Biomarkers and methods for treating NSCLC

Abstract

Biomarkers and methods for the treatment of non-small cell lung cancer (NSCLC). Biomarkers related to patient selection and prognosis, and diagnostic kits for analyzing the above biomarkers. In some embodiments of the methods of the present application, the CD73 antagonist of the above combination treatment is a CD73 antibody. In some embodiments, the CD73 antibody is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.

Description

Technical Field

[0001] Field This application relates to biomarkers and methods for the treatment of non-small cell lung cancer (NSCLC). In particular, biomarkers for patient selection and prognosis, as well as diagnostic kits for analyzing the above biomarkers, are provided.

Background Art

[0002] Background Non-small cell lung cancer (NSCLC) is any type of epithelial lung cancer other than small cell lung cancer (SCLC). It accounts for approximately 85% of all lung cancers. Compared to small cell carcinoma, NSCLC is relatively less sensitive to chemotherapy. The survival rates of stages I-IV NSCLC decrease significantly due to the progression of the disease. For stage I, the 5-year survival rate is 47%, for stage II it is 30%, for stage III it is 10%, and for stage IV it is only 1%.

[0003] Treatment approaches for people with advanced NSCLC aim first to reduce pain and suffering, either through chemotherapy (e.g., cisplatin which targets all rapidly dividing cells indiscriminately), and through more targeted agents that are more regulated against specific genetic abnormalities found within the tumor. However, there is a need for improved treatment approaches.

Summary of the Invention

Means for Solving the Problems

[0004] Summary This application addresses clinical needs.

[0005] In one aspect, a method for treating a subject having NSCLC, the method comprising: (a) determining the level of CD73 in a sample of the subject; (b) evaluating whether the subject is more likely to respond to or benefit from a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73; and (c) administering the combination treatment to the subject if the subject is more likely to respond to or benefit from the combination treatment. The method is provided herein.

[0006] In another aspect, a method for selecting a treatment for a subject having NSCLC, the method comprising: (a) determining the level of CD73 in a sample of the subject; (b) evaluating whether the subject is more likely to respond to or benefit from a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73; and (c) selecting the combination treatment for the subject if the subject is more likely to respond to or benefit from the combination treatment. The method is provided herein.

[0007] In another aspect, a method for identifying that a subject having NSCLC is more likely to respond to or benefit from a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist, the method comprising: (a) determining the level of CD73 in a sample of the subject; (b) evaluating whether the subject is more likely to respond to or benefit from the combination treatment based on the level of CD73; and (c) providing a recommendation for the combination treatment to the subject if the subject is more likely to respond to or benefit from the combination treatment. The method is provided herein.

[0008] A method for predicting the responsiveness of a subject with NSCLC to a combined treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist, the method comprising: (a) determining the level of CD73 in a sample of the subject; (b) evaluating whether the subject is more likely to respond to or benefit from the combined treatment based on the level of CD73; and (c) providing a recommendation for the combined treatment to the subject if the subject is more likely to respond to or benefit from the combined treatment. The method is provided herein.

[0009] In another aspect, a method for identifying that a subject with NSCLC is more likely to respond to or benefit from a combined treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist, the method comprising: (a) determining the level of CD73 in a sample of the subject, wherein an increase in CD73 in the sample relative to that in a reference sample indicates that the subject is more likely to respond to or benefit from the combined treatment; and (b) providing a recommendation that the subject is more likely to respond to or benefit from the combined treatment. The method is provided herein.

[0010] In another aspect, a method for predicting the responsiveness of a subject with NSCLC to a combined treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist, the method comprising: (a) determining the level of CD73 in a sample of the subject, wherein an increase in CD73 in the sample relative to that in a reference sample indicates that the subject is more likely to respond to or benefit from the combined treatment; and (b) providing a recommendation that the subject has an increased likelihood of responding to or benefiting from the combined treatment. The method is provided herein.

[0011] In another aspect, a method for treating a subject having non-small cell lung cancer (NSCLC), the method comprising: (a) identifying an NSCLC subject as likely to respond to or benefit from a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73 in a sample of the subject; and (b) administering the CD73 antagonist and the PD1 / PD-L1 antagonist to the identified subject, is provided herein.

[0012] In another aspect, a method for selecting a treatment for a subject having NSCLC, the method comprising: (a) determining the level of CD73 in a sample of the subject; (b) evaluating whether the subject is likely to respond to or benefit from a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73; and (c) selecting the combination treatment for the subject if it is determined that the subject is likely to respond to or benefit from the combination treatment, is provided herein.

[0013] In another aspect, a method for treating a subject having NSCLC, the method comprising administering a CD73 antagonist and a PD1 / PD-L1 antagonist to a subject having NSCLC and having a level of CD73 in a sample indicative of being likely to respond to or benefit from a combination treatment of the CD73 antagonist and the PD1 / PD-L1 antagonist, is provided herein.

[0014] In another aspect, provided herein is a method for predicting the responsiveness of a subject having NSCLC to a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist, the method comprising: (a) determining the level of CD73 in a sample of the subject; (b) evaluating, based on the level of CD73, whether the subject is likely to respond to or benefit from the combination treatment; and (c) providing a recommendation for the combination treatment to the subject if it is determined that the subject is likely to respond to or benefit from the combination treatment.

[0015] In another aspect, provided herein is a method for identifying a subject having NSCLC as being more likely to respond to or benefit from a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist than a reference patient, the method comprising: (a) determining the level of CD73 in a sample of the subject, wherein an increase in the level of CD73 in the sample relative to that in a reference sample from the reference patient indicates that the subject is more likely to respond to or benefit from the combination treatment than the reference patient; and (b) providing a recommendation that the subject is more likely to respond to or benefit from the combination treatment than the reference patient.

[0016] In another aspect, a method for predicting the responsiveness of a subject with NSCLC to a combined treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist, the method comprising: (a) determining the level of CD73 in a sample of the subject, wherein an increase in the level of CD73 in the sample relative to that in a reference sample derived from a reference patient indicates that the subject is more likely to respond to or benefit from the combined treatment than the reference patient; and (b) providing a recommendation that the subject has an increased likelihood of responding to or benefiting from the combined treatment as compared to the reference patient. The method is provided herein.

[0017] In another aspect, the use of a CD73 antagonist and a PD1 / PD-L1 antagonist for the manufacture of a medicament for treating a subject with NSCLC having a level of CD73 in a sample indicative of being likely to respond to or benefit from the combined treatment of the CD73 antagonist and the PD1 / PD-L1 antagonist is provided herein.

[0018] In another aspect, the use of a CD73 antagonist for the manufacture of a medicament for treating a subject with NSCLC having a level of CD73 in a sample indicative of being likely to respond to or benefit from the combined treatment of the CD73 antagonist and the PD1 / PD-L1 antagonist, wherein the subject is also administered the PD1 / PD-L1 antagonist, is provided herein.

[0019] In another aspect, the use of a PD1 / PD-L1 antagonist for the manufacture of a medicament for treating a subject with NSCLC having a level of CD73 in a sample indicative of being likely to respond to or benefit from the combined treatment of the CD73 antagonist and the PD1 / PD-L1 antagonist, wherein the subject is also administered the CD73 antagonist, is provided herein.

[0020] In some embodiments of the method of the present application, the CD73 antagonist of the above combination treatment is a CD73 antibody. In some embodiments, the CD73 antibody is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, Fab, scFv, diabody, triabody, minibody, VHH and sdAb. In some embodiments, the CD73 antibody comprises (a) HCDR1, HCDR2, and HCDR3 within a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 1, and (b) LCDR1, LCDR2, and LCDR3 within a light chain variable region (VL) having the sequence shown in SEQ ID NO: 2, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 follow the Kabat numbering scheme.

[0021] In some embodiments, the CD73 antibody comprises (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, (2) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, (3) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, (4) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, (5) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and (6) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 8.

[0022] In some embodiments, the CD73 antibody is selected from the group consisting of uliledlimab (I-Mab Biopharma), oleclumab (AstraZeneca), CPI-006 (Corvus Pharma), BMS-986179 (Bristol-Myers Squibb), AB-680 (Arcus Biosciences), NZV-930 (SRF373, Surface Oncolohgy / Novartis), JAB-BX102 (Jacobio), AK119 (Akesobio), Sym024 (Symphogen), IBI 325 (Innovent), BR 101 (Hisun BioRay), and LY3475070 (Eli-Lilly).

[0023] In some embodiments, the PD-1 / PD-L1 antagonist of the above combination treatment is a PD-1 antibody or a PD-L1 antibody. In some embodiments, the PD-1 antibody is pembrolizumab, nivolumab, toripalimab, pidilizumab, semaprimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, dostarlimab, ABBV-181, pemprimumab, genolimzumab, retifanlimab, sasanlimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01. In some embodiments, the PD-L1 antibody is atezolizumab, manelimab, avelumab, cosibelimab, durvalumab, enoblituzumab, socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12, and KY-1003.

[0024] In some embodiments, the combination treatment further includes chemotherapy. In some embodiments, the chemotherapy is selected from the group consisting of platinum agents (e.g., cisplatin, carboplatin), taxane agents (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vincristine, etoposide, pemetrexed, and gemcitabine, and any combination thereof.

[0025] In some embodiments, the combination treatment includes administering the CD73 antagonist and the PD-1 / PD-L1 antagonist simultaneously or separately.

[0026] In some embodiments, the NSCLC is stage III or stage IV NSCLC. In some embodiments, the NSCLC is advanced NSCLC or metastatic NSCLC. In some embodiments, the NSCLC is recurrent NSCLC. In some embodiments, the NSCLC has a squamous histology or a non-squamous histology.

[0027] In some embodiments, the subject is treatment-naive. In some embodiments, the treatment is a first-choice treatment. In some embodiments, the subject is ineligible or refused for the first-choice treatment. In some embodiments, the first-choice treatment is selected from the group consisting of chemotherapy, PD-1 antibody treatment, PD-L1 antibody treatment, and any combination thereof. In some embodiments, the chemotherapy is selected from the group consisting of platinum agents (e.g., cisplatin, carboplatin), taxane agents (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vincristine, etoposide, pemetrexed, and gemcitabine, and any combination thereof. In some embodiments, the PD-1 antibody is pembrolizumab, nivolumab, toripalimab, pidilizumab, semaprilumab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, dostarlimab, ABBV-181, pemprilumab, genolimzumab, retifanlimab, sasanlimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01 Selected from the group consisting of. In some embodiments, the PD-L1 antibody is atezolizumab, manelumab, avelumab, cosibelimab, durvalumab, enobafolimab, socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12 and KY-1003.

[0028] In some embodiments, the level of CD73 is the protein level of CD73. In some embodiments, the protein level is determined by immunohistochemistry (IHC).

[0029] In some embodiments, the sample includes tumor cells, immune cells such as tumor infiltrating immune cells (TIL), stromal cells, and any combination thereof. In some embodiments, the sample is obtained prior to the combination treatment.

[0030] In some embodiments, the level of CD73 includes the proportion (e.g., percentage) of CD73-positive cells among all cells in the sample. In some embodiments, the level of CD73 includes the higher of the proportion of CD73-positive tumor cells (TC) among all cells in the sample or the proportion of CD73-positive immune cells (IC) among all cells in the sample (TC / IC). In some embodiments, a CD73 level (TC / IC) of 30% or higher is identified as a subject being likely to respond to or benefit from the combination treatment. In some embodiments, a CD73 level (TC / IC) of 35%, 40%, 45% or 50% or higher is identified as a subject being likely to respond to or benefit from the combination treatment.

[0031] In some embodiments, the level of CD73 comprises the higher of the percentage of CD73-positive tumor cells (TC(+)) among all cells in the sample or the percentage of CD73-positive immune cells (IC(+)) among all cells in the sample, where the positive is at least moderately positive (TC / IC(+)).

[0032] In some embodiments, a CD73 level of 10% or higher (TC / IC(+)) is identified as potentially having the subject respond to or benefit from combination treatment. In some embodiments, a CD73 level of 15%, 20%, 25%, 30%, 35% or 40% or higher (TC / IC(+)) is identified as potentially having the subject respond to or benefit from combination treatment.

[0033] In some embodiments, the reference sample comprises a referenced tissue or reference cells. In some embodiments, the reference sample is from a healthy subject or a non-diseased sample of the subject.

[0034] In some embodiments, the responsiveness includes a relative increase in one or more of the following: overall survival (OS), progression-free survival (PFS), complete response (CR), partial response (PR), and combinations thereof.

[0035] In another aspect, a kit or article of manufacture for use in the methods of the present application is provided herein, the kit or article of manufacture comprising (1) one or more reagents for determining the level of CD73 in a sample from a subject having NSCLC, and (2) a package insert, wherein the package insert comprises a package insert suggesting treating the subject with a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73.

[0036] In another aspect, a kit or an article of manufacture for use in the methods of the present application, wherein the kit or article of manufacture comprises: (1) one or more reagents for determining the level of CD73 in a sample derived from a subject having NSCLC, and (2) a package insert, wherein the package insert suggests that an increase in the level of CD73 in the sample, as compared to that in a reference sample, indicates that the subject is more likely to respond to or benefit from combination treatment with a CD73 antagonist and a PD1 / PD-L1 antagonist. Kits or articles of manufacture comprising the package insert are provided herein.

[0037] In some embodiments of the kit or the article of manufacture, the reagent is an anti-CD73 antibody. In some embodiments, the antibody is selected from the group consisting of D7F9A (CST, catalog number 13160), 606117 (R&D), EPR6114 (Abcam, catalog number ab133582), 4G6E3 (Abcam, catalog number ab202122), NT5E / 2503 (Abcam, catalog number ab257309), NT5E / 2505 (Abcam, catalog number ab257310), NT5E / 2545 (Abcam, catalog number ab257311), NT5E / 2646 (Abcam, catalog number ab257312), 7G2 (Thermo Fisher, catalog number 1D7), JM11-40 (Thermo Fisher, catalog number JM11-40), CD73 recombinant rabbit monoclonal antibody (1) (Invitrogen, catalog number MA5-29454), BLR054F (Thermo Fisher, catalog number BLR054F), sc-32299 (Santa Cruz, catalog number IE9), and AD2 (Biorad, catalog number AD2).

[0038] It should be understood that one, several, or all of the characteristics of the various embodiments described herein can be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following detailed description.

Brief Description of the Drawings

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Figure 6

Modes for Carrying Out the Invention

[0045] Detailed Description Definitions Before describing the embodiments in detail, it should be understood that the present disclosure is not limited to specific compositions or biological systems, and that these can of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0046] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a molecule” includes, as appropriate, combinations of two or more such molecules.

[0047] The term “about,” as used herein, refers to the ordinary error range for each value readily known to those of ordinary skill in the art. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter itself.

[0048] It is understood that aspects and embodiments of the present disclosure include aspects and embodiments “comprising,” “consisting of,” and “consisting essentially of.”

[0049] As used herein, the term "antibody" is used in its broadest sense and specifically includes intact antibodies (e.g., full-length antibodies), antibody fragments (including, but not limited to, Fab, F(ab')2, scFv, scFv-Fc, single domain antibodies, single heavy chain antibodies, and single light chain antibodies), monoclonal antibodies, and polyclonal antibodies, as long as they exhibit the desired biological activity (e.g., epitope binding).

[0050] As used herein, the term "isolated" antibody may refer to an antibody that is substantially free of other cellular material. In one embodiment, an isolated antibody is substantially free of other proteins from the same species. In another embodiment, an isolated antibody is expressed by cells from a different species and is substantially free of other proteins from said different species. In some embodiments, an "isolated" antibody has been identified and separated and / or recovered from components of its natural environment. Components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Antibodies can be rendered substantially free of associated components (or components associated with the cell expression system used to produce the antibody) by isolation using protein purification techniques well known in the art. In some embodiments, the antibody is (1) greater than 75% by weight, and most preferably greater than 80%, 90%, 95% or 99% by weight, as determined by the Lowry method, of antibody, or (2) homogeneous as determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue staining or preferably silver staining. Isolated antibodies include antibodies in insitu in recombinant cells, since at least one component of the natural environment of the antibody is absent. However, usually, isolated antibodies are prepared by at least one purification step.

[0051] As used herein, the term "native antibodies and immunoglobulins" refers to glycoproteins of approximately 150,000 daltons, usually heterotetramers, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond (also referred to as a "VH / VL pair"), although the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide cross-links. Each heavy chain has at one end a variable domain (VH), followed by several constant domains. Each light chain has at one end a variable domain (VL) and at its other end a constant domain; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the heavy chain variable domain. Certain amino acid residues are thought to form the interface between the light chain variable domain and the heavy chain variable domain. See, e.g., Chothia et al., J. Mol. Biol., 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A., 82:4592 (1985).

[0052] As used herein, the term "variable" refers to the fact that certain portions of the variable domains have extensive sequence variability among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, that variability is not uniformly distributed throughout the variable domains of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions, both in the light chain variable domain and in the heavy chain variable domain. The more highly conserved portions of the variable domains are called frameworks (FRs). The variable domains of the native heavy and light chains each contain four FR regions, predominantly adopt a β-sheet conformation, are connected by three CDRs, which form loops connecting the β-sheet structures and, in some cases, form part of the β-sheet structures. The CDRs in each chain are held together, in proximity by the FR regions, with the CDRs from the other chain and contribute to the formation of the antigen-binding site of the antibody. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., National Institute of Health, Bethesda, Md. (1991). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions (e.g., the involvement of the antibody in antibody-dependent cell-mediated cytotoxicity). The variable region sequences of interest include the humanized variable region sequences for the CD47 antibodies described in detail elsewhere herein.

[0053] The terms "hypervariable region (HVR)" or "complementarity determining region (CDR)" may refer to subregions of the VH and VL domains characterized by enhanced sequence variability and / or defined loop formation. These include three CDRs (H1, H2, and H3) in the VH domain and three CDRs (L1, L2, and L3) in the VL domain. H3 is thought to be extremely important in conferring fine binding specificity, and L3 and H3 exhibit the highest level of diversity. See Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).

[0054] Several CDR / HVR descriptions are known. The Kabat complementarity determining regions (CDRs) are the most commonly used, based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, instead, refers to the positions of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVR represents a compromise between Kabat's HVR and Chothia's structural loops and is used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on the analysis of available complex crystal structures. Residues from each of these HVR / CDRs are noted below. "Framework" or "FR" residues are variable domain residues other than the above HVR / CDR residues.

Table 5

[0055] "Extended" HVRS are also known: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) (Kabat numbering) in VH.

[0056] "Numbering according to Kabat" may refer to the numbering system used for the heavy chain variable domain or the light chain variable domain of antibody editing in Kabat et al. (supra). The actual linear amino acid sequence may contain a few or additional amino acids corresponding to shortening or insertion into the FR or HVR of the variable domain. The Kabat numbering of residues can be determined for a given antibody by alignment in the region of homology of the antibody's sequence with the "standard" Kabat-numbered sequence. Typically, the above Kabat numbering is used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), whereas the EU numbering system or index (e.g., EU index as in Kabat, numbering according to EU IgG1) is generally used when referring to residues in the heavy chain constant region.

[0057] As used herein, the term "antibody fragment" and all its grammatical variations include the antigen-binding site or variable region of an intact antibody and, in certain cases, do not include the heavy chain constant domains of the Fc region of an intact antibody (i.e., CH2, CH3, and / or CH4 depending on the antibody isotype), and are defined as a part of an intact antibody. Examples of antibody fragments include: Fab, Fab’, Fab’-SH, F(ab’)2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single chain polypeptide") ((1) single-chain Fv (scFv) molecules, (2) single-chain polypeptides containing only one light chain variable domain or fragments thereof containing the three CDRs of the light chain variable domain without the associated heavy chain portion, and (3) single-chain polypeptides containing only one heavy chain variable region or fragments thereof containing the three CDRs of the heavy chain variable region without the associated light chain portion), but are not limited thereto); and multispecific or multivalent structures formed from antibody fragments. In antibody fragments containing one or more heavy chains, the heavy chains may include any constant domain sequence found in the non-Fc region of an intact antibody (e.g., CH1 in the IgG isotype), and / or any hinge region sequence found in an intact antibody, and / or may include a leucine zipper sequence fused to or located in the hinge region sequence or constant domain sequence of the heavy chain.

[0058] The Fab fragment also includes the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab sequence by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain that includes one or more cysteines derived from the antibody hinge region. Fab'-SH is the designation herein for Fab' where the cysteine residue of the constant domain has a free thiol group. The F(ab')2 antibody fragment is inherently produced as a pair of Fab' fragments (which have hinge cysteines therebetween). Other chemical couplings of antibody fragments are also known.

[0059] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Each mAb is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture and are likely not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies that will be used in accordance with the present disclosure may be made using immortalized B cells or their hybridomas, or may be made by recombinant DNA methods.

[0060] The monoclonal antibodies herein include, insofar as they exhibit the desired biological activity, hybrid and recombinant antibodies generated by splicing the variable domains (including hypervariable domains) and constant domains (e.g., "humanized" antibodies) of CD47 antibodies, or the light and heavy chains, or chains from one species and chains from another species, as well as fusions with heterologous proteins, regardless of the origin species or immunoglobulin class or subclass designation, and antibody fragments (e.g., Fab, F(ab’)2, and Fv).

[0061] Specifically, the monoclonal antibodies herein include, insofar as they exhibit the desired biological activity, chimeric antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

[0062] As used herein, the term "treatment" refers to a clinical intervention designed to change the natural course of an individual or cell being treated during the process of clinical pathology. Desirable effects of treatment include a decrease in the rate of disease progression, amelioration or palliation of a disease state, and remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated (including, but not limited to, reduction in the growth of cancerous cells (or destruction of cancerous cells), decrease in symptoms resulting from the disease, increase in the quality of life of individuals suffering from the disease, decrease in the dosage of other medications required to treat the disease, and / or extension of the survival of the individual). In some embodiments, "treating" a disease such as cancer refers to delaying the progression of the disease, i.e., deferring, preventing, slowing, retarding, stabilizing, and / or postponing the onset of the disease (e.g., cancer). This delay can vary in duration depending on the disease being treated and / or the medical history of the individual. As will be apparent to those skilled in the art, a sufficient or significant delay may, in essence, include prevention in that the individual does not develop the disease. For example, late-stage cancer (e.g., the occurrence of metastasis) can be delayed.

[0063] "Effective amount" means at least the minimum amount necessary to effect a measurable improvement or prevention of a particular disease (e.g., cancer). The effective amount herein can vary according to factors such as the patient's disease state, age, gender, and weight, as well as the ability of the therapeutic agent (or combination of therapeutic agents) to induce a desired response in an individual. The effective amount is also that in which any toxic or detrimental effects of the above treatment are outweighed by therapeutically beneficial effects. For therapeutic use, beneficial or desired results include a decrease in one or more symptoms resulting from the above disease, an increase in the quality of life of people suffering from the above disease, a decrease in the dosage of other medications required to treat the above disease, enhancement of the effect of another drug therapy such as through targeting, delay in the progression of the above disease, and / or clinical outcomes such as an extension of survival. In the case of cancer or a tumor, the effective amount of a drug is effective in reducing the number of cancer cells; reducing the tumor size; inhibiting the invasion of cancer cells into peripheral organs (i.e., slowing to some extent or preferably stopping); inhibiting tumor metastasis (i.e., slowing to some extent or preferably stopping); inhibiting tumor growth to some extent; and / or reducing to some extent one or more of the symptoms associated with the above disease. The effective amount can be administered in one or more administrations. For the purposes of the present disclosure, the effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve a therapeutic treatment, either directly or indirectly. As understood in a clinical context, the effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, when given alone or in combination with one or more other agents, the desired result can be achieved or is achieved.

[0064] As used herein, the term "subject" for purposes of treatment refers to any animal classified as a mammal (including humans, domesticated animals and livestock, as well as zoo animals, sport animals, or companion animals (e.g., dogs)).

[0065] As used herein, the term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with the signaling resulting from the interaction of PD-1 with one or more of its binding partners (e.g., PD-L1, PD-L2). In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partner. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with the signaling resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In some embodiments, the PD-1 binding antagonist reduces the negative signal mediated by or through cell surface proteins expressed on T lymphocytes and other cells that mediate signaling via PD-1 or PD-L1 so as to render dysfunctional T cells non-dysfunctional.

[0066] As used herein, the term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with the signal transduction resulting from the interaction of PD-L1 with any one or more of its binding partners (e.g., PD-1, B7-1). In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, examples of the PD-L1 binding antagonist include a PD-L1 antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, an oligopeptide, and other molecules that reduce, block, inhibit, suppress, or interfere with the signal transduction resulting from the interaction of PD-L1 with any one or more of its binding partners (e.g., PD-1, B7-1). In some embodiments, the PD-L1 binding antagonist reduces the negative signal mediated by or through cell surface proteins expressed on T lymphocytes and other cells that mediate signal transduction via PD-L1 or PD-1 so as to make dysfunctional T cells non-dysfunctional.

[0067] As used herein, the term "biomarker" refers to an indicator (e.g., predictive, diagnostic, and / or prognostic) that can be detected in a sample. The biomarker can serve as an indicator for a particular subtype of a disease or disorder (e.g., cancer) characterized by a particular characteristic, molecular characteristic, pathological characteristic, histological characteristic, and / or clinical characteristic. Biomarkers include, but are not limited to, polynucleotides, polynucleotide copy number variations, polypeptides, post-translational modifications, carbohydrates, and / or glycolipid-based molecules.

[0068] The "amount" or "level" of a biomarker associated with increased clinical benefit to an individual is a detectable level in a biological sample. These can be measured by methods known to those of skill in the art (e.g., IHC). The level or amount of the biomarker being evaluated can be used to determine the response to the above treatment.

[0069] As used herein, the terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., encoded by a gene and / or epigenetic) is converted into a structure that is present and functional in a cell.

[0070] "Increased expression", "increased expression level" or "increased level" refer to an increased expression or increased level of a biomarker in an individual relative to a reference, e.g., an individual not suffering from the above disease or disorder (e.g., cancer) or an internal control (e.g., a housekeeping biomarker).

[0071] CD73 Over the past several years, many studies have suggested a role for the adenosine pathway in tumor progression and metastasis. Under the tumor microenvironment, increased extracellular adenosine can activate adenosine receptor subtypes A2A and A2B, thereby restricting effector T cell function, inducing immunosuppression, and stimulating angiogenesis.

[0072] Extracellular adenosine production is regulated by many enzymes. The classical pathway of extracellular adenosine production is based on sequential reactions mediated by ectonucleotidases. In particular, the above extracellular adenosine triphosphate is first hydrolyzed by CD39 to adenosine monophosphate (AMP), and then dephosphorylated to adenosine by CD73. Alternatively, CD38 and CD203a can convert NAD+ to ADP-ribose and ADP-ribose to AMP, respectively. Next, AMP is dephosphorylated to adenosine by CD73. In both pathways, CD73 plays an important function in extracellular adenosine production.

[0073] CD73 (surface antigen classification 73) is also known as 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase and is an enzyme that functions to convert AMP to adenosine. CD73 catalyzes the formation of extracellular adenosine that contributes to the immunosuppressive tumor environment. CD73 exists in two forms: one form is tethered to the membrane of many cells or extracellular vesicles via glycosylphosphatidylinositol; the second form is generated upon cleavage from the membrane via the action of proteases or phospholipases and is found in biological fluids.

[0074] CD73 antagonist Preclinical evidence indicates that CD73 inhibition prevents adenosine-mediated lymphocyte suppression, increases the activity of CD8+ effector cells, and reduces both MDSC and Treg. Several anti-CD73 antibodies are being developed as potential anti-cancer agents, but none have been approved for clinical use.

[0075] Any suitable CD73 antagonist can be used in the present application. In some embodiments, the CD73 antagonist is a CD73 antibody. Any CD73 antibody known in the art can be used in the present application. In some embodiments, the CD73 antibody is selected from the group consisting of a full-length antibody, Fab, Fab’, F(ab’)2, scFv, and sdAb. In some embodiments, the CD73 antibody is a full-length antibody.

[0076] In some embodiments, the CD73 antibody comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences of CDR1, CDR2, and CDR3 within the heavy chain variable region (VH) shown in SEQ ID NO: 1. In some embodiments, the CD73 antibody comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences of CDR1, CDR2, and CDR3 within the heavy chain variable region (VH) shown in SEQ ID NO: 1, wherein the CDR1, CDR2, and CDR3 follow the Kabat numbering system. In some embodiments, the CD73 antibody comprises (1) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 1 or more substitutions compared to SEQ ID NO: 3, (2) HCDR2 containing the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 1 or more substitutions compared to SEQ ID NO: 4, and (3) HCDR3 containing the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having 1 or more substitutions compared to SEQ ID NO: 5.

[0077] In some embodiments, the CD73 antibody comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1.

[0078] In some embodiments, the CD73 antibody comprises LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 within the light chain variable region (VL) shown in SEQ ID NO: 2. In some embodiments, the CD73 antibody comprises LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 within the light chain variable region (VL) shown in SEQ ID NO: 2, wherein the CDR1, CDR2, and CDR3 follow the Kabat numbering system. In some embodiments, the CD73 antibody comprises (1) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 1 or more substitutions as compared to SEQ ID NO: 6, (2) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1 or more substitutions as compared to SEQ ID NO: 7, and (3) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 1 or more substitutions as compared to SEQ ID NO: 8.

[0079] In some embodiments, the CD73 antibody comprises a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2.

[0080] In some embodiments, the CD73 antibody comprises HCDR1, HCDR2, and HCDR3, which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 within the heavy chain variable region (VH) shown in SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 within the light chain variable region (VL) shown in SEQ ID NO: 2. In some embodiments, the CD73 antibody comprises HCDR1, HCDR2, and HCDR3, which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 within the heavy chain variable region (VH) shown in SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 within the light chain variable region (VL) shown in SEQ ID NO: 2, wherein the CDR1, CDR2, and CDR3 follow the Kabat numbering system. In some embodiments, the CD73 antibody comprises (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 1 or more substitutions compared to SEQ ID NO: 3, (2) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 1 or more substitutions compared to SEQ ID NO: 4, (3) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having 1 or more substitutions compared to SEQ ID NO: 5, (4) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 1 or more substitutions compared to SEQ ID NO: 6, (5) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1 or more substitutions compared to SEQ ID NO: 7, and (6) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 1 or more substitutions compared to SEQ ID NO: 8.

[0081] In some embodiments, the CD73 antibody comprises: (1) a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1, and (2) a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2.

[0082] In some embodiments, the CD73 antibody is selected from the group consisting of uliledlimab (I-Mab Biopharma), oleclumab (AstraZeneca), CPI-006 (Corvus Pharma), BMS-986179 (Bristol-Myers Squibb), AB-680 (Arcus Biosciences), NZV-930 (SRF373, Surface Oncolohgy / Novartis), JAB-BX102 (Jacobio), AK119 (Akesobio), Sym024 (Symphogen), IBI 325 (Innovent), BR 101 (Hisun BioRay), and LY3475070 (Eli-Lilly). In some embodiments, the CD73 antibody is uliledlimab (I-Mab Biopharma) or oleclumab (AstraZeneca).

[0083] PD-1 / PD-L1 antagonist Any suitable PD-1 / PD-L1 antagonist known in the art can be used in the methods of the present application. In some embodiments, the PD-1 / PD-L1 antagonist comprises a small molecule inhibitor of PD-1 / PD-L1 signaling. In some embodiments, the PD-1 / PD-L1 antagonist comprises an antibody or an antigen-binding fragment thereof (e.g., a PD-1 antibody, a PD-L1 antibody, or an antigen-binding fragment thereof).

[0084] In some embodiments, the PD-1 / PD-L1 signaling inhibitor is a PD-1 antibody or an antigen-binding fragment thereof. Any suitable PD-1 antibody can be used in the present application. Exemplary PD-1 antibodies that can be used in the methods of the present application include, but are not limited to, polyclonal antibodies, monoclonal antibodies, Fab, scFv, diabodies, triabodies, minibodies, VHH, and sdAb. Exemplary PD-1 antibodies include, but are not limited to, pidilizumab, semaprimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, toripalimab, dostarlimab, ABBV-181, pembrolizumab, genolimzumab, retilfanlimab, sasanelimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, nivolumab, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01. In some embodiments, the PD-1 antibody is toripalimab.

[0085] Any suitable PD-L1 antibody can be used in the present application. Exemplary PD-L1 antibodies that can be used in the methods of the present application include, but are not limited to, polyclonal antibodies, monoclonal antibodies, Fabs, scFvs, diabodies, triabodies, minibodies, VHHs, and sdAbs. Exemplary PD-L1 antibodies include, but are not limited to, manelumab, atezolizumab, avelumab, cosibelimab, durvalumab, enoblituzumab, socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12, and KY-1003.

[0086] Combination treatment As used herein, the terms "combination treatment", "combo therapy", "combo treatment", or "combinational therapy" may be used interchangeably and refer to administering to a subject the CD73 antagonist and the PD-1 / PD-L1 antagonist as described herein either simultaneously or separately.

[0087] In some embodiments, the combination treatment includes the CD73 antagonist and the PD-1 / PD-L1 antagonist as described herein. In some embodiments of the combination treatment, the CD73 antagonist and the PD-1 / PD-L1 antagonist are administered intravenously to the subject.

[0088] In some embodiments of the above-described combinatorial treatment, the CD73 antagonist is a CD73 antibody as described herein. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 2 to about 30 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 2 to about 20 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 5 to about 20 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 10 to about 20 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 15 to about 20 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 2 to about 15 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 5 to about 15 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 10 to about 15 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 2 to about 10 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 5 to about 10 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 2 to about 5 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 20 to about 30 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg or about 20 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 20 mg / kg body weight. In some embodiments, the CD73 antibody is administered to the subject at a dose of about 30 mg / kg body weight.

[0089] In some embodiments of the above-described combination treatment, the CD73 antibody is administered to the subject once a week (QW), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), or once a month. In some embodiments, the CD73 antibody is administered to the subject once every three weeks (Q3W). In some embodiments, the CD73 antibody is administered to the subject once every three weeks (Q3W) at a dose of 20 mg / kg. In some embodiments, the CD73 antibody is administered to the subject once every three weeks (Q3W) at a dose of 30 mg / kg.

[0090] In some embodiments of the above-described combination treatment, examples of the CD73 antibody include, but are not limited to, uliledlimab (I-Mab Biopharma), oleclumab (AstraZeneca), CPI-006 (Corvus Pharma), BMS-986179 (Bristol-Myers Squibb), AB-680 (Arcus Biosciences), NZV-930 (SRF373, Surface Oncolohgy / Novartis), JAB-BX102 (Jacobio), AK119 (Akesobio), Sym024 (Symphogen), IBI 325 (Innovent), BR 101 (Hisun BioRay), and LY3475070 (Eli-Lilly). In some embodiments, the CD73 antibody is uliledlimab (I-Mab Biopharma) or oleclumab (AstraZeneca).

[0091] In some embodiments of the above-described combination treatment, the PD-1 / PD-L1 antagonist is a PD-1 antibody or a PD-L1 antibody as described herein. In some embodiments of the above-described combination treatment, examples of the PD-L1 antibody include, but are not limited to, atezolizumab, avelumab, durvalumab, enoblituzumab, embafolimab, socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12, and KY-1003. In some embodiments, the PD-L1 antibody is atezolizumab.

[0092] In some embodiments of the above-described combination treatment, examples of the PD-1 antibody include, but are not limited to, pidilizumab, semaprilumab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, toripalimab, dostarlimab, ABBV-181, pembrolizumab, genolimzumab, retifanlimab, sasanelimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, nivolumab, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01. In some embodiments, the PD-1 antibody is pembrolizumab, nivolumab, or toripalimab.

[0093] In some embodiments, the PD-1 antibody or PD-L1 antibody is administered to the subject once a week (QW), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), or once a month. In some embodiments, the PD-1 antibody or PD-L1 antibody is administered to the subject once every three weeks (Q3W).

[0094] In some embodiments, the combination treatment comprises the step of intravenously administering to the subject uliledrimab at a dose of 20 mg / kg once every three weeks (Q3W) and tripalimumab at a dose of 240 mg flat dose once every three weeks (Q3W). In some embodiments, the combination treatment comprises the step of intravenously administering to the subject orelumab at a dose of 20 mg / kg once every three weeks (Q3W) and tripalimumab at a dose of 240 mg flat dose once every three weeks (Q3W).

[0095] In some embodiments, the combination treatment comprises the step of intravenously administering to the subject uliledrimab at a dose of 30 mg / kg once every three weeks (Q3W) and tripalimumab at a dose of 240 mg flat dose once every three weeks (Q3W). In some embodiments, the combination treatment comprises the step of intravenously administering to the subject orelumab at a dose of 30 mg / kg once every three weeks (Q3W) and tripalimumab at a dose of 240 mg flat dose once every three weeks (Q3W).

[0096] In some embodiments, the combination treatment comprises intravenously administering to the subject, once every three weeks (Q3W), uliledlimab at a dose of 20 mg / kg and, once every three weeks (Q3W), atezolizumab at a dose of 1200 mg uniform dose. In some embodiments, the combination treatment comprises intravenously administering to the subject, once every three weeks (Q3W), orelumab at a dose of 20 mg / kg and, once every three weeks (Q3W), atezolizumab at a dose of 1200 mg uniform dose.

[0097] In some embodiments, the combination treatment comprises intravenously administering to the subject, once every three weeks (Q3W), uliledlimab at a dose of 30 mg / kg and, once every three weeks (Q3W), atezolizumab at a dose of 1200 mg uniform dose. In some embodiments, the combination treatment comprises intravenously administering to the subject, once every three weeks (Q3W), orelumab at a dose of 30 mg / kg and, once every three weeks (Q3W), atezolizumab at a dose of 1200 mg uniform dose.

[0098] In some embodiments, the combination treatment further includes chemotherapy. Exemplary chemotherapies that can be used in the combination treatments of the present application include, but are not limited to, the following: alkylating agents (e.g., thiotepa and cytoxan (registered trademark) cyclophosphamide); alkyl sulfonates (e.g., busulfan, improsulfan, and piposulfan); aziridines (e.g., benzodopa, carbocon, meturedopa, and uredopa); ethyleneimines and methylamelamine (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine); acetogenins (especially, bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard); nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine); antibiotics (e.g., enediynes antibiotics; dynemicin (including dynemicin A); bisphosphonates (e.g., clodronate); esperamicin;and neocarzinostatin chromophore and related pigment protein engyin antibiotics chromophore), actinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, cardinophyllin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodrubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, methotrexate, pteropterin, trimetrexate); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiampurine, thioguanine); pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine); androgens (e.g., calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone); anti-adrenal agents (e.g., aminoglutethimide, mitotane, trilostane); folic acid replenishers, e.g., frolinic acid; aceglatone; aldophosphamide glycoside;Aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoid (e.g., maytansine and ansamitocin); mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; lysofylline; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecene (especially, T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoid (e.g., Taxol (registered trademark) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), Abraxane (registered trademark) paclitaxel Cremophor-free, albumin-engineered nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Ill.), and Taxotere (registered trademark) docetaxel (Rhone-Poulenc Rorer, Antony, France)); chlorambucil; Gemzar (registered trademark) gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;Platinum analogs (e.g., cisplatin, oxaliplatin, and carboplatin); vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine (registered trademark) vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids (e.g., retinoic acid); capecitabine; combretastatin; leucovorin (LV); oxaliplatin (including oxaliplatin treatment regimen (FOLFOX)); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva (registered trademark)) and VEGF-A, and pharmaceutically acceptable salts, acids or derivatives of any of the above).;

[0099] Further non-limiting exemplary chemotherapies include the following: antihormonal agents that act to modulate or inhibit the hormonal action on cancer (e.g., antiestrogens and selective estrogen receptor modulators (SERMs) (e.g., tamoxifen (e.g., Norvadex® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Fareston® toremifene)); aromatase inhibitors that inhibit the enzyme aromatase and regulate estrogen production in the adrenal glands (e.g., 4(5)-imidazole, aminoglutethimide, Megase® megestrol acetate, Arimidex® anastrozole, formestanie, fadrozole, Rivisor® vorozole, Femara® letrozole, and Aromasin® exemestane); and antiandrogens (e.g., flutamide, nilutamide, bicalutamide, leuprolide, and goserelin); and troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways related to adherent cell growth (e.g., PKC-α, Ralf, and H-Ras); ribozymes, e.g., VEGF expression inhibitors (e.g., Angiozyme® ribozyme) and HER2 expression inhibitors; vaccines (e.g., gene therapy vaccines, e.g., Allovectin® vaccine, Leuvectin® vaccine, and Vaxid® vaccine); Proleukin® rIL-2; Lurtotecan® topoisomerase 1 inhibitor; Abarelix® rmRH; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0100] Method In one aspect, a method for treating a subject having NSCLC, the method comprising: (a) determining the level of CD73 in a sample of the subject; (b) evaluating whether the subject is more likely to respond to or benefit from combination treatment with a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73; and (c) administering the combination treatment to the subject if the subject is more likely to respond to or benefit from the combination treatment. The method is provided herein.

[0101] In another aspect, a method for selecting a treatment for a subject having NSCLC, the method comprising: (a) determining the level of CD73 in a sample of the subject; (b) evaluating whether the subject is more likely to respond to or benefit from combination treatment with a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73; and (c) selecting the combination treatment for the subject if the subject is more likely to respond to or benefit from the combination treatment. The method is provided herein.

[0102] In another aspect, a method for identifying that a subject having NSCLC is more likely to respond to or benefit from combination treatment with a CD73 antagonist and a PD1 / PD-L1 antagonist, the method comprising: (a) determining the level of CD73 in a sample of the subject; (b) evaluating whether the subject is more likely to respond to or benefit from the combination treatment based on the level of CD73; and (c) providing a recommendation for the combination treatment to the subject if the subject is more likely to respond to or benefit from the combination treatment. The method is provided herein.

[0103] In another aspect, provided herein is a method for predicting the responsiveness of a subject having NSCLC to a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist, the method comprising: (a) determining the level of CD73 in a sample of the subject; (b) evaluating whether the subject is more likely to respond to or benefit from the combination treatment based on the level of CD73; and (c) providing a recommendation for the combination treatment to the subject if the subject is more likely to respond to or benefit from the combination treatment.

[0104] In some embodiments of the method of the present application, the NSCLC is stage III or stage IV NSCLC. In some embodiments, the NSCLC is stage IV NSCLC. In some embodiments, the NSCLC is advanced NSCLC. In some embodiments, the NSCLC is metastatic NSCLC. In some embodiments, the NSCLC is recurrent NSCLC. In some embodiments, the NSCLC is refractory NSCLC. In some embodiments, the NSCLC has a squamous or non-squamous histological image.

[0105] In some embodiments of the method of the present application, the level of CD73 is the protein level of CD73. Any suitable method known in the art for determining or measuring the protein level can be used in the method of the present application. In some embodiments, the method determines the protein level of CD73 expressed on the cell membrane. In some embodiments, the method determines the protein level of CD73 expressed in the cytosol. In some embodiments, the method determines the protein levels of CD73 expressed on the cell membrane and in the cytosol.

[0106] In some embodiments, the protein level is determined by immunohistochemistry (IHC) that measures the amount of CD73 protein in a cancer tissue sample, e.g., an NSCLC tissue sample. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) sample. In some embodiments, the sample is an archival sample. In some embodiments, the sample is a fresh sample. In some embodiments, the sample is a frozen sample. In some embodiments, the sample is obtained prior to the combination treatment as described herein.

[0107] In some embodiments of the methods of the present application, in an IHC assay, depending on the staining intensity, the level of CD73 protein in each cell (including the cell membrane and cytosol) can be assigned using intensities of 0, 1, 2, or 3 based on the criteria as shown in Table 1. Table 1. CD73 Staining Criteria

Table 1-1

Table 1-2

[0108] The intensity grading can be performed manually by a pathologist with experience based on established conventional criteria. Alternatively, various automated / standardized intensity grading systems have been developed. For example, a quantitative microimmunochemistry assay was published in Kashyap et al., "Quantitative microimmunohistochemistry for the grading of immunostains on tumour tissues", Nature Biomedical Engineering, Volume 3, pages 478 - 490 (2019). The above quantitative assay enables the acquisition of dynamic information through a measurement criterion of the generation of immunohistochemical signals during tissue staining for the quantification of relative antigen density on the tissue surface.

[0109] In some embodiments, the IHC assay determines the percentage of cells having staining intensities of 1, 2, and 3 compared to all cells. In some embodiments, the IHC assay determines the percentage of cells having intensities of 2 and 3 compared to all cells. In some embodiments, the IHC assay determines the percentage of cells having an intensity of 3 compared to all cells.

[0110] In some embodiments, the cells include, but are not limited to, tumor cells, immune cells such as tumor - infiltrating immune cells (TIL), stromal cells, vascular cells, and any combination thereof.

[0111] In some embodiments, the IHC assay determines tumors with staining compared to all tumor cells. In some embodiments, the IHC assay determines the percentage (TC) of tumor cells having staining intensities of 1, 2, and 3 compared to all tumor cells. In some embodiments, the IHC assay determines the percentage (TC(+)) of tumor cells having staining intensities of 2 and 3 compared to all tumor cells. In some embodiments, the IHC assay determines the percentage (TC(++)) of tumor cells having a staining intensity of 3 compared to all tumor cells.

[0112] The H score captures both the intensity and the proportion of the biomarker from the IHC image, includes values between 0 and 300, thereby providing a dynamic range for quantifying the biomarker abundance. In some embodiments of the method of the present application, the IHC assay determines the H score (TCH) of tumor cells. In some embodiments, the H score of tumor cells refers to the sum of TC, TC(+), and TC(++).

[0113] In some embodiments, the IHC assay determines immune cells with staining compared to all immune cells. In some embodiments, the IHC assay determines the percentage (IC) of immune cells having staining intensities of 1, 2, and 3 compared to all immune cells. In some embodiments, the IHC assay determines the percentage (IC(+)) of immune cells having staining intensities of 2 and 3 compared to all immune cells. In some embodiments, the IHC assay determines the percentage (IC(++)) of immune cells having a staining intensity of 3 compared to all immune cells.

[0114] In some embodiments of the method of the present application, the IHC assay determines the H score (ICH) of immune cells. In some embodiments, the H score of immune cells refers to the sum of IC, IC(+), and IC(++).

[0115] In some embodiments, the IHC assay determines tumor cells and immune cells with staining compared to all tumor cells and immune cells. In some embodiments, the IHC assay determines the sum (TC+IC) of the percentage of tumor cells having staining intensities of 1, 2, and 3 compared to all tumor cells and the percentage of immune cells having staining intensities of 1, 2, and 3 compared to all immune cells. In some embodiments, the IHC assay determines the sum (TC+IC(+)) of the percentage of tumor cells having staining intensities of 2 and 3 compared to all tumor cells and the percentage of immune cells having staining intensities of 2 and 3 compared to all immune cells. In some embodiments, the IHC assay determines the sum (TC+IC(++)) of the percentage of tumor cells having a staining intensity of 3 compared to all tumor cells and the percentage of immune cells having a staining intensity of 3 compared to all immune cells.

[0116] In some embodiments, the IHC assay determines tumor cells or immune cells with staining compared to all tumor cells or all immune cells. In some embodiments, the IHC assay determines the higher percentage (TC / IC) of the percentage of tumor cells having staining intensities of 1, 2, and 3 compared to all tumor cells and the percentage of immune cells having staining intensities of 1, 2, and 3 compared to all immune cells. In some embodiments, the IHC assay determines the higher percentage (TC / IC(+)) of the percentage of tumor cells having staining intensities of 2 and 3 compared to all tumor cells and the percentage of immune cells having staining intensities of 2 and 3 compared to all immune cells. In some embodiments, the IHC assay determines the higher percentage (TC / IC(++)) of the percentage of tumor cells having a staining intensity of 3 compared to all tumor cells and the percentage of immune cells having a staining intensity of 3 compared to all immune cells.

[0117] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein, when measured in the IHC assay as described herein, has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% TC in the sample of the subject.

[0118] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein, when measured in the IHC assay as described herein, has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% TC(+) in the sample of the subject.

[0119] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein, when measured in an IHC assay as described herein, has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% TC(++) in the sample of the subject.

[0120] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein has an H score of tumor cells (TCH) of at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or 300.

[0121] In some embodiments of the methods of the present application, the subject that is more likely to respond to or benefit from the combination treatment as described herein, when measured in an IHC assay as described herein, has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% IC in a sample of the subject.

[0122] In some embodiments of the methods of the present application, the subject that is more likely to respond to or benefit from the combination treatment as described herein, when measured in an IHC assay as described herein, has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% IC(+) in a sample of the subject.

[0123] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein, when measured in an IHC assay as described herein, has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% IC(+++) in the sample of the subject.

[0124] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein has an H score of immune cells (ICH) of at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or 300.

[0125] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combinatorial treatment as described herein, when measured in an IHC assay as described herein, has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 195%, or 200% TC+IC in the sample of the subject.

[0126] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein, when measured in an IHC assay as described herein, has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 195%, or 200% TC+IC(+) in a sample of the subject.

[0127] In some embodiments of the method of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein, when measured in the IHC test as described herein, has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 195%, or 200% TC+IC(++) in the sample of the subject.

[0128] In some embodiments of the method of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein, when measured in the IHC test as described herein, has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% TC / IC in the sample of the subject.

[0129] As shown in FIG. 4A, non-responders had a median TC / IC of approximately 20%, while responders had a median TC / IC of approximately 50%. Thus, a TC / IC score of 25% or higher indicates that the corresponding subject may respond to or benefit from the combination treatment. In some embodiments, a TC / IC score of 30%, 35%, 40%, 45%, 50% or 55%, or higher, thus indicates that the corresponding subject may respond to or benefit from the combination treatment.

[0130] In some embodiments of the methods of the present application, the above subjects who are more likely to respond to or benefit from the combination treatment as described herein, when measured in an IHC assay as described herein, have at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% TC / IC(+) in the sample of the subject.

[0131] As shown in FIG. 4B, non-responders had a median TC / IC(+) of approximately 10%, while responders had a median TC / IC(+) of approximately 40%. A TC / IC(+) score of 15% or higher thus indicates that the corresponding subject may respond to or benefit from the combination treatment. In some embodiments, a TC / IC(+) score of 20%, 25%, 30%, 35%, 40% or 45%, or higher, thus indicates that the corresponding subject may respond to or benefit from the combination treatment.

[0132] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combinatorial treatment as described herein, when measured in an IHC assay as described herein, has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% TC / IC(++) in the sample of the subject.

[0133] In another aspect, provided herein is a method for identifying that a subject having NSCLC is more likely to respond to or benefit from a combinatorial treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist, the method comprising: (a) determining the level of CD73 in a sample of the subject, wherein an increase in CD73 in the sample relative to that in a reference sample indicates that the subject is more likely to respond to or benefit from the combinatorial treatment, and (b) providing a recommendation indicating that the subject is more likely to respond to or benefit from the combinatorial treatment.

[0134] In another aspect, a method for predicting the responsiveness of a subject with NSCLC to a combined treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist, the method comprising: (a) determining the level of CD73 in a sample of the subject, wherein an increase in CD73 in the sample relative to that in a reference sample indicates that the subject is more likely to respond to or benefit from the combined treatment; and (b) providing a recommendation that the subject has an increased likelihood of responding to or benefiting from the combined treatment. The method is provided herein.

[0135] In some embodiments, the reference sample comprises a reference tissue or reference cells. In some embodiments, the reference sample is from a healthy subject, a subject who has experienced a first treatment, or a non-diseased sample of the subject.

[0136] In another aspect, a method for treating a subject with non-small cell lung cancer (NSCLC), the method comprising: (a) identifying a subject with NSCLC as likely to respond to or benefit from a combined treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73 in a sample of the subject; and (b) administering the CD73 antagonist and the PD1 / PD-L1 antagonist to the identified subject. The method is provided herein.

[0137] In another aspect, a method for treating a subject with NSCLC, the method comprising administering a CD73 antagonist and a PD1 / PD-L1 antagonist to a subject with NSCLC having a level of CD73 in a sample indicative of being likely to respond to or benefit from a combined treatment of the CD73 antagonist and the PD1 / PD-L1 antagonist. The method is provided herein.

[0138] In another aspect, provided herein is the use of a CD73 antagonist and a PD1 / PD-L1 antagonist for the manufacture of a medicament for treating a subject having NSCLC having a level of CD73 in a sample shown to be responsive to or likely to benefit from a combination treatment of the CD73 antagonist and the PD1 / PD-L1 antagonist.

[0139] In another aspect, provided herein is the use of a CD73 antagonist for the manufacture of a medicament for treating a subject having NSCLC having a level of CD73 in a sample shown to be responsive to or likely to benefit from a combination treatment of the CD73 antagonist and the PD1 / PD-L1 antagonist, wherein the subject is also administered the PD1 / PD-L1 antagonist.

[0140] In another aspect, provided herein is the use of a PD1 / PD-L1 antagonist for the manufacture of a medicament for treating a subject having NSCLC having a level of CD73 in a sample shown to be responsive to or likely to benefit from a combination treatment of the CD73 antagonist and the PD1 / PD-L1 antagonist, wherein the subject is also administered the CD73 antagonist.

[0141] In some embodiments, the increase is determined by TC. In some embodiments, the increase is determined by TC(+). In some embodiments, the increase is determined by the H score of tumor cells. In some embodiments, the increase is determined by TC(++). In some embodiments, the increase is determined by IC. In some embodiments, the increase is determined by IC(+). In some embodiments, the increase is determined by IC(++). In some embodiments, the increase is determined by the H score of immune cells. In some embodiments, the increase is determined by TC / IC. In some embodiments, the increase is determined by TC / IC(+). In some embodiments, the increase is determined by TC / IC(++). In some embodiments, the increase is determined by TC+IC. In some embodiments, the increase is determined by TC+IC(+). In some embodiments, the increase is determined by TC+IC(++).

[0142] In some embodiments of the methods of the present application, the subject is more likely to respond to or benefit from the combination treatment as described herein and has an increase in CD73 of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample.

[0143] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein has an increase in TC of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample. In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein has an increase in TC(+) of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample. In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein has an increase in TC(++) of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample.

[0144] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein has an increase in the H score of tumor cells of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample.

[0145] In some embodiments of the methods of the present application, the subject that is more likely to respond to or benefit from the combination treatment as described herein has an increase in IC of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample. In some embodiments of the methods of the present application, the subject that is more likely to respond to or benefit from the combination treatment as described herein has an increase in IC(+) of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample. In some embodiments of the methods of the present application, the subject that is more likely to respond to or benefit from the combination treatment as described herein has an increase in IC(++) of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample.

[0146] In some embodiments of the methods of the present application, the subject that is more likely to respond to or benefit from the combination treatment as described herein has an increase in the H score of immune cells of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample.

[0147] In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein has an increase in TC / IC of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample. In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein has an increase in TC / IC(+) of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample. In some embodiments of the methods of the present application, the subject who is more likely to respond to or benefit from the combination treatment as described herein has an increase in TC / IC(++) of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500% compared to that in the reference sample.

[0148] In some embodiments of the methods of the present application, the responsiveness includes a relative increase in one or more of the following: overall survival (OS), progression-free survival (PFS), complete response (CR), partial response (PR), stable disease (SD), and combinations thereof. In some embodiments, the responsiveness includes qualification for a procedure such as surgery.

[0149] In some embodiments of the methods of the present application, the subject is treatment-naive. In some embodiments, the treatment is a first-line treatment. In some embodiments, the subject is ineligible for or rejected from the first-line treatment. In some embodiments, the first-line treatment is selected from the group consisting of chemotherapy, PD-1 antibody treatment, PD-L1 antibody treatment, and any combination thereof.

[0150] In some embodiments, the chemotherapy includes, but is not limited to, the following: alkylating agents (e.g., thiotepa and cyclophosphamide (registered trademark) cytoxan); alkyl sulfonates (e.g., busulfan, improsulfan, and piposulfan); aziridines (e.g., benzodopa, carboquone, meturedopa, and uredopa); ethyleneimines and methylamelamine (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine); acetogenins (especially, bullatacin and bullatacinone); camptothecin (including synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189, and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard); nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine); antibiotics (e.g., enediyne antibiotics (e.g., calicheamicin, especially, calicheamicin γ1I and calicheamicin ωI1); dynemicin (including dynemicin A); bisphosphonates (e.g., clodronate); esperamicin;and neocarzinostatin chromophore and related pigment protein enjine antibiotics chromophore), actinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodrubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, methotrexate, pteropterin, trimetrexate); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiampurine, thioguanine); pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine); androgens (e.g., calusterone, drostanolone propionate, epitioestanol, mepitiostane, testolactone); anti-adrenal agents (e.g., aminoglutethimide, mitotane, trilostane); folic acid replenishers, e.g., frolinic acid; aceglatone; aldophosphamide glycoside;Aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoid (e.g., maytansine and ansamitocin); mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecene (especially, T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoid (e.g., Taxol (registered trademark) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), Abraxane (registered trademark) paclitaxel Cremophor-free, albumin-engineered nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Ill.), and Taxotere (registered trademark) docetaxel (Rhone-Poulenc Rorer, Antony, France)); chlorambucil; Gemzar (registered trademark) gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;Platinum analogs (e.g., cisplatin, oxaliplatin, and carboplatin); vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine (registered trademark) vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids (e.g., retinoic acid); capecitabine; combretastatin; leucovorin (LV); oxaliplatin (including oxaliplatin treatment regimen (FOLFOX)); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva (registered trademark)) and VEGF-A, and pharmaceutically acceptable salts, acids or derivatives of any of the above).;

[0151] Additional non-limiting exemplary chemotherapies include antihormonal agents that act to modulate or inhibit the hormonal action on cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs), for example, the following are included: tamoxifen (including Norvadex® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Fareston® toremifene)); aromatase inhibitors that inhibit the enzyme aromatase and regulate estrogen production in the adrenal glands (e.g., 4(5)-imidazole, aminoglutethimide, Megase® megestrol acetate, Arimidex® anastrozole, formestanie, fadrozole, Rivisor® vorozole, Femara® letrozole, and Arimidex® anastrozole, etc.); and antiandrogens (e.g., flutamide, nilutamide, bicalutamide, leuprolide, and goserelin); as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways related to adherent cell growth (e.g., PKC-α, Ralf, and H-Ras, etc.); ribozymes, such as VEGF expression inhibitors (e.g., Angiozyme® ribozyme) and HER2 expression inhibitors; vaccines (e.g., gene therapy vaccines, such as Allovectin® vaccine, Leuvectin® vaccine, and Vaxid® vaccine); Proleukin® rIL-2; Lurtotecan® topoisomerase 1 inhibitor; Abarelix® rmRH; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0152] In some embodiments, exemplary PD-1 antibodies for the first selected treatment include, but are not limited to: pembrolizumab, nivolumab, toripalimab, pidilizumab, semaprimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, dostarlimab, ABBV-181, pemprilumab, genolimzumab, retifanlimab, sasanlimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01.

[0153] In some embodiments, exemplary PD-L1 antibodies for the first selected treatment include, but are not limited to: atezolizumab, manelumab, avelumab, cosibelimab, durvalumab, enoblituzumab, socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12, and KY-1003.

[0154] In another aspect, a combination of a CD73 antagonist and a PD1 / PD-L1 antagonist for use in the treatment of NSCLC in a subject in need thereof, wherein the treatment comprises: (a) determining the level of CD73 in a sample from the subject, wherein an increase in the level of CD73 in the sample relative to that in a reference sample indicates that the subject is more likely to respond to or benefit from the combination treatment; and (b) administering the combination treatment to the subject, is provided herein.

[0155] Kit or article of manufacture In another aspect, a kit or article of manufacture for selecting a treatment for a subject having NSCLC, the kit or article of manufacture comprising: (1) one or more reagents for determining the level of CD73 in a sample from a subject having NSCLC; and (2) a package insert, wherein the package insert comprises a package insert suggesting treating the subject with a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73, is provided herein.

[0156] In another aspect, a kit or article of manufacture for identifying that a subject having NSCLC is more likely to respond to or benefit from a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist, the kit or article of manufacture comprising: (1) one or more reagents for determining the level of CD73 in a sample from a subject having NSCLC; and (2) a package insert, wherein the package insert comprises a package insert suggesting treating the subject with a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73, is provided herein.

[0157] In another aspect, a kit or article of manufacture for use in the methods of the present application, wherein the kit or article of manufacture comprises: (1) one or more reagents for determining the level of CD73 in a sample derived from a subject having NSCLC, and (2) a package insert, wherein the package insert comprises a package insert suggesting treating the subject with a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73 is provided herein.

[0158] In another aspect, a kit or article of manufacture for use in the methods of the present application, wherein the kit or article of manufacture comprises: (1) one or more reagents for determining the level of CD73 in a sample derived from a subject having NSCLC, and (2) a package insert, wherein the package insert comprises a package insert suggesting that an increase in the level of CD73 in the sample relative to that in a reference sample indicates that the subject is more likely to respond to a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist is provided herein.

[0159] In some embodiments, the reagent in the kit or the manufactured article is a CD73 antibody. Exemplary CD73 antibodies that can be used in the kit or the manufactured article include, but are not limited to, D7F9A (CST, catalog number 13160), 606117 (R&D), EPR6114 (Abcam, catalog number ab133582), 4G6E3 (Abcam, catalog number ab202122), NT5E / 2503 (Abcam, catalog number ab257309), NT5E / 2505 (Abcam, catalog number ab257310), NT5E / 2545 (Abcam, catalog number ab257311), NT5E / 2646 (Abcam, catalog number ab257312), 7G2 (Thermo Fisher, catalog number 1D7), JM11-40 (Thermo Fisher, catalog number JM11-40), CD73 recombinant rabbit monoclonal antibody (1) (Invitrogen, catalog number MA5-29454), BLR054F (Thermo Fisher, catalog number BLR054F), sc-32299 (Santa Cruz, catalog number IE9), and AD2 (Biorad, catalog number AD2).

[0160] In some embodiments, the kit or the manufactured article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from various materials such as glass or plastic.

[0161] In some embodiments, the kit or the manufactured article further includes the CD73 antagonist and the PD1 / PD-L1 antagonist of the combination treatment as described herein.

[0162] All publications and patent applications cited herein are hereby incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Example

[0163] The following examples are provided to give those skilled in the art a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure, nor are the following experiments intended to represent all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0164] Example 1. Staining of CD73 by IHC Formalin-fixed paraffin-embedded (FFPE) sections of tumor tissue samples from NSCLC subjects were analyzed via an IHC assay using the Ventana Benchmark Ultra system. Briefly, the FFPE sections were deparaffinized, blocked prior to antigen activation, incubated with the Abcam anti-CD73 antibody EPR6114 (catalog number ab133582), and subsequently incubated with an HRP-labeled secondary antibody. Color development was carried out in the positive regions of the tissue using the UltraView Universal DAB Detection Kit (Roche 760-500), the substrate for HRP.

[0165] In tumor samples, the CD73 staining intensity of cells was present in a dynamic range from faint brown (weak) to dark brown (strong) under a microscope at 4× to 20× magnification. Based on the intensity, the staining can be classified into no staining (0), faint / weak staining (1), moderate / medium staining (2), and prominent / strong staining (3). Exemplary intensities of 0 to 3 are as shown in Figure 1.

[0166] As shown in FIG. 2, the tumor area in the tumor sample usually includes tumor cells and tumor-related stroma (including immune cells such as tumor-infiltrating immune cells (TIL) that infiltrate immediately adjacent to and continuously with the main tumor mass).

[0167] Example 2. Scoring of CD73 Levels by IHC To quantify CD73 in tumor samples, a novel CD73 IHC scoring system was established to measure the CD73 levels in tumor cells and / or immune cells as shown in Table 2 below. Table 2. CD73 IHC Scoring System [Table 2]

[0168] In the ongoing Phase I / II trial of the CD73 antibody uliledlimab developed by I-Mab Biopharma, in the dose expansion stage of the trial, the combination treatment of uliledlimab and the PD-1 antibody toripalimab was included as one of the arms. The subjects enrolled in that arm were divided into two subgroups including the following:

[0169] (I) Subjects who failed the first-choice treatment (including SoC chemotherapy and SoC, followed by PD-1 / PD-L1 antibody treatment), and

[0170] (II) Subjects naive to the first-choice treatment (including subjects who are ineligible for or have refused SoC chemotherapy).

[0171] Before the above treatment, FFPE sections of tumor samples derived from the above subjects were prepared and subjected to IHC staining as described above. The staining results are summarized as shown in Table 3 below. Table 3. IHC Staining of CD73 [Table 3-1]

Table 3-2

[0172] Example 3. Correlation between CD73 level and the response of the subject to the above combination treatment CD73 levels in the samples of the subjects in groups (I) and (II) were analyzed before the above combination treatment. Then, both the subjects in groups (I) and (II) were administered with uliledrimab (20 mg / kg, Q3W) and the PD-1 antibody toripalimab (240 mg, Q3W) over a period of about 42 ± 7 days. After that treatment, the correlation between the response of the subjects to the above combination treatment and their pre-treatment CD73 levels was further analyzed. Here, PR was defined as at least 30% tumor shrinkage from baseline according to RECIST v1.1.

[0173] Figures 3A and 3B show that before the above combination treatment, the mean CD73 levels in groups (I) and (II) were comparable under both the TC / IC and TC / IC(+) scoring systems. No significant difference in CD73 levels was observed between the responding and non-responding individuals among the subjects in the above group (I). Surprisingly, subjects with higher CD73 levels in group (II) showed a significantly stronger response to the above combination treatment of the CD73 antibody and the PD-1 antibody, irrespectively, under the TC / IC (p = 0.091) or TC / IC(+) (p = 0.0019) scoring systems, as shown in Figures 4A and 4B.

[0174] In groups (I) and (II), the correlation between CD73 levels and response to the above combination treatment (measured as the best tumor change from baseline) was further evaluated via Pearson's correlation coefficient. As shown in FIGS. 5A and 5B, CD73 levels had a stronger correlation with the response to the above combination treatment in group (II) compared to that shown in group (I) under both the TC / IC (ρ: -0.38 vs. -0.02) and TC / IC(+) (ρ: -0.53 vs. -0.023) scoring systems. This supports CD73 levels as an appropriate biomarker for selecting naïve subjects for the above combination treatment of anti-CD73 antibody and anti-PD-1 antibody against the first-choice treatment.

[0175] Example 4. Preliminary ROC Assessment A CD73 level cutoff for selecting naïve subjects (group II) for the above combination treatment can be evaluated through receiver operating characteristic (ROC) analysis. FIGS. 6A and 6B show the preliminary ROC curves under the TC / IC and TC / IC(+) scoring systems, respectively. Table 4 below also summarizes the preliminary results of the TC / IC and TC / IC(+) scoring systems from the ROC curves. This indicates that both scoring systems are reliable approaches for subject selection. Table 4. Preliminary Summary of ROC Curves [Table 4] Youden index = sensitivity + specificity - 1

[0176] This disclosure is described with respect to specific embodiments found or proposed by the inventors that include preferred modes for carrying out the present disclosure. In view of the present disclosure, it will be recognized by those skilled in the art that many modifications and changes can be made in the specific embodiments illustrated without departing from the intended scope of the present disclosure. For example, due to codon degeneracy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Further, due to considerations of biological functional equivalence, changes can be made in the protein structure without affecting its biological action in terms of type or amount. All such modifications are intended to be included within the scope of the appended claims. Sequence Listing [Table 6]

Claims

**Claim 1** A method for treating a subject having non - small cell lung cancer (NSCLC), the method comprising: (a) Identifying a subject with NSCLC as potentially responsive to or likely to benefit from a combination treatment of a CD73 antagonist and a PD1 / PD - L1 antagonist based on the level of CD73 in a sample of the subject; and (b) Administering the CD73 antagonist and the PD1 / PD - L1 antagonist to the identified subject. A method as described above. **Claim 2** A method for selecting a treatment for a subject having NSCLC, the method comprising: (a) Determining the level of CD73 in a sample of the subject; and (b) Evaluating whether the subject is likely to respond to or benefit from a combination treatment of a CD73 antagonist and a PD1 / PD - L1 antagonist based on the level of CD73; (c) Selecting the combination treatment for the subject if it is determined that the subject is likely to respond to or benefit from the combination treatment. A method as described above. **Claim 3** A method for treating a subject having NSCLC, the method comprising administering a CD73 antagonist and a PD1 / PD - L1 antagonist to a subject having NSCLC having a level of CD73 in a sample indicating that the subject is potentially responsive to or likely to benefit from a combination treatment of the CD73 antagonist and the PD1 / PD - L1 antagonist. **Claim 4** A method for predicting the responsiveness of a subject having NSCLC to a combination treatment of a CD73 antagonist and a PD1 / PD - L1 antagonist, the method comprising: (a) Determining the level of CD73 in a sample of the subject; (b) Evaluating whether the subject is likely to respond to or benefit from the combination treatment based on the level of CD73; and (c) Providing a recommendation for the combination treatment to the subject if it is determined that the subject is likely to respond to or benefit from the combination treatment. A method as described above. **Claim 5** A method for identifying a subject having NSCLC as being more likely to respond to or benefit from a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist than a reference patient, said method comprising: (a) determining the level of CD73 in a sample of said subject, wherein an increase in the level of CD73 in said sample relative to that in a reference sample derived from said reference patient indicates that said subject is more likely to respond to or benefit from said combination treatment than said reference patient; and (b) providing a recommendation that said subject is more likely to respond to or benefit from said combination treatment than said reference patient. A method comprising the above.

6. A method for predicting the responsiveness of a subject having NSCLC to a combination treatment of a CD73 antagonist and a PD1 / PD-L1 antagonist, said method comprising: (a) determining the level of CD73 in a sample of said subject, wherein an increase in the level of CD73 in said sample relative to that in a reference sample derived from a reference patient indicates that said subject is more likely to respond to or benefit from said combination treatment than said reference patient; and (b) providing a recommendation that said subject has an increased likelihood of responding to or benefiting from said combination treatment as compared to said reference patient. A method comprising the above.

7. The method according to any one of claims 1 to 6, wherein the CD73 antagonist of said combination treatment is a CD73 antibody.

8. The method according to claim 7, wherein the CD73 antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a Fab, an scFv, a diabody, a triabody, a minibody, a VHH, and an sdAb.

9. The CD73 antibody is (a) HCDR1, HCDR2, and HCDR3 within a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 1, and (b) LCDR1, LCDR2, and LCDR3 within a light chain variable region (VL) having the sequence shown in SEQ ID NO: 2, and includes. Here, the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 follow the Kabat numbering scheme, and the method according to claim 7 or 8.

10. The CD73 antibody is (1) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 3, (2) HCDR2 containing the amino acid sequence shown in SEQ ID NO: 4, (3) HCDR3 containing the amino acid sequence shown in SEQ ID NO: 5, (4) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 6, (5) LCDR2 containing the amino acid sequence shown in SEQ ID NO: 7, and (6) LCDR3 containing the amino acid sequence shown in SEQ ID NO: 8, and the method according to any one of claims 7 to 9.

11. The CD73 antibody is selected from the group consisting of uliledlimab (I-Mab Biopharma), oleclumab (AstraZeneca), CPI-006 (Corvus Pharma), BMS-986179 (Bristol-Myers Squibb), AB-680 (Arcus Biosciences), NZV-930 (SRF373, Surface Oncology / Novartis), JAB-BX102 (Jacobio), AK119 (Akesobio), Sym024 (Symphogen), IBI 325 (Innovent), BR 101 (Hisun BioRay), and LY3475070 (Eli-Lilly), and the method according to any one of claims 7 to 10.

12. The PD-1 / PD-L1 antagonist in the combined treatment is a PD-1 antibody or a PD-L1 antibody, and the method according to any one of claims 1 to 11.

13. The PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, toripalimab, pidilizumab, semiprimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balsilimab, dostarlimab, ABBV-181, pemprimab, genolimzumab, retifanlimab, sasanalimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01, the method according to claim 12.

14. The PD-L1 antibody is selected from the group consisting of atezolizumab, manelumab, avelumab, cosibelimab, durvalumab, embafolimab, socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12 and KY-1003, the method according to claim 12.

15. The combined treatment further comprises chemotherapy, the method according to any one of claims 1 to 14.

16. The chemotherapy is selected from the group consisting of platinum agents (e.g., cisplatin, carboplatin), taxane agents (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vincristine, etoposide, pemetrexed and gemcitabine, and any combination thereof, the method according to claim 15.

17. The combined treatment according to any one of claims 1 to 16, which comprises the step of administering the CD73 antagonist and the PD-1 / PD-L1 antagonist simultaneously or separately.

18. The method according to any one of claims 1 to 17, wherein the NSCLC is stage III or stage IV NSCLC.

19. The method according to any one of claims 1 to 18, wherein the NSCLC is advanced NSCLC or metastatic NSCLC.

20. The method according to any one of claims 1 to 19, wherein the NSCLC is recurrent NSCLC.

21. The method according to any one of claims 1 to 20, wherein the NSCLC has a squamous histological pattern or a non-squamous histological pattern.

22. The method according to any one of claims 1 to 21, wherein the subject is treatment-naive.

23. The method according to claim 22, wherein the treatment is a first-line treatment.

24. The method according to claim 23, wherein the subject is ineligible or refused for the first-line treatment.

25. The method according to claim 23 or 24, wherein the first-line treatment is selected from the group consisting of chemotherapy, anti-PD-1 antibody therapy, anti-PD-L1 antibody therapy, and any combination thereof.

26. The method according to claim 25, wherein the chemotherapy is selected from the group consisting of platinum agents (e.g., cisplatin, carboplatin), taxane agents (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vincristine, etoposide, pemetrexed, and gemcitabine, and any combination thereof.

27. The PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, toripalimab, pidilizumab, semaprimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balslimab, dostarlimab, ABBV-181, pemprimab, genolimzumab, retifanlimab, sasanalimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01, the method according to claim 25.

28. The PD-L1 antibody is selected from the group consisting of atezolizumab, manelumab, avelumab, cosibelimab, durvalumab, enobafolimab, socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12 and KY-1003, the method according to claim 25.

29. The level of CD73 is the protein level of CD73, the method according to any one of claims 1 to 28.

30. The protein level is determined by immunohistochemistry (IHC), the method according to claim 29.

31. The sample comprises cells selected from the group consisting of tumor cells, immune cells such as tumor infiltrating immune cells, stromal cells, and any combination thereof, the method according to any one of claims 1 to 30.

32. The method according to any one of claims 1 to 31, wherein the sample is a formalin-fixed paraffin-embedded (FFPE) sample, an archival sample, a fresh sample or a frozen sample.

33. The method according to any one of claims 1 to 32, wherein the sample is obtained prior to the combined treatment.

34. The method according to any one of claims 1 to 33, wherein the reference sample comprises a reference tissue or reference cells.

35. The method according to claim 34, wherein the reference sample is derived from a healthy subject or a non-diseased sample of the subject.

36. The method according to any one of claims 1 to 35, wherein the responsiveness comprises a relative increase in one or more of the following: overall survival (OS), progression-free survival (PFS), complete response (CR), partial response (PR) and combinations thereof.

37. The method according to any one of claims 1 to 36, wherein the level of CD73 comprises the proportion of CD73-positive cells among all cells in the sample.

38. The method according to claim 37, wherein the level of CD73 comprises the higher of the proportion of CD73-positive tumor cells among all tumor cells in the sample or the proportion of CD73-positive immune cells among all immune cells in the sample.

39. The method according to claim 38, wherein a CD73 level of 30% or higher identifies the subject as potentially responsive to or likely to benefit from the combined treatment.

40. The method according to claim 38, wherein a CD73 level of 35%, 40%, 45% or 50% or higher identifies the subject as potentially responsive to or likely to benefit from the combined treatment.

41. The method according to claim 37, wherein the level of CD73 comprises the higher of the proportion of CD73-positive tumor cells among all tumor cells in the sample or the proportion of CD73-positive immune cells among all immune cells in the sample, wherein the positive is at least moderately positive.

42. The method according to claim 41, wherein a CD73 level of 10% or higher identifies the subject as potentially responsive to or likely to benefit from the combined treatment.

43. A level of CD73 of 15%, 20%, 25%, 30%, 35% or 40% or higher identifies the subject as potentially responsive to or likely to benefit from the combination treatment, according to the method of claim 41.

44. A kit or article of manufacture for use in the method according to any one of claims 1 to 43, wherein the kit or article of manufacture comprises (1) one or more reagents for determining the level of CD73 in a sample derived from a subject having NSCLC, and (2) a package insert, wherein the package insert suggests treating the subject with a CD73 antagonist and a PD1 / PD-L1 antagonist based on the level of CD73. A kit or article of manufacture comprising the above.

45. A kit or article of manufacture for use in the method according to any one of claims 1 to 43, wherein the kit or article of manufacture comprises (1) one or more reagents for determining the level of CD73 in a sample derived from a subject having NSCLC, and (2) a package insert, wherein the package insert suggests that an increase in the level of CD73 in the sample, compared to that in a reference sample, indicates that the subject is more likely to respond to or benefit from combination treatment with a CD73 antagonist and a PD1 / PD-L1 antagonist. A kit or article of manufacture comprising the above.

46. The kit or article of manufacture according to claim 44 or 45, wherein the reagent is a CD73 antibody.

47. The CD73 antibody is selected from the group consisting of D7F9A (CST, catalog number 13160), 606117 (R&D), EPR6114 (Abcam, catalog number ab133582), 4G6E3 (Abcam, catalog number ab202122), NT5E / 2503 (Abcam, catalog number ab257309), NT5E / 2505 (Abcam, catalog number ab257310), NT5E / 2545 (Abcam, catalog number ab257311), NT5E / 2646 (Abcam, catalog number ab257312), 7G2 (Thermo Fisher, catalog number 1D7), JM11-40 (Thermo Fisher, catalog number JM11-40), CD73 recombinant rabbit monoclonal antibody (1) (Invitrogen, catalog number MA5-29454), BLR054F (Thermo Fisher, catalog number BLR054F), sc-32299 (Santa Cruz, catalog number IE9), and AD2 (Biorad, catalog number AD2), the kit or article of manufacture according to claim 46.

48. Use of a CD73 antagonist and a PD1 / PD-L1 antagonist for the manufacture of a medicament for treating a subject having NSCLC having a level of CD73 in a sample shown to be responsive to or likely to benefit from a combination treatment of the CD73 antagonist and the PD1 / PD-L1 antagonist.

49. Use of a CD73 antagonist for the manufacture of a medicament for treating a subject having NSCLC having a level of CD73 in a sample shown to be responsive to or likely to benefit from a combination treatment of the CD73 antagonist and the PD1 / PD-L1 antagonist, wherein the subject is also administered the PD1 / PD-L1 antagonist.

50. Use of a PD1 / PD-L1 antagonist for the manufacture of a medicament for treating a subject having NSCLC having a level of CD73 in a sample shown to be responsive to or likely to benefit from a combination treatment of the CD73 antagonist and the PD1 / PD-L1 antagonist, wherein the subject is also administered the CD73 antagonist.

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