PD-1 / TIGIT binding protein for cancer treatment

Abstract

This disclosure relates to a method for treating cancer by administering to a subject a binding protein containing antibodies that bind to programmed death-1 ("PD-1") and T cell immune receptors having Ig and ITIM domains ("TIGIT") in doses ranging from approximately 70 mg to approximately 1500 mg.

Description

[Technical Field]

[0001] This disclosure provides a method for treating cancer in a subject, comprising administering to the subject in an amount of approximately 70 mg to approximately 1500 mg of a bispecific binding protein that specifically binds to programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT). This disclosure further provides a method for producing such a bispecific binding protein and a composition comprising a pharmaceutical composition containing such bispecific protein. [Background technology]

[0002] Programmed Cell Death-1 (PD-1) is an approximately 31kD type I membrane protein that is a member of the extended CD28 / CTLA4 family of T cell regulators (see Ishida et al., Induced Expression of PD-1, A Novel Member of the Immunoglobulin Gene Superfamily, Upon Programmed Cell Death, EMBO J. 1992, 11: 3887-95). PD-1 is expressed on activated T cells, B cells, and monocytes, and at low levels in natural killer (NK) T cells. PD-1 is a well-validated target for immunomediated therapy in oncology. Antagonistic suppression of the PD-1 / PD-L1 interaction increases T cell activation and enhances the recognition and elimination of tumor cells by the host immune system.

[0003] T-cell immune receptors (TIGITs) possessing Ig and ITIM domains are immune receptors present on several T cells and natural killer (NK) cells. TIGITs are upregulated by immune cells, including activated T cells, natural killer cells, and regulatory T cells. [Overview of the project]

[0004] This disclosure provides a method for treating cancer in a subject, comprising administering a bispecific binding protein that specifically binds to PD-1 and TIGIT in an amount ranging from approximately 70 mg to approximately 1500 mg. The bispecific binding protein comprises: a) a first binding domain that specifically binds to PD-1, the first binding domain comprising a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, and HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 4, LCDR2 having the amino acid sequence of SEQ ID NO: 5, and LCDR3 having the amino acid sequence of SEQ ID NO: 6; and b) a second binding domain that specifically binds to TIGIT, the second binding domain comprising a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 11, HCDR2 having the amino acid sequence of SEQ ID NO: 12, and HCDR3 having the amino acid sequence of SEQ ID NO: 13, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 14, LCDR2 having the amino acid sequence of SEQ ID NO: 15, and LCDR3 having the amino acid sequence of SEQ ID NO: 16.

[0005] In some embodiments, the amount of bispecific binding protein administered is approximately 70 mg, 150 mg, 210 mg, 450 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1250 mg, or 1500 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 750 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 1500 mg.

[0006] In some embodiments, the bispecific binding protein is administered once per treatment cycle. In some embodiments, the treatment cycle is approximately 7 days, 14 days, 21 days, 28 days, or 35 days. In some embodiments, the treatment cycle is approximately 7 days. In some embodiments, the treatment cycle is repeated for up to 35 cycles.

[0007] In some embodiments, the bispecific binding protein is administered to the subject as monotherapy. In some embodiments, the bispecific binding protein is administered by intravenous infusion (IV). In some embodiments, the subject has not received any prior systemic therapy. In some embodiments, the subject has not received any prior chemotherapy.

[0008] In some embodiments, cancer includes cancer cells that express PD-L1.

[0009] In some embodiments, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 9.

[0010] In some embodiments, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9.

[0011] In some embodiments, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain having the amino acid sequence of SEQ ID NO: 8 and a light chain having the amino acid sequence of SEQ ID NO: 10.

[0012] In some embodiments, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10.

[0013] In some embodiments, the second binding domain of the bispecific binding protein that specifically binds to TIGIT comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 19.

[0014] In some embodiments, the second binding domain of the bispecific binding protein that specifically binds to TIGIT comprises a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19.

[0015] In some embodiments, the second binding domain of the bispecific binding protein that specifically binds to TIGIT comprises a heavy chain having the amino acid sequence of SEQ ID NO: 18 and a light chain having the amino acid sequence of SEQ ID NO: 20.

[0016] In some embodiments, the second binding domain of the bispecific binding protein that specifically binds to TIGIT comprises a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 18 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20.

[0017] In some embodiments, the bispecific binding protein is a human or humanized bispecific antibody or an antigen-binding fragment thereof.

[0018] In some embodiments, the bispecific binding protein includes a variant Fc region. In some embodiments, the variant Fc region of the bispecific binding protein is numbered according to the EU index defined by Kabat, as follows: 221K, 221Y, 225E, 225K, 225W, 228P, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235E, 235F, 236E, 237L, 237M, 237 P, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240 M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245 A, 247L, 247V, 247G, 250E, 250Q, 251F, 252L, 252Y, 254S, 254T, 255L, 256 E, 256F, 256M, 257C, 257M, 257N, 262I, 262A, 262T, 262E, 263I, 263A, 263T , 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265A, 265G , 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T , 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P , 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 296G, 297S , 297D, 297E, 298A, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V , 299H, 299F, 299E, 305I, 308F, 313F, 316D, 318A, 318S, 320A, 320S, 322A , 322S, 325Q, 325L, 3251, 325D, 325E, 325A, 325T, 325V, 325H, 326A, 326D , 326E, 326G, 326M, 326V, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E,It comprises at least one substitution selected from 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 333A, 333D, 333G, 333Q, 333S, 333V, 334A, 334E, 334H, 334L, 334M, 334Q, 334V, 334Y, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 428L, 428F, 433K, 433L, 434A, 434W, 434Y, 436H, 440Y and 443W.

[0019] In some embodiments, the variant Fc region of the bispecific binding protein comprises one or more amino acid substitutions at positions selected from 428 and 434 when numbered according to the EU index defined by Kabat. In some embodiments, the variant Fc region of the bispecific binding protein comprises one or more amino acid substitutions selected from 428L, 428F, 434A, 424F, 434W, and 434Y.

[0020] In some embodiments, the variant Fc region of the bispecific binding protein comprises the YTE mutation (M252Y / S254T / T256E). In some embodiments, the Fc variant region of the bispecific binding protein comprises the L234F / L235E / P331S triple mutation (TM).

[0021] In some embodiments, the Fc region of the bispecific binding protein is aglycosylated. In some embodiments, the Fc region of the bispecific binding protein is deglycosylated.

[0022] In some embodiments, the Fc region of the bispecificity binding protein has reduced fucosylation or is not fucosylated at all.

[0023] In some embodiments, the bispecificity-binding protein includes a kappa light chain constant region. In some embodiments, the bispecificity-binding protein includes a lambda light chain constant region.

[0024] In some embodiments, the bispecificity-binding protein is an antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is humanized.

[0025] In some embodiments, the cancer is one or more of the following: ovarian cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, pancreatic cancer, renal cell carcinoma, and lung cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is advanced or metastatic.

[0026] In some embodiments, subjects have a PD-L1 tumor percentage score of 1% or higher. In some embodiments, subjects have a PD-L1 tumor percentage score of 50% or higher. In some embodiments, subjects are checkpoint inhibitor (CPI) naive.

[0027] In some embodiments, the disclosure further provides pharmaceutical compositions comprising bispecific binding proteins that specifically bind to PD-1 and TIGIT in amounts ranging from about 70 mg to about 1500 mg. In some embodiments, the bispecific binding protein comprises: a) a first binding domain that specifically binds to PD-1, wherein the first binding domain comprises a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, and HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 4, LCDR2 having the amino acid sequence of SEQ ID NO: 5, and LCDR3 having the amino acid sequence of SEQ ID NO: 6; and b) a second binding domain that specifically binds to TIGIT, wherein the second binding domain comprises a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 11, HCDR2 having the amino acid sequence of SEQ ID NO: 12, and HCDR3 having the amino acid sequence of SEQ ID NO: 13, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 14, LCDR2 having the amino acid sequence of SEQ ID NO: 15, and LCDR3 having the amino acid sequence of SEQ ID NO: 16.

[0028] In some embodiments, the pharmaceutical composition contains approximately 70 mg, approximately 150 mg, approximately 210 mg, approximately 450 mg, approximately 750 mg, approximately 800 mg, approximately 850 mg, approximately 900 mg, approximately 950 mg, approximately 1000 mg, approximately 1250 mg, or approximately 1500 mg of bispecific binding protein. In some embodiments, the pharmaceutical composition contains approximately 750 mg of bispecific binding protein. In some embodiments, the pharmaceutical composition contains approximately 1500 mg of bispecific binding protein.

[0029] In some embodiments where the cancer is NSCLC, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 9.

[0030] In some embodiments, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9.

[0031] In some embodiments, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain having the amino acid sequence of SEQ ID NO: 8 and a light chain having the amino acid sequence of SEQ ID NO: 10.

[0032] In some embodiments, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10.

[0033] In some embodiments, the second binding domain of the bispecific binding protein that specifically binds to TIGIT includes a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 19.

[0034] In some embodiments, the second binding domain of the bispecific binding protein that specifically binds to TIGIT includes a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19.

[0035] In some embodiments, the second binding domain of the bispecific binding protein that specifically binds to TIGIT includes a heavy chain having the amino acid sequence of SEQ ID NO: 18 and a light chain having the amino acid sequence of SEQ ID NO: 20.

[0036] In some embodiments, the second binding domain of a bispecific binding protein that specifically binds to TIGIT includes a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 18 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20.

[0037] This disclosure further provides a kit comprising any of the above-described pharmaceutical compositions. In some embodiments, the kit further includes instructions for administering the pharmaceutical composition.

[0038] This disclosure further provides the pharmaceutical compositions defined above for use in the treatment of cancer.

[0039] In some embodiments of the pharmaceutical composition for use, the cancer is one or more of the following: ovarian cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, pancreatic cancer, renal cell carcinoma, and lung cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is advanced or metastatic.

[0040] In some embodiments of the pharmaceutical composition for use, the cancer is NSCLC and has a PD-L1 tumor percentage score of 1% or higher. In some embodiments of the pharmaceutical composition for use, the cancer is NSCLC and has a PD-L1 tumor percentage score of 50% or higher.

[0041] In some embodiments of the pharmaceutical composition for use, the cancer has not been previously treated with a checkpoint inhibitor. [Brief explanation of the drawing]

[0042] The following drawings form part of this specification and are included to further illustrate the exemplary aspects of this disclosure.

[0043] [Figure 1]Figure 1 shows the predicted receptor occupancy percentages for PD-1 and TIGIT in tumors. Trough concentrations were used for prediction; the model assumes that the impact of ADA on PK is limited. Low / high tumor PC = 5% / 50%, low / high tumor PD-1 RC = 3 / 30nM, low / high tumor TIGIT RC = 4 / 40nM, ADA = anti-drug antibody; Ig = immunoglobulin; PC = partition coefficient; PD-1 = programmed cell death-1; PK = pharmacokinetics; RC = receptor concentration; RO = receptor occupancy; TIGIT = T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domain.

[0044] [Figure 2] Figure 2 is a schematic diagram illustrating an example of a treatment method that includes a dose escalation phase (Part A) and three dose expansion phases (Parts B-D).

[0045] [Figure 3] Figure 3 shows the adverse events that occurred in more than 5% of the 80 subjects evaluated in the Part A and Part B trials described in Example 8 below. Adverse events related to AZD2936 are plotted on the right side of the figure.

[0046] [Figure 4] Figure 4 shows the adverse events that occurred in more than 5% of the 54 subjects who received AZD2936, as evaluated in Part A and Part B trials described in Example 8 below. Adverse events related to AZD2936 are plotted on the right side of the figure.

[0047] [Figure 5] Figure 5 shows the adverse events that occurred in more than 5% of the 83 subjects who received AZD2936, as evaluated in the Part A and Part B expanded studies described in Example 11 below. Treatment-induced adverse events (TEAEs) are plotted on the left side of the figure, and treatment-related adverse events (TRAEs) are plotted on the right side.

[0048] [Figure 6]Figure 6 shows the adverse events that occurred in 54 subjects who received the recommended Phase 2 dose (RP2D) of AZD2936, as evaluated in the Part A and Part B expanded studies described in Example 11 below. TEAEs are plotted on the left side of the figure, and TRAEs are plotted on the right side.

[0049] [Figure 7-1] Figure 7 is a waterfall plot of post-baseline scans for the 83 subjects described in Example 11. The plot shows the best percentage change in target lesion size from baseline. [Figure 7-2] Same as above.

[0050] [Figure 8] Figure 8 plots the duration of exposure to AZD2936 for each subject, measured on a weekly basis, corresponding to the results in Figure 7.

[0051] [Figure 9A] Figures 9A–9C show the pharmacodynamics of two subjects treated with PR2D. Figures 9A and 9B show the radiographic response at week 9 for the first and second subjects, respectively. Figure 9C is a plot of the two subjects who showed a 100% reduction in circulating tumor deoxyribonucleic acid (ctDNA) at week 6. Abbreviations in Figures 9A–9C: ctDNA, circulating tumor DNA; LN, lymph node; mVAF, mean variant allele frequency; PD-L1, programmed cell death ligand-1; RP2D, recommended phase 2 dose; TL, target lesion. [Figure 9B] Same as above. [Figure 9C] Same as above.

[0052] [Figure 10] Figure 10 shows the adverse events that occurred in more than 10% of the 54 subjects who received AZD2936 at a dose of 750 mg Q3W in the Part A and Part B trials described in Example 8 below. Adverse events related to AZD2936 are plotted on the right side of the figure.

[0053] [Figure 11] Figure 11 shows the adverse events that occurred in more than 10% of 19 subjects who received AZD2936 at a dose of 750 mg Q3W, as evaluated in the Part C trial described in Example 12 below. Adverse events related to AZD2936 are plotted on the right side of the figure.

[0054] [Figure 12] Figure 12 shows the adverse events that occurred in more than 10% of the 13 subjects who received AZD2936 at a dose of 1500 mg Q3W, as evaluated in the Part D study described in Example 13 below. Adverse events related to AZD2936 are plotted on the right side of the figure.

[0055] [Figure 13] Figure 13 shows the adverse events that occurred in more than 10% of the 14 subjects who received AZD2936 at a dose of 750 mg Q3W in the Part D study described in Example 13 below. Adverse events related to AZD2936 are plotted on the right side of the figure.

[0056] [Figure 14] Figure 14 shows the best percentage change from baseline in target lesion size in 19 subjects who received AZD2936 at 750 mg and 1500 mg Q3W, as evaluated in Part C and D trials described in Examples 12 and 13 below. Each bar corresponds to one subject and is color-coded to reflect the best overall response.

[0057] [Figure 15] Figure 15 shows the change in target lesion size over time in 19 subjects who received AZD2936 at 750 mg and 1500 mg Q3W, as evaluated in the Part C and D trials described in Examples 12 and 13 below. Each line corresponds to a subject and is color-coded to reflect the best overall response. [Modes for carrying out the invention]

[0058] definition Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings that are ordinarily understood by those skilled in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms.

[0059] As used herein, "a" or "an" may mean one or more. As used herein, when used with the word "comprising," "a" or "an" may mean one or more. As used herein, "another" or "a further" may mean at least two or more.

[0060] This disclosure supports the definitions of "choices only" and "and / or," however, the use of the term "or" in a claim is used to mean "and / or" unless explicitly indicated to refer to "choices only" or the choices are mutually exclusive.

[0061] As used herein, the terms “comprising” (and any variations or forms of “comprising,” such as “comprise” and “comprises”), “having” (and any variations or forms of “having,” such as “have” and “has”), “including” (and any variations or forms of “including,” such as “includes” and “include”), or “containing” (and any variations or forms of “containing,” such as “contains” and “contain”) are inclusive or open-ended and do not exclude any additional, undescribed elements or processes of method.

[0062] Throughout this application, the term “about” is used to indicate a numerical value that includes the inherent variation in the error of the method / device employed to determine the value, or the variation present among the objects under test. Typically, the term “about” means to include, depending on the context, variation of less than or greater than (which may be “greater” or “less” than) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, a person skilled in the art will understand the level of variation indicated by the term “about” from the context in which it is used herein. The use of the term “about” should be understood to also include the values ​​specifically stated.

[0063] The use of “for example” and its corresponding abbreviation “e.g.” (which may or may not be italicized) means that the specific terms described are representative examples and aspects of the disclosure and are not intended to be limited to the specific examples referenced or cited unless expressly stated otherwise.

[0064] The ranges provided herein, of any kind, include all numerical values ​​within a given range and values ​​approximately to the values ​​at the ends of that range. Where used herein, "between" refers to a range that includes the ends of the range. For example, the numbers between x and y explicitly include the numbers x and y, as well as any numbers within the range of x and y.

[0065] As used herein, the term "antibody" refers to a protein that can recognize and specifically bind to an antigen. Conventional mammalian antibodies typically consist of a tetramer of two identical pairs of polypeptide chains, with each pair consisting of one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50 - 70 kDa). The terms "heavy chain" and "light chain" as used herein refer to any immunoglobulin polypeptide having a variable domain sequence sufficient to confer specificity to a target antigen. The amino-terminal portion of each light and heavy chain typically contains a variable domain of about 100 to 110 or more amino acids that is responsible for antigen recognition. The carboxyl-terminal portion of each chain typically defines a constant domain responsible for effector functions. Thus, in a naturally-derived antibody, a full-length heavy-chain immunoglobulin polypeptide contains a variable domain (V H ) and three constant domains (C H1 , C H2 and C H3 ) and a hinge region between C H1 and C H2 , with the V H ]> domain present at the amino-terminus of the polypeptide and the C H3 domain present at the carboxyl-terminus. A full-length light-chain immunoglobulin polypeptide contains a variable domain (V L ) and a constant domain (C L ), with the V L domain present at the amino-terminus of the polypeptide and the C L domain present at the carboxyl-terminus. One of ordinary skill in the art will recognize, however, that the domain positions in a naturally-derived antibody can be altered in certain antibody-like binding protein formats without loss of antigen-binding ability. The classes of human light chains are called kappa and lambda light chains.

[0066] In some embodiments, the light-chain constant region is a kappa chain. In some embodiments, the light-chain constant region is a lambda chain.

[0067] Within the full-length light and heavy chains, the variable and constant domains are typically linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 10 or more amino acids. The variable region of each light / heavy chain pair typically forms an antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure as a relatively conserved framework region (FR) linked by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs of the two chains in each pair are typically aligned by the framework region and may be able to bind to specific epitopes. From the amino-terminus to the carboxyl-terminus, both the light and heavy chain variable domains typically contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains.

[0068] The term "antibody fragment" refers to a complete or full-length chain or a portion of an antibody, generally known as a target-binding or variable region. Examples of antibody fragments include, but are not limited to, F ab F ab’ F (ab’)2 and F v Fragments are included. As used herein, the term “functional fragment” is generally synonymous with “antibody fragment,” and with respect to antibodies, F v F ab F (ab’)2 This could refer to antibody fragments such as those mentioned above.

[0069] The numbering of amino acid residues described herein is based on the EU numbering system (as also described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0070] The term "human antibody," as used herein, includes antibodies having variable and constant regions substantially corresponding to human germline immunoglobulin sequences. In some embodiments, human antibodies are produced in non-human mammals, including, but not limited to, rodents such as mice and rats, and rabbits such as rabbits. In other embodiments, human antibodies are produced in hybridoma cells. In yet another embodiment, human antibodies are produced by recombination. In some embodiments, the bispecific binding protein is a human antibody or humanized antibody.

[0071] As used herein, the terms “antigen” or “target antigen” refer to a molecule or part of a molecule that can be recognized or bound by the binding protein of this disclosure. A target antigen can be used in animals that produce antibodies capable of binding to the epitope of the antigen. A target antigen may have one or more epitopes.

[0072] As used herein, the term “epitope” refers to a region or structural element of an antigen that is recognized and bound by the binding protein of this disclosure. More precisely, an epitope is a specific structure to which a binding protein’s CDR is bound. Epitopes may also include protein structural elements, carbohydrates, or parts of lipid structures found in membranes. A binding protein is said to bind specifically to an antigen when it preferentially recognizes its antigen target in a mixed complex of proteins and / or macromolecules. The term “specific binding” refers to a binding protein that specifically binds to a molecule or a fragment of it (e.g., an antigen). A binding protein that specifically binds to a molecule or a fragment of it may bind to other molecules with lower affinity, as determined, for example, by an immunoassay, BIAcore, or other assay known in the art. In particular, an antibody or fragment that specifically binds to at least one molecule or fragment of it can competitively remove molecules to which it nonspecifically binds. This disclosure specifically includes antibodies with multiple specificities (e.g., antibodies with specificity to two or more different antigens). For example, a bispecific antibody can bind to two adjacent epitopes on a single target antigen, or it can bind to two different antigens.

[0073] As used herein, the term “native Fc” refers to a molecule containing a sequence of non-antigen-binding fragments resulting from the digestion of an antibody or otherwise produced, in monomeric or polymeric form, and potentially containing a hinge region. The source of the immunoglobulin from which native Fc originates is preferably of human origin and may be any immunoglobulin. Native Fc molecules consist of monomeric polypeptides that can be linked into dimeric or polymeric forms by covalent (i.e., disulfide bonds) and non-covalent interactions. The number of intermolecular disulfide bonds between monomeric subunits of a native Fc molecule depends on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2) and ranges from 1 to 4. An example of native Fc is a disulfide-bonded dimer resulting from the papain digestion of IgG. As used herein, the term “native Fc” is generally applied to monomeric, dimeric, and polymeric forms.

[0074] As used herein, the term “Fc variant” refers to a molecule or sequence modified from native Fc, but still containing a binding site to the salvage receptor, FcRn (embryonic Fc receptor). Exemplary Fc variants and their interactions with salvage receptors are known in the art. Therefore, the term “Fc variant” may include molecules or sequences humanized from non-human native Fc. Furthermore, native Fc may contain regions in which specific residues providing structural features or biological activity not required for the binding proteins of this disclosure can be removed or mutated to create modified Fc variants. Therefore, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues, or have one or more Fc sites or residues modified, which affect or involve (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity in expression in selected host cells, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cell-mediated cytotoxicity (ADCC).

[0075] As used herein, the term "Fc domain" encompasses the native Fc, Fc variants, and sequences defined above. Similar to Fc variants and native Fc molecules, the term "Fc domain" includes monomeric or polymeric molecules, whether digested from the whole antibody or produced by other means.

[0076] The term “to treat” or “treatment” refers to administering a compound or pharmaceutical composition to a subject in order to bring about a change or improvement in a disease, disorder, or condition in that subject. As used herein, the term “treatment” or “to treat” may refer to both therapeutic treatments and disease-preventive or preventive measures. Subjects requiring treatment include those having a disease or condition, as well as those susceptible to the disease or condition, or those for whom the disease or condition should be prevented.

[0077] The term “dose” refers to a specific amount of a compound or medicinal substance delivered in a single administration or within a specific period. In some embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in some embodiments, where subcutaneous administration is desired, the desired dose may require a volume that cannot be easily contained in a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In some embodiments, a dose may be administered in two or more injections to minimize injection site reactions in the individual. In other embodiments, the compound or medicinal substance is administered by infusion over an extended period or continuously. A dose may be expressed as the amount of medicinal substance per hour, day, week, or month.

[0078] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to mammalian subjects. In one embodiment, “subject” is a human, a domesticated animal, a livestock animal, a sports animal, and a zoo animal, such as a human, a non-human primate, a dog, a cat, a guinea pig, a rabbit, a rat, a mouse, a horse, a cow, etc. In one embodiment, the subject is a cynomolgus macaque (Macaca fascicularis). In a preferred embodiment, the subject is a human. In the method of the present invention, the subject may not have been previously diagnosed with cancer. Alternatively, the subject may have been previously diagnosed with cancer. The subject may be a subject exhibiting disease risk factors, or a subject with asymptomatic cancer. The subject may have cancer in progress, or a subject at risk of cancer progression. Thus, in one embodiment, the method of the present invention can be used to confirm the presence of cancer in a subject. For example, the subject may have been previously diagnosed with cancer by other means. In one embodiment, the subject has previously received cancer treatment. A subject being "checkpoint inhibitor naive" or "CPI naive" means a subject whose cancer has not been previously treated with a checkpoint inhibitor (CPI).

[0079] The term "effectiveness" refers to the ability to produce a desired effect. A "therapeutic effective dose" or "therapeutic dose" is the amount sufficient to produce the desired clinical outcome (i.e., achieve therapeutic effectiveness). A therapeutic effective dose can be administered in one or more doses.

[0080] The term “adverse reaction” means any physiological disorder and / or condition resulting from a procedure other than the desired effect. In some embodiments, adverse reactions include injection site reactions, abnormal liver function tests, abnormal kidney function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, myopathy, and fatigue. For example, elevated serum aminotransferase levels may indicate hepatotoxicity or abnormal liver function. For example, elevated bilirubin may indicate hepatotoxicity or abnormal liver function. “Disease” or “condition” means any condition from which a person can benefit from a procedure using the methods of this disclosure. “Disease” and “condition” are used interchangeably herein and include chronic and acute disorders or diseases, including pathological conditions that make a patient susceptible to the disorder in question. In some embodiments, disease is a tumor. In some embodiments, disease is a solid tumor. In some embodiments, disease is cancer. In some embodiments, the cancer is one or more of the following: ovarian cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, pancreatic cancer, renal cell carcinoma, and lung cancer. In some embodiments, the disease is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is advanced or metastatic. In some embodiments, advanced NSCLC is stage III or stage IV NSCLC.

[0081] As used herein, the terms “administer” or “to administer” refer to providing, contacting, and / or delivering one or more compounds by any suitable route to achieve the desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-arterial, intra-synovial, intra-sternal, intrathecal, intra-lesional, or intracranial injection), percutaneous, topical, buccal, rectal, vaginal, nasal, ocular, inhalation, and implantation.

[0082] As used herein, the terms “pharmaceutical composition” or “therapeutic composition” refer to a compound or composition that, when appropriately administered to a subject, can induce a desired therapeutic effect. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the binding protein of the present disclosure.

[0083] As used herein, the terms “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” refer to one or more formulation materials suitable for accomplishing or enhancing the delivery of one or more binding proteins of this disclosure.

[0084] Cancer treatment methods This disclosure relates to a method for treating cancer in a subject, comprising administering to the subject a bispecific binding protein that specifically binds to PD-1 and TIGIT in an amount ranging from approximately 70 mg to approximately 1500 mg. This disclosure also provides compositions and kits comprising a pharmaceutical composition containing such bispecific protein.

[0085] As used herein, a bispecific binding protein has binding specificity to at least two independent antigens (or targets) or different epitopes within the same antigen. An exemplary bispecific binding protein may bind to two different epitopes of a target or to two different targets. Other such binding proteins may combine a second binding site for another target with a first target binding site. In some embodiments, the binding protein is a bispecific antibody.

[0086] In some embodiments, bispecific antibodies provide additional and / or synergistic therapeutic effects derived from simultaneously targeting two antigens with the administration of a single manufactured molecule.

[0087] In some embodiments, the antibodies provided herein are monovalent bispecific antibodies (MBabs). The monovalent bispecific antibody scaffolds described herein provide an excellent platform for bispecific antibody production that satisfies all the benefits associated with bispecific antibodies while reducing the potential therapeutic risks described above due to their monovalent nature. Furthermore, the MBabs provided herein are likely to be readily expressed, stable, and have low immunogenicity. The term “monovalent bispecific,” which may be abbreviated as “MBab” as used herein, refers to a bispecific antibody in which each arm can specifically bind to a different target antigen, and for a given pair of different target antigens (A and B), the MBab can bind to one of them. In certain embodiments, a monovalent bispecific antibody can specifically bind to two independent antigens (or targets) or two independent epitopes on the same antigen. Typically, a monovalent bispecific antibody contains two different variable regions. In some embodiments, the binding affinities to the two independent antigens are nearly the same. In some embodiments, the binding affinities to the two independent antigens are different.

[0088] In some embodiments, the bispecific binding protein comprises: a) a first binding domain that specifically binds to PD-1, wherein the first binding domain comprises a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, and HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 4, LCDR2 having the amino acid sequence of SEQ ID NO: 5, and LCDR3 having the amino acid sequence of SEQ ID NO: 6; and b) a second binding domain that specifically binds to TIGIT, wherein the second binding domain comprises a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 11, HCDR2 having the amino acid sequence of SEQ ID NO: 12, and HCDR3 having the amino acid sequence of SEQ ID NO: 13, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 14, LCDR2 having the amino acid sequence of SEQ ID NO: 15, and LCDR3 having the amino acid sequence of SEQ ID NO: 16.

[0089] In some embodiments, the first binding domain that specifically binds to PD-1 includes a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the first binding domain that specifically binds to PD-1 includes a heavy chain having the amino acid sequence of SEQ ID NO: 8 and a light chain having the amino acid sequence of SEQ ID NO: 10.

[0090] In some embodiments, the first binding domain that specifically binds to PD-1 includes a heavy chain variable domain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the first binding domain that specifically binds to PD-1 includes a light chain variable domain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 9.

[0091] In some embodiments, the first binding domain that specifically binds to PD-1 includes a heavy chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first binding domain that specifically binds to PD-1 includes a light chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 10.

[0092] In some embodiments, the second binding domain that specifically binds to TIGIT includes a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 19. In some embodiments, the second binding domain that specifically binds to TIGIT includes a heavy chain having the amino acid sequence of SEQ ID NO: 18 and a light chain having the amino acid sequence of SEQ ID NO: 20.

[0093] In some embodiments, the second binding domain that specifically binds to TIGIT includes a heavy chain variable domain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the second binding domain that specifically binds to TIGIT includes a light chain variable domain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 19.

[0094] In some embodiments, the second binding domain that specifically binds to TIGIT includes a heavy chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the second binding domain that specifically binds to TIGIT includes a light chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 20.

[0095] In some embodiments, the first binding domain that specifically binds to PD-1 includes a heavy chain variable domain encoded by the nucleic acid sequence of SEQ ID NO: 21 and a light chain variable domain encoded by the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the first binding domain that specifically binds to PD-1 includes a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 22 and a light chain encoded by the nucleic acid sequence of SEQ ID NO: 24.

[0096] In some embodiments, the first binding domain that specifically binds to PD-1 includes a heavy chain variable domain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 21, and a light chain variable domain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 23.

[0097] In some embodiments, the first binding domain that specifically binds to PD-1 includes a heavy chain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 22, and a light chain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 24.

[0098] In some embodiments, the second binding domain that specifically binds to TIGIT includes a heavy chain variable domain encoded by the nucleic acid sequence of SEQ ID NO: 25 and a light chain variable domain encoded by the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the second binding domain that specifically binds to TIGIT includes a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 26 and a light chain encoded by the nucleic acid sequence of SEQ ID NO: 28.

[0099] In some embodiments, the second binding domain that specifically binds to TIGIT includes a heavy chain variable domain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 25, and a light chain variable domain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 27.

[0100] In some embodiments, the second binding domain that specifically binds to TIGIT includes a heavy chain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 26, and a light chain encoded by a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 28.

[0101] In some embodiments, the Fc region is a domain or contains one or more Fc regions derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0102] In some embodiments, bispecific binding proteins include a variant Fc region. Fc region engineering is widely used in the art to extend the half-life of therapeutic antibodies and protect them from in vivo degradation. In some embodiments, the Fc region or antigen-binding fragment of an IgG antibody can be modified to increase the affinity of the IgG molecule to the embryonic Fc receptor (FcRn), which mediates IgG metabolism and protects the IgG molecule from degradation.

[0103] In some embodiments, the variant Fc region of the bispecific binding protein is numbered according to the EU index defined by Kabat as 221K, 221Y, 225E, 225K, 225W, 228P, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 2 35P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235E, 235F, 236E, 237L, 237M, 237P, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 250E, 250Q, 251F, 252L, 252Y, 254S, 254T, 255L, 25 6E, 256F, 256M, 257C, 257M, 257N, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265A, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 26 5T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 296G, 297S, 297D, 297E, 298A, 298H, 298I, 29 8T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 308F, 313F, 316D, 318A, 318S, 320A, 320S, 322A, 322S, 325Q, 325L, 3251, 325D, 325E, 325A, 325T, 325V, 325H, 326A, 326D, 326E, 326G, 32 6M, 326V, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H,328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331 W, 331K, 331Q, 331E, 331S, 331V, 3311, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 3 Includes at least one substitution selected from 32T, 332H, 332Y, 332A, 333A, 333D, 333G, 333Q, 333S, 333V, 334A, 334E, 334H, 334L, 334M, 334Q, 334V, 334Y, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 428L, 428F, 433K, 433L, 434A, 434W, 434Y, 436H, 440Y, and 443W.

[0104] In some embodiments, the variant Fc region includes one or more modifications at positions selected from 428 and 434 when numbered according to the EU index defined in Kabat. In some embodiments, the variant Fc region includes one or more amino acid substitutions at positions selected from 428 and 434 when numbered according to the EU index defined in Kabat. In some embodiments, the variant Fc region includes one or more amino acid substitutions selected from 428L, 428F, 434A, 424F, 434W, and 434Y.

[0105] In some embodiments, the variant Fc region of the bispecific binding protein contains one or more amino acid substitutions at positions selected from 428 and 434 when numbered by the EU index defined in Kabat. In some embodiments, the variant Fc region of the bispecific binding protein contains one or more amino acid substitutions selected from 428L, 428F, 434A, 424F, 434W, and 434Y. In some embodiments, the variant Fc region of the bispecific binding protein contains a YTE mutation (M252Y / S254T / T256E).

[0106] In some embodiments, the Fc variant antibody or its conjugated fragment exhibits reduced antibody-dependent cell-mediated cytotoxicity (ADCC) when administered in vivo. In some embodiments, the Fc variant antibody or its conjugated fragment exhibits reduced ADCC compared to an antibody or its conjugated variant containing a wild-type Fc region. In some embodiments, the Fc variant antibody or its conjugated fragment does not trigger ADCC when administered in vivo. In some embodiments, the Fc variant antibody or its conjugated fragment induces reduced ADCC when administered in vivo. In some embodiments, the Fc variant antibody or its conjugated fragment, which has reduced ADCC activity or does not have ADCC activity, contains the L234F / L235E / P331S triple mutation (TM) within the variant Fc region.

[0107] In some embodiments, an antibody or its conjugated fragment having reduced CDC activity exhibits reduced toxicity when administered to a subject. In some embodiments, an antibody or its conjugated fragment having reduced ADCC activity exhibits reduced toxicity when administered to a subject.

[0108] In some embodiments, the Fc region of the bispecific binding protein is aglycosylated. In some embodiments, the Fc region of the bispecific binding protein is deglycosylated. In some embodiments, the Fc region of the bispecific binding protein has reduced fucosylation or is afucosylated.

[0109] In some embodiments, the bispecificity-binding protein includes a kappa light chain constant region. In some embodiments, the bispecificity-binding protein includes a lambda light chain constant region.

[0110] In some embodiments, the bispecificity-binding protein is an antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is humanized.

[0111] In certain embodiments, the Disclosure provides methods for inducing an immune response in a subject by administering a protein, nucleic acid molecule, and / or composition to the subject, and for treating or preventing tumors and / or cancer in the subject.

[0112] In certain embodiments, the present invention provides a method for inducing an immune response in a subject, comprising administering a bispecific protein described herein to the subject. In one embodiment, the present invention provides a method for inducing an immune response in a subject, comprising administering a nucleic acid described herein to the subject. In one embodiment, the present invention provides a method for inducing an immune response in a subject, comprising administering a pharmaceutical composition described herein to the subject.

[0113] In one embodiment, the materials provided herein are bispecific proteins as defined herein for therapeutic use. In another embodiment, the materials provided herein are bispecific proteins as defined herein for use in the treatment of cancer.

[0114] In one embodiment, what is provided herein is the use of a bispecific protein as defined herein in the manufacture of a pharmaceutical product for the treatment of cancer.

[0115] In one embodiment, the nucleic acids provided herein are defined for therapeutic use. In another embodiment, the nucleic acids provided herein are defined for use in the treatment of cancer.

[0116] In one embodiment, what is provided herein is the use of nucleic acids as defined herein in the manufacture of a pharmaceutical product for the treatment of cancer.

[0117] In some embodiments, the binding proteins disclosed herein can be formulated as pharmaceutical compositions with pharmaceutically acceptable carriers, excipients, or stabilizers. In certain embodiments, such pharmaceutical compositions are suitable for administration to humans or non-human animals via any one or more routes of administration using methods known in the art. The term “pharmaceutically acceptable carrier” means one or more non-toxic substances that do not interfere with the efficacy of the biological activity of the active ingredient. Such preparations typically contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic substances. Such pharmaceutically acceptable preparations may also contain compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans. Other conceivable carriers, excipients, and / or additives available in the formulations described herein include, for example, flavorings, antimicrobials, sweeteners, antioxidants, antistatics, lipids, serum albumin, protein excipients such as gelatin and casein, and salt-forming counterions such as sodium. These additional known pharmaceutically acceptable carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art and are cited, for example, in “Remington: The Science & Practice of Pharmacy,” 2nd ed., Lippincott Williams & Wilkins, (2005) and “Physician's Desk Reference,” 60th ed., Medical Economics, Montvale, NJ (2005). Pharmacochemically acceptable carriers can be selected to suit the desired or required dosage form, solubility, and / or stability.

[0118] In some embodiments, the amount of bispecific binding protein administered is approximately 50 mg to approximately 2000 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 70 mg to approximately 1500 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 100 mg to approximately 1400 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 200 mg to approximately 1250 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 500 mg to approximately 1000 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 600 mg to approximately 900 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 700 mg to approximately 800 mg.

[0119] In some embodiments, the amount of bispecific binding protein administered is approximately 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 850 mg, 900 mg, and 950 mg. These amounts are approximately 1000mg, 1050mg, 1100mg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1440mg, 1450mg, 1500mg, 1550mg, 1600mg, 1650mg, 1700mg, 1750mg, 1800mg, 1850mg, 1900mg, 1950mg, or 2000mg.

[0120] In some embodiments, the amount of bispecific binding protein administered is approximately 70 mg, 150 mg, 210 mg, 450 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1250 mg, or 1500 mg.

[0121] In some embodiments, the amount of bispecific binding protein administered is approximately 750 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 1500 mg.

[0122] In some embodiments, the amount of bispecific binding protein administered is 70 mg, 150 mg, 210 mg, 450 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1250 mg, or 1500 mg.

[0123] In some embodiments, the amount of bispecific binding protein administered is 750 mg. In some embodiments, the amount of bispecific binding protein administered is 1500 mg.

[0124] In some embodiments, the bispecific binding protein is administered once per treatment cycle. In some embodiments, the bispecific binding protein is administered twice per treatment cycle. In some embodiments, the bispecific binding protein is administered three times per treatment cycle.

[0125] In some embodiments, the treatment cycle is approximately 7, 14, 21, 28, or 35 days. In some embodiments, the treatment cycle is approximately 7 days. In some embodiments, the treatment cycle is approximately 21 days.

[0126] In some embodiments, the treatment cycle is repeated for a maximum of approximately 10 to 100 cycles. In some embodiments, the treatment cycle is repeated for a maximum of approximately 20 to 50 cycles. In some embodiments, the treatment cycle is repeated for a maximum of approximately 30 to 40 cycles. In some embodiments, the treatment cycle is repeated for a maximum of 10, 15, 20, 25, 30, 35, 40, 45, or 50 cycles. In some embodiments, the treatment cycle is repeated for a maximum of 35 cycles.

[0127] In some embodiments, the bispecific binding protein is administered to the subject as monotherapy or in combination therapy.

[0128] In one embodiment, the method involves administering to a subject a therapeutically effective amount of the binding protein disclosed herein in combination with an additional anticancer compound. In some embodiments, the anticancer compound is a small molecule drug. In some embodiments, the anticancer compound is pemetrexed, carboplatin, gemcitabine, cisplatin, paclitaxel, or a combination thereof. In some embodiments, the binding protein and the additional anticancer treatment are administered simultaneously. In some embodiments, the binding protein and the additional anticancer treatment are not administered simultaneously but are administered within the same treatment cycle.

[0129] In some embodiments, the bispecific binding protein is suitable for oral administration, parenteral administration such as subcutaneous or intravenous administration, or intramuscular injection or infusion. In some embodiments, the bispecific binding protein is administered by intravenous infusion (IV).

[0130] In some embodiments, the subjects have not received prior line of systemic therapy. In some embodiments, the subjects have not received prior chemotherapy. In some embodiments, chemotherapy includes platinum-based chemotherapy. In some embodiments, the prior line of systemic therapy includes checkpoint inhibitor (CPI) therapy.

[0131] In some embodiments, cancer comprises cancer cells expressing PD-L1. In some embodiments, cancer is one or more of ovarian cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, pancreatic cancer, renal cell carcinoma, and lung cancer.

[0132] In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is advanced or metastatic.

[0133] In the embodiment where the cancer is NSCLC, subjects have a PD-L1 tumor percentage score of 1% or higher. In some embodiments where the cancer is NSCLC, subjects have a PD-L1 tumor percentage score of 50% or higher.

[0134] In some embodiments, the subjects are checkpoint inhibitor (CPI) naive, meaning that the subjects have not previously been administered a CPI.

[0135] Pharmaceutical composition In some embodiments, the disclosure further provides pharmaceutical compositions comprising bispecific binding proteins that specifically bind to PD-1 and TIGIT in amounts ranging from about 70 mg to about 1500 mg. In some embodiments, the bispecific binding protein comprises: a) a first binding domain that specifically binds to PD-1, wherein the first binding domain comprises a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, and HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 4, LCDR2 having the amino acid sequence of SEQ ID NO: 5, and LCDR3 having the amino acid sequence of SEQ ID NO: 6; and b) a second binding domain that specifically binds to TIGIT, wherein the second binding domain comprises a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 11, HCDR2 having the amino acid sequence of SEQ ID NO: 12, and HCDR3 having the amino acid sequence of SEQ ID NO: 13, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 14, LCDR2 having the amino acid sequence of SEQ ID NO: 15, and LCDR3 having the amino acid sequence of SEQ ID NO: 16.

[0136] In some embodiments, the amount of bispecific binding protein administered is approximately 50 mg to approximately 2000 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 70 mg to approximately 1500 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 100 mg to approximately 1400 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 200 mg to approximately 1250 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 500 mg to approximately 1000 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 600 mg to approximately 900 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 700 mg to approximately 800 mg.

[0137] In some embodiments, the amount of bispecific binding protein administered is approximately 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 850 mg, 900 mg, and 950 mg. These amounts are approximately 1000mg, 1050mg, 1100mg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1440mg, 1450mg, 1500mg, 1550mg, 1600mg, 1650mg, 1700mg, 1750mg, 1800mg, 1850mg, 1900mg, 1950mg, or 2000mg.

[0138] In some embodiments, the amount of bispecific binding protein administered is approximately 70 mg, 150 mg, 210 mg, 450 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1250 mg, or 1500 mg.

[0139] In some embodiments, the amount of bispecific binding protein administered is approximately 750 mg. In some embodiments, the amount of bispecific binding protein administered is approximately 1500 mg.

[0140] In some embodiments, the amount of bispecific binding protein administered is 70 mg, 150 mg, 210 mg, 450 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1250 mg, or 1500 mg.

[0141] In some embodiments, the amount of bispecific binding protein administered is 750 mg. In some embodiments, the amount of bispecific binding protein administered is 1500 mg.

[0142] In some embodiments, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9.

[0143] In some embodiments, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain having the amino acid sequence of SEQ ID NO: 8 and a light chain having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10.

[0144] In some embodiments, the second binding domain of a bispecific binding protein that specifically binds to TIGIT includes a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 19. In some embodiments, the second binding domain of a bispecific binding protein that specifically binds to TIGIT includes a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19.

[0145] In some embodiments, the second binding domain of a bispecific binding protein that specifically binds to TIGIT includes a heavy chain having the amino acid sequence of SEQ ID NO: 18 and a light chain having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the second binding domain of a bispecific binding protein that specifically binds to TIGIT includes a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 18 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20.

[0146] In some embodiments, the pharmaceutical compositions disclosed herein can be formulated with pharmaceutically acceptable carriers, excipients, or stabilizers. In certain embodiments, such pharmaceutical compositions are suitable for administration to humans or non-human animals via any one or more routes of administration using methods known in the art. The term “pharmaceutically acceptable carrier” means one or more non-toxic substances that do not interfere with the efficacy of the biological activity of the active ingredient. Such preparations typically contain salts, buffers, preservatives, suitable carriers, and optionally other therapeutic substances. Such pharmaceutically acceptable preparations may also contain suitable solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans. Other conceivable carriers, excipients, and / or additives available in the formulations described herein include, for example, flavorings, antimicrobials, sweeteners, antioxidants, antistatics, lipids, serum albumin, gelatin, protein excipients such as casein, and salt-forming counterions such as sodium. These additional known pharmaceutically acceptable carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art and are cited, for example, in “Remington: The Science & Practice of Pharmacy,” 2nd ed., Lippincott Williams & Wilkins, (2005) and “Physician's Desk Reference,” 60th ed., Medical Economics, Montvale, NJ (2005). Pharmacochemically acceptable carriers can be selected to suit the desired or required dosage form, solubility, and / or stability.

[0147] This disclosure further provides pharmaceutical compositions for use in the treatment of cancer, as defined above. In some embodiments, the cancer is one or more of ovarian cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, pancreatic cancer, renal cell carcinoma, and lung cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is advanced or metastatic.

[0148] kit In some embodiments, the Disclosure further provides a kit comprising any of the above-described pharmaceutical compositions. In some embodiments, the kit comprises instructions for administering the pharmaceutical compositions. In some embodiments, the kit comprises additional anticancer substances as described herein.

[0149] Bispecific binding molecule sequence Aspects of this disclosure include DuetMab-format bispecific binding proteins that bind to PD-1 and TIGIT, prepared using the sequences in Table 1 below. The CDRs in Table 1 are determined based on Kabat's system. Table 1: Sequence of PD-1 / TIGIT bispecific binding molecules [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0150] The use of anti-TIGIT / anti-PD-1 bispecific antibodies is an innovative approach that simultaneously targets both TIGIT / PD-1 receptors. This approach offers several potential advantages compared to the simultaneous administration of anti-TIGIT and anti-PD-1 / PD-L1 antibodies separately. In addition to the ease of drug delivery (two doses can be replaced with one), it provides certainty of uniform distribution in the body of a single molecule that targets both receptors. Furthermore, the design of the bispecific antibody allows for two potentially different modes of action: one targets both receptors located in close proximity on the same cell (cis effect), and the other targets two receptors on two adjacent cells, simultaneously inhibiting checkpoints and forming persistent immune synapses (trans effect).

[0151] All documents cited herein, including patents, patent applications, papers, textbooks, etc., and documents cited within them, are incorporated herein by reference in their entirety to the extent not already incorporated.

[0152] Without limiting the scope of this disclosure, several aspects of this disclosure are described herein for illustrative purposes. [Examples]

[0153] The following embodiments are illustrative of specific aspects of the Disclosure and various uses thereof. They are provided for illustrative purposes only and should not be construed as limiting the scope of the Disclosure in any way.

[0154] AZD2936 is provided for treatment in the examples. AZD2936 is a monovalent, bispecific, humanized immunoglobulin G (IgG)1 monoclonal antibody (mAb) (IgG1 triple mutant) having a crystallizable fragment (Fc) domain engineered to reduce Fc effector function. AZD2936 is part of a complex system of cell surface receptors that specifically bind to human T cell immunoreceptors and programmed cell death protein 1 (PD-1), possessing immunoglobulin and immunoreceptor tyrosine-based repressive motif domains (TIGIT), and provide co-inhibitory signals to T cells and modulate their activity when bound to the corresponding ligand. In preclinical studies, dual inhibition of TIGIT and PD-1 by AZD2936 enhanced human T cell function and promoted an anti-tumor immune response. AZD2936 is used in the treatment of subjects with advanced or metastatic non-small cell lung cancer (NSCLC) expressing programmed cell death ligand 1 (PD-L1; tumor percentage score [TPS] ≥ 1%). AZD2936 is described in the international patent application publication WO2022 / 229919, which is incorporated herein by reference.

[0155] Example 1: Prediction of TIGIT and PD-1 receptor occupancy using AZD2936 in patients with solid tumors. As described in the examples below, AZD2936 monotherapy was tested at dose levels ranging from 70 to 1500 mg administered intravenously over Q3W. High intratumoral receptor occupancy (RO) is considered necessary for efficacy in patients. Model simulations can be used to predict the RO of PD-1 and TIGIT at different dose levels and to support recommended phase 2 doses (RP2D) and dose optimization approaches for AZD2936.

[0156] The model was constructed by linking a two-compartment PK model representing the clearance and transport of AZD2936 in the central and peripheral compartments, a tumor compartment, and two independent target-mediated pharmacokinetic (TMDD) modules in the central and tumor compartments that capture drug concentration and receptor binding dynamics. Two-compartment PK model parameters were estimated using separate population PK analyses. The tumor compartment was considered an effect compartment, and it was assumed that the distribution of AZD2936 to tumors does not affect its blood concentration.

[0157] To identify the mechanisms by which PD-1 and TIGIT significantly affect intratumoral RO, sensitivity analyses were performed. Simulations were conducted to predict RO within the patient population, incorporating variabilities contributing to these mechanisms in addition to PK variabilities. The predicted RO was compared to approved anti-PD-1 compounds at different AZD2936 dose levels.

[0158] The model was constructed by linking a two-compartment PK model representing the clearance and distribution of AZD2936 in the central and peripheral compartments, a tumor compartment, and two independent target-mediated pharmacokinetic (TMDD) modules in the central and tumor compartments to capture drug concentration and receptor binding dynamics. The tumor compartment was considered an effect compartment, and it was assumed that the distribution of AZD2936 to the tumor did not affect its blood concentration. An overall schematic diagram of the PK / PD model is shown in Figure 1. The model was then used to predict PD-1 and TIGIT receptor occupancy in both blood and tumors after IV administration of AZD2936 at various dose levels.

[0159] PD1 and TIGIT expression were measured in peripheral blood mononuclear cells (PBMCs). The concentrations of four receptors were estimated from PBMC data. First, the concentrations of T cells, NK cells, and other PBMCs expressing PD1+TIGIT+ / PD1+TIGIT- / PD1-TIGIT+ receptors were calculated. Combining the analysis of TIGIT PD1 on PBMCs with the previous results suggests that an equivalent number of PD1 or TIGIT are expressed on the cells in which they co-express (see Johnston et al., Cancer Cell, 26(6):923-937, 2014). The ratio of the initial concentrations of PD1 and TIGIT on PD1+TIGIT+ cells (referred to as RA0 and RB0), the initial concentration of PD1 on PD1+TIGIT- cells (RC0), and the initial concentration of TIGIT on PD1-TIGIT+ cells (RD0) was calculated to be 1:1:2:3.

[0160] The receptor concentrations in the central compartment (RA0, RB0, RC0, RD0) were calculated by multiplying the concentrations of PD1+TIGIT+ / PD1+TIGIT- / PD1-TIGIT+ T cells, NK cells, and other PBMCs by the number of receptors per cell, and were assumed to be between 10,000 and 1,000,000. The result for the PD1 receptor concentration RA0 in PD1+TIGIT cells was 0.001-0.1 nM. The concentrations of the other receptors (RB0, RC0, RD0) were calculated using the aforementioned ratio of 1:1:2:3.

[0161] Analysis of PBMCs can be deduced that approximately 78% of T cells in blood PBMCs are PD1-TIGIT-. (i) This percentage is lower in the tumor microenvironment (TME) due to the induction of PD1 / TIGIT expression, (ii) more receptors per cell are induced in the TME compared to the blood (M. Mkrtichyan et al., J Immunol, 189(5):2338-2347, 2012), and (iii) assuming that the ratio of PD1+TIGIT+, PD1+TIGIT-, and PD1-TIGIT+ T cells remains the same as in the blood, the percentages of these cell types can be calculated. The total tumor T cell volume density can be calculated from the area density (which can be measured by digital pathological analysis derived from patient tumor samples) using the stereologic formula from J.-P. Royet, Progress in Neurobiology, 37(5):433-474, 1991.

[0162] The AZD2936 analysis dataset included 72 patients who received either 70 mg, 210 mg, 750 mg, or 1500 mg at Q3W. The PK of AZD2936 was reasonably described using a two-compartment model with first-order elimination. Body weight was applied to PK using an allometric scaling approach (0.75 for elimination clearance (CL) and intercompartmental clearance (Q); 1 for central volume of distribution (VC) and peripheral volume of distribution (VP)). Inter-subject variability was estimated for CL, VC, and VP. Parameter estimates were CL: 0.392 L / day; VC: 3.63 L; VP: 2.94 L; Q: 0.935 L / day, with an additive error of 2.11 μg / mL and a proportional error of 29.05%.

[0163] AZD2936 administration was planned as an IV infusion in Q3W. Dose levels for simulation were set at 70, 210, 500, 750, and 1500 mg. 1000 hypothetical patients were simulated for each of the four combinations of partition coefficient (PC) and receptor concentration (RC).

[0164] Five parameters were varied among 1000 virtual patients using the Latin Hypersquare Sampling (LHS) sampling scheme: RA 0,VC RA 0,VT , k int,PD1 , k eq,AZD2936 , and η1. The parameters were sampled from a log-normal distribution in which 95% of the values ​​were between 1 / 2 and 2 times their respective mean values. In addition to these five parameters, and independently of them, VC, VP, and CL were sampled based on the between-subject variability (BSV).

[0165] The model was built using Matlab SimBiology (version 2021b). The simulation was run on an SCP cluster using Matlab (version 2019b).

[0166] result Sensitivity analysis was performed to evaluate the relative influence of key parameters predicting tumor receptor occupancy. This analysis revealed that, in the dose ranges where complete receptor occupancy (750 and 1500 mg) is most likely to be achieved, the predicted tumor receptor occupancy of PD-1 and TIGIT is most influenced by the tumor partition coefficient (PC), which determines the amount of drug that can penetrate tumor tissue (i.e., tumor penetration), and the initial receptor concentration (RC) in the tumor microenvironment. These are also influenced by internalization rates and the equilibrium rate constant.

[0167] Of these four parameters, PC and RC vary considerably within the patient population, while the internal transfer rate and equilibrium rate constant are subject to some degree of uncertainty based on internal literature findings. Due to insufficient information to provide a probability distribution of PC and RC in patients, four groups consisting of combinations using high and low PC and RC values ​​were tested. int,PD1 and k eq,AZD2936 For each group, we sampled them from the distributions described in Section 2.5 to reflect the best insights while taking into account the uncertainty of the values.

[0168] When receptor occupancy was predicted based on tumor penetrant (PC=5% or 50%) and tumor RC (3nM or 30nM for PD-1; 4nM or 40nM for TIGIT, which can be calculated using RA0, VT=1 or 10nM, ψBA=1, ψCA=2, ψDA=3), the hypothetical patients were divided into four subgroups: "high PC, low RC", "high PC, high RC", "low PC, low RC", and "low PC, high RC". The number of receptors per cell was 100,000, and the T cell area density in the tumor ranged from 400 to 4,000 cells / mm³. 2 High / low tumor PD-1 / TIGIT receptor concentrations were calculated based on the assumption that the ratio between PD-1+TIGIT+ / PD-1+TIGIT- / PD-1-TIGIT+ cells remains the same between peripheral and tumor cells. The 50% figure in the high-PC group is supported by experimental investigations using radiolabeled mAbs to measure tumor uptake. Since these data do not have sufficient granularity to quantitatively describe the spatial heterogeneity of mAb concentrations within tumor lesions, conservative estimates were applied to approximate the scenario in the low-PC group, resulting in a tumor concentration of ~5% relative to blood (TR Li et al., Clinical Pharmacology & Therapeutics, 110(1):200-209, 2021). The number of receptors per cell is 100,000, and the T cell surface density in tumors ranges from 500 to 5,000 cells / mm³. 2 This is within the range of PD-1 + TIGIT + / PD-1 + TIGIT - / PD-1 - TIGIT + High / low tumor PD-1 / TIGIT receptor concentrations were calculated based on the assumption that the cell-to-cell ratio remains the same between peripheral and tumor cells.

[0169] The model predicts ≥90% receptor occupancy for both PD-1 and TIGIT in the blood over the entire dosing interval in ≥90% of patients starting with a 70 mg dose, regardless of patient subgroup. Predicted receptor occupancy for PD-1 and TIGIT in the tumor microenvironment after IV administration of AZD2936 at 70, 210, 750, and 1500 mg Q3W is shown in Figure 1 and Table 2. The 70 and 210 mg Q3W doses are likely to result in suboptimal receptor occupancy (particularly PD-1) in patients with low tumor distribution coefficients (i.e., low drug penetration into the tumor). The 750 mg Q3W dose level is predicted to achieve ≥90% intratumoral receptor occupancy in most patients across a wide range of conditions. The 1500 mg Q3W dose level is predicted to achieve better receptor occupancy than 750 mg Q3W, particularly in the "low PC, high RC" group. Table 2: Results of occupancy rates obtained by receptor modeling [Table 2]

[0170] The predicted RO of AZD2936 against PD-1 in tumors at different dose levels was compared with other anti-PD-1 agents (i.e., nivolumab and pembrolizumab). Comparisons were performed in the same four scenarios for PC / RC combinations. Binding affinity and population PK parameters for nivolumab and pembrolizumab were obtained from the literature and regulatory reports listed below, while other system parameters and variability were maintained as in the AZD2936 simulations.

[0171] Simulations show that when nivolumab is administered at 240 mg Q2W and pembrolizumab at 200 mg Q3W, tumor PD-1 RO at trough concentrations is maintained above 90% in all four patient groups. In AZD2936, the 210 mg Q3W dose was predicted to have suboptimal receptor occupancy compared to nivolumab and pembrolizumab in the same hypothetical patient group, particularly in patients with a low tumor distribution coefficient.

[0172] overview We predicted the TIGIT and PD1 receptor occupancy in the blood and tumors of a hypothetical patient treated with AZD2936. The conclusions of this analysis are as follows: The model predicts a ≥90% receptor occupancy rate for both PD-1 and TIGIT in the blood across the entire dosing interval in ≥90% of patients starting with a 70 mg dose, regardless of patient subgroup. Sensitivity analysis indicates that tumor RO is significantly influenced by tumor penetration and receptor concentration, and can vary considerably from patient to patient. Since probability distribution data for these two parameters in patients are unavailable, tumor RO was evaluated at different dose levels within each subgroup. • At doses of 70 and 210 mg Q3W, suboptimal PD-1 receptor occupancy is predicted in patients with low tumor penetration. The 750 mg Q3W dose level is predicted to achieve ≥90% intratumoral receptor occupancy in most patients across a wide range of conditions. The 1500 mg Q3W dose level is predicted to achieve better receptor occupancy than 750 mg Q3W, particularly in patients with low tumor penetration and high receptor concentrations.

[0173] Example 2: Test Design and Definition Several solid malignancies, including NSCLC, are thought to be sensitive to immune checkpoint inhibitors. However, even in patients who initially responded to immuno-oncology (IO) therapy, disease progression may occur due to primary or secondary resistance mediated by multiple mechanisms, such as T cell depletion, insufficient T cell priming, and abnormal antigen presentation. TIGIT was identified as a key inhibitor of the antitumor response that can disrupt multiple stages of the cancer immune cycle. In vitro and in vivo studies have shown that AZD2936 inhibits TIGIT and PD-1 from binding to their major ligands (CD155 and PD-L1, respectively), thereby enhancing the immune-mediated antitumor response compared to targeting PD-1 alone. Therefore, targeting both PD-1 and TIGIT with AZD2936 offers clinical benefit to patients with advanced or metastatic NSCLC.

[0174] Figure 2 shows the trial flowchart for the study outlined in the following examples. The study is a first-in-one (FTIH), open-label, multi-center, multi-part, dose-escalation and dose-expansion study to evaluate the safety, pharmacokinetics, pharmacodynamics, and efficacy of AZD2936 in adult subjects with unresectable stage III or stage IV NSCLC.

[0175] Each trial, Parts A through D, includes a screening period of -28 to -1 day. The screening includes a single visit by a healthcare professional, i.e., V1. During the screening period, subjects will be evaluated, which may include recording of medical history, performing a physical examination, blood and urine tests for safety, evaluation of biomarkers, and collection of tumor tissue.

[0176] As shown in the schematic diagram in Figure 2, Parts A through D consist of four parts and are a continuous treatment test without masking.

[0177] Part A evaluates dose escalation of AZD2936 in patients with unresectable stage III or stage IV NSCLC, where the tumor expresses PD-L1 with a TPS of ≥1%, exhibits primary or secondary resistance to CPI-inducing regimens, and has experienced second-line or higher (2L+) checkpoint inhibitors (CPIs), to determine the maximum tolerated dose (MTD), optimal biomedical dose (OBD), or maximum administerable dose (MFD), and recommended phase II dose (RP2D). Dose escalation will consist of up to 12 patients per dose level, following a modified toxicity probability interval -2 (mTPI-2) algorithm. Intermediate dose levels will also be investigated if necessary due to the acquisition of safety, pharmacokinetic (PK), pharmacodynamic, biomarker, and response data. Patients will be evaluated for dose-limiting toxicity (DLT) during a 21-day DLT evaluation period.

[0178] Part BD will commence once the MTD, OBD, or MFD and RP2D are determined in Part A (dose escalation). Parts B and C will evaluate the safety, tolerability, and antitumor activity of AZD2936 at the RP2D determined during Part A (dose escalation) in two cohorts.

[0179] Part B (dose escalation) will be conducted in patients with unresectable stage III or stage IV NSCLC whose tumor expresses PD-L1 with a TPS of ≥1% and who have experienced 2L+ CPI. The eligibility criteria are the same as for Part A (dose escalation), and patients who have experienced primary and secondary CPI will be enrolled.

[0180] Part C (dose expansion) is performed in CPI-naive patients with tumors expressing PD-L1 with a TPS of ≥1% and having stage IV NSCLC.

[0181] Part D incorporates a randomized design to evaluate the safety, tolerability, and antitumor activity of AZD2936 at 750 mg and 1500 mg Q3W. Part D is designed to assess whether a dose-efficacy plateau is reached at 750 mg Q3W and to enable the selection of the optimal dose to be investigated in future trials. If a cohort for Part C is already open, Part D will be conducted in parallel with Part C; if a cohort for Part C is not open, Part D will replace Part C. Part D (dose expansion) will be conducted in CPI-naive subjects with stage IV NSCLC, PD-L1-expressing tumors with TPS ≥ 50%. Subjects will be randomized 1:1 to either AZD2936 750 mg Q3W (RP2D) or AZD2936 1500 mg Q3W.

[0182] Definitions In the tests described in the following examples, the following terms are used. Dose-limiting toxicity (DLT). DLTs are evaluated in Part A (dose escalation). The DLT evaluation period is 21 days from the first dose of AZD2936 on day 1 of cycle 1. DLTs are defined as ≥ Grade 3 toxicities occurring during the DLT evaluation period in Part A (dose escalation), including modifications and exceptions. Toxicity clearly attributable to the primary disease, other concomitant medications, disease-related processes under investigation, or other non-drug-related causes (not related to the treatment in the study intervention) are excluded from this definition. All DLTs must be recorded as adverse events (AEs). All AEs are evaluated according to NCI CTCAE v5.0.

[0183] Maximum Tolerable Dose (MTD). The MTD is selected from all tested dose levels that have not been previously judged as "DU (the current dose shows unacceptable toxicity)" by the mTPI-2 algorithm and have not been publicly declared unsafe. Due to this constraint, the MTD is determined as the dose level where the estimated DLT is closest to the target toxicity level of 30%.

[0184] For dose levels with estimated toxicity equal to the target toxicity of 30% (tie dose levels), the following approach is used (Ji Y, et al., A modified toxicity probability interval method for dose-finding trials, Clin Trials, 2010, 7(6):653-63): If >30% toxicity is not estimated at any dose level, the highest dose level among all tie dose levels with target toxicity ≤30% is selected; otherwise, the lowest dose level is selected.

[0185] Efficacy assessment. Tumor response is evaluated according to RECIST v1.1 (Eisenhauer et al, 2009) according to the plan (Q9W [±7 days] for 54 weeks starting from the day of the first dose [day 1 of cycle 1], followed by Q18W [±14 days] until disease progression or initiation of other anticancer therapies).

[0186] Tumor assessment. Tumor assessment includes physical examination and cross-sectional imaging using CT (preferred) or MRI scans. A CT scan of the chest, as well as CT or MRI scans of the abdomen and pelvis, should be performed at screening (with contrast unless the subject has known intolerances). The preferred method for systemic disease assessment is CT with contrast; if CT with contrast is contraindicated, CT without contrast is preferred over MRI. The preferred method for brain imaging is MRI over CT (with contrast unless the subject has known intolerances). The preferred method for bone imaging is bone scanning. At screening, bone imaging is applicable only to subjects with clinical suspicion of bone metastasis or confirmed bone metastasis. Follow-up brain and bone imaging should be performed as clinically necessary if there are no metastases at baseline, and at each restaging scan if brain or bone metastases are present at baseline. The same method should be used for all subsequent tumor assessments. If tumor assessment is performed within six months of the initiation of treatment, a pre-baseline tumor assessment may also be requested, if possible. Where available, circulating tumor deoxyribonucleic acid (ctDNA) and tumor antigen markers should be used as exploratory markers in the assessment of disease response.

[0187] ECOG performance status is assessed at specific points in time.

[0188] Adverse Events. An adverse event (AE) is the occurrence of any adverse medical event in a patient or subject in a clinical trial to which a drug has been administered, and which is not necessarily causally related to the treatment. Therefore, an AE may be any undesirable unintended sign (e.g., abnormal test results), symptom (e.g., nausea, chest pain), or disease that is temporally related to the use of the drug, whether or not it is related to the drug.

[0189] The term AE is used to include both serious and non-serious AEs, and may include exacerbations of pre-existing medical events. AEs may occur at any point, including during the induction or washout period, even if no treatment is being performed for the study.

[0190] A serious adverse event (SAE) is an AE that occurs during any trial period (i.e., induction, treatment, washout, or follow-up) and meets one or more of the following criteria: (1) death; (2) immediate life-threatening; (3) requiring hospitalization of the subject or extension of the current hospitalization; (4) resulting in persistent or significant disability or dysfunction; (5) a birth defect or abnormality; or (6) a serious medical event that could endanger the subject or require medical intervention to prevent one of the above outcomes.

[0191] Disease progression. Disease progression can be considered a deterioration of the subject's condition due to the disease targeted by the study drug. This may be an increase in severity and / or an increase in disease symptoms under study conditions. The occurrence of new metastases of the primary cancer or progression of existing metastases under study conditions should be considered disease progression, not an adverse event (AE). Events that are clearly caused by disease progression should not be reported as AEs during the study.

[0192] New cancers. The occurrence of new cancers should be considered SAEs. New primary cancers are those that were not primarily caused by the implementation of the trial intervention and were discovered after the subjects were enrolled in this trial. These are not included as metastases from the original cancer.

[0193] Example 3: Formulation and administration of AZD2936 This example illustrates the formulation and administration of AZD2936. AZD2936 is provided in Parts A through D as either a lyophilized product or a liquid product.

[0194] Lyophilized AZD2936 is supplied as a sterile, white to off-white, lyophilized product in glass vials with a nominal fill volume of 5.0 mL for injection after reconstitution. Each vial contains 250 mg (nominal) of the novel active substance. After reconstitution with 5.0 mL of sterile water for injection (sWFI), each vial contains 50 mg / mL of AZD2936. The reconstituted solution should be clear to opalescent, colorless to slightly yellow, and free from visible particles. After reconstitution with sWFI, AZD2936 is further diluted with 0.9% (w / v) saline.

[0195] Liquid AZD2936 is supplied as a sterile solution in glass vials with a nominal fill volume of 15.0 mL for injection. Each vial contains 750 mg (nominal) of the novel active substance.

[0196] AZD2936 product vials should be stored away from light. Test product vials should be stored at 2°C to 8°C (36°F to 46°F) until ready for use.

[0197] AZD2936 is administered via IV infusion.

[0198] The dose of AZD2936 for administration should be prepared using sterile techniques. The total time from needle insertion of the AZD2936 vial to the start of administration should not exceed 24 hours at 2°C to 8°C (36°F to 46°F) or 4 hours at room temperature up to 25°C (77°F). The standard infusion time for AZD2936 is over 1 hour (maximum total of 90 minutes); however, even with interruptions during infusion, the permitted total infusion time should not exceed 4 hours at room temperature. Do not administer other drugs simultaneously through the same infusion line. If either the preparation time or the infusion time exceeds the limit, prepare a new dose from a new vial. AZD2936 contains no preservatives.

[0199] The doses administered using IV bags are prepared as follows: Doses of AZD2936 between 70 mg and 1500 mg are administered using IV bags containing 0.9% (w / v) saline, at a final AZD2936 concentration ranging from 1 to 24 mg / mL, and delivered via IV administration with a 0.2- or 0.22-μm filter. If the nominal AZD2936 drug product concentration is 50 mg / mL, the required amount (mL) of AZD2936 to be added to the IV bag at each dose level is calculated using the following formula:

number

[0200] Example 4: Initial Risk / Benefit Assessment The study will evaluate the risks of AD2936 administration and provide appropriate doses of AZD2936 for Parts A through D.

[0201] First, the no-adverse-effect level (NOAEL)-based starting dose for FTIH in humans was determined to be 70 mg, based on the Good Laboratory Practice cynomolgus monkey toxicology study.

[0202] Next, an initial risk assessment was performed using a dose escalation method based on a specified starting dose of 70 mg. A total of 64 2L+ subjects with NSCLC received AZD2936 therapy across five dose cohorts: Cohort A1, 70 mg (4 subjects); Cohort A2, 210 mg (11 subjects); Cohort A3, 750 mg (13 subjects); Cohort A4, 1500 mg (7 subjects); and Cohort B, 750 mg (29 subjects).

[0203] The results of this initial assessment indicate that AZD2936 was well tolerated across all doses evaluated (including up to 1500 mg Q3W and 1500 mg Q3W). The safety profile of AZD2936 was similar across dose levels, with no DLTs reported up to regimens including up to 1500 mg Q3W and 1500 mg Q3W; therefore, the MTD was not reached during dose escalation. Additional safety assessments were conducted and are described below.

[0204] Preliminary signals of efficacy were observed in the first cohort of 46 2L+ subjects with NSCLC, with partial response achieved in two subjects (one subject [9.1%] in cohort A2 and one subject [8.3%] in cohort A3). A total of 16 subjects achieved stable disease at ≥8 weeks; of these, nine subjects (75%) were in cohort A3 and were treated with AZD2936 750 mg Q3W.

[0205] Using non-compartmental analysis, preliminary PK data were evaluated in 32 subjects who received AZD2936 monotherapy at 70, 210, 750, and 1500 mg Q3W during the dose-escalation part of this study. Systemic exposure to AZD2936 increased approximately proportionally to the dose from 70 to 1500 mg. Targeted-mediated pharmacokinetics were not observed at the 70, 210, 750, and 1500 mg Q3W dose levels. After a single dose IV administration, the mean terminal elimination half-life (t) was approximately 8 to 9 days. 1 / 2 The levels of AZD2936 were reduced in a biphasic manner. Steady state was reached in approximately 3 cycles, and minimal accumulation of AZD2936 was observed after repeated administration. max The average accumulation ratio for is in the range of 1.14 to 1.3.

[0206] Based on the clinical samples analyzed in this study, AZD2936 achieved ≥90% PD-1 and TIGIT RO in peripheral T cells at all dose levels, including up to 1500 mg, and PK / PD modeling predicted that optimal intratumor PD-1 and TIGIT RO (≥90%) would be achieved at doses of ≥750 mg.

[0207] Example 5: Test Subject This embodiment provides criteria for selecting subjects for the examinations in Parts A through D.

[0208] Inclusion criteria for Parts A-D: 1. Applicants must be 18 years of age or older at the time of registration for the exam. 2. Histologically or cytologically confirmed squamous / non-squamous NSCLC that is not an indication for surgical or radiotherapy, as defined below (according to the International Society for Lung Cancer's Staging Manual for Thoracic Oncology, 8th Edition): Parts A and B: Stage III unresectable NSCLC and Stage IV squamous / non-squamous NSCLC Parts C and D: Stage IV squamous / non-squamous NSCLC only. 3. PD-L1 confirmed by IHC meets the following criteria: Part A: Local reports indicate PD-L1 TPS ≥ 1% Part B: Local reports indicate PD-L1 TPS ≥ 1% Part C: Local reports indicate PD-L1 TPS ≥ 1% Part D: Local reports indicate PD-L1 TPS ≥ 50% Part C: One of the following definitions must be met: (i) Not previously treated for NSCLC, high PD-L1 expression (TPS ≥ 50%) making them candidates for CPI monotherapy, and not requiring rapid disease control with a regimen including chemotherapy, or (ii) Not having previously received treatment for NSCLC, not being eligible for or not consenting to platinum-based chemotherapy or combination regimens, or (iii) The patient has previously received treatment for NSCLC with a single regimen consisting solely of chemotherapy. Part D: One of the following definitions must be met: (i) Not having previously received treatment for NSCLC and not requiring rapid disease control with a regimen including chemotherapy, or (ii) The patient has previously received treatment for NSCLC with a single regimen consisting solely of chemotherapy. 5. Body Mass Index ≥ 17. 6. The group's (ECOG) performance status at the time of enrollment is 0 or 1. 7. Predicted life expectancy is ≥12 weeks. 8. There must be at least one lesion measurable by RECIST v1.1. (a) For subjects undergoing biopsy at screening and / or treatment, the biopsy lesion must be different from any lesion used for evaluation by RECIST v1.1. 9. Organ and bone marrow function measured within 28 days prior to the first dose is sufficient. Detailed criteria are shown in Table 3. Table 3: Criteria for adequate organ and bone marrow function [Table 3]

[0209] Example 6: Part A Dose escalation This embodiment describes a Part A dose escalation study to determine the dosage of AZD2936 in Parts B and C.

[0210] The primary objectives of Part A dose escalation include evaluating safety and tolerability, characterizing DLT, and determining the MTD, OBD, or MFD, and RP2D of AZD2936 in subjects with PD-L1 (TPS ≥ 1% as measured by PD-L1 immunohistochemical staining (IHC) assay) and unresectable stage III / IV NSCLC who have previously received 2L+CPI. Endpoints include the percentage of subjects experiencing adverse events (AEs) and immune-mediated AEs (imAEs), serious AEs (SAEs), DLT, vital signs, and abnormal laboratory parameters; and the discontinuation rate of AZD2936 administration due to toxicity.

[0211] A secondary objective of Part A dose escalation is to determine the preliminary antitumor activity of AZD2936 in patients with PD-L1 with a TPS of ≥1% and unresectable stage III / IV NSCLC who have previously received 2L+CPI. Endpoints include ORR, disease control rate (DCR), duration of response (DoR), and sustained response rate (DRR) according to RECIST v1.1.

[0212] A secondary objective of Part A dose escalation is to evaluate the targeted engagement of AZD2936 in peripheral blood. The endpoints include measuring the receptor occupancy (RO) of TIGIT and PD-1 on peripheral blood T cells.

[0213] In all of Parts A-D, a secondary objective is to evaluate the suitability of the pharmacokinetic (PK) profile of AZD2936 administered Q3W in subjects with stage III / IV unresectable NSCLC who have previously received 2L+CPI and in CPI-naive subjects. Endpoints include serum concentrations and PK parameters (where possible) of AZD2936; the PK parameters to be evaluated include, but are not limited to, the maximum measurable concentration (C max ), area under the concentration-time curve (AUC), clearance, and terminal elimination half-life (t 1 / 2 ) is included.

[0214] In all parts A through D, a secondary objective is to evaluate the immunogenicity of AZD2936. The evaluation items include the incidence of anti-drug antibodies (ADAs) against AZD2936 in serum.

[0215] After an mTPI-2 dose escalation design with up to six dose levels, subjects were enrolled in Part A. Dose escalation to the next dose level was performed using the mTPI-2 algorithm with a target DLT rate of 30% and equivalence intervals (25%, 35%). At least three subjects had to complete the DLT evaluation period before deciding on an escalation. The escalation consisted of at least three planned dose levels of AZD2936. Intermediate dose levels may be investigated if required by the acquisition of safety, PK, pharmacodynamic, biomarker, and efficacy data. Recommendations for dose escalation and dose reduction followed the mTPI-2 algorithm. The mTPI-2 algorithm employs a simple beta-binomial Bayes model. The prior distribution at all dose levels is Beta(1,1). The posterior density of toxicity probabilities is divided into multiple intervals of equal length. These intervals are classified as low dose, appropriate dose, and high dose from a toxicity perspective. The low-dose interval corresponds to dose escalation, the high-dose interval corresponds to dose decrease, and the appropriate dose corresponds to remaining at the current dose. Given an interval and a probability distribution, the unit probability mass of the interval is defined as the probability distribution of the interval divided by the length of the interval. The dose escalation design of this study uses a target DLT rate of 30% and equivalence intervals (25%, 35%) for dose escalation / decrease decisions and MTD determination. If the probability of exceeding the target DLT rate of 30% in at least three subjects treated and evaluated at the same dose level is estimated to be 95% or greater (i.e., probability [DLT>30% data] ≥ 95%), that dose level is considered unsafe, and no additional subjects will be enrolled at that dose level.

[0216] Part A used four dose levels: 70 mg, 210 mg, 750 mg, and 1500 mg.

[0217] Following an initial screening period of up to 28 days, a Part A intervention period was planned. The Part A intervention period consisted of up to 35 cycles of 21 days each, with AZD2936 administered every three weeks (Q3W), i.e., once every 21-day cycle.

[0218] Eligible subjects will receive AZD2936 intravenously (IV) at a selected dose, starting on day 1 of cycle 1, for up to 35 cycles by Q3W. Subjects will be treated with the study intervention until disease progression, unacceptable toxicity, at the discretion of the study investigators, completion of up to 35 treatment cycles, or withdrawal of consent. All subjects will be tracked for survival until the end of the study. Dose reduction of AZD2936 will not be permitted at any time.

[0219] After the intervention period (invention period), the subjects will be further evaluated for disease progression, their condition at the end of treatment, and their condition at follow-up.

[0220] Current data obtained during Part A indicate that preliminary PK was nearly dose-proportional (70, 210, 750, and 1500 mg Q3W), with limited ADA effects. In peripheral blood, all tested doses of AZD2936 achieved a return on action (RO) of ≥90% for both PD-1 and TIGIT. Of the doses tested in dose escalation, 750 mg Q3W was estimated to be the minimum dose at which intratumor receptor occupancy reached ≥90% for both PD-1 and TIGIT in the majority of subjects across a wide range of conditions. Based on PK / PD modeling analysis and acquisition of the safety profile of AZD2936, 750 mg Q3W was identified as the RP2D to be further evaluated in the expansion phase of this trial (Parts B and C).

[0221] Example 7: Part B Dose Expansion In this example, the Part B study period was conducted using a 750 mg dose of AZD2936 to test dose expansion.

[0222] Part BD (dose escalation) is initiated after the establishment of the MTD, OBD, or MFD and RP2D in Part A (dose escalation).

[0223] The primary objective of Part B dose escalation is to evaluate the safety and tolerability of AZD2936 in RP2D in subjects with PD-L1 with TPS ≥ 1% and who have previously received 2L+CPI and have unresectable stage III / IV NSCLC. Endpoints include the percentage of subjects with AEs and imAEs, SAEs, DLT-like events, vital signs, and abnormal laboratory parameters; and the discontinuation rate of AZD2936 due to toxicity.

[0224] A primary objective of the Part B dose expansion is to determine the preliminary antitumor activity of AZD2936 in RP2D in subjects with PD-L1 (TPS ≥ 1% using PD-L1 IHC assay) and unresectable stage III / IV NSCLC who have previously received 2L+CPI. The endpoint will include the objective response rate (ORR) according to the Response Evaluation Criteria for Solid Tumors (RECIST) v1.1.

[0225] A secondary objective of the Part B dose expansion is to determine the preliminary antitumor activity of AZD2936 in RP2D in patients with PD-L1 with TPS ≥ 1% and who have previously received 2L+CPI and have unresectable stage III / IV NSCLC. Endpoints include DCR, DoR, DRR, and progression-free survival (PFS) according to RECIST v1.1.

[0226] A secondary objective of Part B is to evaluate the targeted engagement of AZD2936 with RP2D in peripheral blood. Endpoints include the measurement of TIGIT and PD-1 RO on peripheral blood T cells.

[0227] A sample size of at least 30 participants was selected to obtain a preliminary ORR with a standard error (SE) of 0.1 or less. Two-sided 80% confidence intervals (CIs) using the Clopper-Pearson method for the range of possible response rates for the 30 participants are provided below: 3% ORR (1 / 30 response); 80% CI [0.4%, 12%] 7% ORR (2 / 30 response); 80% CI [2%, 17%] 10% ORR (3 / 30 response); 80% CI [4%, 21%] 13% ORR (4 / 30 response); 80% CI [6%, 25%] 20% ORR (6 / 30 response); 80% CI [11%, 32%].

[0228] Based on the decision framework (Frewer P, et al., Decision-making in early clinical drug development, Pharm Stat, 2016, 15(3):255-63), if the target ORR (TV) is 15%, then the conclusion is reached that there is no evidence of target activity if an objective RECIST response of ≤1 (confirmed complete response [CR] or partial response [PR]) is observed (ORR ≤3%). If the true response rate is 15%, the probability of observing a response of ≤1 in 30 evaluable subjects is ≤10%.

[0229] Based on PK / PD modeling analysis and the acquisition of a safety profile for AZD2936, 750 mg Q3W was identified as a RP2D to be further evaluated during the expansion phases of Parts B and C.

[0230] After a screening period of up to 28 days, a Part B intervention period was planned. The Part B intervention period consisted of up to 35 cycles of 21 days each, with AZD2936 administered every three weeks (Q3W), i.e., once every 21-day cycle.

[0231] In particular, eligible subjects will receive AZD2936 via intravenous (IV) infusion at RP2D (750 mg Q3W) for up to 35 cycles at Q3W, starting on day 1 of cycle 1. Subjects will be treated with the study intervention until disease progression, unacceptable toxicity, at the discretion of the study investigators, completion of up to 35 treatment cycles, or withdrawal of consent.

[0232] After the intervention period, participants will be further evaluated for disease progression, their condition at the end of treatment, and their condition at follow-up.

[0233] Example 8: Preliminary results from Part A and Part B In parts A and B of the experiment described in the above example, 83 subjects were evaluated.

[0234] Table 4 shows statistics for subjects evaluated in Parts A and B. Table 5 shows the medical conditions of the 83 subjects evaluated in Parts A and B. As shown in Table 5, most subjects had metastatic disease and non-squamous tissue. Adverse events that occurred in more than 5% of subjects in the preliminary analysis are plotted in Figure 3. Adverse events related to AZD2936 are shown on the right side of the figure. Adverse events that occurred in more than 5% of subjects who received 750 mg of AZD2936 in the preliminary analysis are plotted in Figure 4. Adverse events that occurred in more than 10% of subjects who received 750 mg of AZD2936 in Q3W are plotted in Figure 10. Adverse events related to AZD2936 are shown on the right side of the figure. Nine serious adverse events (SAEs) potentially related to AZD2936 were observed: one case of elevated alanine aminotransferase (occurring in Part A4-JPN), one case of elevated aspartate aminotransferase (occurring in Part A4-JPN), one case of colitis (occurring in Part A4), one case of diarrhea (occurring in Part A4), one case of pneumonitis (occurring in Part B), one case of rash (occurring in Part B), and one case of suspected immune system disorder (occurring in Part A3); one case of acute hepatitis and one case of malaise (both occurring in Part B). Table 4: Statistics of the evaluated subjects

Table 4

Table 5-1

Table 5-2

[0235] The results of efficacy are provided in Table 6. Table 6: Initial Efficacy Results in the Test

Table 6

Table 7

[0236] As seen in Table 6, in the administration of 750 mg Q3W, the overall response rate (ORR) was 5.6%, 24 subjects achieved stability, and the disease control rate (DCR) was 50.0%. The difference in DCR at 750 mg between Part A and Part B would be explained by the heterogeneity of the patient population, in which patients with poor disease characteristics were concentrated in Part B.

[0237] Tables 6 and 7 show the summary of the initial results obtained from participants who had received second-line or greater (2L+) checkpoint inhibitors (CPIs) in Part A and Part B. Eighty-three subjects with pretreated NSCLC were treated as described above. AZD2963 continued to show a favorable safety profile, which may further change with longer-term follow-up and the transition to CPI-naïve subjects.

[0238] Example 9: Part C Dose Escalation This embodiment describes the use of a 750 mg dose of AZD2936 for further dose expansion studies.

[0239] The primary objective of Part C is to evaluate the safety and tolerability of AZD2936 in RP2D in CPI-naive subjects with PD-L1 (TPS ≥ 1% using PD-L1 IHC assay) and stage IV NSCLC. Endpoints include the percentage of subjects with AEs and imAEs, SAEs, DLT-like events, vital signs, and abnormal laboratory parameters; and the discontinuation rate of AZD2936 administration due to toxicity.

[0240] A primary objective of Part C is to determine the preliminary antitumor activity of AZD2936 in RP2D in CPI-naive subjects with PD-L1 TPS ≥ 1% and stage IV NSCLC. Endpoints include ORR according to RECIST v1.1.

[0241] A secondary objective of Part C is to further characterize the preliminary antitumor activity of AZD2936 in RP2D in CPI-naive subjects with PD-L1 TPS ≥ 1% and stage IV NSCLC. Endpoints include DCR, DoR, DRR, and PFS according to RECIST v1.1.

[0242] With a sample size of at least 30 subjects, a preliminary ORR of less than 0.1 SE should be obtained. Below are two-sided 80% CIs using the Clopper-Pearson method for the range of possible response rates in the 30 subjects: 20% ORR (6 / 30 response); 80% CI [11%, 32%] 30% ORR (9 / 30 response); 80% CI [19%, 43%] 40% ORR (12 / 30 response); 80% CI [28%, 53%] 47% ORR (14 / 30 response); 80% CI [34%, 60%] 60% ORR (18 / 30 response); 80% CI [47%, 72%].

[0243] Based on the decision framework (Frewer et al, 2016), if the ORR TV is 40%, then if ≤8 objective RECIST responses (confirmed CR or PR) are observed (ORR ≤ 27%), the conclusion is reached that there is no evidence of target activity. If the true response rate is 40%, the probability of observing ≤8 responses in 30 evaluable subjects is ≤10%.

[0244] During the implementation of Part C, the emergence of toxicity that is not clearly attributable to the primary disease, other concomitant medications, the disease-related process under investigation, or another non-drug-related cause (not related to the treatment in the study intervention) will trigger the automatic termination rule. In this scenario, if any of the following conditions occur, enrollment of new subjects in the study will be immediately suspended until the Safety Assessment Committee evaluates the benefits / risks of AZD2936. 1. If one Grade 5-related toxicity occurs at any point during the implementation of Part C. 2. If, at any point during the implementation of Part C, two cases of Grade 4-related toxicity occur in two different subjects. 3. If, in the initial 10 enrolled subjects, more than 4 serious adverse drug reactions (SADRs) occur in different subjects (i.e., if more than 2 occur in the same subject, it is counted as only one), or if more than 7 SADRs occur in the initial 20 subjects.

[0245] Participants will be enrolled in Part C. After an initial screening period of up to 28 days, eligible participants will receive AZD2936 Q3W via intravenous (IV) infusion, starting on day 1 of cycle 1, for up to 35 cycles. Participants will be treated with the study intervention until disease progression, unacceptable toxicity, at the discretion of the study investigators, completion of up to 35 treatment cycles, or withdrawal of consent. All participants will be tracked for survival until the end of the study. Dose reduction of AZD2936 will not be permitted at any time.

[0246] Following the screening period, a Part C intervention period will be planned. The Part C intervention period will consist of up to 35 cycles of 21 days each, with AZD2936 administered every three weeks (Q3W), i.e., once every 21-day cycle.

[0247] In particular, eligible subjects will receive AZD2936 Q3W via intravenous (IV) infusion, starting on day 1 of cycle 1, for a maximum of 35 cycles. Subjects will be treated with the study intervention until disease progression, unacceptable toxicity, at the discretion of the study investigators, completion of a maximum of 35 treatment cycles, or withdrawal of consent. All subjects will be tracked for survival until the end of the study. Dose reduction of AZD2936 will not be permitted at any time.

[0248] After the intervention period (invention period), the subjects will be further evaluated for disease progression, their condition at the end of treatment, and their condition at follow-up.

[0249] Example 10: Part D Dose Expansion This embodiment describes the use of AZD2936 at 750 mg (Part D1) and 1500 mg (Part D2) doses for dose expansion studies.

[0250] The primary objective of Part D is to evaluate the safety and tolerability of AZD2936 at 750 mg and 1500 mg every three weeks (Q3W) in CPI-naive subjects with PD-L1 TPS ≥ 50% and stage IV NSCLC. Endpoints include the percentage of subjects with AEs and imAEs at each dose level, SAEs, DLT-like events, vital signs, and abnormal laboratory parameters; and the discontinuation rate of AZD2936 administration due to toxicity at each dose level.

[0251] The primary objective of Part D also includes evaluating the preliminary anti-tumor activity of AZD2936 at 750 mg and 1500 mg Q3W in CPI-naïve subjects with stage IV NSCLC who have PD-L1 with TPS ≥ 50%. The evaluation items include the ORR at each dose level according to RECIST v1.1.

[0252] The secondary objective of Part D also includes further characterizing the preliminary anti-tumor activity of AZD2936 at 750 mg and 1500 mg Q3W in CPI-naïve subjects with stage IV NSCLC who have PD-L1 (TPS ≥ 50% using the PD-L1 IHC assay). The evaluation items include, at each dose level, DCR, DoR, DRR, and PFS according to RECIST v1.1.

[0253] Subjects are randomized 1:1 to either AZD2936 750 mg Q3W (RP2D; n = 30) or AZD2936 1500 mg Q3W (n = 30) and are enrolled in the primary analysis of Part D. With a sample size of at least 30 subjects at each dose level, a preliminary ORR with SE ≤ 0.1 will be obtained. The two-sided 80% CIs for the possible response rate range of 30 subjects using the Clopper-Pearson method are provided below: 20% ORR (6 / 30 responders); 80% CI [11%, 32%], 30% ORR (9 / 30 responders); 80% CI [19%, 43%], 40% ORR (12 / 30 responders); 80% CI [28%, 53%], 47% ORR (14 / 30 responders); 80% CI [34%, 60%], 60% ORR (18 / 30 responders); 80% CI [47%, 72%].

[0254] Based on the decision framework (Frewer, et al, 2016), if the ORR TV is 45%, then if ≤9 objective RECIST responses (confirmed CR or PR) are observed (ORR ≤ 30%), the conclusion is reached that there is no evidence of target activity. If the true response rate is 45%, the probability of observing ≤9 responses in 30 evaluable subjects is ≤10%.

[0255] During the implementation of Part D, the emergence of toxicity that is not clearly attributable to the primary disease, other concomitant medications, the disease-related process under investigation, or another non-drug-related cause (not related to the treatment in the study intervention) will trigger the automatic discontinuation rule (hereinafter referred to as the discontinuation rule). In this scenario, if any of the following conditions occur, enrollment of new subjects in the study will be immediately stopped until the Safety Assessment Committee evaluates the benefits / risks of AZD2936. 1. If one case of Grade 5-related toxicity occurs at any point during the implementation of Part D. 2. If, at any point during the implementation of Part D, two cases of Grade 4-related toxicity occur in two different subjects at the same dose level. 3. If, in the initial 10 enrolled subjects, ≥4 SADR events occur in different subjects (i.e., ≥2 events in the same subject are counted as only 1), or if ≥7 SADR events occur in the initial 20 subjects at each dose level.

[0256] Following the screening period, a Part D intervention period will be planned. The Part D intervention period will consist of up to 35 cycles of 21 days each, with AZD2936 administered every three weeks (Q3W), i.e., once every 21-day cycle.

[0257] In particular, eligible subjects will receive AZD2936 Q3W via intravenous (IV) infusion at a dose of 750 mg or 1500 mg Q3W (1:1 randomization), starting on day 1 of cycle 1, for up to 35 cycles. Subjects will be treated with the study intervention until disease progression, unacceptable toxicity, investigator's judgment, completion of up to 35 treatment cycles, or withdrawal of consent. All subjects will be followed up for survival until the end of the study. After the intervention period, subjects will be further evaluated for disease progression, status at the end of treatment, and status at follow-up.

[0258] As described in Part A above, AZD2936 750 mg Q3W was identified as RP2D for further evaluation in the expansion phase of Part D. Furthermore, AZD2936 at a higher dose of 1500 mg Q3W will be evaluated in parallel with the 750 mg Q3W, which is PR2D. The 1500 mg dose was indicated as safe and tolerable by SRC. The 1500 mg dose was selected based on preliminary analysis showing similar peripheral blood target engagement with ≥90% RO at 750 mg and was predicted to achieve ≥90% intratumor RO in both PD-1 and TIGIT. On the other hand, as shown in Example 1 above, the lower dose of 210 mg was predicted to achieve suboptimal intratumor RO compared to 750 mg and 1500 mg. In investigating the 1500 mg dose level, Part D will assess whether a dose-efficacy plateau is reached at 750 mg Q3W, enabling the selection of the optimal dose to investigate in future trials.

[0259] In Part D, potential bias is reduced through the following process: All subjects are assigned to a randomized trial intervention using Interactive Response Technology (IRT) / Randomization and Trial Supply Management.

[0260] Example 11: Expanded results of the Part A and Part B dose escalation study The Part A dose escalation and Part B dose expansion studies of AZD2936 described above were continued. The inclusion criteria for subjects in Parts A and B were as provided in Example 5 above, and included: (i) having previously received both CPI and platinum-based chemotherapy (in combination or on a separate line); (ii) having confirmed disease progression while receiving treatment with a regimen including CPI; (iii) not having known genomic alterations with epidermal growth factor receptor (EGFR) mutations, anaplastic lymphoma kinase (ALK) fusions or other first-line approved targeted therapies; and (iv) not having previously discontinued CPI treatment due to toxicity. Abbreviations are as defined in Example 2 above. 83 subjects were enrolled and treated: 51 in Part A and 32 in Part B. Subjects had PD-L1 with TPS ≥ 1% and unresectable stage III or stage IV NSCLC that had been previously treated with CPI.

[0261] AZD2936 was administered intravenously once every three weeks (Q3W). Statistics for the subjects are provided in Table 8. Table 8: Baseline statistics and disease characteristics [Table 8]

[0262] Primary resistance is defined as resistance resulting from <6 months of exposure to CPI therapy, and secondary resistance is defined as resistance resulting from ≥6 months of exposure to CPI therapy. ECOG PS stands for Group Performance Status of Clinical Trials on the US East Coast. As can be seen in Table 8, most subjects had achieved resistance to previous CPI treatment.

[0263] At the time of data collection, the median treatment duration was 14 weeks (range: 1.7–95.6) in Part A and 10.7 weeks (range: 5.9–60.7) in Part B. No dose-limiting toxicities (DLTs) were observed during dose escalation (Part A). The RP2D was 750 mg based on the Part A data, and this was also supported by modeling analysis of predicted intratumor receptor occupancy. A summary of the study's safety is provided in Table 9. Treatment-induced adverse events (TEAEs) occurred in 90.4% of subjects at doses of 70–1500 mg, with 33.7% being grade 3 or higher, and in 94.4% of subjects at the 750 mg dose, with 31.5% being grade 3 or higher. Treatment-related adverse events (TRAEs) occurred in 53.0% of subjects in the 70-1500 mg range, with 8.4% being grade 3 or higher. In the 750 mg range, TRAEs occurred in 59.3% of subjects, with 9.3% being grade 3 or higher. Table 9: Safety Overview [Table 9]

[0264] Figure 5 summarizes the TEAEs and TRAEs that occurred in >5% of 83 patients treated with AZD2936 (20-1500 mg Q3W). Figure 6 summarizes the TEAEs and TRAEs that occurred in >5% of a subset of 54 patients treated with AZD2936 at the recommended Phase 2 dose (750 mg Q3W). As seen in the plots in Figures 5 and 6, no Grade 4 or 5 TRAEs occurred. Thirteen patients (15.7%) received steroids to treat TRAEs. Three patients (3.6%), all receiving 750 mg, discontinued AZD2936 due to AEs. These AEs were determined to be treatment-related in two patients and both recovered with steroids: myocarditis (Grade 1) and acute hepatitis (Grade 3). All of these cases occurred in Part A, with four patients (4.8%) experiencing fatal adverse events: ileus (n=2), pneumonia (n=1), and respiratory failure (n=1). None of these were determined to be related to AZD2936.

[0265] The efficacy of AZD2936 was analyzed by measuring the change in target lesion size. Figure 7 is a waterfall plot of the best percentage change from baseline in target lesion size in 82 subjects analyzed for all doses. Table 10 summarizes the antitumor activity in all 83 subjects. Overall, the median PFS was 2.1 months and the ORR was 6.0%. Table 10: Antitumor activity in subjects with CPI-resistant NSCLC [Table 10]

[0266] Figure 8 shows the percentage change from baseline in target lesion size over time in a subset of 54 patients treated with the recommended phase 2 dose (750 mg Q3W, parts A and B), summarized in the third column of Table 10. The median PFS was 3.8 months, and the ORR was 5.6%. Response and stability were generally sustained at this dose.

[0267] Based on the efficacy results described above, 20 patients remained stable for at least 6 months, most of whom (n=18) were administered a dose of 750 mg or 1500 mg; their PD-L1 TPS was 1-49% (n=10) or ≥50% (n=10).

[0268] Pharmacodynamics were measured in two subjects. Figures 9A and 9B show subjects treated with the recommended phase 2 dose, for which a radiological response was confirmed at week 9, respectively. Patient 1 (Figure 9A) was a 59-year-old woman with adenocarcinoma, PD-L1 ≥ 50%, exhibiting primary resistance and having previously received two lines of treatment. Patient 2 (Figure 9B) was a 53-year-old man with adenocarcinoma, PD-L1 ≥ 50%, exhibiting secondary resistance and having previously received eight lines of treatment. Figure 9C shows that ctDNA decreased by corresponding 100% at week 6 in both patients. Common features of these patients included a sustained PR of ≥ 6 months, PD-L1 TPS ≥ 50%, and ECOG PS 0.

[0269] In conclusion, this study demonstrated the following: • AZD2936 was safe across all tested doses. No dose-limiting time (DLT) was observed during dose escalation, and the medium-term dose (MTD) was not reached. Antitumor activity was observed, including at the recommended phase 2 dose, in patients with CPI-resistant advanced / metastatic NSCLC treated with AZD2936, who had received multiple previous treatments. The safety and preliminary efficacy observed in previously treated patients provide rationale for further testing of AZD2936 in CPI-naive patients with NSCLC.

[0270] Example 12: Preliminary Part C Results In Part C of the experiment described in the above example, 19 subjects were evaluated.

[0271] Table 11 shows the available statistics for the seven subjects evaluated in Part C. Table 12 shows the available medical conditions for the seven subjects evaluated in Part C. As shown in Table 12, most subjects had metastatic disease and squamous epithelial tissue type. Adverse events that occurred in more than 10% of the subjects in Part C are plotted in Figure 11. Adverse events related to AZD2936 are plotted on the right side of the figure. One case of SAE potentially related to AZD2936 was observed: immune-mediated hepatitis. Table 11: Statistics of subjects evaluated in Part C [Table 11] Table 12: Medical conditions of subjects evaluated in Part C [Table 12]

[0272] The efficacy results are provided in Table 13. Table 13: Results of the initial effectiveness of the test in Part C [Table 13] Table 14: Summary of Results [Table 14]

[0273] As shown in Table 13, the overall response rate (ORR) with 750 mg Q3W administration was 57.1%, all three subjects remained stable, and the disease complete response (DCR) was 100%.

[0274] Tables 13 and 14 summarize the initial results obtained in Part C in CPI-naive subjects. Nineteen subjects with NSCLC were treated as described above, and seven were evaluable for efficacy. The preliminary AZD2936 safety profile remains favorable, and the initial efficacy is promising.

[0275] Example 13: Preliminary Part D Results In Part D of the study described in the above example, 27 subjects were evaluated. This included 14 patients on D1 (750 mg Q3W) and 13 patients on D2 (1500 mg Q3W).

[0276] Table 15 shows the available statistics for the 11 subjects evaluated in Part D. Table 16 shows the available medical conditions for the 11 subjects evaluated in Part D. As shown in Table 16, most subjects had metastatic disease, with squamous cell carcinoma and adenocarcinoma being the most common histological types. Adverse events that occurred in more than 10% of subjects treated with AZD2936 1500 mg Q3W are plotted in Figure 12. Adverse events that occurred in more than 10% of subjects treated with AZD2936 750 mg Q3W are plotted in Figure 13. Adverse events related to AZD2936 are shown to the right of both plots. One potentially AZD2936-related SAE was observed: elevated alanine aminotransferase (occurred in Part D2 with 15000 mg Q3W). Table 15: Statistics of subjects evaluated in Part D [Table 15] Table 16: Medical conditions of subjects evaluated in Part D [Table 16]

[0277] The results of the efficacy study are provided in Table 17. Table 17: Results of the initial efficacy of the trial [Table 17] Table 18: Summary of Results [Table 18]

[0278] As shown in Table 17, with 750 mg Q3W administration, the ORR was 28.6%, with 5 subjects remaining stable and a DCR of 100%. With 1500 mg Q3W administration, the ORR was 20%, with 2 subjects remaining stable and a DCR of 60%.

[0279] Tables 17 and 18 summarize the initial results obtained in Part D for CPI-naive subjects. Of the 27 subjects with NSCLC, the treatment was as described above, and 12 were evaluable for efficacy. The difference in ORR observed between cohorts across Parts C and D likely reflects sample size and will change over time.

[0280] Figure 14 shows the best percentage change from baseline in target lesions for all 19 subjects with CPI-naive NSCLC who received AZD2936 at 750 mg and 1500 mg Q3W in Parts C and D. Figure 15 shows the change in target lesion size over time for the 19 subjects with CPI-naive NSCLC who received AZD2936 at 750 mg and 1500 mg in Parts C and D. The preliminary AZD2936 safety profile remains favorable, and initial efficacy is promising.

Claims

1. A method for treating cancer in a subject, comprising administering to the subject in an amount of approximately 70 mg to approximately 1500 mg a bispecific binding protein that specifically binds to programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domain (TIGIT), wherein the bispecific binding protein a) A first binding domain that specifically binds to PD-1, wherein the first binding domain comprises a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, and HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 4, LCDR2 having the amino acid sequence of SEQ ID NO: 5, and LCDR3 having the amino acid sequence of SEQ ID NO: 6; and b) A second binding domain that specifically binds to TIGIT, wherein the second binding domain comprises a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 11, HCDR2 having the amino acid sequence of SEQ ID NO: 12, and HCDR3 having the amino acid sequence of SEQ ID NO: 13, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 14, LCDR2 having the amino acid sequence of SEQ ID NO: 15, and LCDR3 having the amino acid sequence of SEQ ID NO:

16. Methods that include...

2. The method according to claim 1, wherein the amount of bispecific binding protein administered is approximately 70 mg, approximately 150 mg, approximately 210 mg, approximately 450 mg, approximately 750 mg, approximately 800 mg, approximately 850 mg, approximately 900 mg, approximately 950 mg, approximately 1000 mg, approximately 1250 mg, or approximately 1500 mg.

3. The method according to claim 2, wherein the amount of bispecific binding protein administered is approximately 750 mg.

4. The method according to claim 2, wherein the amount of bispecific binding protein administered is approximately 1500 mg.

5. The method according to any one of the preceding claims, wherein the bispecific binding protein is administered once per treatment cycle.

6. The method according to claim 5, wherein the treatment cycle is approximately 7 days, approximately 14 days, approximately 21 days, approximately 28 days, or approximately 35 days.

7. The method according to claim 5, wherein the treatment cycle is approximately 7 days.

8. The method according to any one of claims 5 to 7, wherein the treatment cycle is repeated for a maximum of 35 cycles.

9. The method according to any one of the preceding claims, wherein a bispecific binding protein is administered to a subject as monotherapy.

10. The method according to any one of the preceding claims, wherein the bispecific binding protein is administered by intravenous infusion (IV).

11. The method according to any one of the preceding claims, wherein the subject has not received a prior treatment line for systemic therapy.

12. The method according to any one of claims 1 to 10, wherein the subject has previously received chemotherapy.

13. The method according to any one of the preceding claims, wherein the cancer comprises cancer cells expressing PD-L1.

14. The method according to any one of claims 1 to 13, wherein the first binding domain of a bispecific binding protein that specifically binds to PD-1 comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having the amino acid sequence of SEQ ID NO:

9.

15. The method according to any one of claims 1 to 13, wherein the first binding domain of a bispecific binding protein that specifically binds to PD-1 includes a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

9.

16. The method according to any one of claims 1 to 13, wherein the first binding domain of a bispecific binding protein that specifically binds to PD-1 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 8 and a light chain having the amino acid sequence of SEQ ID NO:

10.

17. The method according to any one of claims 1 to 13, wherein the first binding domain of a bispecific binding protein that specifically binds to PD-1 comprises a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

10.

18. The method according to any one of claims 1 to 17, wherein the second binding domain of a bispecific binding protein that specifically binds to TIGIT comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain having the amino acid sequence of SEQ ID NO:

19.

19. The method according to any one of claims 1 to 17, wherein the second binding domain of a bispecific binding protein that specifically binds to TIGIT comprises a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

19.

20. The method according to any one of claims 1 to 17, wherein the second binding domain of a bispecific binding protein that specifically binds to TIGIT comprises a heavy chain having the amino acid sequence of SEQ ID NO: 18 and a light chain having the amino acid sequence of SEQ ID NO:

20.

21. The method according to any one of claims 1 to 17, wherein the second binding domain of a bispecific binding protein that specifically binds to TIGIT comprises a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 18 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

20.

22. The method according to any one of the preceding claims, wherein the bispecific binding protein is a human or humanized bispecific antibody or its antigen-binding fragment.

23. The method according to any one of the preceding claims, wherein the bispecific binding protein comprises a variant Fc region.

24. When the variant Fc region of the bispecific binding protein is numbered according to the EU index defined by Kabat, the following are obtained: 221K, 221Y, 225E, 225K, 225W, 228P, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235E, 235F, 236E, 237L, 237M, 237P, 239D, 239E, 239N, 23 9Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E , 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 250E, 2 50Q, 251F, 252L, 252Y, 254S, 254T, 255L, 256E, 256F, 256M, 257C, 257M, 257 N, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 2 64R, 264F, 264M, 264Y, 264E, 265A, 265G, 265N, 265Q, 265Y, 265F, 265V, 265 I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 29 6T, 296L, 296I, 296H, 296G, 297S, 297D, 297E, 298A, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 308F, 313F, 316D, 31 8A, 318S, 320A, 320S, 322A, 322S, 325Q, 325L, 3251, 325D, 325E, 325A, 325T, 325V, 325H, 326A, 326D, 326E, 326G, 326M, 326V, 327G, 327W, 327N, 327L, 32 8S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F,329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331 Q, 331E, 331S, 331V, 3311, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 33 The method according to claim 23, comprising at least one substitution selected from 2Y, 332A, 333A, 333D, 333G, 333Q, 333S, 333V, 334A, 334E, 334H, 334L, 334M, 334Q, 334V, 334Y, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 428L, 428F, 433K, 433L, 434A, 434W, 434Y, 436H, 440Y, and 443W.

25. The method according to claim 23, wherein the variant Fc region of the bispecific binding protein comprises one or more amino acid substitutions at positions selected from 428 and 434 when numbered by the EU index defined in Kabat.

26. The method according to any one of claims 23 to 25, wherein the variant Fc region of the bispecific binding protein comprises one or more amino acid substitutions selected from 428L, 428F, 434A, 424F, 434W, and 434Y.

27. The method according to any one of claims 23 to 26, wherein the variant Fc region of the bispecific binding protein contains a YTE mutation.

28. The method according to claim 23 or 24, wherein the Fc variant region of the bispecific binding protein contains the L234F / L235E / P331S triple mutation (TM).

29. The method according to any one of claims 23 to 28, wherein the Fc region of the bispecific binding protein is not glycosylated (aglycosylated).

30. The method according to any one of claims 23 to 28, wherein the Fc region of the bispecific binding protein is deglycosylated.

31. The method according to any one of claims 23 to 28, wherein the Fc region of the bispecific binding protein has reduced fucosylation or is not fucosylated.

32. The method according to any one of the preceding claims, wherein the bispecific binding protein comprises a kappa light chain constant region.

33. The method according to any one of claims 1 to 32, wherein the bispecific binding protein includes a constant region of the lambda light chain.

34. The method according to any one of the preceding claims, wherein the bispecific binding protein is an antibody.

35. The method according to claim 34, wherein the antibody is an IgG antibody.

36. The method according to claim 35, wherein the antibody is an IgG1 antibody.

37. The method according to any one of claims 34 to 36, wherein the antibody is humanized.

38. The method according to any one of the preceding claims, wherein the cancer is one or more of ovarian cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, pancreatic cancer, renal cell carcinoma, and lung cancer.

39. The method according to claim 38, wherein the cancer is non-small cell lung cancer (NSCLC).

40. The method according to claim 39, wherein NSCLC is progressing or has metastasized.

41. The method according to claim 39, wherein the subject has a PD-L1 tumor percentage score of 1% or more.

42. The method according to claim 39, wherein the subject has a PD-L1 tumor percentage score of 50% or more.

43. The method according to any one of claims 39 to 42, wherein the subject is checkpoint inhibitor (CPI) naive.

44. A pharmaceutical composition comprising a bispecific binding protein that specifically binds to PD-1 and TIGIT in an amount of approximately 70 mg to approximately 1500 mg, wherein the bispecific binding protein is a) A first binding domain that specifically binds to PD-1, wherein the first binding domain comprises a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, and HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 4, LCDR2 having the amino acid sequence of SEQ ID NO: 5, and LCDR3 having the amino acid sequence of SEQ ID NO: 6; and b) A second binding domain that specifically binds to TIGIT, wherein the second binding domain comprises a heavy chain variable domain including HCDR1 having the amino acid sequence of SEQ ID NO: 11, HCDR2 having the amino acid sequence of SEQ ID NO: 12, and HCDR3 having the amino acid sequence of SEQ ID NO: 13, and a light chain variable domain including LCDR1 having the amino acid sequence of SEQ ID NO: 14, LCDR2 having the amino acid sequence of SEQ ID NO: 15, and LCDR3 having the amino acid sequence of SEQ ID NO:

16. A pharmaceutical composition containing the following:

45. The pharmaceutical composition according to claim 44, wherein the pharmaceutical composition comprises approximately 70 mg, approximately 150 mg, approximately 210 mg, approximately 450 mg, approximately 750 mg, approximately 800 mg, approximately 850 mg, approximately 900 mg, approximately 950 mg, approximately 1000 mg, approximately 1250 mg, or approximately 1500 mg of bispecific binding protein.

46. The pharmaceutical composition according to claim 44, wherein the pharmaceutical composition comprises about 750 mg of bispecific binding protein.

47. The pharmaceutical composition according to claim 44, wherein the pharmaceutical composition contains about 1500 mg of bispecific binding protein.

48. The pharmaceutical composition according to any one of claims 44 to 47, wherein the first binding domain of a bispecific binding protein that specifically binds to PD-1 comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having the amino acid sequence of SEQ ID NO:

9.

49. The pharmaceutical composition according to any one of claims 44 to 47, wherein the first binding domain of a bispecific binding protein that specifically binds to PD-1 comprises a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

9.

50. The pharmaceutical composition according to any one of claims 44 to 47, wherein the first binding domain of a bispecific binding protein that specifically binds to PD-1 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 8 and a light chain having the amino acid sequence of SEQ ID NO:

10.

51. The pharmaceutical composition according to any one of claims 44 to 47, wherein the first binding domain of a bispecific binding protein that specifically binds to PD-1 comprises a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

10.

52. The pharmaceutical composition according to any one of claims 44 to 47, wherein the second binding domain of a bispecific binding protein that specifically binds to TIGIT comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain having the amino acid sequence of SEQ ID NO:

19.

53. The pharmaceutical composition according to any one of claims 44 to 47, wherein the second binding domain of a bispecific binding protein that specifically binds to TIGIT comprises a heavy chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

19.

54. The pharmaceutical composition according to any one of claims 44 to 47, wherein the second binding domain of a bispecific binding protein that specifically binds to TIGIT comprises a heavy chain having the amino acid sequence of SEQ ID NO: 18 and a light chain having the amino acid sequence of SEQ ID NO:

20.

55. The pharmaceutical composition according to any one of claims 44 to 47, wherein the second binding domain of a bispecific binding protein that specifically binds to TIGIT comprises a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 18 and a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

20.

56. A kit comprising the pharmaceutical composition according to any one of claims 44 to 55.

57. The kit according to claim 56, further comprising instructions for administering a pharmaceutical composition.

58. A pharmaceutical composition according to any one of claims 44 to 55 for use in the treatment of cancer.

59. The pharmaceutical composition for use according to claim 58, wherein the cancer is one or more of ovarian cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, pancreatic cancer, renal cell carcinoma, and lung cancer.

60. The pharmaceutical composition for use according to claim 59, wherein the cancer is non-small cell lung cancer (NSCLC).

61. A pharmaceutical composition for use according to claim 60, wherein NSCLC is progressing or has metastasized.

62. A pharmaceutical composition for use according to claim 60 or 61, wherein the cancer has a PD-L1 tumor percentage score of 1% or more.

63. A pharmaceutical composition for use according to claim 60 or 61, wherein the cancer has a PD-L1 tumor percentage score of 50% or more.

64. A pharmaceutical composition for use according to any one of claims 60 to 63, wherein the cancer has not been previously treated with a checkpoint inhibitor.

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