were snap-frozen in liquid nitrogen and stored at -80°C at the time of collection in the animal unit and shipped on dry ice to Novogene. Tissue processing and RNA extraction were performed by Novogene Co. 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. Then, after adding gDNA Eliminator solution and chloroform, the homogenate was separated into an aqueous phase and an organic phase by centrifugation. The upper aqueous phase containing RNA was collected, and the RNA was purified using an RNeasy spin column. RNA quality control (QC) was performed by 1% agarose gel electrophoresis, the quantity and purity were measured by Nanodrop, and the RNA integrity number (RIN) was obtained using a Bioanalyzer Agilent 2100.

[0184] Library preparation for sequencing was performed using the Ultra RNA Library Preparation Kit (catalog number E7530L). Samples were sequenced using Illumina PE150 (50 M reads / sample) bulk RNA-seq, and downstream bioinformatics analysis was performed.

[0185] RNASeq Data Analysis

[0186] Reads were mapped to the Mus musculus genome (mm10) using Star (Dobin A et al., Bioinformatics 2013; 29: 15-21). Unique mapped reads were counted using htseq-count (Putri GH et al., Bioinformatics 2022; 38: 2943-5). DESeq2 (Love MI et al., GenomeBiol 2014; 15) was used to normalize the counts with size factors and to identify differentially expressed genes in different conditions with a threshold of absolute (log2FC) > 1 and adjusted p-value < 0.05.

[0187] Gene set enrichment analysis was performed using the hallmark gene sets of the mouse MSigDB (Subramanian A. et al., Proc Natl Acad Sci USA 2005; 102: 15545-50) with the R package "fGSEA" (Korotkevich G et al., bioRxiv 2021; 060012). 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 adjusted p-value < 0.05. KEGG pathway enrichment analysis was performed using the R package clusterProfiler (Wu T. et al., The Innovation 2021; 2: 100141). The immune cell abundances of each sample and condition were estimated using the immune gene markers in MCPCounter with the MCP counter tool (Becht E. et al., Genome Biol 2016; 17: 1-20).

[0188] Tissue Preparation for Mass Spectrometry Imaging (MSI)

[0189] The tumor was snap-frozen in liquid nitrogen immediately after resection and the frozen tissue was embedded in HPMC / PVP hydrogel as described previously (Dannhorn A. et al., Anal Chem 2020; 92: 11080-8). Sections were made on a CM3050 S cryostat (Leica Biosystems, Nussloch, Germany) at a thickness of 10 μm. The tissue sections were immediately thaw-fixed, dried under a nitrogen stream, and sealed in a vacuum bag to maintain the metabolic integrity of the sections. Tissue sections for DESI-MSI and IMC were thaw-fixed on Superfrost microscope slides (VWR, catalog number 630-2863), while sections prepared for MALDI-MSI were thaw-fixed on conductive indium tin oxide (ITO)-coated slides (Bruker Daltonik, catalog number 8237001). Polyvinylpyrrolidone (PVP) and (hydroxypropyl) methylcellulose (HPMC) were purchased from Merck (catalog numbers PVP360; H8384). Methanol (catalog number 1 5624680), isopentane (catalog number 1 5692830), and isopropanol (catalog number 10674732) were obtained from Fisher Scientific.

[0190] Mass Spectrometry Imaging (MSI)

[0191] 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 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 set to 70 μm. A homemade Swagelok DESI sprayer was operated with a mixture of 95% methanol and 5% water, delivered at a flow rate of 2721.5 μL / min and nebulized with nitrogen at a backpressure of 6 bar. The resulting.raw files were converted to.mzML files using ProteoWizard msConvert (version 3.0.4043), and subsequently 274 compiled to generate.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).

[0192] MALDI-MSI analysis was performed on a RapifleX Tissuetyper instrument (Bruker Daltonik, Bremen, Germany) operating in negative ion detection mode. 9-Aminoacridine (9-AA) prepared in 80:20 methanol:water was used as the MALDI matrix, and spray deposition was carried out 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 accumulated for each 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 in the SCiLS Lab (version 2021b) software package. All DESI and MALDI data and images were normalized to the total ion current (TIC) to compensate for signal variations during the experiment. Data analysis performed in the SCiLS Lab software package included classifying the dataset based on histological guidance for manual annotation of MSI data to identify "tumor" and "necrosis", while removing the background (where applicable). Partial least squares discriminant analysis (PLS-DA) was used for tissue classification. The "necrosis margin" was delineated and distinguished from "viable tumor" by subjecting the "tumor" tissue cluster to pixel-by-pixel principal component analysis (PCA). The principal component (PC) loadings highlighting tissue compartments near the necrosis region were extracted and used to create an unsupervised segmentation peak list based on a binary K-means classifier. All data shown were extracted from the "viable tumor" cluster.

[0193] Imaging Mass Cytometry (IMC)

[0194] Imaging mass cytometry was performed on slides that had undergone DESI-MSI analysis. The antibodies used for IMC staining are shown in Table 1.

[0195]

[0196] According to the manufacturer's instructions, antibodies were purchased pre-conjugated with heavy metal tags or unlabeled antibodies were conjugated internally using the Fluidigm Maxpar Antibody Labeling Kit. The slides were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes. The slides were washed 3×5 minutes in PBS, permeabilized with Triton X-100 diluted 1:1000 in casein solution for 5 minutes, washed 3×5 minutes in PBS, and blocked with casein solution for 30 minutes. Antibodies were diluted to the appropriate concentration and the slides were incubated overnight at 4°C with the antibody solution. The slides were washed 3×5 minutes in PBS, stained with the DNA intercalator iridium diluted 1:400 in PBS for 30 minutes. The slides were washed 3×5 minutes in PBS, washed in deionized water for 30 seconds, and then dried at room temperature for storage until analysis. Regions for IMC analysis were selected using consecutive H&E-stained sections and MSI results. Regions approximately 1.5 mm × 1.5 mm to 2.0 mm × 2.0 mm were selected for analysis, including necrosis, the necrosis margin, and viable tumor regions. IMC analysis was performed using a Hyperion instrument (Fluidigm Corporation, San Francisco, CA, USA) with ablation energy of 4 db and ablation frequency of 100 Hz.

[0197] IMC data were analyzed using Halo v.3.3.2541.366 (Indica Laboratories, Albuquerque, NM, USA). Tissue regions were classified into viable tumor, necrosis, necrosis margin, and outside of tissue using random forest. Analysis was performed using HighplexFL v4.0.4. Cell segmentation and thresholds were optimized manually. Nuclear segmentation was performed using the 193Ir DNA intercalator channel. IMC images were adjusted and extracted using the built-in graph generation tool.

[0198] Immunohistochemistry

[0199] A portion of each tumor was immersion-fixed in 10% neutral-buffered formalin and then processed into paraffin using conventional methods. The tissue was sectioned at 4 mm thickness and immunohistochemically stained using a rabbit monoclonal antibody against CD73 (D7F9A, Cell Signaling Technology) diluted at 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).

[0200] The resulting slides were digitally scanned at 20x magnification using an Aperio scanner (Leica Biosystems). For analysis, the Halo image analysis software (Indica Labs) was used to measure the CD73-positive area and the hematoxylin-positive area. Briefly, the tumor areas were manually annotated, and the DAB and hematoxylin-positive areas of the annotated regions were measured using a Halo area quantification algorithm suitable for tissue staining characteristics. The percentages of the CD73-positive area and the hematoxylin-positive area within the total tumor area were obtained. The final data output used was the ratio of CD73 to the hematoxylin area, with the hematoxylin area considered a representative indicator of cellularity (since it mainly stains cell nuclei), normalizing the CD73-positive area to the overall cellular density of the tumor.

[0201] Statistical Analysis

[0202] All in vivo data were collated in an Excel spreadsheet and transferred to GraphPad Prism 9.00 (GraphPad Software Inc.) for graphical representation and statistical analysis. After outlier identification and the Shapiro-Wilk test for normal distribution, if the data were normally distributed, statistical significance was determined by one-way ANOVA, followed by Dunnett multiple comparisons for comparisons of more than two groups. If the data were not normally distributed, the Kruskal-Wallis test and Dunn multiple comparison test were used, as detailed in the figure legends. Survival studies were analyzed using the log-rank (Mantel-Cox) test, comparing only two survival curves at a time. P-values were not adjusted for multiple testing.

[0203] Example 1: Combined Anti-CD73, Anti-PD-L1 Antibody and 5FU+OHP Treatment in a Syngeneic Mouse Model Results in Enhanced Complete Response

[0204] In two murine syngeneic models of cancer - CT26 (p = 0.005) and MCA205 (p = 0.008) - combination treatment with anti-CD73 (aCD73) and anti-PD-L1 (aPD-L1) antibodies, except for 5-fluorouracil (5FU) + oxaliplatin (OHP), led to enhanced efficacy and complete responses, as Figure 1 B and Figure 1 C show (Kruskal-Wallis test). Treatment of CT26- or MCA205-bearing mice with aCD73 as a single therapy resulted in negligible benefits in terms of tumor growth inhibition. Anti-PD-L1 treatment delayed the tumor growth rate in some mice bearing CT26 tumors, but showed minimal activity in the MCA205 model ( Figure 2)。In both models, 5FU+OHP chemotherapy inhibits the tumor growth rate, but the combination of 5FU+OHP with immuno-oncology (IO) agents (anti-CD73 or anti-PD-L1) has minimal further improvement in efficacy. Treatment with the combination of anti-CD73 and anti-PD-L1 antibodies (aCD73+aPD-L1) provides a level of tumor growth control similar to that of anti-PD-L1 alone, highlighting the importance of the chemotherapy component in the chemotherapy+IO combination( Figure 1 and Figure 2 ).

[0205] Example 2: Effect of aCD73 on Chemotherapy-Induced Cytotoxicity in Cell Cultures

[0206] Meanwhile, to investigate whether the effects seen in vivo are driven by the tumor microenvironment (TME), it was determined whether anti-CD73 could enhance the direct cytotoxic effect of 5FU+OHP on the cell line itself. However, no evidence was found that this occurred in the in vitro environment( Figure 4 ). The cytotoxic effect of docetaxel on cultured CT26 cells was also investigated to see if it was affected by the presence of aCD73. No effect of the antibody was detected.

[0207] Example 3: CD8 Cell Depletion Impairs the Efficacy of the Quadruple (aCD73+ aPD-L1+5FU+OHP) Combination in MCA205 Tumor-Bearing Mice

[0208] To test the hypothesis that CD8 T cells play a role in the enhanced efficacy of the combination, starting on day 17 after tumor implantation, clone 53-6.7 was used for IP injection to selectively deplete CD8 T cells in the MCA205 model (schematic Figure 5 A). This experiment confirmed a significant reduction (>50%) in tumor control and long-term survival in the combination (5FU+OHP+aCD73+aPD-L1) treatment group (log-rank test, p = 0.026, Figure 5 C). These CD8 depletion data indicate that cell-mediated immunity plays an important role in the observed activity of the chemotherapy-IO combination. This does not exclude the contribution of other immune system components or the "conventional" cytotoxic or cytostatic effects mediated by 5FU+OHP.

[0209] 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 Figure 3

[0210] The pharmacodynamic activity of the combination of aCD73 and aPD-L1 plus 5FU+OHP chemotherapy was evaluated using immunohistochemistry (IHC) and imaging mass cytometry (IMC) to understand the early changes mediated by CD73 blockade. As expected, the murine surrogate antibody of oleclumab decreased CD73 levels in CT26 tumors. IHC-based CD73 detection uses an antibody that does not compete with the murine surrogate antibody of oleclumab; thus, the lower levels of CD73 protein are consistent with oleclumab's known ability to internalize CD73. IMC highlighted that CT26 tumors from aCD73-treated mice tended to have a lower frequency of cells expressing markers known to be associated with cancer-associated fibroblasts and inhibitory tumor-associated macrophages. Mass spectrometry imaging (MSI) revealed a significant inhibitory trend of adenosine, inosine, and xanthine in CT26 tumors from anti-CD73-treated mice ( Figure 5 and Example 5: Pharmacodynamic Effects of aCD73+aPD-L1+5FU+OHP on the CT26 Transcriptome ). In contrast, adenosine monophosphate (AMP) (the main substrate of CD73) in tumor tissues from mice treated with the combination of chemotherapy and anti-CD73 appeared to be elevated relative to other treatment groups. These pharmacodynamic data are consistent with the proposed mechanism of action of oleclumab; in terms of CD73 targeting, enzymatic inhibition, and adenosine pathway regulation.

[0211] Figure 11

[0212] To further understand the mechanistic basis for the enhanced activity of the quadruple combination, RNAseq was used to explore the changes within the CT26 transcriptome. Using the DESeq2 and fGSEA packages, minimal changes in the CT26 transcriptome were identified after treatment with aCD73 or aPD-L1 as single therapies (see, Table 2 below). On the other hand, 5FU+OHP was highly perturbing; causing significant upregulation of 277 genes and significant downregulation of an additional 158 genes relative to control tumors. Gene ontology (GOBP) base pair enrichment and KEGG pathway analysis of these data highlighted significant effects on genes associated with immune response, leukocytes, NK cell and T cell activation, T cell receptor signaling, and interferon (type 1 and type 2) production (adjusted p-value < 0.05). Individual genes upregulated in line with this included: CCL3, CCL4, CCL8, CCL17, CXCL10, GDF15, CD8a, IFNγ, perforin, granzyme, Lag-3, and PD-1. CXCL2 expression was decreased ( Figure 11 ). These RNAseq data highlight the significant perturbation of genes associated with immune function by 5FU+OHP treatment in the murine CT26 mouse model of cancer.

[0213] In contrast to the minor transcriptomic changes observed with monotherapy consisting of, the combination of aCD73 and aPD-L1 resulted in 1236 differentially expressed genes. The 5FU+OHP+aCD73+aPD-L1 combination achieved the most significant and extensive transcriptomic changes; compared to untreated tumors, it caused 1490 genes to be upregulated and 128 genes to be downregulated (Table 2).

[0214]

[0215]

[0216] KEGG pathway and gene ontology enrichment analyses highlighted chemotactic mobilization, 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 7A Among the genes upregulated by 5FU+OHP treatment, there were several significant increases, including (but not limited to): CD38, CD39, CXCL1, CXCL3, CXCL5, CD163, CTLA4, CXCR3, granzyme A, ICAM1, Il6, P2RY1, TNF-a, IL2a, Il1a, ALOX15, SLC7a2, Ear2, Havc2.

[0217] Including treatment groups representing the various components in the combination allowed for deconvolution of the contributions of the individual treatment components ( Figure 7B , Figure 11 and Figure 7A , Table 2). This analysis highlighted the key role of 5FU+OHP in driving the activation of interferon pathways (type 1 and type 2) and the activation of T cells and NK cells, cytotoxic activity, and IL2 / STAT5 pathway signaling. The effects of including 5FU+OHP were significantly different from those mediated by adding either IO drug component to the treatment, as demonstrated by the fact that the 5FU+OHP component uniquely upregulated 360 genes and downregulated 122 genes. Including 5FU+OHP in the combined aCD73+aPD-L1 treatment regimen served to increase the expression of key immune-related genes such as interferon gamma, TRIM6, CCL17, granzyme B, GDF15, perforin, and Lag3. Conversely, IL-10, IL-1b, CXCL2, and S100A8 were downregulated when 5FU+OHP was included.

[0218] Adding CD73 blockade to the combination of 5FU+OHP and aPD-L1 upregulated 510 genes and downregulated 8 genes that were not regulated in the other combination iterations, as Figure 7CAs shown. Genes significantly affected include CXCR3, H2-AB1, Itgae, CXCL3, Mgl2, CXCR5, CD4, and Cybb. This also drives even 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 the new and important role of adenosine pathway inhibition in chemotherapy / checkpoint inhibitor combinations. In particular, individual genes and markers related to myeloid cell and B cell biology. When chemotherapy plus anti-PD-L1 group is enhanced with anti-CD73, two genes, CD38 and P2Y1, known to be related to the adenosine pathway itself, are also significantly upregulated. Removal of aPD-L1 from the 5FU+OHP plus aCD73 combination treatment is detrimental in upregulating genes related to inflammation, immune response, and interferon γ pathway activation. Notably, ALOX15 (a gene related to macrophage function and efferocytosis) and IL-1b are affected.

[0219] Next, we examined whether gene perturbation of this magnitude also leads to changes in cell population counts. For this purpose, we used the MCP counter tool to estimate the abundance of immune cell populations in treated CT26 tumors (Becht E et al., Genome Biol 2016;17:1-20). This computational analysis highlights the significant role of oxaliplatin and 5-fluorouracil chemotherapy in increasing lymphocyte representation; consistent with the significantly elevated levels of pro-inflammatory and chemotactic chemokine / cytokine gene expression after this treatment ( Example 6: Combined aCD73, aPD-L1, and Docetaxel Treatment in a CT26 Syngeneic Mouse Model Results in Enhanced ). Lymphocyte infiltration in CT26 tumors from mice treated with single therapies and combinations that did not include the chemotherapy component was significantly less. The 5FU+OHP+aCD73+aPD-L1 treatment was significantly prominent due to increased tumor abundance of both lymphocyte (cytotoxic T cells, NK cells, B cells) and myeloid cell populations (including monocyte dendritic cells); a feature that contributes to improved tumor control and increased overall survival time in cancer models (Petitprez F et al., Nature2020577:7791; Voss MH et al., JCO20203815_suppl50252020;38:5025-5025; Chambers AM et al., Front Immunol 2018;9:2533: Mastelic-Gavillet B et al., JImmunotherCancer 2019;7:1-16).

[0220] Complete Response Figure 8

[0221] Importantly, the synergistic combination of 5FU+OHP was found to extend to a second chemotherapy class; namely docetaxel (DTX). The combination approach was superimposed onto an established tolerable dosing regimen for this chemotherapy. In this context, the combination of DTX, aPD-L1, and aCD73 resulted in significantly improved tumor growth inhibition, and compared to a maximum of 3 out of 12 (25%) (p = 0.001) in the aPD-L1 plus docetaxel combination group, 7 out of 12 (58%) had a complete response (p = 0.0001), Kruskal-Wallis test, as Example 7: Enhanced Fractionated Radiotherapy by Combined aCD73, aPD-L1 in an MC38 Syngeneic Mouse Model shown.

[0222] Figure 9

[0223] Previously published preclinical data have highlighted the role of CD73 in cancer radiotherapy response (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). It was explored whether adding aCD73 + aPD-L1 treatment would also enhance radiotherapy response in line with the chemotherapy data. The MC38 model of colorectal cancer was used to test the effect of an established fractionated radiotherapy regimen, and in this experiment, a simultaneous approach was adopted, i.e., all treatments were started on the same day (schematic Figure 9 A). Once the tumor reached 70 mm 3 -120 mm 3 , the tumors were included for intervention. The data confirmed the effective effect of RTx + aCD73 + aPD-L1 treatment in MC38 tumor-bearing mice (p = 0.0001), Kruskal-Wallis test, as Example 8: Optimal Scheduling of aCD73, aPD-L1 in an MC38 Syngeneic Mouse Model shown in B.

[0224] Figure 12

[0225] The effect of aCD73, aPD-L1, and radiotherapy timing was also determined using the MC38 syngeneic mouse model. 5×10 5 cells were implanted into six groups of mice, as Figure 1 shown. As Figure 14 shown in 3, mice treated simultaneously with aCD73, aPD-L1, and RTx showed the highest level of tumor inhibition and subsequent survival probability. This was also associated with the induction of a protective memory response after rechallenge using the B16F10 and MC38 mouse models ( Figure 1 ).

[0226] To determine whether the timing of administration of single aCD73, aPD-L1, and radiotherapy has an impact on tumor volume, MC38 cells were implanted as previously described, and the timing of single therapies was plotted as shown in Figure 1 Figure 5. Interestingly, administration of aCD73 therapy prior to aPD-L1 and RTx showed the greatest reduction in tumor volume, which was associated with the highest survival probability ( Figure 7A Figure 6).

[0227] Increasing evidence supports the immunosuppressive role of extracellular adenosine within the tumor microenvironment; adenosine-related gene signatures have been associated with poor outcomes and reduced responses to T cell checkpoint inhibitor drugs in several indications (Sidders et al.; and Allard D et al., Immunol Lett 2019; 205: 31–9). Molecules targeting extracellular nodes in the adenosine production pathway have advanced in the clinical development arena, and currently, the combination of oleclumab and durvalumab has entered phase 3 clinical development in patients with stage 3 NSCLC (non-small cell lung cancer) previously treated with chemoradiotherapy (Global Study to Evaluate the Impact of Durvalumab With Oleclumab or Durvalumab With Monalizumab After Concurrent Chemoradiotherapy in Patients With Stage III Unresectable Non-Small Cell Lung Cancer - Full View - ClinicalTrials.gov).

[0228] Although it is generally believed that CD73 inhibition in combination with cytotoxic therapies that promote extracellular ATP release via cell death is beneficial, there is a lack of published preclinical data to support this. The data presented herein explore the synergy of CD73 inhibition with chemotherapy and PD-L1 inhibition, highlighting additive effects and novel biological effects mediated by including CD73 blockade. To this end, the combination of aCD73 + aPD-L1 + 5FU + OHP provided enhanced efficacy in two murine models of cancer (colorectal and sarcoma). Judging from the effect of the CD8 depletion antibody in the MCA205 model, the activity of the combination therapy depends on CD8 T cells. These data infer an important contribution of the cell-mediated arm of the murine immune system in the resulting anti-tumor effect. Consistent with this, RNAseq analysis confirmed that aCD73 + aPD-L1 + 5FU + OHP drives an increase in the tumor abundance of cytotoxic lymphocytes and other key immune cells such as myeloid dendritic cells and B cells. The replacement of OHP + 5FU with a taxane backbone (i.e., DTX) was also explored and a similar enhanced efficacy profile was confirmed in the CT26 model. These data support the complementarity of oleclumab and an aPD-L1 antibody with platinum- and taxane-based chemotherapy backbones. The data also highlight the additive effect of radiotherapy in the MC38 model; extending the findings of Wennerberg et al. (Cancer Immunol Res 2020;8:465-78) to a colorectal model and enhancing the intervention strategy to inhibit the PD-1 / PD-L1 axis.

[0229] Mechanistically, the aCD73 antibody rapidly reduces the expression of CD73 within CT26 tumors and modulates extracellular adenosine levels in a manner consistent with its proposed mechanism of action. However, imaging mass cytometry of the same samples did obtain 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 the phenotypes of tumor macrophages and fibroblasts (Magagna I et al., Cancers (Basel) 2021;13.; Yu M et al., Nat Commun 2020;11).

[0230] The single-agent 5FU+OHP treatment delayed tumor growth in a subset of drug recipients bearing CT26 / MCA205 tumors. A notable finding of the current study was the breadth of immune pathway gene regulation following 5FU+OHP treatment of CT26 tumors. These included type 1 and type 2 interferons, gene signatures associated with cytotoxic lymphocytes, and effector molecules and 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 the findings regarding in vivo effects (Dosset M et al., Oncoimmunology 2018;7). In particular, the data presented herein have identified that 5FU+OHP drives the type I interferon pathway, which is known to have broad effects on immune and cancer cells within the tumor microenvironment (Zitvogel L et al., Nat Rev Immunol 2015;15:405-14). Specific genes regulated by the single-agent 5FU+OHP treatment included IFN-γ, CCL3, CCL8, Lag-3, and granzyme B (upregulated), as well as downregulation of CXCL2, IL-1b, CD103, and XCR1. The 5FU+OHP treatment elevated the expression of the ARORA2 gene, although the ARORA2 upregulation was offset when aPD-L1 or aCD73 Mab was combined with the 5FU+OHP treatment. Gene signatures associated with pro-inflammation, STAT5 pathway activation, and chemotaxis were only significantly upregulated following co-application of CD73 blockade with the OHP+5FU treatment (Table 2). GDF-15 is an emerging pleiotropic cytokine of interest in cancer (Wischhusen J et al., Front Immunol 2020;11), and the GDF15 transcript levels in CT26 tumors were significantly elevated by treatment containing 5FU+OHP; reflecting findings from other mouse models and human cancer patients regarding platinum therapy (Breen DM et al., Cell Metab 2020;32:938-950.e6).

[0231] Compared to tumor RNAseq data obtained from mice treated with a single IO agent (e.g., as monotherapy), the combination of aCD73 and aPD-L1 resulted in 1236 differentially expressed genes; highlighting broader transcriptomic changes associated with antibody-mediated targeting of multiple inhibitory checkpoints within the tumor microenvironment. IO "doublets" are known for activating pathways and gene families associated with 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 very different from those affected by 5FU + OHP treatment; and have the potential for complementarity if superimposed in a combination treatment modality. Notably, the "doublet" IO combination (aCD73 + aPD-L1) provides lower efficiency activation of type 1 interferon pathway genes; known to be associated with favorable disease outcomes in patients with multiple cancers and mediating a range of beneficial immunomodulatory effects within the tumor microenvironment (Zitvogel L et al., Nat Rev Immunol 2015;15:405-14).

[0232] Despite the detected transcriptomic changes, it is clear that the "pure" small / large molecule approaches (5FU+OHP chemotherapy or IO combinations) failed to achieve the same efficacy levels provided by the combination of 5FU+OHP with aCD73 and aPD-L1. Therefore, in-depth understanding of the specific transcriptomic differences that may explain this would be informative, as these genes and these markers may be useful beyond the context of adenosine pathway regulation. Intriguingly, the addition of aCD73 to 5FU+OHP+aPD-L1 significantly upregulated CXCR3 in the CT26 tumor microenvironment. CXCR3 is the cognate receptor for the IFN-induced chemokines CXCL9-11 on activated T cells; its upregulation was consistent with increased T cell abundance and interferon-activated chemokine expression in CT26 tumors of mice receiving 5FU+OHP or combinations containing this chemotherapy. 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., JImmunother Cancer 2021;9; Qu Y et al., Cell Rep2020;32; Chow MT et al., Immunity 2019;50:1498-1512.e5). Elevated Pdcd1 (PD-1) expression inferred that tumor T cells in those CT26 tumor-bearing mice may be activated and / or exhausted, and strongly supported the inclusion of aPD-L1 to counteract adaptive immune resistance against antitumor effects. Notably, the combination containing aPD-L1 and IO significantly upregulated 15-lipoxygenase (15-LOX), which is involved in various macrophage functions, including efferocytosis and ferroptosis. Interestingly, Snodgrass et al. (FrontImmunol2018;9) identified a new role of ALOX15 in CCL17 production in human macrophages; it is important to note that CCL17 expression was significantly increased in the most protective form of treatment.

[0233] Regulation of genes associated with dendritic cell biology and antigen presentation (MHC II, Itgae, Itgax, DCstamp, TARM1, CD301) is of particular interest and is generally consistent with other work exploring CD73 inhibition in the context of radiotherapy4 and adenosine pathway blockade. Wennerberg et al. (Cancer Immunol Res. 2020) have highlighted the key role of radiotherapy-induced type I interferons in remodeling the tumor microenvironment, particularly cDC1. The same group has emphasized the complementarity of CD73 blockade and radiotherapy, with aCD73 treatment playing a key role in tumors with suboptimal radiotherapy-induced type I interferon induction. The data presented herein appear to be generally consistent with their findings. Consistent with this, genes for MHCII molecules expressed on DCs and macrophage galactose C-type lectin (MGL / CD301) are upregulated within the CT26 TME. Due to the monosaccharide specificity of CD301 for Gal and N-acetylgalactosamine30, CD301 is thought to be involved in the recognition of molecules from altered self and pathogens. T cell interaction activating receptor on myeloid cells-1 (TARM1; gene symbol Tarm1) is a member of the LILR family encoded within the recently identified leukocyte receptor complex. TARM1 is expressed by DCs and is required for DC activation; after induction of CIA in Tarml+ / - mice, Tarm1 is highly expressed in inflammatory-type (I-A / I-E+Ly6C+CD11b+CD11c+) DCs of the draining LN (dLN)31. Another novel finding is the unique ability of combinations containing aCD73 to 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;43:546-63). Emerging evidence suggests an immunomodulatory role for extracellular RNase molecules, thus acting as alarmin (Lu L et al., Front Immunol 2018;9:1012). Similarly, combination therapy significantly increases the expression of tumor RNase 2a. The effect on NOX2 (gene Cybb) is also significant and potentially notable, considering its fundamental role in conferring the ability of macrophages to respond to extracellular ATP stimulation and strong cellular oxidative changes (Moore SF et al., Journal of Immunology 2009;183:3302-8), as well as its role in regulating ATM kinase activation and radiotherapy efficacy in macrophages (Wu Q et al., Cell Death Differ 2017;24:1632-44).Macrophage galectin-like oxidized LDL receptor-1 (LOX-I / OLR1) is also upregulated in CT26 tumors from mice treated with 5FU+OHP+aCD73+aPD-L1. This receptor is known to sense heat shock proteins and is significantly upregulated by TLR agonists and other pro-inflammatory stimuli.

[0234] The effect on B cell phenotypes within CT26 tumors is also worthy of comment ( Figure 7C and Figure 11 ). When aCD73 is included in the 5FU+OHP+aPD-L1 combination, B cell-related genes such as JChain, CXCR5, and CXCL13 are significantly regulated ( Sequences ). Although the impact of B cell biology in cancer is less well understood than that of CD8, there is increasing 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). Murine B cell-related data is also consistent with recent publications on the direct effects of CD73 inhibitory 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 well consistent with the enhanced survival and tumor growth control characteristics in animals administered 5FU+OHP+aCD73+aPD-L1.

[0235] ​

[0236] SEQ ID NO: 1 Heavy chain CDR1 amino acid sequence; anti-PD-L1 antibody durvalumab

[0237] GFTFSRYWMS

[0238] SEQ ID NO: 2 Heavy chain CDR2 amino acid sequence; anti-PD-L1 antibody durvalumab

[0239] NIKQDG SEKYYVD SVKG

[0240] SEQ ID NO: 3 Heavy chain CDR3 amino acid sequence; anti-PD-L1 antibody durvalumab

[0241] EGGWFGELAFDY

[0242] SEQ ID NO: 4 Amino Acid Sequence of Light Chain CDR1; Avelumab, an Anti-PD-L1 Antibody

[0243] RASQRVS S SYLA

[0244] SEQ ID NO: 5 Amino Acid Sequence of Light Chain CDR2; Avelumab, an Anti-PD-L1 Antibody

[0245] DASSRAT

[0246] SEQ ID NO: 6 Amino Acid Sequence of Light Chain CDR3; Avelumab, an Anti-PD-L1 Antibody

[0247] QQYGSLPWT

[0248] SEQ ID NO: 7 Amino Acid Sequence of Heavy Chain Variable Domain (VH); Avelumab, an Anti-PD-L1 Antibody

[0249] EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQM[NSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVS S

[0250] SEQ ID NO: 8 Amino Acid Sequence of Light Chain Variable Domain (VL); Avelumab, an Anti-PD-L1 Antibody

[0251] EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK

[0252] SEQ ID NO: 9 Amino Acid Sequence of Heavy Chain CDR1; Olalizumab, an Anti-CD73 Antibody

[0253] SYAYS

[0254] SEQ ID NO: 10 Amino Acid Sequence of Heavy Chain CDR2; Olalizumab, an Anti-CD73 Antibody

[0255] AISGSGGRTYYADSVKG

[0256] SEQ ID NO: 11 Amino Acid Sequence of Heavy Chain CDR3; Olalizumab, an Anti-CD73 Antibody

[0257] LGYGRVDE

[0258] SEQ ID NO: 12 Amino acid sequence of light chain CDR1; Oleclumab, an anti-CD73 antibody

[0259] SGSLSNIGRNPVN

[0260] SEQ ID NO: 13 Amino acid sequence of light chain CDR2; Oleclumab, an anti-CD73 antibody

[0261] LDNLRLS

[0262] SEQ ID NO: 14 Amino acid sequence of light chain CDR3; Oleclumab, an anti-CD73 antibody

[0263] ATWDD SHPGWT

[0264] SEQ ID NO: 15 Amino acid sequence of heavy chain variable domain (VH); Oleclumab, an anti-CD73 antibody

[0265] EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAYSWVRQAPGKGLEWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLYLQM[NSLRAEDTAVYYCARLGYGRVDEWGRGTLVTVS S

[0266] SEQ ID NO: 16 Amino acid sequence of light chain variable domain (VL); Oleclumab, an anti-CD73 antibody

[0267] QSVLTQPPSASGTPGQRVTISCSGSLSNIGRNPVNWYQQLPGTAPKLLIYLDNLRLSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDSHPGWTFGGGTKLTVL

[0268] SEQ ID NO: 17 Amino acid sequence of heavy chain; Oleclumab, an anti-CD73 antibody

[0269] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAYSWVRQAPGKGLEWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLYLQM[NSLRAEDTAVYYCARLGYGRVDEWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0270] SEQ ID NO: 18 Light chain amino acid sequence; Olaratumab, an anti-CD73 antibody

[0271] QSVLTQPPSASGTPGQRVTISCSGSLSNIGRNPVNWYQQLPGTAPKLLIYLDNLRLSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDSHPGWTFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Claims

1. 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 a reduced level of CD73 protein or CD73 activity compared to a normal subject.

2. 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 a reduced level of CD73 protein or CD73 activity compared to a normal subject.

3. 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 a reduced level 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 a therapeutically effective amount 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 a therapeutically effective amount of a CD73 inhibitor, a PD-L1 inhibitor, and chemotherapy and / or radiation therapy.

6. 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 CD73 inhibitor, a PD-L1 inhibitor, and chemotherapy and / or radiation therapy.

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

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

9. The method according to any one of claims 4 to 6, wherein the CD73 inhibitor is administered before the PD-L1 inhibitor and the chemotherapy and / or radiotherapy.

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

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

6.

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

8.

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

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

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

14.

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

16.

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

18.

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

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

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

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

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